Neurodegeneration_(1617611190)

NEUROSCIENCE RESEARCH PROGRESS

NEURODEGENERATION: THEORY, DISORDERS AND TREATMENTS No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services.

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NEUROSCIENCE RESEARCH PROGRESS

NEURODEGENERATION: THEORY, DISORDERS AND TREATMENTS

ALEXANDER S. MCNEILL EDITOR

Nova Science Publishers, Inc. New York

Copyright © 2011 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com

NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers‟ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Additional color graphics may be available in the e-book version of this book. Library of Congress Cataloging-in-Publication Data Neurodegeneration : theory, disorders, and treatments / editors, Alexander S. McNeill. p. ; cm. Includes bibliographical references and index. ISBN 978-1-61324-178-3 (eBook) 1. Nervous system--Degeneration. I. McNeill, Alexander S. [DNLM: 1. Neurodegenerative Diseases. WL 359] RC365.N453 2010 616.99'48--dc22 2010026969

Published by Nova Science Publishers, Inc. † New York

Contents Preface Chapter 1

Chapter 2

Chapter 3

Chapter 4

Chapter 5

Chapter 6 -

Chapter 7

vii Oxidative Stress-Mediated Neurodegeneration: A Tale of Two Models Paul K. Y. Wong, Jeesun Kim, Soo Jin Kim, Xianghong Kuang and William S. Lynn

1

Mechanisms of the Motoneuron Stress Response and Its Relevance in Neurodegeneration Mac B. Robinson, David J. Gifondorwa and Carol Milligan

45

Methylene Blue Induces Mitochondrial Complex IV and Improves Cognitive Function and Grip Strength in old Mice Afshin Gharib and Hani Atamna

63

Receptor Specific Features of Excitotoxicity Induced Neurodegeneration Sergei M. Antonov and Dmitrii A. Sibarov

87

Mitochondrial Uncoupling Proteins – Therapeutic Targets in Neurodegeneration? Susana Cardoso, Cristina Carvalho, Sónia Correia, Renato X. Santos, Maria S. Santos and Paula I. Moreira

107

Targeting Caspases in Neonatal Hypoxic Ischemic Brain Injury and Traumatic Brain Injury Xin Wang, Rachna Pandya, Jiemin Yao, He Ma and Jianmin Li

125

Alterations in N-Methyl-D-Aspartate (NMDA) Receptor Function and Potential Involvement in AnestheticInduced Neurodegeneration Cheng Wang, Xuan Zhang, Fang Liu, Merle G. Paule and William Slikker Jr.

155

vi Chapter 8

Contents Genetics and Molecular Biology of Alzheimer's Disease and Frontotemporal Lobar Degeneration: Analogies and Differences Daniela Galimberti, Chiara Fenoglio and Elio Scarpini

Chapter 9

The Cholinergic Neuron in Alzheimer‟s Disease Christian Humpel and Celine Ullrich

Chapter 10

Retinal Neurodegeneration Is an Early Event in the Pathogenesis of Diabetic Retinopathy: Therapeutic Implications Rafael Simó and Cristina Hernández

Chapter 11

Index

Molecular Imaging and Parkinson‟s Disease Valentina Berti, Cristina Polito, Maria T. R. De Cristofaro and Alberto Pupi.

173 189

203 215

221

Preface Neurodegeneration is the umbrella term for the progressive loss of structure or function of neurons, including the death of neurons. Many neurodegenerative diseases including Parkinson‟s, Alzheimer‟s, and Huntington‟s occur as a result of neurodegenerative processes. This book presents current research in the study of neurodegeneration, including oxidative stress-mediated neurodegeneration; preserving motoneuron viability and function during disease or after traumatic injury; research in aspects of excitotoxicity mechanisms; uncoupling proteins as therapeutic targets in stroke and neurodegenerative diseases; the genetics and molecular biology of Alzheimer's Disease; and retinal neurodegeneration in diabetic retinopathy. Chapter 1 - Maintenance of a balanced redox status within cells provides a healthy environment for cellular functions and is critical to the fate of the cell. Alterations in cellular redox status affect many redox sensitive activities including signal transduction, DNA and protein synthesis, and protein folding. Significant or prolonged deviations in the intracellular redox status disrupt cellular processes leading to numerous disease conditions. This chapter focuses on two mouse models of two human diseases that display disruption of the redox status of the cells leading to oxidative stress-mediated neurodegeneration (ND). One model represents the human childhood genetic disorder ataxia telangiectasia (A-T) lacking a functional ATM (A-T mutated) protein kinase. A-T is primarily a neurodegenerative disorder that also affects other systems in the human body. Since one of the key functions of the ATM protein is to maintain normal cellular redox status, the absence of a functional ATM in cells of the central nervous system (CNS) results in chronic oxidative stress leading to ND. A second model represents the human HIV-associated dementia (HAD) and other neurological diseases associated with the accumulation of misfolded proteins. This model uses a murine retrovirus called ts1 (a mutant of Moloney murine leukemia virus) that causes oxidative stress, endoplasmic reticulum (ER) stress, and mitochondrial impairment as a result of virus infection and accumulation of misfolded viral envelope protein in the ER of astrocytes. Neurons are not productively infected by retroviruses thus neuronal loss induced by these retroviruses is not directly due to productive infection of neurons, but rather due to the infection of other cells in the CNS, including astrocytes, oligodendrocytes, microglia and endothelial cells. The goals are to understand the pathogenic mechanisms for both of these diseases thereby helping to develop drugs to prevent neuronal cell loss. Recently the authors have found that neurological symptoms of both disease models can be prevented by treatment with redox-

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Alexander S. McNeill

active drugs, notably phthalazine dione, without repairing the initial causes. This finding suggests that these two animal models share oxidative stress in the CNS as a common mechanism of neuropathogenesis, although they have different initial causes, one from genetic mutation and the other from viral infection. This chapter brings together two important translational topics: elucidation of neurodegenerative mechanisms and development of therapeutic treatment. These two models will provide insight into the pathology of oxidative stress-mediated cell death and demonstrate how mouse models can help in understanding human diseases. Insights from this understanding may enable us to progress toward improved treatments in humans, not only for neurodegeneration (ND) but also other related debilitating diseases resulting from oxidative stress. Chapter 2 - Preserving motoneuron viability and function during disease or after traumatic injury is an intense area of research focusing on both the molecular mechanisms of degeneration and therapeutic interventions to prevent it. Understanding how motoneurons sense and respond to injury or pathology may help us identify potential targets for therapeutic intervention. The motoneuron stress response or heat stress response (HSR) has been an area of investigation spanning now well over a decade and has explored the role of heat shock protein (HSP) expression during physiological stress and in animal models of neurodegenerative disease. What we have found from these studies is that, in the midst of a physiological stress, motoneurons rarely activate a classical stress response as characterized by increased expression of Hsp70. It has been proposed that this lack of stress response activation could contribute to pathological motoneuron dysfunction and degeneration. Understanding the molecular mechanisms responsible for this phenomenon may provide insights as to why motoneurons are the pathological hallmark in amyotrophic lateral sclerosis (ALS) and other neurodegenerative conditions. Chapter 3 - Methylene blue (MB) is very effective in delaying cellular senescence and enhancing mitochondrial activity of primary human embryonic fibroblasts. At nanomolar concentrations, MB increased the activity of mitochondrial cytochrome c oxidase (complex IV), heme synthesis, cell resistance to oxidants, and oxygen consumption. MB is the most effective among the many agents that has been are reported to delay cellular senescence. The authors extended these in vitro findings to the investigation of the effect of long-term intake of MB in old mice. The authors administered MB, in the drinking water (250 µM), to old mice for 90 days. In vivo, MB prevented the age-related decline in cognitive function and spatial memory. MB also prevented the age-related decline in grip strength. Interestingly, MB resulted in 100 % and 50 % increases in complex IV activities in the brains and hearts of old mice, respectively. The age-related decline in protein content of the brain was prevented by MB. We also found a 39 % decrease in brain monoamine oxidase (MAO) activity in old mice treated with MB while aging or MB did not affect the activity of brain NQO1. The findings suggest that the in vitro model for cell senescence may be used for fast and reliable screening for mitochondria-protecting candidate agents before testing in animal models. The study also demonstrates simultaneous enhancement of mitochondrial function, improvement of the cognitive function, and improvement of grip strength in old mice by a drug. Since these are three major concerns in human aging, MB may be a useful agent for delaying neurodegeneration and physical impairments associated with aging. Chapter 4 - Excitotoxicity is a term that describes the neuronal death caused by neurotoxic effects of glutamate, which is the most abundant excitatory neurotransmitter in the

Preface

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vertebrate central nervous system. Glutamate is well known to be involved in cognitive functions like learning and memory, but its excessive accumulation in extracellular space can lead to neuronal damages and eventual cell death via necrosis and apoptosis. As a result excitotoxicity contributes to pathogenesis of numerous neurodegenerative diseases. Both normal function and pathological action imply an activation of the same glutamate receptors particularly of NMDA- (N-methyl-D-aspartate), AMPA- (α-anino-3-hydroxyl-5-methyl-4isoxazole-propionate) and KA- (kainate) subtypes. Many achievements in the mechanisms of neurodegeneration were obtained using different experimental approaches on primary neuronal cultures. Double successive acridine orange and ethidium bromide staining combined with confocal microscopy offers fast, easy, sensitive and reproducible method by which necrosis and apoptosis can be recognized and quantified in a population of living neurons. Together with immunostaining they provide many research advantages and allow analysis of protein expression patterns. The growing quantity of evidence reveals the diversity of apoptosis cascades. Whereas our data show the same profiles of excitotoxicity for NMDA and KA, we found receptor subtype specific differences in neuronal death mechanisms. For example, apoptosis caused by prolonged NMDA receptors activation develops through the caspase-independent cascades via release of apoptosis inducing factor (AIF) from mitochondria and its direct action on nuclear chromatin. In contrast AMPA and KA receptors mediated apoptosis includes caspasedependent pathway. On the basis of our data and literature the chapter will review the contemporary state of research concerning the aspects of excitotoxicity mechanisms discussed above. Chapter 5 - Uncoupling proteins (UCPs) are mitochondrial inner membrane proteins that uncouple electron transport from ATP production by dissipating protons across the inner membrane. UCP1 was the first uncoupling protein described and is present in brown adipose tissue being involved in the non-shivering thermogenesis. Subsequent studies demonstrated that neurons express at least three UCPs isoforms including the widely expressed UCP2 and the neuron-specific UCP4 and UCP5. UCPs control the mitochondrial membrane potential, free radicals production and calcium homeostasis and thereby influence neuronal function. Given that mitochondrial energy impairment and free radicals production are thought to be central players in neurodegeneration, recent data suggest that UCPs may have an important role in neuroprotection and neuromodulation. The function of neuronal UCPs and their impact on the central nervous system are attracting an increased interest as potential therapeutic targets in several disorders including neurodegenerative diseases. Here the authors will discuss the uncoupling process as an intrinsic mechanism of mitochondria physiology. The role of UCPs in healthy and pathological brain conditions will be also considered. Finally, they will discuss UCPs as potential therapeutic targets in stroke and neurodegenerative diseases. Chapter 6 - Mounting evidence implicates apoptosis in the pathogenesis of both acute and chronic neurological disorders. The caspase family of cysteine proteases plays a central role in the initiation and execution of neuronal apoptosis. So far the caspase family has been expanded to 18 cysteine protease members. About two decades of investigation involving the caspase family has produced a wealth of information. Studies indicate that targeting the caspase family can prevent neuronal cell death in neurological disorders. This chapter will discuss the role of the caspase family in experimental models of neonatal hypoxia-ischemia brain injury and traumatic brain injury in vivo and in vitro, as well as in human neonatal

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hypoxic-ischemic encephalopathy and traumatic brain injury. Given that elucidation of the roles of individual caspases could yield multiple points of possible therapeutic intervention, from the drug discovery and treatment perspective, the review will summarize what is currently known about the beneficial effects of targeting caspases using a variety of treatments against neonatal hypoxia-ischemia brain injury and traumatic brain injury. It will focus on commonalities in the inhibition of caspase in the cell death receptor pathway, the mitochondrial death pathway and the endoplasmic reticulum death pathway. Chapter 7 - Advances in pediatric and obstetric surgery have resulted in an increase in the duration and complexity of anesthetic procedures. It is known that the most frequently used general anesthetics have either NMDA receptor blocking or γ-aminobutyric acid (GABA) receptor activating properties. It is also known that anesthetic agents can cause widespread and dose-dependent apoptotic neurodegeneration in the developing brain. Exposure of developing mammals to NMDA-type glutamate receptor antagonists affects the endogenous NMDA receptor system and enhances neuronal cell death. The NMDA receptor regulates a calcium channel and calcium influx that overwhelms the mitochondrial buffering capacity can result in increased production of reactive oxygen species (ROS) and cell death. Meanwhile, stimulation of immature GABA receptors is thought to be excitatory early in development but inhibitory in mature neurons. Stimulation of immature neurons by GABA agonists is thus thought to increase overall nervous system excitability and may contribute to NMDA receptor-associated increased excitability during early development. This increased excitability may contribute to abnormal neuronal cell death during development. The type of excitotoxic insults that lead to neuronal apoptosis or necrosis are not adequately understood but surely depend upon animal species, the concentration of stressors, durations of exposures, the receptor subtypes activated and the stage of development or maturity of a particular cell type at the time of exposure. It has been proposed that prolonged blockade of the NMDA receptor in the developing brain by NMDA receptor antagonists such as the dissociative anesthetics ketamine or phencyclidine (PCP) causes a compensatory upregulation of NMDA receptors. Neurons bearing these up-regulated receptors are subsequently more vulnerable to the excitotoxic effects of endogenous glutamate, because this up-regulation of NMDA receptors allows for the influx of toxic levels of intracellular Ca2+ under normal physiological conditions. Although many more studies will be necessary in order to develop adequate quantitative models to explain the relationships between altered NMDA receptor function and anestheticinduced neurodegeneration, a general hypothesis has been constructed and tested in an interactive manner using carefully selected agents as defined by their pharmacological and physiological properties. The integrative and iterative evaluation of these kinds of models will lead to a better understanding of the potential neurotoxicity of NMDA antagonists and GABA agonists in the developing human. Chapter 8 - Alzheimer‟s disease (AD) is the most common cause of dementia in the elderly, whereas Frontotemporal Lobar Degeneration (FTLD) is the most frequent neurodegenerative disorder with a presenile onset. The two major neuropathologic hallmarks of AD are extracellular Amyloid beta (A) plaques and intracellular neurofibrillary tangles (NFTs). Conversely, in FTLD the deposition of tau has been observed in a number of cases, but in several brains there is no deposition of tau but instead a positivity for ubiquitin.

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In some families these diseases are inherited in an autosomal dominant fashion. Genes responsible for familial AD include the Amyloid Precursor Protein (APP), Presenilin 1 (PS1) and Presenilin 2 (PS2). The majority of mutations in these genes are often associated with a very early onset (40-50 years of age). Regarding FTLD, the first mutations described are located in the Microtubule Associated Protein Tau gene (MAPT). Tau is a component of microtubules, which represent the internal support structures for the transport of nutrients, vesicles, mitochondria and chromosomes within the cell. Mutations in MAPT are associated with an early onset of the disease (40-50 years), and the clinical phenotype is consistent with Frontotemporal Dementia (FTD). Recently, mutations in a second gene, named progranulin (GRN), have been identified in some families with FTLD. Progranulin is expressed in neurons and microglia and displays anti-inflammatory properties. Nevertheless, it can be cleaved into granulins which, conversely, show inflammatory properties. The pathology associated with these mutations is most frequently characterized by the immunostaining of TAR DNA Binding Protein 43 (TDP-43), which is a transcription factor. The clinical phenotype associated with GRN mutations is highly heterogeneous, including FTD, Progressive Aphasia, Corticobasal Syndrome, and AD. Age at disease onset is variable, ranging from 45 to 85 years of age. The majority of cases of AD and FTLD are however sporadic, and likely several genetic and environmental factors contribute to their development. Concerning AD, it is known that the presence of the 4 allele of the Apolipoprotein E gene is a susceptibility factor, increasing the risk of about 4 fold. A number of additional genetic factors, including cytokines, chemokines, Nitric Oxide Synthases, contribute to the susceptibility for the disease. Some of them also influence the risk to develop FTLD. In this chapter, current knowledge on molecular mechanisms at the basis of AD and FTLD, as well as the role of genetics, will be presented and discussed. Chapter 9 - Alzheimer´s disease (AD) is a chronic brain disorder characterized by cognitive decline, neuronal and synaptic loss, beta-amyloid-containing plaques, neurofibrillary tangles, inflammation and cerebrovascular damage. Numerous studies revealed that cholinergic neurons in the basal forebrain (septum, diagonal band of Broca, basal nucleus of Meynert) are affected in AD and a loss of acetylcholine directly correlates with memory dysfunction. (1) We will give an overview on the cholinergic neurons in the basal forebrain and discuss the role of the key enzyme choline acetyltransferase (ChAT). (2) We review the protective role of nerve growth factor (NGF) to support the cholinergic phenotype. (3) We demonstrate different in vitro and in vivo models, which are used to study cholinergic CNS neurons. (4) We reconsider if cholinergic neurons degenerate in AD or if cholinergic neurons only downregulate the key enzyme ChAT. (5) Finally, our review will summarize recent therapeutic strategies on augmenting cholinergic neurotransmission to improve or reverse cognitive deficits in AD. In summary our review focuses on the cholinergic CNS neurons and their role in AD. Alzheimer‟s disease is a severe and chronic degenerative disorder characterized by a progressive neurodegeneration, amyloid-containing plaques, neurofibrillary tangles, as well as cognitive dysfunction. Cholinergic neurons in the basal forebrain are located in six main central nuclei (Ch1-Ch6). The key enzymes for the cholinergic system, choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) can be used for immunohistochemical staining and characterization of the system. Essential for the development and survival of cholinergic neurons in the basal forebrain is the nerve growth

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factor (NGF). The cholinergic neurotransmitter system in the basal forebrain is severely affected in AD and loss of the neurotransmitter acetylcholine directly correlates with cognitive dysfunction (Perry et al., 1981; Francis et al., 1985). Basic research of the neuropathologic hallmarks and treatment strategies in AD is a fundamental goal, due to immense costs of caring for patients with AD. The current review will highlight present knowledge of the cholinergic dysfunction in AD and will demonstrate different models, which are used to study AD, as well as possible therapeutic approaches. Chapter 10 - Diabetic retinopathy (DR) remains the leading cause of blindness among working-age individuals in developed countries. Although tight control of both blood glucose levels and hypertension are essential to prevent or arrest progression of the disease, the recommended goals are difficult to achieve in many patients and, consequently, DR develops during the evolution of the disease. Therefore, new therapeutic strategies based on the understanding of the pathophysiological mechanisms of DR are needed. DR has been classically considered to be a microcirculatory disease of the retina due to the deleterious metabolic effects of hyperglycemia per se, and the metabolic pathways triggered by hyperglycemia. However, before any microcirculatory abnormalities can be detected in ophthalmolscopic examination, retinal neurodegeneration is already present. The two main features of retinal neurodegeneration are apoptosis and glial activation. Most of the information regarding retinal neurodegeneration has been obtained from rats with streptozotocin-induced diabetes (STZ-DM). Streptozotocin (STZ) is a potent neurotoxic agent and is able to produce neural degeneration. Therefore, neurodegeneration observed in rats with STZ-DM could be due to STZ itself rather than the metabolic pathways related to diabetes. However, the recent observation that both apoptosis and glial activation also occur in the retina of diabetic patients, even before any microvascular abnormality could be detected in ophthalmologic examination, reinforces the concept that neurodegeneration is a crucial pathogenic factor of DR. Neuroretinal damage produces functional abnormalities such as the loss of both chromatic discrimination and contrast sensitivity. These alterations can be detected by means of electrophysiological studies in diabetic patients with less than two years of diabetes duration, that is before microvacular lesions can be detected in ophthalmologic examination. In addition, neuroretinal degeneration subsequently initiates and/or activates several metabolic and signaling pathways which participate in the microangiopathic process, as well as in the disruption of the blood-retinal barrier (a crucial element in the pathogenesis of DR). Therefore, the study of the mechanisms that lead to neurodegeneration will be essential for identifying new therapeutic targets in the early stages of DR. Chapter 11 - Parkinson‟s disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic (DA) terminals in the striatum, resulting in functional changes in frontostriatal circuits. DA transporter imaging ([123I]FP-CIT SPECT imaging) and brain metabolic imaging 18F ([ ]FDG PET imaging) have been broadly employed to explore the biological substrate of PD, and together they could highlight the pathological processes occurring in early stages of PD. To evaluate the functional association between DA degeneration and cortical metabolism we performed both [123I]FP-CIT SPECT and [18F]FDG PET in the same PD sample; through a multiple regression analysis with SPM we explored the correlation between putaminal DA degeneration and cortical metabolic rate of glucose.

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In the putamen, which is the first and most affected striatal region in PD, the severity of dopaminergic impairment is directly related to cortical hypometabolism in premotor, dorsolateral prefrontal, anterior prefrontal and orbitofrontal cortices. [123I]FP-CIT SPECT and [18F]FDG PET allow to identify the early functional alterations in the frontostriatal circuits involved in PD.

In: Neurodegeneration: Theory, Disorders and Treatments ISBN: 978-1-61761-119-3 Editor: Alexander S. McNeill © 2011 Nova Science Publishers, Inc.

Chapter 1

Oxidative Stress-Mediated Neurodegeneration: A Tale of Two Models Paul K. Y. Wong, Jeesun Kim, Soo Jin Kim, Xianghong Kuang and William S. Lynn

Department of Carcinogenesis, The University of Texas, MD Anderson Cancer Center, Science Park-Research Division, Smithville, Texas, USA

Abstract Maintenance of a balanced redox status within cells provides a healthy environment for cellular functions and is critical to the fate of the cell. Alterations in cellular redox status affect many redox sensitive activities including signal transduction, DNA and protein synthesis, and protein folding. Significant or prolonged deviations in the intracellular redox status disrupt cellular processes leading to numerous disease conditions. This chapter focuses on two mouse models of two human diseases that display disruption of the redox status of the cells leading to oxidative stress-mediated neurodegeneration (ND). One model represents the human childhood genetic disorder ataxia telangiectasia (A-T) lacking a functional ATM (A-T mutated) protein kinase. A-T is primarily a neurodegenerative disorder that also affects other systems in the human body. Since one of the key functions of the ATM protein is to maintain normal cellular redox status, the absence of a functional ATM in cells of the central nervous system (CNS) results in chronic oxidative stress leading to ND. A second model represents the human HIV-associated dementia (HAD) and other neurological diseases associated with the accumulation of misfolded proteins. This model uses a murine retrovirus called ts1 (a mutant of Moloney murine leukemia virus) that causes oxidative stress, endoplasmic reticulum (ER) stress, and mitochondrial impairment as a result of virus infection and accumulation of misfolded viral envelope protein in the ER of astrocytes. Neurons are not productively infected by retroviruses thus neuronal loss induced by these retroviruses is

2

Paul K. Y. Wong, Jeesun Kim, Soo Jin Kim et al. not directly due to productive infection of neurons, but rather due to the infection of other cells in the CNS, including astrocytes, oligodendrocytes, microglia and endothelial cells. Our goals are to understand the pathogenic mechanisms for both of these diseases thereby helping to develop drugs to prevent neuronal cell loss. Recently we have found that neurological symptoms of both disease models can be prevented by treatment with redox-active drugs, notably phthalazine dione, without repairing the initial causes. This finding suggests that these two animal models share oxidative stress in the CNS as a common mechanism of neuropathogenesis, although they have different initial causes, one from genetic mutation and the other from viral infection. This chapter brings together two important translational topics: elucidation of neurodegenerative mechanisms and development of therapeutic treatment. These two models will provide insight into the pathology of oxidative stress-mediated cell death and demonstrate how mouse models can help in understanding human diseases. Insights from this understanding may enable us to progress toward improved treatments in humans, not only for neurodegeneration (ND) but also other related debilitating diseases resulting from oxidative stress.

Introduction Oxidative stress is a destructive consequence of many disease states, particularly those involving the CNS. Oxidative stress in the nervous system has multiple causes, including genetic mutations, viral infection, energy or thiol deprivation, aging, and extreme environmental conditions. These disease conditions all exhibit oxidative stress, especially in ER and mitochondria and these events are tightly linked (Figure 1). Different organelles in the cells could be the sources for reactive oxygen species (ROS) production. Mitochondrial dysfunction is believed to be the major cause of increased ROS. Another source of ROS could be the result of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) in plasma and ER membrane.Extensive accumulation of misfolded protein in the ER lumen can also lead to production of ROS. If redox balance is not restored in the ER, Ca2+ is rapidly released from ER stores and picked up by mitochondria. This could disrupt the mitochondrial electron transport chain resulting in increased production of ROS. On the other hand, accumulation of ROS disrupts redox homeostasis in the ER, which impairs protein folding that facilitates the accumulation of misfolded proteins, leading back into a vicious cycle with further accumulation of misfolded protein in ER and severe ER stress. Collectively, these events eventually activate the cellular apoptotic cascade. This chapter focuses on two animal models for neurodegenerative diseases. One is a genetic disease with mutated Atm gene as a mouse model for human A-T. The other is a murine retrovirus mouse model for human HIV-associated dementia and other neurodegenerative syndromes. In both models, intracellular oxidative stress and oxidative stress-initiated pathways cause cell dysfunction and death in the CNS. The common involvement of oxidative stress in these two different diseases contributes to our understanding of shared mechanisms in human degenerative diseases. Our goals are to understand the pathogenic mechanisms for both of these diseases, to develop drugs to protect the viability of CNS cells and to prevent neuronal cell loss, thereby ameliorating NDs. Potential treatments with a unique antioxidant and anti-inflammatory phthalazine dione drug,

Oxidative Stress-Mediated Neurodegeneration: A Tale of Two Models

3

monosodium luminol (MSL or GVT) and combination drug treatment with short-chain fatty acid chemical chaperones for these diseases will be addressed.

Oxidative Stress, ER Stress and Mitochondria Impairment ROS, or free radicals, are highly reactive molecules due to the existence of unpaired electrons. In addition, in the presence of Fe++, hydrogen peroxide (H2O2) can be converted into highly toxic hydroxyl radicals and lipid hydroperoxides. At low physiological levels H2O2 modulates cell signaling events, including those responsible for cell proliferation and cell death [1-7]. At high concentrations, however, H2O2 and free radicals can damage cells by oxidizing proteins, DNA, and lipids, ultimately leading to cell death. Oxidative stress occurs in cells when the production of ROS exceeds intracellular antioxidant defenses [8, 9]. Oxidative stress is generally accompanied by thiol depletion in cells, because thiol-mediated antioxidants such as glutathione (GSH) are consumed when antioxidant defenses are mobilized [10, 11]. Oxidative stress contributes to many human neurodegenerative diseases [12], including Alzheimer‟s disease (AD) [13, 14], Parkinson‟s disease (PD) [15], multiple sclerosis (MS) [16], amyotrophic lateral sclerosis (ALS) [17], Charcot-Marie-Tooth disease (CMT) [16], Vanishing White matter Disease [16], HIV-associated dementia (HAD) [18-22], neuropathy associated with endogenous human retroviruses [23] and other viral infections.

Figure 1.

4

Paul K. Y. Wong, Jeesun Kim, Soo Jin Kim et al.

Sources of ROS Sources of ROS are variable for tissue, cell type, cell organelle and for each stressful situation. Stress on the redox-sensitive translation system in ER generates hydrogen peroxide. Stress on the mitochondrial electron transport system liberates oxygen free radical, primarily superoxide and hydroxyl radicals if free iron is around. Stress in plasma membrane activates NOX to generate H2O2 (Figure 2). Thus, cells are abundantly equipped to produce ROS in different organelles. The different sources of ROS are presented below. The endoplasmic reticulum: protein folding (disulfide-bond formation) in the ER can lead to formation of H2O2 and ER-generated oxidative stress It has been estimated that up to 25% of the total ROS generated by eukaryotic cells is a consequence of oxidative protein folding in the ER (Tu BP and Weissman JS), Oxidative protein folding in eukaryotes: mechanisms and consequences, JBC, 2004). Protein folding in the ER is an energy-consuming process, and oxidizing conditions are required for the formation of intramolecular and intermolecular disulfide bonds [24-26], a process catalyzed by protein disulfide isomerase (PDI) and ER oxidoreductase (ERO1) [27]. PDI accepts and donates electrons from protein-folding substrates, resulting in formation of the disulfide bond. To maintain proper protein thiol redox potentials, the flavoenzyme ERO1 uses a flavindependent electron bypass reaction FAD (flavin adenine dinucleotide) to transfer electrons from flavin to molecular oxygen (O2), resulting in the production of H2O2 [28]. In healthy cells H2O2 levels are normally low, but with excessive turnover of this thiol redox system, and increased H2O2 levels, both proliferation (at low levels) or senescence (at high levels) occur [reviewed in 29, 30]. As nascent proteins fold to their proper conformations, disulfide bonds are broken and reformed several times, to achieve proper folding. These isomerization reactions are catalyzed by the PDI [29]. Reduced and oxidized glutathione (GSH/GSSG ratio) in the ER lumen serve as the redox catalysis for PDI [30]. Redox stress, caused by an excess of misfolded proteins with inappropriate disulfide bond formation and/or breakage, disturbs the thiol redox potentials. As a result, GSH levels in the ER are reduced by ERO1 and transfer of the electron to O2 causes the production of H2O2. These conditions, with the production of H2O2 during protein oxidation, together with GSH depletion by reduction of abnormal disulfides, can exacerbate oxidative stress in the cell, leading to the release of Ca2+ from ER stores, and activation of mitochondrial apoptotic pathways (Figure 2). These conditions can also affect the ER environment, such as disruption of ER redox status, leading to further accumulation of proteins in the ER, causing ER stress. As an adaptive measure response to ER stress, the ER possesses a signaling network that senses and responds to the presence of accumulated misfolded proteins and targets them to be degraded by proteolytic systems such as the proteasome [31]. This signaling network is collectively termed the unfolded protein response (UPR), or the ER stress response. Irreversibly misfolded proteins are either retained within the ER lumen, in complexes with molecular chaperones, or they are disposed of by the ubiquitin-proteasome system, in a process called ER-associated degradation (ERAD). The activities of the ER surveillance components are highly dependent on the redox environment of the ER [32, 33]. GSH, the principal thiol compound of the ER, has been shown to play a critical role in maintaining the ER thiol redox environment [34]. GSH can


5

also assist in disulfide-bond reduction, when there is an accumulation of misfolded proteins due to inappropriate disulfide bonds [35].

Figure 2. Cellular components involved in production of H2O2 and ROS Protein folding within the ER is carried out by a family of protein disulfide isomerases (PDI) and ER oxidoreductases (ERO1) that catalyze disulfide bond formation and isomerization. Accumulation of misfolded proteins in the ER lumen can cause ER stress. ER stress, in turn, causes an increase in the formation of incorrect intra and/or intermolecular disulfide bonds that require breakage (unfolding) and reformation (refolding) for proteins to attain the appropriate folded conformation. PDI catalyzes disulfide bond formation and isomerization, whereas GSH reduces improperly paired disulfide bonds. Reoxidation is mediated by ERO1 with ROS production in the process. Thus, accumulation of misfolded protein in the ER lumen is sufficient to produce ROS. If redox balance is not restored, Ca2+ stored in the ER is released. The excess Ca2+ is taken up into the inner membranes of the mitochondria, thereby disrupting the electron transport chain. This diverts the electrons off-course and allows their release from the mitochondria, to react with molecular oxygen in the cytoplasm, producing ROS. The ROS produced during these events can cause further Ca2+ release from the ER, resulting in amplified accumulation of ROS. Excess Ca2+ can also activate NOX with production of ROS, which damages mtDNA, followed by activation of poly (ADP-ribose) polymerase 1 (PARP) that depletes ATP. Together, these events result in permeability transition pore (PTP) opening, leading to activation of apoptotic pathways. Nox at the plasma membrane can also be activated by ligand-receptor interaction, resulting in generation of H2O2. H2O2, at appropriate (low) levels functions as a signaling molecule.

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Mitochondria: The major source of ROS production Mitochondria not only play a central role in cellular energy and metabolisms, but are also the major source of free radical production, particularly when the cell is subjected to redox stress conditions. Critical for their function is the oxidation-reduction (redox) reactions, which is essential for cellular respiration and ATP production. This process produces free radical intermediates. Defects in mitochondrial functions result in many diseases, especially those involved in metabolism and the nervous system [reviewed in 36, 37]. These and other disease syndromes are likely to stem from the nature of the electron transfer processes that underlie the oxidative phosphorylation complex mechanism. These may include the increased production of toxic levels of ROS by the electron transport chains and altered ion homeostasis [reviewed in 38]. Mitochondria and ER are physically and physiologically interconnected. A subset of mitochondria is found in close proximity to the ER, at the opening of the inositol 1,4,5triphosphate (IP3)-sensitive Ca2+ channel. The Ca2+ released from ER is rapidly sequestered by mitochondria. In healthy cells, large amounts of Ca2+ are stored in the ER. Under steadystate conditions, the ER releases small amounts of free Ca2+ during signal transduction events that occurs in many cellular activation processes. This free Ca2+ is returned to ER stores by ATP-dependent pumps and remains in the ER in a bound state. Thus, cytosolic free Ca2+ is only present at very low levels. However, when certain triggering events occur such as the initiation of the UPR in response to ER stress, there is a large net release of Ca2+ from these ER stores. Much of this Ca2+ is taken up into the inner membranes of the mitochondria and disrupts the electron transport chain. During normal energy production, electrons in the mitochondrial inner membrane flow down the mitochondrial electron transport chain, until they are joined by two single oxygen ions to form water. When the electron transport is disrupted, electrons in the transport chain are diverted off-course, and are released from the mitochondria to react with molecular oxygen in the cytoplasm, producing ROS. The ROS produced during these events can cause further Ca2+ release from the ER, resulting in amplified accumulation of toxic levels of ROS (Figure 2). Increased Ca2+ levels could also stimulate NADPH oxidase (NOX) activation to produce ROS. ROS damages mitochondrial DNA (mtDNA), activating poly(ADP-ribose) polymerase 1 (PARP), which depletes ATP [36]. ROS at toxic levels also activates mitochondrial apoptotic programs causing mitochondrial transmembrane potential (m) dissipation. This together with ATP decline is followed by activation of mitochondrial collapse and apoptosis via opening of the permeability transition pore (PTP) [36, 37, 39-43]. Thus, it is clear that ER stress and mitochondrial stress are intricately linked [44]. Ultimately, the consequence of these stresses is amplification of apoptotic signals leading to cell death. As noted above, numerous studies have linked ER stress and mitochondrial dysfunction to almost all NDs [reviewed in [14, 36, 37]. NADPH oxidase (NOX Complex): Source of H2O2 Another source of ROS could be the result of NOX action at the cellular membrane. Originally discovered in neutrophils and phagocytic cells, NOX complex provides host cellular defense against bacteria via a rapid “respiratory burst” of ROS. This involves reduction of molecular oxygen to produce the superoxide anion. Superoxide then is converted to H2O2. Use of cell-free systems for subunits of NOX complex, including p47phox, p67phox, Rac and p40phox have been identified. Phosphorylation of p47 phox leads to a conformational


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change allowing its interaction with p22phox at the membrane. This in turn allows p47phox to bring p67phox into contact with Nox2. When this occurs, the GTP bound Rac interacts with the Nox2 and subsequently interacts with p67phox. The assembled complex can generate superoxide by transferring electrons from NADPH to oxygen [reviewed in 45] . Recent studies have shown that in addition to neutrophils NOX subunits are present in many cell types, including endothelial cells, neurons and astrocytes. There is growing evidence supporting the idea that ROS produced by NOX are causative factors in several neurodegenerative diseases. For example, deletion of Nox2 in mouse models for AD [46], PD [47], and ALS [48, 49] slows down disease progression and improves cell survival. Recently, Brennan et al [50] found that neurons exposed to glutamate modulate ROS levels through NOX rather than mitochondria. Unlike mitochondria, NOX enzymes do not generate energy when they produce ROS. One electron is released in the cytoplasm when each electron is transported. The translocation of negatively charged electrons may change ion fluxes. NOX resides not only at the plasma membrane but also the ER membrane. NOX enzyme can produce superoxide into the lumen of ER and extracellular environment [51]. In particular, the ER is highly permeable to protons and could sustain NOX activity suggesting that this may be another mechanism for the ER to generate ROS. NOX activation is also interrelated to mitochondria. Recent studies investigating PINK1associated Parkinson‟s disease, suggests that an initial defect in calcium mishandling by mitochondria leads to activation of NOX resulting in increased ROS in the cytoplasm, which damages the glucose transporter leading to respiratory impairment [52]. Cellular defense against oxidative stress Cellular defense responses to oxidative stress occur in a controlled sequence. The first level of cellular defense involves upregulation of superoxide dismutases (SODs) and catalase to counteract ROS buildup. If ROS overload causes significant cysteine and GSH depletion, the second line of cellular defense is deployed via activation and nuclear translocation of the transcription factor NF-E2 related factor 2 (Nrf2) [53-55]. Nrf2 transcriptional activity is regulated by several mechanisms, including protein interactions, protein stability, nuclear cytoplasmic shuffling, and phosphorylation [56]. Under normal conditions, Nrf2 is sequestered in the cytoplasm by the actin-bound regulatory protein Kelch-like Ech-associated protein 1 (Keap1) [57]. Multiple cysteine residues on the redox-sensitive Keap1 molecule allow it to respond to intracellular accumulation of ROS, with release of Nrf2 from its complex with Keap-1. This change allows Nrf2 phosphorylation and activation, which is followed by Nrf2 nuclear translocation. In the nucleus, Nrf2 activates the expression of genes via the antioxidant response element (ARE) promoter sequences. The genes that are activated include many detoxification enzymes, antioxidant enzymes, and reducing molecules, such as GSH [11, 58-60]. These products protect the cell from oxidative damage. In the cytoplasm of resting cells, Nrf2 remains complexed with Keap-1, where it is cyclically ubiquitinated and degraded through proteasome pathways [61]. Thus, Keap1 serves as both an adaptor protein docking Nrf2 for ubiquitination and as a sensor for oxidative stress. Another protein that binds to Nrf2 to keep it in the cytoplasm ready for phosphorylation but not yet translocated into the nucleus is DJ-1 [62]. Interestingly oxidation of a critical residue of DJ-1 causes relocation of the protein to the mitochondria sensor of oxidative stress. Mutations in this protein result in impaired response to oxidative damage and increased cell death in a PD cell model [63]. Under basal conditions some Nrf2 is also present in the

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nucleus, mediating constitutive expression of Nrf2 target genes, such as NADPH quinone oxidoreductase 1 (Nqo1) and hemoxygenase 1 (HO-1). Activation of Nrf2 in the brain has been shown to mitigate the effects of oxidative stress in many NDs, including AD and PD [62, 64, 65]. A recent report shows that Nrf2 activation in astrocytes with resultant increase secretion of GSH prevents neuronal cell death and ND in mouse models of amyotrophic lateral sclerosis (ALS) [17]. Thus, Nrf2/ARE pathway has been implicated to be a potential therapeutic target against NDs. A key product of the Nrf2 pathway is GSH. In addition to its role in detoxification to protect cells from oxidative stress, GSH provides the main redox buffer for cells and as such functions as a net reductant in the ER, either by maintaining ER oxidoreductase in a reduced state or by directly reducing disulfide bonds in substrate folding proteins [30]. Thus, GSH has a major role not only in the protein folding process but also in balancing redox reactions thereby protecting cells from oxidative stress.

Mouse Genetic Model of A-T Associated Neurodegeneration A-T is an autosomal recessive genetic disease in which the Atm gene is mutated. Humans with A-T display pleiotropic phenotypes, including cancer predisposition, immunodeficiency, and progressive ND, with development of ataxia, telangiectasia, and premature aging. However, A-T is primarily a neurological disorder. Symptoms of A-T are usually manifested in the first few years of life, when children exhibit ataxia or wobbly gait. Loss of neuromuscular control or coordination is relentless and, by ten years of age, children are usually confined to a wheelchair. A-T patients are also more susceptible to infection, radiation, pulmonary failure, and /or lymphoid cancer in the second decade of life [66, 67]. Despite increasing interest in recent years, the mechanisms underlying ND in human A-T are still poorly understood. For this reason, treatments for A-T ND are not available. Gaining knowledge of how ATM works is key to understanding the molecular basis of A-T associated NDs and in development of therapeutic treatment for A-T ND.

ATM Regulates Cellular Redox Status and Maintains Redox Balance The ATM protein is a large kinase that plays critical roles in regulation of cell cycling, DNA repair, and control of cellular redox status. In unstressed cells, ATM exists as an inactive form, but ATM becomes enzymatically active by autophosphorylation [68-70]. Until recently, the molecular mechanisms that trigger ATM activation remain unclear. The cellular activity of many kinases is known to be redox-sensitive, and this redox sensitivity is primarily dependent on reactive cysteine residues in the proteins [4, 71]. For this reason, it has been postulated that ATM might be activated in response to increased levels of H2O2 in cells, e.g., as a result of cell metabolism and oxidants generated during postnatal development. At moderate levels H2O2 acts as a protein-modifying signaling molecule [4]. H2O2 is readily diffusible intracellularly, and induces protein activation rapidly. This could be one way in which oxidative stress is “sensed” by redox-sensitive kinases, including ATM (Figure 3). It


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has been shown that H2O2 is directly and specifically responsible for oxidation-induced disulfide bond formation that leads to conformational alteration and autophosphorylation of ATM [72]. ATM might regulate cellular ROS levels by (a) increasing production of reductant precursors, such as NAD+ and NADP+ [73], (b) decreasing energy consumption and ROS production by cell cycling arrest [74], and (c) slowing electron transport in mitochondria, thus decreasing ROS production [75]. Cells lacking ATM have increased rates of mitochondrial respiration, particularly in the brain [73]. This suggests that the absence of ATM leads to upregulation of intracellular ROS from mitochondria. The brain consumes about 20% of inhaled oxygen, and energy production by neurons is heavily dependent upon mitochondrial respiration. This situation makes the brain highly susceptible to oxidative stress [74, 76-83]. This could explain why loss of ATM results in oxidative stress. Persistent oxidative stress in ATM-deficient cells probably overcomes cellular antioxidant defense systems, resulting in dysregulation of signaling pathways and a neurological phenotype associated with oxidative stress-induced damage. Until now, the most convincing evidence linking oxidative stress to neurological phenotypes in A-T has been obtained with Atm knockout (Atm-/-) mice. ATM-deficient mice exhibit genomic instability and hypersensitivity to ionizing radiation and other treatments that generate ROS [77]. Overexpression of SODs that generate H2O2 in these mice exacerbated certain features of A-T phenotypes [84].

Figure 3. ATM is a redox-sensitive protein and this redox sensitivity is primarily dependant on reactive cysteine residues in the protein. ATM might be activated in response to increases in H2O2 levels in the cell. Oxidation of the two SH groups in cysteine results in disulfide bond formation, thereby leading to conformational alteration and activation of the protein. The activated ATM then performs multiple functions including DNA repair, cell cycle checkpoint maintenance, redox homeostasis and initiation of signal transduction.

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ROS levels are intrinsically upregulated in Atm-/- neural stem cells (NSCs), astrocytes [81, 82, 85] and thymocytes [85], as well as hematopoietic stem cells [3, 86] suggesting that ATM-deficient cells are constitutively under oxidative stress. In cultured normal NSCs and astrocytes, addition of H2O2 results in a rapid and dose-dependent phosphorylation of ATM [81, 82] that can be suppressed by antioxidants (unpublished data from our lab). Together, these findings strongly support the notion that activation of ATM can be linked to increased ROS levels in cells. When ATM is activated, it exerts a systemic influence in the cell, involving a large number of substrates that control cell functions [87]. Of these, the two bestknown functions have been cell cycle checkpoint control and DNA repair [78]. In recent years, however, mounting evidence suggests that ATM can be activated by conditions that increase intracellular ROS independent of its respond to DNA damage [72, 79, 88, 89].

ATM Deficiency and ER Stress Although ATM deficiency has been shown to induce ER stress through oxidative stress [77, 86], no conclusive evidence has been documented showing that ATM suppresses ER stress. We have previously shown that ATM deficiency also induces ER stress in astrocytes with increased levels of ER stress markers, glucose-regulated protein 78 (GRP78), or BiP, and activation of caspase-12 cleavage [80]. These markers are also upregulated in cerebella of Atm-/- mice. In keeping with this we also observe that BiP and phosphorylated alpha subunit of eukaryotic translation initiation factor 2 (p-eIF2alpha) are upregulated in Atm-/- thymocytes relative to Atm+/+ thymocytes [90]. In another study, using ATM deficient cells or cells treated with Atm siRNA, He and coworkers [91] show that ATM blocks ER stress induced by the ER stress inducer tunicamycin. Together, these findings showing ATM regulation of ER stress substantiates the notion that ATM plays a crucial role in controlling stress-mediated disease conditions. These data also suggest that oxidative stress is linked to ER stress since ROS levels are constitutively upregulated in ATM deficient cells. As mentioned above, prolonged ER stress makes the ER membrane more permeable to Ca2+ and this in turn results in perturbation of intracellular Ca2+ concentration. In view of the close apposition of ER and mitochondria, perturbed Ca2+ signaling could lead to mitochondrial collapse and apoptosis via mitochondrial membrane permeabilization and opening of the permeability transition pore [39, 40].

Lack of ATM Expression Causes Mitochondrial Dysfunction A newly uncovered function for ATM is that it may regulate mitochondrial homeostasis [38]. Shadel and coworkers show that fibroblasts from A-T patients exhibit conditional mitochondria DNA (mtDNA) depletion independent of DNA damage. These cells also fail to promote increases in mtDNA when DNA damage was induced by ionizing radiation [92]. Tissue-specific alterations in mtDNA copy number were also observed in Atm-/- mouse tissue. In addition, the structural organization of mitochondria in A-T cells is abnormal compared to wild type [73]. Moreover, ATM-deficient cells harbor a much larger population of mitochondria with decreased membrane potential than control cells [93]. Thus, ATM


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apparently plays a key role in mitochondrial function under normal growth conditions. Since ATM also regulates p53 function [94], and since p53 has also been shown to regulate mitochondrial respiration [95, 96], it is likely that some of the effects of ATM deficiencymediated mitochondrial dysfunction may be due to disruption of p53 in the absence of ATM. This newly uncovered ATM function suggests that mitochondrial impairment may be at least in part involved in the pathogenic mechanism of ATM deficiency. In addition to the hallmark characteristics of ND in A-T, some A-T patients are associated with metabolic syndrome and premature aging, both of which are linked to mitochondrial dysfunction. Mitochondrial dysfunction also leads to the release of ROS resulting in oxidative stress and apoptosis. Thus, further investigation of the effects of ATM on mitochondrial function is warranted. This also may open the avenue for novel therapeutic treatment targeted to mitochondria for A-T patients.

ATM Deficiency Results in Defective Self-Renewal and Proliferation of NSCs through an Oxidative Stress-Mediated Neurodegenerative Signaling Pathway In the normal brain, the number of NSCs is the result of a tightly controlled balance among self-renewal, differentiation, and death [97]. NSCs undergo asymmetric self-renewing division to produce neuronal and glial progenitor cells, which differentiate to neurons, astrocytes, and oligodendrocytes. Thus, proper control of these events is critical in maintaining the normal numbers of neurons, astrocytes, and oligodendrocytes in the brain [98]. ATM expression is abundant in NSCs in the normal brain, but is gradually downregulated as the cells differentiate [99], suggesting that ATM may play an essential role in NSC survival and proliferation during development. In the absence of ATM, abnormal neuronal and astrocytic development occurs [80, 99, 100]. This could be the result of abnormal differentiation of NSCs. ROS levels are constitutively high in NSCs of Atm-/- mice and elevated ROS levels are associated with defective self-renewal and proliferation of these cells. Treatment with the antioxidant antioxidant N-acetyl cysteine (NAC) restores normal renewal and proliferation for these cells. The elevated ROS in Atm-/- NSCs results in phosphorylation of p38MAPK (here after called p38), which is correlated with decreased levels of p-Akt and Bmi-1 [82]. Bmi-1 is a component of the polycomb repressor complex 1 (PRC1) that represses p21CIP1 (hereafter called p21) by chromatin modification. Thus, downregulation of Bmi-1 function results in p21 upregulation. Furthermore, treatment of the Atm-/- NSCs with the p38 inhibitor SB203580, or with NAC, restores normal levels of p21 and normal proliferation of Atm-/NSCs [82]. These results suggest that ATM is required in NSCs to maintain normal intracellular redox homeostasis. In the absence of ATM, chronic oxidative stress results in activation of the p38-Akt-Bmi-1-p21 pathway in NSCs. Bmi-1 can also separately regulate mitochondrial function and redox homeostasis [101] by reducing the intracellular levels of ROS [102]. Thus, downregulation of Bmi-1 and ATM in cells both result in oxidative stress. Whether downregulation of ATM results in downregulation of Bmi-1 is unclear at the present time, although Bmi-1 is downregulated in Atm-/- NSCs, or when normal NSCs are treated with H2O2 [82]. Interestingly, Bmi-1 deficient

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mice exhibit a progressive postnatal depletion of NSCs, leading to neurological abnormalities and ataxia [103]. Conversely, NSCs over expressing Bmi-1 have increase self-renewal and proliferation capacities [104] (Kim and Wong unpublished data). Collectively, these observations strongly support the notion that downregulation of Bmi-1, like ATM deficiency, contributes to decreased proliferation and self-renewal of NSCs. Until now, the identity of upstream effectors that might control the levels or function of Bmi-1 has been unclear. It has been shown that Bmi-1 could be phosphorylated by 3pk (MAPKAP kinase 3), which is a downstream effector of p38 [105]. Phosphorylation of Bmi-1 by 3pk or by p38 reduces Bmi-1‟s ability to bind to chromatin, thus reducing its suppressive effect on p21. Another way in which p38 may regulate Bmi-1 levels is via downregulation of Akt since oxidative stress-induced activation of p38 attenuates insulin-like growth factor stimulation of Akt [106]. On the other hand, Bmi-1 may be a substrate of Akt, and upregulation of p-Akt coincides with upregulation of p-Bmi-1 (Kim and Wong, unpublished data) probably by stabilizing Bmi-1. However, the question whether Akt can phosphorylate and upregulate Bmi-1 remains to be addressed. Finally, a recent report has shown that inhibition of ATM by an ATM-specific inhibitor KU-60019 reduces phosphorylation of Akt and cell proliferation [107]. Together, these data and the findings described above indicate that Bmi-1 also plays a critical role in NSC self-renewal and downregulation of Bmi-1 greatly affects cerebellum development [103], supporting the notion that in the absence of ATM, chronic oxidative stress results in activation of the p38-Akt-Bmi-1-p21 pathway leading to defective proliferation and self-renewal of NSCs (Figure 4). This may at least in part contribute to the defective cerebellum development and neurodegenerative phenotype in Atm-/- mice.

Figure 4. One of the major functions of ATM is to regulate cellular redox status. In the absence of ATM, reactive oxygen species (ROS) levels are intrinsically high in different cell types. This could lead to oxidative/ER/mitochondrial stress with activation of cell death pathways. In Atm-/- NSCs the elevated ROS results in upregulation of p-p38, which is correlated with decreased levels of p-Akt and Bmi-1. Bmi-1 is a component of the polycomb repressor complex 1 (PRC1) that represses p21 by chromatin modification. Thus, downregulation of Bmi-1 function results in p21 upregulation, which suppresses cell proliferation. In Atm-/- astrocytes increased ROS activates the MEK-ERK pathways also resulting in downregulation of Bmi-1 with repression of p16, leading to suppression of cell proliferation


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Astrocytes Lacking ATM Results in Oxidative Stress-Mediated ERK1/2 Activation, Downregulation of Bmi-1 and Upregulation of P16 Leading to Retardation of Growth Interestingly, like NSC from Atm-/- mice, primary astrocytes isolated from Atm-/- mice proliferate more slowly than do those from Atm+/+ mice. Atm-/- astrocytes can be passed only a few times before they become flat and enlarge, with increased intracellular vacuolation, proliferative arrest and death. Atm-/- astrocytes, like Atm-/- NSCs, not only proliferated much slower than Atm+/+ astrocytes, but their growth was severely arrested [80]. When normal (Atm+/+) astrocytes were treated with H2O2, with the ATM inhibitor KU55933, or with siRNA-ATM, they grew significantly more slowly than do untreated C1 astrocytes (Kim J, 2009, unpublished data). This retardation of growth could be corrected, to a large extent, by the MEK inhibitor or by NAC [81]. Cultured astrocytes from Atm-/- mouse brains show signs of increased levels of an oxidative stress-regulating kinase ERK1/2 or extracellular signal-regulating kinases1/2 [81]. Phosphorylated extracellular signal-regulating kinases1/2, and their downstream mediators, are now known to be major contributors to CNS pathology in a number of neurodegenerative diseases, including AD [108-110]. In mice carrying a human tau gene (an animal model of AD), a specific inhibitor of ERK2 reduces tau phosphorylation, and corrects the animals‟ motor impairments [109]. Interestingly, ATM downregulates p-ERK1/2 for cell survival after IR [111]. Thus, in the absence of ATM, oxidative stress-induced upregulation of p-ERK1/2 may have detrimental effects in cells. Consistent with this notion, astrocytes from Atm-/- mice show signs of oxidative stress and increased levels of p-ERK1/2 in vivo [80]. In Atm-/- mice the specialized cerebellar Bergmann astrocytes have increased oxidative stress markers and increased p-ERK1/2 [80]. These astrocytes are primary supporting cells for the Purkinje neurons that control movement, and their dysfunction is likely to compromise their support function to these neurons. One of the downstream events that follow ERK1/2 activation is upregulation of p16 expression [112], which like p21, is a suppressor of cell cycling and a major marker of aging [113, 114]. Since p16 expression is regulated at the transcriptional level, p-ERK1/2 is unlikely to act directly on p16 expression. Instead, as in Atm-/- NSCs, Bmi1 acts as the bridge for this gap. Phosphorylation of BMI-1, by p-ERK1/2, may lift the normal repression of the p16 gene by Bmi-1, allowing p16 expression to inhibit astrocyte cell cycling and proliferation. Phosphorylation of Bmi-1 could also be inhibited with the MEK inhibitor. Not surprisingly, p16 levels are constitutively increased in Atm-/-astrocytes, but when Atm-/astrocytes are exposed to H2O2 in culture, p16 is further elevated and the elevated levels persist for up to 16h. In Atm+/+ astrocytes, by contrast, addition of H2O2 caused a brief upregulation of p16 at 4 h, but then this was followed by a return to normal levels at 16 h. This means that oxidative stress due to increased H2O2 is reversible when ATM is present. In the Atm+/+ cells, it seems likely that the brief expression of p16 shuts down cell cycling, allowing time for the cells to repair any damage. Once this task is complete, p16 levels returned to normal, as a result of ATM‟s redox balancing action. However, if oxidative stress was prolonged, as it is in cells lacking ATM, upregulation of p-ERK1/2 persistently increased p16 expression, resulting in prolonged cell cycle arrest and retardation of cell proliferation inhibition of p-ERK1/2 activation with MEK inhibitor reduces p16 levels and 4 h after H2O2 treatment [81]. Together, the above findings suggest that chronic oxidative stress results in

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the activation of MEK-ERK-Bmi-1 and p16 pathways leading to defective proliferation of astrocytes (Figure 4). Whether the two signaling pathways presented above in ATM-deficient astrocytes and NSCs are linked is at present unclear. Notably, in both Atm-/- and NSCs and astrocytes,the MAPK p38 and ERK pathways converge to downregulate Bmi-1 with resultant defective proliferation of these cell types. Furthermore, whether these signaling pathways are also involved in neurons lacking ATM is also unclear. What is clear, however, is that in the absence of ATM, ROS levels are intrinsically high in all these cell types, which substantiates the notion that the defects in these cell types are associated with chronic oxidative stress in the absence of ATM. Interestingly, convergence of the ERK and p38 pathways is also observed in AD [115].

Mouse Model of Retrovirus Induced Neurodegenerations Retrovirus-Mediated ND Is Caused by Oxidative Stress, ER Stress and Mitochondria Impairment Oxidative stress appears to also play a critical role in the neurovirulence caused by many viruses. These include Epstein-Barr Virus [116], respiratory syncytial virus [117], Japanese encephalitis virus [118-120]. Three groups of retroviruses that cause ND are HIV in humans (see below), simian immunodeficiency virus (SIV) in nonhuman primates [121], feline immunodeficiency virus (FIV) [122] and certain murine retroviruses (see below). This chapter focuses on NDs caused by murine retroviruses and HIV. This approach may yield a number of important insights into the pathogenesis of NDs induced by these viruses via oxidative/ER stress and mitochondrial impairment. In addition, using mouse models for human diseases can bridge the gap between them, and provide better understanding of the mechanism of pathogenesis and insights into improved treatment not only of HAD but also other deliberating NDs associated with oxidative stress, ER stress and mitochondrial impairment.

Retroviruses Retroviruses are major health hazards because of their ability to cause persistent infection of the nervous, immune and other systems in our body. A number of retroviruses, including the murine leukemia viruses (MuLV), feline immunodeficiency virus, simian immunodeficiency virus and human HIV-1 [123, 124], as well as human endogenous retroviruses [125], are associated with neurological and systemic disorders. A recently identified retrovirus, called xenotropic MuLV-related virus, is linked to chronic fatigue syndrome in humans [126][Lo SC et. al, Detection of MLV-related virus gene sequences in blood of patients with chronic fatigue syndrome and healthy blood donors, 2010 PNAS, epub ahead of print]. Despite many years of study however, the mechanisms that underlie the pathogenesis of retrovirus-induced NDs are not completely understood.


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Since retroviruses were discovered, a considerable amount of information about their biology has become available from studies on the murine retroviruses. In fact, before HIVAIDS appeared on the world landscape, retrovirus-associated ND was discovered as a pathological manifestation of infection by some MuLV strains [reviewed in 127]. Therefore, goals for HIV-AIDS research should be broadened toward a more basic understanding of pathogenic mechanisms and disease progression [128] including neuropathogenic MuLVs in mice.

Neurovirulent Murine Retrovirus A group of neurovirulent murine retroviruses, represented by the ts1-Moloney murine leukemia virus, a mutant derived from MoMuLV [127] and FrCasNC, derived from the CasBrE retrovirus [129, 130] cause spongiform encephalopathy in infected mice. The determinant of neurovirulence of both viruses results from genetic variation in the viral env gene [130-133]. For both retroviruses, there is now strong evidence for oxidative stress, ER stress and mitochondrial impairment in the neuropathology that they cause. One puzzling aspect of these retrovirus-mediated ND is the cellular specificity of the cytotoxicity. These viruses infected all the cell types, except neurons, in the CNS. However, in all cell types, except microglia, signs of apoptosis are detected [Wong PK and Yuen PH, 1994, Histol Histopathol 9:845].

ts1 MoMuLV Model In ts1 MoMuLV, a single point mutation (Val to Ile) in the env gene results in misfolding of the envelope precursor protein gPr80env. The misfolded gPr80env accumulates in the ER, because it cannot be transported from the ER to the plasma membrane. ts1 infects many cell types, but gPr80env accumulation occurs mainly in infected T lineage lymphocytes in the immune system and astrocytes (and perhaps oligodendrocytes) in the CNS [123, 134, 135]. This results in a disease that resembles HIV infection in several important ways [123, 124, 136, 137]. The characteristic features of both HIV- and ts1-induced disease include T cell depletion [138-140] and cell death in astrocytes and neurons in the CNS [124, 136, 141-149] (see Table 1). T cells and macrophages are primary peripheral targets for both HIV and ts1 [138, 139, 150]. In the CNS, both HIV and ts1 infect microglia, astrocytes, oligodendrocytes, and endothelial cells, but not neurons [123, 139, 141, 151-153]. Thus, neuronal loss induced by these retroviruses is not directly due to productive infection of neurons, but rather to astroglial neuronal support impairment, or the secretion of neurotoxic factors by infected or activated glia [141, 142, 153-156]. In the CNS, accumulation of the misfolded precursor envelope protein of ts1 in the ER of astrocytes results in UPR, leading to oxidative stress and ER stress, which in turn results in mitochondria-mediated cell death [42, 157]. As noted above, since neurons are not directly infected by ts1 but they die alongside infected astrocytes, neuron death is most likely due to reduced thiol support from dysfunctional astrocytes (causing oxidative stress and ER stress

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and thiol deficiency in neurons) and/or to neurotoxic factors produced by ts1 infected astrocytes and microglia or due to loss of support from ER- stressed oligodendrocytes. Table 1. Retrovirus-mediated cell death and NDs are associated with oxidative stress, ER stress and mitochondrial cell death pathways in glial and neurons

Oxidative stress Antioxidants defense ER stress Dysregulation of calcium homeostasis Mitochondria impairment Upregulation of xCTOxidative stress with accumulation protein other than viral protein NOX activation Elevation of iNOS Elevated COX-2 levels Increase in FGF Protective effect of Nrf2 Protective effect of Bcl2 Protective effect of minocycline and other antioxidants Astrocytes and neuronal death Role of microglia

HIV [18, 21, 147, 172-176] [146, 178, 179] [144, 180] [175, 183-185] [180, 184, 186] [187] [188-192]

ts1 [11, 161, 167, 177] [11, 161] [42, 181, 182] Unpublished data [42, 181] [11] [142]

[193] [194] [195] [197] [199, 200] [158] [201]

Unpublished data [157] [196] [198] [11, 167] [159, 161] [167, 177]

[144-149, 202-204] [123]

[42, 141, 142, 177, 181] [123, 141, 153, 205]

In ts1-Infected Astrocyte Cultures and Brainstem Tissues of ts1-Infected Mice, Oxidative Stress Is Associated with ER and Mitochondrial Stress, with Initiation of Apoptotic Pathways In ts1-infected astrocytes increased expression of ER and mitochondrial stress biomarkers including upregulation of the chaperone proteins GRP78 (BiP), and mitochondrial degeneration [42] are observed. The UPR is initiated, as shown by activation of the ERresident transmembrane protein kinase PERK, which upregulates both the initiation factor-2 (eIF2) and CHOP. Interestingly, studies by others have shown that CHOP downregulates Bcl2 [158], a protein that has protective capability against ts1-induced [159] and HIVinduced [158] ND. The ER stress-specific enzyme caspase-12 is also activated, cleavage of procaspase-9 occurs, and caspase-3 is activated leading to apoptosis. Evidence for mitochondrial involvement in ts1 neuronal death comes from a recent reports showing p53 accumulation in neurons in the ts1-infected CNS [160] and that overexpression of Bcl2 in astrocytes confers resistance to ts1-induced cell death [161] and to ts1-mediated ND in infected mice [159]. In the brainstems of ts1-infected mice, activated caspase-3 and damaged mitochondria are present in astrocytes. Therefore, it appears that


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oxidative, ER, and mitochondrial stress-related apoptotic pathways are involved in ts1induced astrocytic death [11, 42].

Nrf2 Mediates Antioxidant Defenses in ts1 Infection, and It Promotes Cell Survival As noted above, ts1 infection of astrocytes induces thiol (i.e., GSH and cysteine) depletion and ROS accumulation, in parallel with viral envelope precursor gPr80env accumulation [11]. ts1-infected cultured astrocytes apparently mobilize their antioxidative defenses by upregulating their levels of Nrf2, and levels of its target genes, including the xCT- cystine/glutamate antiporter, -glutamylcysteine ligase, and glutathione peroxidase. Thiol depletion appears to accelerate astrocyte cell death, while thiol supplementation promotes survival of ts1-infected astrocytes. Together, these data substantiate the notion that ts1 infection may damage astrocytes by oxidative and ER stress, which can be alleviated by Nrf-2-mediated thiol antioxidant defenses [11].

A Subpopulation of ts1-Infected Cultured Astrocytes Survives by Upregulating Antioxidant Defenses As previously mentioned, ts1-infected astrocyte cultures show increased levels of ROS. Despite this, however, only about half of infected astrocytes die in these cultures. The surviving cells continue to proliferate and produce virus. To determine how these resistant cells survive ts1 infection in culture, we established and characterized a subline of these cells (C1-ts1-S). C1-ts1-S cells proliferate more slowly than do C1 cells, and produce fewer viruses than do infected C1 cells. They also show reduced H2O2 levels, increased uptake of cystine, and higher levels of both GSH and cysteine, compared to acutely infected (nonsurviving) cells or to uninfected C1 cells [161]. C1-ts1-S cells also upregulate their thiol antioxidant defenses by activation of Nrf2 and its target genes and Bcl2, a mitochondrial protector. We conclude that some astrocytes can survive ts1 infection by successfully mobilizing their antioxidant defenses via Nrf2 activation and by upregulation of Bcl2 [161].

ts1-Associated Caspase 8 Activation in Astrocytes Is Caused by Intracellular Events In ts1-infected astrocytes, caspase 8 is activated by an intrinsic pathway, which starts with elevation of the death receptor DR5 and the C/EBP homologous protein (GADD153/CHOP), an ER stress-initiated transcription factor, rather than through TNF2 and TNF-R1 interaction on the cell surface. Upregulation of CHOP that inhibits Bcl2 may explain why Bcl2 is downregulated in ts1 infected primary astrocyte cultures. Activated caspase 8 cleaves Bid into tBid, initiating mitochondria-driven apoptosis via tBid translocation. This in turn amplifies ER stress, contributing to oxidative stress-induced apoptosis. Treatment of ts1infected astrocytes with a specific caspase 8 inhibitor reduces ER stress responses. This

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occurs because the inhibitor reduces both caspase 8 activation and cleavage of the ERassociated membrane protein BAP31 into BAP20, of which overexpression exacerbates the ER stress response. These findings suggest for the first time that the caspase 8- and ER and mitochondria stress-associated apoptotic pathways are linked [44]. These findings suggest that caspase 8 could be a new target for treatment of stress-related diseases in humans.

Retrovirus Infection Promotes ROS Production through NOX Following ts1 infection of astrocytes, levels of NADPH are decreased and levels of superoxide are increased when compared with uninfected astrocytes, which suggests NOX activation (Kim and Wong, unpublished data). Moreover, NOX inhibition induces the antiapoptotic Bcl-2 and BclxL proteins, and suppresses activation of the pro-apoptotic enzyme caspase 3 (Kim and Wong, unpublished data). As noted above, ER stress may be related to intracellular ROS induction in ts1-infected cells. Due to mutation, the precursor viral envelope protein of ts1 gPr80env is unable to fold properly and unable to proceed to Golgi. The UPR may activate NOX, which might be localized in the ER membrane. Although Nox2 distribution in ER membranes of astrocytes has not been well studied, Nox4 in endothelial cells is predominantly expressed in the ER membrane. Chen et al reported that Nox4-dependent ROS inactivates protein tyrosine phosphatase (PTP)1B in the ER. PTP1B then serves as a regulatory switch for epidermal growth factor (EGF) receptor trafficking [51]. Enveloped retroviruses such as HIV and MLV may use ROS induction of cells as a tool to invade host cells. The viral envelope makes contact with its receptor on the plasma membrane, and this is followed by viral entry. Notably, retroviral entry could be modified by the local redox climate [162, 163]. Ryser et al [164] proposed that receptor-bound envelope glycoprotein gp120 of HIV is reduced by host surface-associated PDI which is colocalized with CD4 [165]. Inhibition of PDI activity prevents the entry of HIV [166]. Reducing disulfide bonds causes conformational changes in gp120 and these changes may enhance viral fusion to the host membrane. In summary, the key findings in these studies have been that ts1 infection causes oxidative stress, ER stress, and mitochondrial impairment, all of which play critical roles both in ts1-induced ND [167-169]. Interestingly, overwhelming evidence now shows that oxidative stress, ER stress and mitochondrial dysfunction also play a key role in HIV neuropathogenesis (see Table 1).

Advantages of the ts1 Model for HAD Despite extensive research on HAD, the mechanisms by which HIV causes death in neurons of AIDS patients remain unclear. The main problems have been the expense and ethical costs of human studies and of primate models of HIV infection. These limitations and a lack of suitable alternative animal models have hampered full understanding of the mechanisms involved in HIV-induced CNS cell death.


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ts1 is a well-characterized murine retrovirus that causes a rapidly developing disease syndrome. Disease severity and latency are well defined and can be manipulated by the virus strains used, the dosage of virus, the age of the host when infected, and the mouse strains employed [123, 127, 132, 136, 137, 170]. ts1-mediated disease is dramatic, easily induced, and reproducible [127, 132]. With the ts1 model, we can perform both cell biology and systemic studies of retroviral-induced disease. Because of the availability of gene knockout and transgenic mouse models, such as mice overexpressing Nrf2 [17] or Bcl-2 [159], this model can be used to focus on specific proteins involved in ts1-induced ND. These technologies have the distinct advantage of allowing analysis of disease states within the context of the whole animal, which more accurately mimic the human disease [171]. Although ts1 does not completely reproduce all pathologic features of HIV infection, and although the sources of ROS overproduction in ts1 and HIV infection may not be identical, important similarities exist between the ts1-induced ND model and HAD, as described above (Table 1). Since oxidative stress, ER stress, mitochondria impairment and downstream events are involved in both ts1-mediated ND and HAD, similar cell death mechanisms may contribute to these diseases. Knowledge gained from the studies with animal models should therefore shed light on the pathogenic mechanisms and on potential therapeutic treatment for HAD in humans. Furthermore, a better understanding of the cellular and molecular mechanisms of oxidative stress, ER stress and mitochondrial dysfunction induced by various agents, including protease inhibitors used in HAART for HIV, may provide useful information for the development of new therapeutic strategies against these stressful conditions.

Oxidative Stress in HAD HIV induces chronic systemic oxidative stress in AIDS patients, which occurs even in early stages of the disease [206, 207]. Later in the disease course, signs of oxidative damage have been observed in dying neurons and astrocytes in the brains of HIV associated dementia HAD patients [18-21, 149, 208]. HIV-induced oxidative stress causes apoptosis in cultured astrocytes and neurons [146] (see Table 1). In addition, ROS generation by glial cells is linked to neural cell death induced by HIV in vitro [22]. In general, individuals infected by HIV-1 show decreased systemic antioxidant defenses suggesting that the nutrient substrates for oxidant defense are being depleted by ongoing oxidative stress [178]. The exact mechanisms by which HIV induces oxidative stress, however, remain unclear. Several HIV-1 proteins, in particular gp120 and Tat, have been associated with ROS production and oxidative stress in cultured infected astrocytes [146, 147, 202]. Although HIV does not infect neurons [175, 183, 185], both HIV gp120 and Tat disrupt neuronal calcium homeostasis by perturbing the Ca2+ regulating system in the plasma membrane and ER, leading to oxidative stress and mitochondrial dysfunction, which together cause neuronal death. When the HIV-1 protein Tat is added to HIV-1 target cells in culture, levels of ceramide, an oxidative stress marker, are increased. Ceramide levels are also increased in the brains of HAD patients [209]. Upregulation of xCT- after Tat exposure has been documented for human retinal pigment epithelial cells and retinas of Tat-transgenic mice [187]. In addition, Tat itself activates oxidative and inflammatory pathways in the brain vascular endothelium [210]. It has been

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reported that human astrocytic cell line exposed to HIV-1 underwent apoptosis as a result of HIV-1 mediated oxidative stress, and that antioxidant compound NAC prevents these HIVinduced cellular damages [146]. Furthermore, it has been shown that HIV gp 120 is toxic to astroglial cells, by lipid peroxidation as a result of oxidative stress. This effect of gp120 on astroglial cells can also be counteracted by NAC [202]. Together, these studies suggest that oxidative stress may play a critical role in the pathogenesis of HAD.

ER Stress in HAD Chronic ER stress is a primary component in the neuropathology of a wide variety of NDs [211, 212]. As noted above, in the oxidative environment of the ER, elevated oxidative stress can directly cause alterations in protein folding [213, 214]. In addition, accumulation of misfolded protein may itself alter the redox status of the ER, causing other proteins to misfold [215, 216]. These conditions can also generate disulfide bond-mediated intermolecular protein aggregation [217]. Generally, protein misfolding leads to protein accumulation, ER stress and proteinopathy (accumulation of misfolded proteins), which contribute to many human NDs, including β-amylold in AD [13], α-synuclein in PD [15], MS [16], ALS [218], Prion disease [219] and neuropathy associated with retrovirus infection and endogenous human retroviruses [23]. Thus, although NDs can have multiple causes, they all seem to share common mechanisms involving oxidative stress, ER stress, mitochondrial impairment and proteinopathy (Figure 1). Since both ts1-ND and HAD are associated with oxidative stress, it is possible that ER stress-initiated oxidative stress and cell damage is a common culprit in cell death in retrovirus-mediated NDs. As shown above, ER stress and cell death occur in both astrocytes and neurons in the CNS of patients with HAD [144, 149, 180]. Although the precursor envelope protein of HIV gp160 accumulation has not been reported for HIV-infected cells in the CNS, retention of gp160 in the ER of T cells [220, 221] suggests a likely relationship between retention of gp160 in ER and cytopathic effects in HIV infection [222, 223]. The gp160 molecule requires complex and time-consuming folding in the ER, and is prone to misfolding and accumulation [220]. Because of this, there is a high incidence of unusual cysteine variants in HIV envelope proteins in individual patients and this results in aberrant disulfide bond formation and gp160 accumulation [224]. Thus, it is possible that accumulation of gp160 in HIV-infected cells results in oxidative and ER stress. Even though gp160 has not been shown to accumulate in HIV-infected astrocytes, ER stress may still be a possible cause of astrocyte damage and neuronal death, if oxidative stress occurs in the cells and ER redox state is disturbed by HIV infection. The fact that accumulation of β-amyloid occurs in HAD brain [189, 190, 192] and inside neurons [188] provides strong evidence that redox imbalance leading to global protein misfolding in HIV-infected CNS cells. Recent studies also demonstrate that exposure of endothelial cells to HIV results in acute and significant increases in their intracellular βamyloid levels. Although the mechanisms underlying this phenomenon are unclear, a primary factor is likely to be HIV-mediated oxidative stress and inflammation [191]. Thus, gp160 and other redox sensitive proteins could misfold as a result of HIV-mediated oxidative stress/inflammation.


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It is important to note that at therapeutic concentrations, HIV protease inhibitors (PIs), used for the highly activated antiretroviral therapy (HAART) for HIV-1 infection, are now known to activate the UPR in macrophages. This results in ER stress, depletion of ER calcium stores and activation of apoptosis in these cells [225]. Although the mechanisms underlying this effect of HAART are unclear, a recent report has demonstrated enhanced βamyloid levels in the brains of HAART-treated HAD patients. Thus, a better understanding of the cellular and molecular mechanisms of UPR activation and ER stress and the events that follow may provide useful insights for development of new therapeutic strategies for HAD in HIV-AIDS.

Role of Astrocytes in HAD CNS Cell Death Astrocytes comprise more than 50% of total cells in the CNS and they have crucial homeostatic and redox regulatory functions that maintain neuron integrity and essential brain function [226, 227]. Increasing evidence suggests that astrocytes play a prominent role in HIV neuropathogenesis. In the CNS, HIV affects astrocyte functions by distinct pathways [228, 229]. Astrocytes are natural host cells for HIV-1, particularly in advanced HAD [230]. Using state of the art technology, Churchill and coworkers demonstrated that astrocyte infection is extensive in human patients with HAD [203], occurring up to 19% of astrocytes. Moreover, astrocytes infection is frequently correlated with the severity of neuropathological changes, emphasizes the important role of astrocyte infection in HAD. Since the majority of cells in the CNS are astrocytes, they are a significant reservoir of latent HIV infection. Latent HIV infection results in global changes in astrocyte gene expression [228]. In addition, a subpopulation of latently infected astrocytes undergo apoptosis that correlates with severity of HAD [149, 203]. In the brain, astrocytes are exposed constantly to whole HIV particles, gp120 alone, Tat alone and other substances produced by HIV-1-infected microglia. It has been shown that HIV gp120 is toxic to astroglial cells by lipid peroxidation as a result of oxidative stress. In addition, HIV-1 efficiently binds to astrocytes and induces neuroinflammatory responses [231]. Since neurons are not infected by retrovirus, neuronal death is most likely due to reduced thiol support from dysfunctional astrocytes (causing oxidative stress and thiol deficiency in neurons). It has been reported that productive infection of astrocytes with HIV-1 leads to oxidative stress and cell death and neuropathology in a mouse model for HIV infection [232-234]. Upregulation of BiP has been documented in astrocytes in the CNS of HIV-positive individuals [144]. Additionally, Tat released from HIV-infected astrocytes induces mitochondrial dysfunction and neuronal death [208, 235-237]. Tat also impairs glutamate uptake in astrocytes, exposing neurons to glutamate excitotoxicity. Microarray analysis reveals HIV effects on gene expression in both human and mouse astrocytes [204]. It was demonstrated that similar changes were found in HIV-1-exposed mouse and human astrocytes in vitro, underscoring the usefulness of the mouse model for studying HIV-1 pathogenesis. These findings strongly suggest that changes in gene expression of astrocytes are a major component of the overall molecular profile of disease in the brains of HIV-1-infected patients, and also suggest that exposure of human or mouse

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astrocytes to HIV-1 in culture can be a useful tool for investigating the molecular and functional changes, involved in the development of HIV-associated dementia. Proteomic modeling of HIV-1 infected cells, in a study of astrocyte-microglia interactions, has shown that astrocytes have a profound effect on protein expression in HIVinfected microglia. This finding provides novel insights into the influence of astrocytes on the onset and progression of HAD [229]. This and similar studies also provide a new perspective on previously undisclosed pathogenic mechanisms for retrovirus-induced NDs and on the importance of CNS cell-cell interactions. Together the above observations underscore potential implications for therapeutic approaches toward treating HAD. While HAART reduces HIV-1 viral load and raises CD4+ T cell counts in the peripheral lymphoid system, neurologic damage is not significantly reduced in treated patients [238-240]. As noted above, this may be due to the fact that these drugs themselves activate the UPR and induce apoptosis [225] and thus could have adverse effects. It is possible that these drugs may not have therapeutic effects on HAD mechanisms, including oxidative stress [18, 21, 172, 174, 176]. Another possibility, as mention above, is that HAART targets replicating HIV in the CNS produced by macrophage-lineage cells but does not target HIV latent infection in astrocytes, thereby only providing partial clinical benefit to HAD patients.

Therapeutic Intervention Several important criteria must be satisfied before a candidate drug against NDs mediated by oxidative stress can be deemed suitable. 1) The drug must be able to attenuate, rather than completely shut-off ROS production, because low (appropriate) levels of ROS are beneficial to cells. 2) The drug should not only lower oxidation reactions but also increase the cell‟s capacity to cope with oxidative stress, by attenuating stress signals. 3) The drug must be nontoxic, stable, and suitable for long-term use because of the slow and progressive courses of most neurodegenerative diseases. 4) The drug should have a global effect in the cell. 5) The drug should also be orally bioavailable, and be able to cross the blood-brain barrier (BBB) as well as penetrate cell membranes. 6) The drug should be able to upregulate or stabilize Nrf2 and restore GSH levels in the cells by supplying cysteine, the precursor of GSH. This is because cells can develop thiol deficits even during antioxidant treatment, since neurodegenerative syndromes typically are diagnosed after they are well underway, at a time when CNS cells are already thiol-depleted, and unable to refill their stores with their own reducing equivalents. We have been working with two drugs that in combination may meet these requirements. An antioxidant and redox buffer, MSL, and the antioxidant/thiol replenishing agent N-acetylcysteine-amide (AD4) may act together to restore the redox state and to replenish the deleted GSH.


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1. Using the Antioxidant Drug MSL Alone, or in Combination with other drugs such as AD4, for Therapeutic Intervention Earlier work from our laboratory has shown that ts1-induced damage to the CNS and thymus of infected mice is suppressed by treatment of infected animals with N-acetyl cysteine (NAC) [170]. While NAC inhibits ts1-associated thymic atrophy, it has relatively limited effects against ts1-induced ND. This could be due to its limited ability to cross the BBB [241, 242].

Figure 5.

Recently, we have reported that MSL is much more effective than NAC, or other antioxidant that we have test in preventing oxidative stress mediated ND in ts1-infected mice [167, 177]. MSL is unique among many antioxidants and therefore investigating its mechanism of action in retrovirus-mediated NDs is highly significant. MSL is a phthalazine dione redox-buffering compound (Figure 5) with a proven non-toxic quality that modulates intracellular redox status by being able to accept and donate electrons and by scavenging free radicals, especially superoxide and peroxinitrite. The position of the amine group on the MSL phenolic ring enables MSL to enter cells. In cells and in animals MSL has both antioxidant and anti-inflammatory effects. MSL scavenges free radicals (ROS and RNS) and converts their energy into light (luminol and H2O). This process is reversible, and therefore the MSL molecule can be recycled (reusable). Luminol is also a well-recognized iron chelator and thereby preventing iron-catalyzed oxidative stress. Experiments in mice with oxidative stress related diseases reveal that MSL: 1) is relatively nontoxic, well absorbed and rapidly excreted upon systemic administration [243, 244]; 2) can balance disordered redox states in stressed cells by resetting proper redox potentials. This is accomplished by redox buffering actions, by its ability to scavenge free radicals and upregulate Nrf2 for antioxidant defense [245]; 3) decreases intracellular ROS levels in primary astrocyte cultures infected with ts1 [167]; 4) reacts with ONOO- to prevent protein nitration and oxidation in microglia cells (Qiang and Wong, unpublished data), and reduces markers of lipid peroxidation in CNS and thymus of ts1-infected mice; 5) prevents

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microglia-induced neuronal damage [246]; 6) restores mitochondrial membrane potential and mitochondrial-induced cell death in ts1-infected astrocytes and brain slice culture (unpublished data); 7) upregulates neuroprotective proteins such as vascular endothelial growth factor (VEGF) and Bcl2, an anti-apoptotic factor [247]; and finally, prevents oxidative stress not only in the CNS [167], but also in the thymus and intestine of ts1-infected mice [168, 169]. Since cystine/cysteine availability is rate limiting for GSH production in cells, it is possible that drugs that replenish cysteine are crucial for full protection of ts1-infected astrocytes. As a thiol amide, NAC-amide (AD4) is less toxic than NAC and can penetrate the BBB, cross cell membrane and supply cysteine to cells to overcome GSH depletion under oxidative stress [241]. Therefore, this effective, penetrative and nontoxic thiol provider could be suitable for long-term use in treating chronic NDs. AD4 has been shown to be neuroprotective in three different mouse models of NDs [242, 248, 249]. AD4 has also been shown to delay ts1-induced ND (Kim and Wong, unpublished data). As mentioned above, oxidative stress is also associated with neuropathogenesis of A-T. Several studies by others have shown that anitoxidants, such as NAC [250], EUK-189 [251], Tempol [252] and 5-carboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl (CTMIO) [253] improve motor deficits in Atm-/- mice. We have also administered MSL to Atm-/- mice via drinking water and found that MSL also improves motor performance in these mice (unpublished data). These results suggest that oxidative stress is closely linked to ND in A-T, and that antioxidant treatment could be an effective therapeutic approach for ND in A-T.

2. Using Chemical Chaperones and Proteostasis Regulators for Disease Intervention Cellular proteins face constant challenges to their homeostasis or proteostasis (refers to the stability, conformation, integrity, location and function of individual proteins making up the proteome of the cell). Defects in proteostasis occurring as a result of ER and oxidative stress may contribute to many diseases, including NDs, immunodeficiency, atherosclerosis [254] and cancer. Chemical or molecular resolution of ER stress can prevent misfolded protein accumulation and facilitate protein secretion. For example, certain small molecules that act as proteostasis regulators have been shown to ameliorate proteostasis dysregulation in some of the most challenging diseases [255]. Administration of chemical chaperone phenylbutyrate (PBA), which improves ER folding capacity and trafficking, reduces ER stress and restores glucose homeostasis in a mouse model of type 2 diabetes [256]. PBA also restores proteostasis of the misfolding-prone cystic fibrosis transductance regulator, so it is currently being tested in clinical trials to treat cystic fibrosis [255]. Proteostasis regulators that selectively target the folding of viral proteins have been suggested as antiviral drugs that may prevent evolution of drug resistance [257]. Our published data indicate that PBA reduces the accumulation of gPr80env in ts1-infected astrocytes and reduces the levels of the ER stressrelated chaperone BiP [Kuang et al, Neurochem Int 2010]. As a result, levels of the survival factor Bcl2 increases and the apoptotic markers Bax and caspase 3 decrease allowing extended survival of ts1-infected cells. PBA also delays the onset of ND in ts1-infected mice [Kuang et al 2010 Neurochem Int [258]. The advantage of chemical chaperones is that they are not disease specific, but instead that they target a common pathway to many diseases (i.e.


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proteostasis imbalance). Thus, restoration of proteostasis, mediated by proteostasis regulators, may ameliorate some of the most important diseases of our era, including HAD and AIDS. Interestingly, small molecules have been identified that target HIV envelope proteins for ERassociated protein degradation, and they have been shown to be effective for HIV therapy [259]. Taken into consideration of the different effects of MSL, AD4 and PBA (Figure 6) a combined treatment with these three drugs with different targets may provide better protection relative to treatment with any one of the drugs alone, not only for survival but also in the major marker analysis.

Figure 6.

3. Potential Therapeutic Intervention Using NSC Transplantation and Antioxidants Since self-renewal and proliferation of neural stem cells are defective in ATM deficiency, a NSC-based treatment may be beneficial to this genetic mutation-related ND. NSCs possess a range of actions that can be potentially used for therapy [260]. They are capable of selfrenewal, and can differentiate into cells of astroglial and neuronal lineages in the CNS [261]. In addition, they can readily proliferate ex vivo, and when transplanted into diseased brains, where they migrate and differentiate according to cues from host tissues, appear to capable of affecting host cells in the recipient brain. Recent studies imply that NSCs may hold promise for therapeutic treatment of human genetic diseases resulting in NDs, such as in A-T. The first study involves the nervous (nr) mutant mice, in which, like in ATM deficiency, the Purkinje neurons (PN) become abnormal and dysfunctional and a majority of these cells die by the fifth week [262]. By transplanting normal NSCs into the cerebellum of nr mutant mice, PN function is repaired by the transplanted NSCs, not just by cell replacement, but also by rectifying their gene expression and restoring defective molecular homeostasis due to the gene defect. In another study, intracranial transplantation of normal NSCs was used to treat mice in a model of the human neurodegenerative disease called Sandhoff disease [260]. This study shows that the transplantation of normal NSCs into disease brains delays disease onset, preserves motor function, and prolongs survival of the diseased mice. These two studies show that NSCs may have a broad repertoire of therapeutic actions, of which neuronal replacement is but one. Thus, NSCs may also help in formulating a rational multi-factorial strategy, including combination with antioxidants, for treatment of NDs.

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Conclusion This chapter focuses on two animal models of NDs that show some commonality involving oxidative stress, ER stress, and mitochondrial dysfunction. These findings also suggest that ROS signals can engage specific pathways to generate their effects in different cell types in the CNS. This is not only true for these two particular models, but increasing bodies of evidence suggest that multiple human NDs and other diseases are affected or regulated by this culmination of cellular stress. Thus, the inevitable problem in almost all NDs is accumulation of both ROS with impaired redox homeostasis and accumulation of abnormal misfolded proteins with stress on ER and mitochondrial functions. Activation of inflammatory response, apoptotic pathways, and cell death is the ultimate result. Understanding theses multifaceted pathways and the intricate parts involved with this mechanism will be beneficial for us to develop basic therapeutic strategies against ND in general. We can target the oxidative stress, or ER stress, or mitochondrial stress directly and many therapies may need a combination of these. Small molecule nontoxic drugs that can effectively pass the blood brain barrier and alleviate these stresses may be extremely beneficial in the maintenance and treatment of several NDs and promote neuronal cell survival. We have shown that treatment with the redox-active small molecules, the phthalazine dione (MSL) fully prevent the neurodegenerative syndrome induced by infection with the murine retrovirus mutant ts1. Both the oxidative stress and the ER stress with excessive accumulation of viral mutated proteins in ER as well as the neuronal loss and gliosis were fully prevented by these treatments. In the murine model of A-T, we have also observed that the global oxidative stress with neuronal damage and the accompanying gliosisis can also be fully prevented by treatment with MSL. Potential therapy treatment using drugs that supply cysteine to cells to overcome GSH depletion under oxidative stress, as well as chemical chaperone treatment against NDs are discussed. Further understanding of the mechanism of action of these drugs singly or in combination can more specifically target these drugs for more effective treatment. Finally although many questions remain to be fully addressed, stem cell-based therapy represents a potential highly rewarding treatment of genetic-related NDs. Therefore further research into this area may provide rationale for new therapies in the future.

Acknowledgments The authors thank Drs.Virginia Scofield and Mingshan Yan as well as Dr. Joanne Ajmo and Mr. Mark Henry from Bach Pharma for their helpful support and discussion. The authors also thank Ms. Shawna Johnson for her assistance of the preparation of this chapter. This work was supported by NIH Grants MH071583 and NS043984 (awarded to Dr. Paul Wong) the University of Texas MD Anderson Cancer Center Support Grant CA16672. Support was also provided by the Longevity Foundation of Austin, Texas and The A-T Children‟s Project of Deerborne Florida.


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[251] Browne, SE; Roberts, LJ, 2nd; Dennery, PA; Doctrow, SR; Beal, MF; Barlow, C; Levine, RL. Treatment with a catalytic antioxidant corrects the neurobehavioral defect in ataxia-telangiectasia mice. Free Radic Biol Med, 2004, 36, 938-42. [252] Schubert, R; Erker, L; Barlow, C; Yakushiji, H; Larson, D; Russo, A; Mitchell, JB; Wynshaw-Boris, A. Cancer chemoprevention by the antioxidant tempol in Atmdeficient mice. Hum Mol Genet, 2004, 13, 1793-802. [253] Gueven, N; Luff, J; Peng, C; Hosokawa, K; Bottle, SE; Lavin, MF. Dramatic extension of tumor latency and correction of neurobehavioral phenotype in Atm-mutant mice with a nitroxide antioxidant. Free Radic Biol Med, 2006, 41, 992-1000. [254] Hotamisligil, GS. Endoplasmic reticulum stress and atherosclerosis. Nat Med, 16, 3969. [255] Balch, WE; Morimoto, RI; Dillin, A; Kelly, JW. Adapting proteostasis for disease intervention. Science, 2008, 319, 916-9. [256] Ozcan, U; Yilmaz, E; Ozcan, L; Furuhashi, M; Vaillancourt, E; Smith, RO; Gorgun, CZ; Hotamisligil, GS. Chemical chaperones reduce ER stress and restore glucose homeostasis in a mouse model of type 2 diabetes. Science, 2006, 313, 1137-40. [257] Geller, R; Vignuzzi, M; Andino, R; Frydman, J. Evolutionary constraints on chaperonemediated folding provide an antiviral approach refractory to development of drug resistance. Genes Dev, 2007, 21, 195-205. [258] Liu, N; Qiang, W; Kuang, X; Thuillier, P; Lynn, WS; Wong, PK. The peroxisome proliferator phenylbutyric acid (PBA) protects astrocytes from ts1 MoMuLV-induced oxidative cell death. J Neurovirol, 2002, 8, 318-325. [259] Jejcic, A; Daniels, R; Goobar-Larsson, L; Hebert, DN; Vahlne, A. Small molecule targets Env for endoplasmic reticulum-associated protein degradation and inhibits human immunodeficiency virus type 1 propagation. J Virol, 2009, 83, 10075-84. [260] Lee, JP; Jeyakumar, M; Gonzalez, R; Takahashi, H; Lee, PJ; Baek, RC; Clark, D; Rose, H; Fu, G; Clarke, J; McKercher, S; Meerloo, J; Muller, FJ; Park, KI; Butters, TD; Dwek, RA; Schwartz, P; Tong, G; Wenger, D; Lipton, SA; Seyfried, TN; Platt, FM; Snyder, EY. Stem cells act through multiple mechanisms to benefit mice with neurodegenerative metabolic disease. Nat Med, 2007, 13, 439-47. [261] Parker, MA; Anderson, JK; Corliss, DA; Abraria, VE; Sidman, RL; Park, KI; Teng, YD; Cotanche, DA; Snyder, EY. Expression profile of an operationally-defined neural stem cell clone. Exp Neurol, 2005, 194, 320-32. [262] Li, J; Imitola, J; Snyder, EY; Sidman, RL. Neural stem cells rescue nervous purkinje neurons by restoring molecular homeostasis of tissue plasminogen activator and downstream targets. J Neurosci, 2006, 26, 7839-48. [263] Lo SC, Pripuzova N, Li B, Komaroff AL, Hung GC, Wang R, Alter HJ. Detectio of MLV-related virus gene sequences in blood of patients with chronic fatigue syndrome and healthy blood donors. PNAS, 2010, epub ahead of print. [264] Tu BP, Weissman JS. Oxidative protein folding in eukaryotes: mechanisms and consequences. JCB, 2004, 164, 341-346 [265] Kuang X, Hu W, Yan M and Wong PK. Phenylbutyric acid suppresses protein accumulation-mediated ER stress in retrovirus-infected astrocytes and delays onset of paralysis in infected mice. Neurochem Int. 2010. In press.


Chapter 2

Mechanisms of the Motoneuron Stress Response and Its Relevance in Neurodegeneration Mac B. Robinson, David J. Gifondorwa and Carol Milligan* Dept. of Neurobiology and Anatomy, Wake Forest University School of Medicine, Winston-Salem, North Carolina, USA

Abstract Preserving motoneuron viability and function during disease or after traumatic injury is an intense area of research focusing on both the molecular mechanisms of degeneration and therapeutic interventions to prevent it. Understanding how motoneurons sense and respond to injury or pathology may help us identify potential targets for therapeutic intervention. The motoneuron stress response or heat stress response (HSR) has been an area of investigation spanning now well over a decade and has explored the role of heat shock protein (HSP) expression during physiological stress and in animal models of neurodegenerative disease. What we have found from these studies is that, in the midst of a physiological stress, motoneurons rarely activate a classical stress response as characterized by increased expression of Hsp70. It has been proposed that this lack of stress response activation could contribute to pathological motoneuron dysfunction and degeneration. Understanding the molecular mechanisms responsible for this phenomenon may provide insights as to why motoneurons are the pathological hallmark in amyotrophic lateral sclerosis (ALS) and other neurodegenerative conditions.

Keywords: motor neuron, stress response, heat shock proteins, amyotrophic lateral sclerosis, neurodegenerative disease, heat shock protein 70.

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Mac B. Robinson, David J. Gifondorwa and Carol Milligan

Introduction The ability of a cell to properly sense and respond to stress is critical for the cells‟ and indeed the organism‟s ultimate survival. One way cells respond to stress is through the activation of intracellular signaling pathways that, through a series of subsequent phosphorylation events, influences macromolecular interactions and de novo protein synthesis that are meant to abrogate the deleterious effects of the stress, or in some cases exacerbate them; therefore, facilitating the death of the cell. One beneficial response that is influenced by these intracellular signaling pathways is the heat stress response (HSR). Heat shock proteins (HSPs), the main product of the HSR, protect cellular proteins from damage by acting as molecular chaperones; binding to and refolding the damaged proteins, hence preserving their function. Alternatively, Hsps may facilitate the removal of irreparably damaged proteins preventing their toxic accumulation (Luders et al., 2000; Kaushik and Cuervo, 2006). While initially discovered in response to elevations in the thermal environment (Ritossa, 1962; Ritossa, 1996), the HSR is activated in response to other stresses including injury, oxidative stress, exposure to heavy metals, antibiotic treatment, infection, and UV radiation exposure (Schlesinger, 1990; Welch, 1992; Morimoto et al., 1997; Kregel, 2002). Since its discovery, a field of research has developed focusing on the products and mechanisms of the HSR in virtually all realms of the biological sciences. Work in various cell types following numerous insults has provided results leading to an understanding of some of the mechanisms involved in the HSR (see figure 1). With continued investigations, however, a number of examples have been found that suggest that the HSR is not as homogenous as once thought. It is now known that not all cells respond to a particular stress in the same manner, where the response can vary from eliciting a partial response to not exhibiting a stress response at all (Morimoto and Fodor, 1984; Manzerra and Brown, 1992; Mathur et al., 1994; Tacchini et al., 1995; Marcuccilli et al., 1996; Goldbaum and RichterLandsberg, 2001; Kaarniranta et al., 2002; Kalmar et al., 2002; Batulan et al., 2003; Robinson et al., 2005). The extent to which neurons, and specifically motoneurons mount an HSR is variable and atypical (Manzerra and Brown, 1992; Kalmar et al., 2002; Batulan et al., 2003; Robinson et al., 2005). The molecular events modulating this lack of response or altered response have yet to be elucidated, but current data suggest a number of possibilities including but not limited to aberrant regulation of the intracellular signaling cascades associated with activation of the HSR, insufficient HSF activation and stress buffering. The clinical relevance of this line of study is certainly one of the more compelling examples given the pathology of motoneuron diseases including amyotrophic lateral sclerosis (ALS) that can include protein aggregation and increased ROS, all of which can lead to motoneuron cell death, but can be ameliorated to some extent by HSPs. It has been proposed that, if motoneurons were able to readily initiate a full HSR, it may positively influence the outcome of motoneurons during disease (Okado-Matsumoto and Fridovich, 2002). Indeed, as will be discussed, attempts to modulate the HSR have shown promise in mitigating the pathology in animal models of ALS and may be a promising therapeutic avenue. However, the effect of HSR modulation at this point is global and not targeted directly at the motoneuron; therefore, discussion will also be made of the importance of the knowledge we gain from these studies and where the protective effect may be derived with treatments that induce the stress response.

Mechanisms of the Motoneuron Stress Response and Its Relevance…

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Figure 1. During the cell‟s basal state or unstressed condition, the heat shock transcription factor, HSF, and heat shock proteins, mainly Hsp70 and Hsp90 reside in the cytoplasm in a heterocomplex. This relationship renders the HSF in an inactive monomeric state. However, at the outset of a stress, this complex dissociates, most likely due intracellular signal and protein misfolding. With the HSF/HSP complex broken the HSF is free to trimerize and translocate to the nucleus. The prototypical heat stress response is characterized by the activation of a number of signal transduction pathways. These molecules can act to phosphorylate the HSF trimer, most likely while bound to the heat shock element in the nucleus. This inductive phosphorylation creates a transcriptionally competent HSF trimer. Transcription of HSPs ensues, increasing the free pool of HSPs and potentially mitigating the stress on a number of levels from refolding, protecting or degrading irreparably damaged proteins to intercepting both apoptotic and non-apoptotic cell death pathways. Mechanisms of termination are still a matter of debate; however, there is recurring evidence of regulation by the inducible HSPs, especially Hsp70 and/or Hsp90 in conjunction with p23. Motoneuron HSR dysfunction may occur on a number of levels. Signaling activation in motoneurons appears to be dampened compared to other cell types. Additionally, when it is activated, it appears unique. Activation of p38 appears to be a repeatable event during neurodegeneration and may be an attempt to activate Hsp25 to modulate antioxidant defenses. Dampened signaling could result in a lack of inducible phosphorylation. In support of this, overexpression of wtHSF does not facilitate activation of the response. Furthermore, increased basal levels of Hsc70 may act as an endogenous stress buffer to instantly protect motoneurons from most stress, allowing the large cell to conserve much needed resources in the midst of a stressor. See Manzerra and Brown, 1992; Batulan, et al. 2003; Morimoto, 1993; Pirkkala, et al. 2001; Saleh, et al.2000; Beere, et al. 2000, and Ruchalski, et al. 2006.

Signaling Influences on the Stress Response There are 4 main steps involved in the activation of the HSR: 1) release of the heat shock transcription factor (HSF) from multi-chaperone complexes, 2) homotrimerization and nuclear translocation of HSF, 3) binding of HSF to the heat shock element (HSE), and 4)

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transcriptional activation of HSF by phosphorylation and transcription of heat stress genes. Some of these steps appear to be independent events in the activation of heat stress genes and can be uncoupled through the use of certain chemicals (Jurivich et al., 1992). Additionally, intracellular signaling molecules play a critical role in the activation and repression of HSF. HSR activation and subsequent production of Hsps is a transcriptionally regulated event though the kinetics of the activation can vary between cell types. Additionally, at least one cell line has shown a translational mechanism of activation (Kaarniranta et al., 2002), but this seems an exceedingly rare event. The transcription factor responsible for HSP activation is the HSF. Mammals have 3 HSFs, HSF1, HSF-2 and HSF-4 with HSF-1 being the stress inducible HSF and HSF-2 being involved in development (Pirkkala et al., 2001). HSF-4 appears to be expressed in a cell type specific manner. These proteins can be regulated by alternative splicing and as recent reports indicate, by heterocomplexing within the HSF family (Ostling, et al. 2007). Most organisms harbor the stress activated HSF, HSF-1, some other stress activated HSF, or an HSF (Drosophila only has one) that can function in all aspects of cell development and stress (Birch-Machin et al., 2005). Other proposed stress activated HSFs, like HSF-3 in the embryonic chick, are most likely regulated similarly. HSF1 and HSPs are in heterocomplex when the cell is in a basal, unstressed state. This complex prevents the homotrimerization step of HSF-1 activation (Santoro, 2000; Pirkkala et al., 2001). The molecular mechanisms that dissociate this complex are not completely understood, but presumably, once damage occurs to cellular proteins, the free pool of Hsps becomes depleted with chaperones being bound to misfolded or damaged proteins (Morimoto, 1993). This depletion triggers a release of chaperones from the HSF/HSP complexes liberating HSF-1. Subsequently, HSF-1, through what is proposed to be a conserved redox mechanism, associates into trimers. (Zhong et al., 1998; Ahn and Thiele, 2003). These trimers translocate to the nucleus and associate with the heat shock elements (HSEs) of stress inducible genes containing the consensus sequence nGAAnnTTCnnGAAn (Morimoto, 1993). This process seems not only highly conserved, but also reproducible under a number of circumstances. Purified HSF-1 can trimerize and bind to the HSE in vitro in response to increased calcium, heat, or reactive oxygen species (ROS), (Mosser et al., 1990) suggesting this initial step is fairly liberal in its execution to provide ample response to cover a diversity of insults. Additionally, a recent report has described a role for HSF-2 in stress induced HSR activation. Traditionally, thought of as an HSF that was critical during development rather than mitigating stress, this HSF may associate with HSF1 to modulate expression of heat stress inducible genes (Ostling et al., 2007). HSF1 associating with other HSFs is not unheard of. In avian cells, HSF3 and HSF1 have been shown to be in association, though the role of this association is unclear, but most likely a regulatory event (Nakai et al., 1995; Tanabe et al., 1998). However, this may be relegated to embryonic and undifferentiated cells. Another report indicates that in the mature avian system HSF3 may be utlized by mature blood cells while HSF1 is the main player in the brain (Shabtay and Arad, 2006). This would suggest that, in addition to cell type and transcriptional factors controlling HSF activation, regulation may also occur on a developmental level. One of the most interesting examples of a complex family of HSFs lies in Arabidopsis thaliana, an organism with nearly 21 HSFs organized in to 3 groups and 14 classes (Nover et al., 2001). A recent study increased that number to 22 and also found 25 HSFs in rice (Guo, et al. 2008). While physical translocation to the nucleus and binding of HSF to the HSE are obvious critical steps, this is a transcriptionally impotent arrangement and further modification


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through phosphorylative modification of serine residues in the HSF-1 regulatory domain is needed to fully activate the system. For such regulation, one looks toward the many intracellular kinases within the cell responsible for phosphorylative modifications and generation of intracellular signals. Regulation of HSF-1 activity may be attributed to a number of intracellular kinases including, but not limited to extracellular signal regulated kinase (ERK), phosphatidylinositol 3 kinase (PI-3K)/protein kinase B (Akt), glycogen synthase kinase -3beta (GSK-3B), calmodulin dependent kinases II and IV (CAMKII and CAMKIV), p38 mitogen activated protein kinase (MAPK) and c-jun N-terminal kinase (JNK) (Bijur and Jope, 2000; Park and Liu, 2001; Pirkkala et al., 2001; Taylor et al., 2007). Serines 303, 307, and 363 are thought to be constitutively phosphorylated and exert a repressive effect on HSF-1. However, phosphorylation of serine 230 appears to be associated with transcriptional competence (Pirkkala et al., 2001). The activity of PI-3K and p38 may result in positive regulation of the HSR. Negative regulation, however, comes from a number of sources including ERK, GSK-3B, and CAMKII suggesting a fairly tight regulation of the activation of the response and an even tighter redundant negative regulation (Soncin et al., 2000). In addition to phosphorylation, sumoylation of lysine 298 on HSF also appears to be critical in activating the response (Hong et al. 2001). Interestingly, the repressive phosphorylation site S303 must be phosphorylated for this activating sumoylation to occur (Hietakangas, et al. 2003). The idea of stress kinase activation (pJNK and p38) and subsequent, linear activation of the HSR provides interesting discussion simply because, at least in the case of JNK, the relationship is not linear. Indeed, JNK is thought to be involved as a negative regulator of HSF activity. Overexpression of JNK1 and JNK2 negatively regulates HSF-1 activity. Paradoxically, overexpression of JNK1 and JNK2 also induces the expression of reporter constructs carrying an Hsp70 promoter. (Park and Liu, 2001). Yet, SP600125, a JNK phosphorylation inhibitor, can decrease the amounts of hyperphosphorylated HSF-1 (Park and Liu, 2001; Kim et al., 2005). These contrasting results with JNK manipulation make this kinase an interesting subject in the study of the HSR. Future experiments utilizing JNK knock-out mice may be very informative in assigning JNKs ultimate role in activation of the response. The MAPK, p38 is also an important component in the response. For one, it may be a first line of defense given a triggering event of the response is oxidative stress and Hsp27 expression has the ability to modulate other antioxidant molecules (Arrigo, 2001). Additionally, p38 may be required for activation of pAkt that may fully activate the response (Mustafi, et al. 2009). This would suggest that the stress response, at least in some cell types, may be a biphasic event. This data, and other data to be discussed, suggest a level of complexity previously not thought of in terms of the levels of activation. Once the HSF-1 trimer is activated, Hsp production proceeds. The termination of the response in regulated on some level by the induced chaperones themselves (Mosser et al., 1993; Lee and Schoffl, 1996; Satyal et al., 1998). It appears levels of the inducible Hsp70 may be an important factor in limiting the intensity of the response. Indeed, overexpression of Hsp70 prior to heat stress results in a dampened response. Once free pools of HSPs accumulate to high levels, they being to associate with free HSF-1. In addition to other proteins, namely HSBP1, the HSF1 trimer is dephophorylated, released from the HSE and complexed with chaperones returning the cell to a basal state.

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Heat Shock Transcription Factors and Regulation of the HSR in the Nervous System The heat shock transcription factors (HSFs) appear to be responsible for regulating Hsp expression and the HSR. While there is conserved identity in protein sequence, the HSFs are not functionally interchangeable, and regulation of HSF activity is not consistent for each. For example, There are four HSFs, and all four have demonstrated roles in the nervous system. HSF1 becomes functional as a trimer, whereas HSF2 resides as a nonfunctional dimer then trimerizes under the appropriate stimulus (There are four characterized HSFs). HSF1 appears to regulate stress induced expression of HSPs. HSF1 null mice develop and are born alive, and endogenous expression of HSPs is consistent with wild-type animals. The animals or cells isolated from the animals; however, cannot mount an HSR in response to heat stress. HSF3 is unique to avians and appears to function cooperatively with avian HSF1. HSF2 does not appear to be involved in increased expression of Hsps in response to stress, but rather may dictate developmental expression of the proteins. In the CNS, HSF1 null adult mice exhibit progressive loss of myelin and astrogliosis. These events are enhanced when HSF2 or 4 are also knocked-out (Homma et al, 2007). In rodents, HSF1 and HSF2 are expressed in neurons, astrocytes and oligodendrocytes with HSF2 appearing critical for CNS development (Walsh et al., 1997; Stacchiotti et al., 1999; Wang et al., 2003; Chang et al., 2006). HSF4 is expressed in the developing CNS and certain neuronal populations (Hu and Mivechi, 2003), and mutations in the DNA binding domain of HSF4 are associated with inherited cataract formation (Bu et al., 2002). The specific roles of each HSF are unclear at this point; however, it appears as if each regulates distinct gene expression during development or in response to stressful stimuli. The genetic regulation is further enhanced when HSFs work in concert or in opposition (reviewed in Akerfelt et al., 2007). Furthermore, it is not known if the CNS abnormalities observed in the mutant mice are because of insufficient HSP or other gene expression.

The Motoneuron Stress Response Examination of motoneuron signaling and the HSR are associated with some unique challenges. The main hurdle is the difficulty in culturing motoneurons in sufficient amounts to perform the biochemical and molecular analysis to fully characterize motoneuron signaling and the motoneuron HSR. However, using immunological approaches and in situ hybridization in vivo and in vitro systems have provided a relatively informative picture of how motoneurons respond to loss of trophic support, axotomy, hyperthermia, and in neurodegenerative disease (Manzerra and Brown, 1992; Kalmar et al., 2002; Batulan et al., 2003; Batulan et al., 2005; Batulan et al., 2006). As for in vitro approaches, some investigators have used dissociated spinal cord or spinal cord slice cultures (Batulan et al., 2003; Batulan et al., 2005; Batulan et al., 2006; Taylor et al., 2007). This has allowed for some extrapolation as to how certain processes may be executed; however, experiments to fully examine the mechanistic features of process in specific cell types, namely motoneurons are difficult in these systems. Another approach is to use a culture of purified motoneurons. While the cultures allow for more biochemical analysis including western blots, RNA analysis, and subcellular


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fractionations, they are very time-consuming in order to collect sufficient material for analysis. Many investigators utilize chick motoneuron cultures because they are amenable in yielding sufficient material for analysis and are more economically feasible than similar experiments using mammalian systems. With regard to the HSR, however, interpretations of results must consider that heat shock factor-3 (HSF-3) appears to be the main stress inducible transcription factor used in the embryonic avian system, rather than heat shock factor-1 (HSF1) that is used in mammals. The significance of the use of HSF1 vs. HSF3 is not known because there appears to be cooperation between the two factors, atleast in embryonic development (Nakai et al., 1995; Kawazoe et al., 1999). Intracellular signaling is highly conserved however, so some of the mechanisms involved in activation the HSR in chick may be conserved in mammals (Pirkkala et al., 2001). For instance, cultured muscle cells from the chick and from the mouse appear to respond to heat stress in a similar manner albeit at a different threshold (unpublished observations).

Figure 2. HSFs regulate gene expression independent of HSR genes during development and for physiological homeostasis. Heat Shock Transcription Factors, especially HSF1 are thought to be key regulators of the HSR and expression of Hsps. These factors are also key regulators of other gene expression specific to developmental and physiological events as illustrated in the figure (reviewed in Akerfelt et al., 2007)

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Figure 3. Diagram to illustrate the complexity of the cellular stress response. Motoneurons, like many other cell types encounter potential toxic changes in the extracellular environment. These changes result in alterations in plasma membrane structure (1), signal transduction pathways (2), generation of intracellular ROS and Ca+2 imbalances (3), and protein denaturation or unfolding (4). The response to these challenges include increased expression of HSR genes, including Hsps regulated in part by interactions between the HSFs and other binding proteins (5). The Hsps are critical to refolding of denatured proteins (6). Denatured proteins not refolded by Hsps are degraded by the ubiquitinproteasome pathway (7). When this pathway become damaged, perhaps by being overloaded with denatured proteins, protein aggregation can occur (8). Protein aggregation may contribute to damage to intracellular organelles, further compromising the cell. Additionally, intracellular ROS and Ca+2 imbalances can contribute to mitochondrial and ER stress (red arrows). The ER can respond with the unfolded protein response (9) that may contribute to increased expression of HSR proteins; however, it also results in an overall decrease in protein translation. See Pirkkala et al., 2001, Boyce and Yuan, 2006 and Lindholm et al., 2006 and Prahlad and Morimoto, 2009 for reviews

Some observations of induction or non-induction of a motoneuron HSR suggest that HSF1 may be inappropriately or not phosphorylated sufficiently to confer transcriptional competence (Batulan et al., 2003). Another theory is that alternate signaling mechanisms exist for specific Hsp regulation in motoneurons. Constitutively active CAMKIV in motoneurons appears to regulate Hsp70 expression, whereas this kinase has no effect on Hsp70 expression in fibroblasts (Taylor et al., 2007). Surprisingly, co-injection of a construct encoding wtHSF1, in addition to CA-CAMKIV, abolished this effect. This would suggest that CAMKIV may be activating Hsp70 expression independent of HSF1. Motoneurons are one of the largest cells in an organism and therefore thought to have a higher metabolic demand than most cells. During embryonic and neonatal development, motoneuron survival is dependent on target derived trophic support and in the absence of trophic support they die (Oppenheim et al., 1988). Motoneurons, especially those that appear


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to be susceptible in ALS do not express Ca+2 binding proteins calbindin-D28k and parvalbumin, suggesting that they have a diminished ability to buffer cytosolic Ca+2(Shaw and Eggett, 2000). Motoneurons also express the Ca+2 permeable AMPA receptors. They also appear to be very vulnerable to mitochondrial dysfunction (reviewed in von Lowinski and Keller, 2005). Given these facts, one would expect this particular cell to have the HSR molecular machinery poised for activation. Motoneurons in culture without trophic support die; however, it takes approximately 16 hours for cell death associated events to occur (Li et al., 2001). If availability of trophic support is so critical for survival, one might reason that during the initial 16 hours, the cells may mount a HSR in an attempt to promote survival. This however, does not appear to be the case (Robinson et al., 2005). Additionally, motoneurons barely initiate an HSR in response to normal heat stress and no response to H2O2 treatment (Robinson et al., 2005; 2007). Furthermore, stressed induced induction of the HSR in motoneurons does not appear to be protective. For example, after exposure at 45oC motoneurons will increase expression of Hsp27, 40 and 90, but not 70 and therefore are not considered to mount a HSR (Robinson et al., 2005). If however, the cells are exposed to 50oC, all Hsps, including 70 have increased expression. Despite the increased expression of Hsp70, there is no protection conferred when the cells are exposed to a subsequent stress such as H2O2 (Robinson et al., 2007). Motoneurons express high amounts of endogenous Hsc70 and this has been proposed as a reason why motoneurons seem resistant to HSR activation (Manzerra and Brown, 1996). Many functions of both the inducible and constitutively expressed proteins are redundant and the ability of free Hsc70 to dampen the stress response may be one of those redundant functions. Additionally, increased Hsc70 could act as a stress buffer. It could make sense for a cell the size of a motoneuron to have a large amount of free chaperone available for use so not to deplete resources for the physiological needs of the cell. Interestingly, one molecular event observed during the HSR, translocation of Hsc70 to the nucleus, is retained in motoneurons (Manzerra and Brown, 1996). Therefore, motoneurons still recognize and, in part, respond to stress in what would be a typical manner early in the execution of the HSR, yet a molecular trigger is not occurring to sufficiently activate the HSR system.

The Heat Stress Response in Motoneuron Injury and Pathology Probably the most common in vivo motoneuron injury model is sciatic nerve axotomy. Motoneurons, like DRG, the other neuronal component affected by axotomy, respond through the activation of JNK, p38, MAPK and PI-3K after axotomy (Murashov et al., 2001; Yang et al., 2006). Additionally, downstream effectors like c-jun exhibit increased phosphorylation (Brecht et al., 1997). What does this mean for Hsp expression? It appears that in the case of axotomy, Hsp27 or Hsp25 and Hsp90, but not Hsp70 are upregulated in the motoneuron (Murashov et al., 2001; Kalmar et al., 2002; Tidwell et al., 2004). Interestingly, p38 appears to be a critical link to this stress response in that Hsp25 expression can be inhibited by the p38 inhibitor, SB203580 (Murashov et al., 2001). These data clearly show that motoneurons do respond to injury and Hsp expression can be increased. However, Hsp70 appears to not be a component of this response.

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Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder affecting both upper and lower motoneurons, resulting in progressive muscle weakening and loss of motoneuron function, ultimately leading to paralysis and death within 3-5 years of diagnosis. The pathology of the disease can be mimicked in transgenic mice harboring a mutation in the Cu/Zn superoxide dismutase-1 (SOD-1) gene. Altered Hsp levels during the disease process have been observed; however these changes do not, in most cases, appear to coincide with any overt signaling changes. Aggregated proteins may act as a chaperone sink and may be one mechanism by which neurodegerative diseases may alter HSP levels (Okado-Matsumoto and Fridovich, 2002). The free pools of HSPs are depleted by misfolded or damaged proteins and sequestered in the pathological aggregates. Hsp70 is a component of these aggregates as are other chaperones including glucose regulated protein 78/BiP (GRP78), Hsp25 and Hsp 105. (Yamashita et al., 2007) (Maatkamp et al., 2004; Strey et al., 2004). Individual chaperone levels can decrease as much as 50 %. Despite this there appears to be no compensatory activation of the response to restore the free pools of chaperone resulting in an arguably pathological situation. These data punctuate the fact that despite cellular stresses that would likely cause the demise of the neuron, motoneurons seem to be refractory to activating a stress response. In the mutant SOD1 mouse model there are numerous events that suggest activation of an HSR. Stresses including mitochondrial swelling and vacuolization and mutant SOD1 inclusions/aggregates and toxicity providing ample opportunity for cellular dysfunction and cell stress (Manfredi and Xu, 2005). While an overt HSR including increased expression of Hsp70 does not appear to be activated, an unfolded protein response has been suggested to play a role. The endoplasmic reticulum (ER) and Golgi are thought to be potential targets of mutant SOD1 toxicity by its accumulation of misfolded mutant SOD1 that then initiates an ER stress response that may contribute to pathogenesis in ALS (Tobisawa et al., 2003; Atkin et al., 2006; Kikuchi et al., 2006; Nagata et al., 2007; Urushitana et al., 2008). Although these previous studies have focused on the putative role of ER stress in the progression vs. the onset of disease in ALS mice, a recent study has provided evidence consistent with ER stress being a contributor to early disease onset in mouse models of ALS as well as playing a role in the selective early vulnerability of motoneurons (Saxena et al., 2009).

The HSR as a Therapeutic Overexpression of individual chaperones, particularly Hsp70, was one of the first experimental measures used to assess the therapeutic role for Hsps. The emphasis on the Hsp70 family of proteins stems from its abundance during the HSR and the multitude of effects these proteins have on not only cell death pathways, but also many physiological processes such as pathological aggregation (Sharp et al., 1999; Muchowski et al., 2000; Muchowski, 2002). Additionally, therapeutic preconditioning, a means by which one stresses cells to increase the levels of HSPs in the cell, but maintain cell viability, has also been used to demonstrate the beneficial effects of the stress response under conditions which would normally be cytotoxic. Overexpression of mutant superoxide dismutase-1 (mSOD1) and Hsp70 in the same cell show increased survival and reduced cellular aggregation formation (Bruening et al., 1999). These and other studies have contributed to a wealth of literature


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demonstrating the protective affects of Hsp70 and providing some hope that modulating the levels of Hsp70 may be beneficial in particular neurodegenerative diseases. However, when mSOD1 mice are crossed with mice that ubiquitously overexpress Hsp70 no benefit was observed (Liu et al., 2005). Overexpression of another potent anti-apoptotic chaperone, Hsp27, has also been investigated and this chaperone also did not increase their lifespan (Krishnan et al., 2008). However, a recent study from our laboratory using purified Hsp70 showed a benefit to mSOD1 G93A mice when Hsp70 was given by intraperitoneal injection (Gifondorwa et al., 2007). This raises the common question of the method of administration and presents the possibility of an unknown effect exerted by extracellular Hsp70. Another question is whether overexpression of a singular chaperone is sufficient to rescue the mSOD1 phenotype. In support of multiple chaperone overexpression, when Hsp70 and Hsp27 are overexpressed together a delay in pathology in the G93A mouse is observed (Patel et al., 2005). Additionally, a recent study using arimoclomol, a hydroxyl-amine derivative and coinducer of the HSR, to increase multiple Hsps at the time of disease onset in the mSOD1 G93A did result in some rescue of motor function and pathology (Kalmar et al., 2008). Additionally, this study used a sufficient number of animals to properly control for background noise influencing the results, a recent criticism of past mSOD1 mouse studies (Benatar, 2007). The arimoclomal study suggests that not only may regulation of multiple Hsps be critical, but intervention with the proper pharmacological treatment at disease onset may be plausible. This leads us to an area of study that has gained momentum recently and that is the use of molecules that can alter cell physiology in a way to make a cell more responsive to stress or allow a stress response to be activated pharmacologically. The molecules that perform this function are known as coinducers. A number of molecules have been shown to be coinducers of the HSR including antibiotics, hydroxylamines, alcohols, and non-steriodal antiinflammatory drugs (NSAIDS) (Jurivich et al., 1992; Kalmar et al., 2002; Kieran et al., 2004; Batulan et al., 2006; Salehi et al., 2006). The overexpression of multiple Hsps by the use of coinducers in a primary cell culture model of ALS has been shown to be highly neuroprotective in some instances (Batulan et al., 2006). However, some of these molecules can be toxic to motoneurons so care must be taken in the risk/benefit analysis in drug choice. Given this, co-inducers are promising pharmacological intervention in the treatment of neurodegenerative disease. Interestingly, a recent report indicates that HSF1, the inducible transcription factor for Hsps, is a target of Riluzole, the only FDA approved drug to treat ALS (Yang et al., 2008). Whether this targeting will have any effect on an affected motoneuron is unclear. While the study shows Riluzole can activate a reporter construct driven by an Hsp70 promoter and may increase levels of Hsp70 under heat stress conditions in HELA cells, the motoneuron is clearly a unique cell and it relation to a cell line mechanism may be fairly disparate.

Discussion Our current understanding of the motoneuron HSR suggests that these cells do not mount a response that is sufficient or beneficial for protecting the cell. This logic may be faulty, however; because our understanding of the mechanisms regulating HSRs in motoneurons and

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other cells is not complete. Our definition of HSR in motoneurons is therefore limited and perhaps incorrect. Signal transduction regulation of HSF activation/inactivation varies between cells, and even within the same cell under different conditions. Furthermore, the HSFs regulate expression not only of genes involved in the response to potentially toxic stimuli, but also genes required for cell maintenance and developmental, differentiation events (Westerheide and Morimoto, 2005; Akerfelt et al., 2007; Morimoto, 2008). In fact, a recent study in Drosophila, an organism with only one HSF, shows that during heat stress the HSF targets nearly 200 genes (Birch-Machin et al., 2005). Additionally, Hsp expression and function appears to be developmentally regulated and cell type specific. For example, in rodents, Hsp70 expression is not induced until the third postnatal week. In chick lymphocytes show increased expression of all Hsps while reticulocytes only show increased Hsp70. Furthermore, in quail Hsp25 is not produced in myotubes, but is expressed in undifferentiated myoblasts (reviewed in Linquist 1986). It is also possible and perhaps probable, that a motoneuron HSR may differ depending on if the potentially toxic stimuli is delivered extracellularly (e.g., H202 administration glutamate toxicity) or intracellularly (e.g., overexpressing of a mutant protein). Therefore, while examining the expression of HSPs in the stress response is a good first step, clearly we have a long way to go until we fully understand the scope and necessity of the protein constituents activated by a cell with multiple HSFs. Nonetheless, careful characterization of these responses in individual cell populations and the role these responses play in neurodegenerative disorders is the critical first step if therapeutic approahes are going to be developed.

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Ritossa, F. (1996). Discovery of the heat shock response. Cell Stress Chaperones, 1, 97-98. Robinson, M. B., Tidwell, J. L., Gould, T., Taylor, A. R., Newbern, J. M., Graves, J., Tytell, M. & Milligan, C. E. (2005). Extracellular heat shock protein 70: a critical component for motoneuron survival. J Neurosci, 25, 9735-9745. Salehi, A. H., Morris, S. J., Ho, W. C., Dickson, K. M., Doucet, G., Milutinovic, S., Durkin, J., Gillard, J. W. & Barker, P. A. (2006). AEG3482 is an antiapoptotic compound that inhibits Jun kinase activity and cell death through induced expression of heat shock protein 70. Chem Biol, 13, 213-223. Santoro, M. G. (2000). Heat shock factors and the control of the stress response. Biochem Pharmacol, 59, 55-63. Satyal, S. H., Chen, D., Fox, S. G., Kramer, J. M. & Morimoto, R. I. (1998). Negative regulation of the heat shock transcriptional response by HSBP1. Genes Dev, 12, 19621974. Schlesinger, M. J. (1990). Heat shock proteins. J Biol Chem, 265, 12111-12114. Shabtay, A. & Arad, Z. (2006). Reciprocal activation of HSF1 and HSF3 in brain and blood tissues: is redundancy developmentally related? Am J Physiol Regul Integr Comp Physiol, 291, R566-572. Sharp, F. R., Massa, S. M. & Swanson, R. A. (1999). Heat-shock protein protection. In: Trends Neurosci, 97-99. Shaw, P. J. & Eggett, C. J. (2000). Molecular factors underlying selective vulnerability of motor neurons to neurodegeneration in amyotrophic lateral sclerosis. J Neurol, 247, Suppl 1, I17-27. Soncin, F., Asea, A., Zhang, X., Stevenson, M. A. & Calderwood, S. K. (2000). Role of calcium activated kinases and phosphatases in heat shock factor-1 activation. Int J Mol Med, 6, 705-710. Strey, C. W., Spellman, D., Stieber, A., Gonatas, J. O., Wang, X., Lambris, J. D. & Gonatas, N. K. (2004). Dysregulation of stathmin, a microtubule-destabilizing protein, and upregulation of Hsp25, Hsp27, and the antioxidant peroxiredoxin 6 in a mouse model of familial amyotrophic lateral sclerosis. In: Am J Pathol, 1701-1718. Tacchini, L., Pogliaghi, G., Radice, L., Anzon, E. & Bernelli-Zazzera, A. (1995). Differential activation of heat-shock and oxidation-specific stress genes in chemically induced oxidative stress. Biochem J, 309, 453-459. Tanabe, M., Kawazoe, Y., Takeda, S., Morimoto, R. I., Nagata, K. & Nakai, A. (1998). Disruption of the HSF3 gene results in the severe reduction of heat shock gene expression and loss of thermotolerance. Embo J, 17, 1750-1758. Taylor, D. M., De Koninck, P., Minotti, S. & Durham, H. D. (2007). Manipulation of protein kinases reveals different mechanisms for upregulation of heat shock proteins in motor neurons and non-neuronal cells. Mol Cell Neurosci, 34, 20-33. Tidwell, J. L., Houenou, L. J. & Tytell, M. (2004). Administration of Hsp70 in vivo inhibits motor and sensory neuron degeneration. Cell Stress Chaperones, 9, 88-98. Welch, W. J. (1992). Mammalian stress response: cell physiology, structure/function of stress proteins, and implications for medicine and disease. Physiol Rev, 72, 1063-1081. Xiao, X. Z., Zuo, X. X., Davis, A. A., McMillan, D. R., Curry, B. B., Richardon, J. A. & Benjamin, I. J. (1999). HSF1 is required for extra-embryonic development, postnatal growth and protection during inflammatory responses in mice. EMBO J., 18, 5943-5942.


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Yamamoto, N., Takemori, Y., Sakurai, M., Sugiyama, K. & Sakurai, H. (2009). Differential recognition of heath shock elements by members of the heat shock transcription factor family. FEBS J., 276, 1962-1974. Yamashita, H., Kawamata, J., Okawa, K., Kanki, R., Nakamizo, T., Hatayama, T., Yamanaka, K., Takahashi, R. & Shimohama, S. (2007). Heat-shock protein 105 interacts with and suppresses aggregation of mutant Cu/Zn superoxide dismutase: clues to a possible strategy for treating ALS. In: J Neurochem. Yang, J., Bridges, K., Chen, K. Y. & Liu, A. Y. (2008). Riluzole increases the amount of latent HSF1 for an amplified heat shock response and cytoprotection. PLoS ONE, 3:e2864. Yang, Y., Xie, Y., Chai, H., Fan, M., Liu, S., Liu, H., Bruce, I. & Wu, W. (2006). Microarray analysis of gene expression patterns in adult spinal motoneurons after different types of axonal injuries. Brain Res, 1075, 1-12. Zhong, M., Orosz, A. & Wu, C. (1998). Direct sensing of heat and oxidation by Drosophila heat shock transcription factor. Mol Cell, 2, 101-108.

In: Neurodegeneration: Theory, Disorders… Editor: Alexander S. McNeill

ISBN: 978-1-61761-119-3 © 2011 Nova Science Publishers, Inc.

Chapter 3

Methylene Blue Induces Mitochondrial Complex IV and Improves Cognitive Function and Grip Strength in old Mice

1

Afshin Gharib2,3 and Hani Atamna1*

Department of Basic Sciences, Neuroscience, The Commonwealth Medical College, Scranton, PA 18510 2 Children's Hospital Oakland Research Institute (CHORI), CA 94609. 3 Dominican University of California, San Rafael, CA 94901

I. Abstract Methylene blue (MB) is very effective in delaying cellular senescence and enhancing mitochondrial activity of primary human embryonic fibroblasts. At nanomolar concentrations, MB increased the activity of mitochondrial cytochrome c oxidase (complex IV), heme synthesis, cell resistance to oxidants, and oxygen consumption. MB is the most effective among the many agents that has been are reported to delay cellular senescence. We extended these in vitro findings to the investigation of the effect of longterm intake of MB in old mice. We administered MB, in the drinking water (250 µM), to old mice for 90 days. In vivo, MB prevented the age-related decline in cognitive function and spatial memory. MB also prevented the age-related decline in grip strength. Interestingly, MB resulted in 100 % and 50 % increases in complex IV activities in the brains and hearts of old mice, respectively. The age-related decline in protein content of the brain was prevented by MB. We also found a 39 % decrease in brain monoamine oxidase (MAO) activity in old mice treated with MB while aging or MB did not affect the activity of brain NQO1. Our findings suggest that the in vitro model for cell senescence may be used for fast and reliable screening for mitochondria-protecting candidate agents before testing in animal models. The study also demonstrates simultaneous enhancement *

Corresponding author: Assistant Professor of Biochemistry&Neuroscience, Department of Basic Sciences , The Commonwealth Medical College, Tobin Hall, 501 Madison Avenue, Scranton, PA 18510, Office: (570) 5049643, [email protected]

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Afshin Gharib and Hani Atamna of mitochondrial function, improvement of the cognitive function, and improvement of grip strength in old mice by a drug. Since these are three major concerns in human aging, MB may be a useful agent for delaying neurodegeneration and physical impairments associated with aging.

KeyWords: Mitochondria, aging, brain, methylene blue, senescence, complex IV, Alzheimer‟s diseases.

II. Introduction II.a. Mitochondria and Aging Physical and cognitive impairments in age-related disorders are often ascribed to impaired mitochondrial function [1-3]. Impaired mitochondrial function interferes with energy and intermediary metabolism, increases the production of oxidants, and the risk for tissue dysfunction. The aging brain has a limited capacity for self repair, increased mitochondrial dysfunction, impairment in energy metabolism, and oxidative stress [4]. For example, the decline in the activity of mitochondrial complex IV, energy hypometabolism, and increased oxidative stress are associated with the early signs of Alzheimer‟s disease (AD). Therefore, mitochondria-protecting agents may be potential drugs to prevent or delay age-related neurodegernation (e.g., Alzheimer‟s disease). Physical and cognitive declines in age-related disorders are widespread medical problems with mounting social and economic implications [5]. Like humans [6], aging rodents show an age-associated decline in spatial memory in addition to declines in muscle strength and physical independence [7]. These declines are linked, in part, to age-associated changes in mitochondria in neuronal and muscle cells leading to impaired hippocampal or muscular functions, respectively [4, 8].

II.b. Aging and Cognition In humans [9, 10] and rodents [11, 12] aging is associated with changes in both muscle strength and performance on a variety of cognitive tasks. A long established test for muscle strength in rodents is to measure the grip strength of the animal as it holds on to a bar attached to a strain gauge [13]. With age, grip strength declines [11]. In rodents, the age-associated change in cognitive ability has been particularly well studied in terms of spatial memory. The standard test for spatial memory in rats and mice is the Morris water maze [14], in which the animal is placed in a pool of water and has to locate a hidden platform using the spatial cues located around the pool and around the testing room. Over a number of trials, cognitively unimpaired animals become faster at finding the platform. There is a well established decline in water maze performance with age [15].

Methylene Blue Induces Mitochondrial Complex IV and Improves…

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II.c. Methylene Blue; new use for an Old Drug Research interest on aging is directed at finding pharmacological or nutriceutical agents that could be used to improve the quality of life in the elderly by delaying the onset of agerelated neurodegenration and physical disability due to dementia, sarcopenia, or other factors [3, 16]. We recently showed that Methylene blue (MB) improves mitochondrial function and delays cell senescence by interacting with the electron transport complexes of mitochondria [17]. MB extended the life span of primary cells at nanomolar concentrations by more than 50% [17], which makes MB the most effective among the many agents reported to delay cell senescence [18]. Methylene blue has been in clinical use for about a hundred years to treat a variety of pathological conditions and diseases [19]. One of the most common uses is the chronic treatment of congenital methemoglobinemia, which is due to methemoglobin reductase deficiency. MB is also used to treat methemoglobinemia caused by cyanide, CO, or nitrate poisoning [19]. Recent clinical uses for MB include preventing the side effects of chemotherapy (e.g., ifosfamide-induced encaphelopathy [20, 21]), and preventing hypotension in septic shock [22, 23]. MB is also used in the treatment of some psychiatric disorders because of its anxiolytic and antidepressant properties [24-26]. MB has been shown to protect against endotoxin-induced lung injury, bacterial lipopolysaccharide-induced fever [27, 28], cyclosporin injury to the kidney [29], and streptozotocin injury to the pancreas [30]. MB also protects from ischemic-reperfusion injury [31], radiation [32, 33], and enhances -oxidation of long chain fatty acids [34]. MB inhibits the aggregation of amyloid- peptide [35] and choline esterase [36]; both are implicated in Alzheimer‟s disease. A single high dose of MB improves the escape response in rats and increases the activity of cytochrome c oxidase (complex IV) by 25 % in the brain [37, 38]. MB also protects from methylmalonate-induced seizures [39] and lowers retinal injury induced by rotenone [40]. MB has no side effects when used at the clinically recommended dose. Although when exposed to high intensity of UV light MB causes oxidative damage to isolated DNA, no such toxicity has been shown in humans [41], presumably because it requires high exposure to UV and most MB in vivo is in the reduced form of MBH2, which does not have photodynamic activity [42]. MB has been proposed to act by inhibiting the NO-activating soluble guanylate cyclase (sGC) [43] (though the basal activity of sGC is not affected), inhibiting nitric oxide synthase (NOS) [44], inhibiting monoamine oxidase (MAO) [45], or acting as an antioxidant precursor [46]. These effects of MB were measured at concentrations greater than 10 µM. However, recent studies showed effects of MB that are not consistent with the above proposed mechanisms [17, 47]. These discrepancies may be caused by MB exhibiting different effects at different concentrations (>10 µM vs., nM concentrations). We have proposed a new mechanism that may explain, in part, some of the biological actions of MB that we observed when using MB at nM concentrations [17]. This mechanism requires MB cycling between oxidized (MB) and reduced (MBH2) forms (Scheme 1).

Afshin Gharib and Hani Atamna

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Methylene Blue (MB) N (H3C)2N

S +

N(CH3)2

Reduction H

N (H3C)2N

S

+

N(CH3)2 H Leucomethylene Blue (colorless)

Scheme 1: The oxidized (MB) and reduced (MBH2) forms of Methylene blue.

II.d. Objectives of the Current Study The objectives of the study we describe here are to test if MB exhibits positive effects on specific organs and activities in old mice. Furthermore, we wanted to also examine if an in vitro finding from cell culture [17] can be extended to aging in vivo. Using old mice, we demonstrate that MB exhibits mitochondria-protecting activity in vivo, which might be relevant for preventing specific age-related diseases. Furthermore, our findings suggest that in vitro models of cell senescence may be useful to evaluate specific potential mitochondria protecting agents.

III. Experimental Procedures III.a. Experimental Design We used male C57BL/6 mice (Harlan, NIA). The Children‟s Hospital Oakland Research Institute (CHORI) approved the protocol for the experimentation using MB and mice. Methylene blue was purchased from Fluka (through Sigma, St. Louis, MO) and purified by crystallization as described in [48]. In a pilot experiment directed at examining if MB can be administered in drinking water for extended period of time and determining the highest tolerated concentration of MB (an 100, 250, 500, or 1000 µM) by old mice, we found that 250 µM added to drinking water was the best tolerated dose. In addition, we found that the MB at this dose improved the grip strength in old mice (data not shown). We then performed a


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second larger study using 25 old (starting at age 21 months) and ten young (starting at age 3 months) mice. Animals were housed in groups of 4–5 and maintained on a 12/12 h light/dark cycle. Because the mice had been on a chow diet, all mice were switched to an AIN93 diet for a month before starting the treatment with MB. At this time, 12 old mice (now 22 months old) were maintained on the AIN93 diet and water (control group, old), while the remaining 13 were maintained for three months on the AIN93 diet and water supplemented with 250 µM MB (MB group). Ten mice from the old control group and 9 mice from the MB group survived until the end of the study. All the young mice (now 4 months) were given the AIN93 diet without MB and used as controls (control group, young). All mice had free access to food and water. All mice were tested for grip strength for 4 days (2 trials per day) after 2 month of treatment. Mice were also given spatial memory tests in the water-maze after 2 months of treatment. All behavioral testing occurred during the light phase.

III.b. Measuring Cognitive Function Spatial memory is frequently tested in rodents using the Morris Water Maze. The pool consists of a circular plastic tank (1.5 m diameter by 0.5 m height) with a removable circular white platform (13.5 cm diameter). The water (25 °C) is made opaque by the addition of a non-toxic, water-soluble white dye. The platform is 30 cm above the floor of the pool and the water level is 1 cm above the platform. The pool is divided into four equally sized imaginary quadrants and the platform is placed approximately 30 cm from the pool wall in one of the quadrants. A digital camera suspended over the pool and computer software (Columbus Instruments, VideoMex-V) recorded in real time the distance, speed, and location of the animal during the swim trial. Animals were trained for 6 days. During each day of training, animals received four trials. For each trial, the animal was released with its head facing the opposite pool wall from one of four possible quadrant boundary lines. Each mouse was allowed to swim until 1) locating the platform or 2) 60 seconds passed without finding the platform. The time to locate the platform was recorded. Mice that failed to locate the platform were carried and placed on the platform. All mice were allowed to stay on the platform at the end of each trial for 30 seconds. After the 4th trial, mice were housed in a cage which rested on heating pads 10 – 20 minutes (with access to drinking water) until the animal was dry. After 6 days of training, a single 60 second probe trial was given. During this trial, the platform was removed and the total distance swam in the platform quadrant as well as the number of times the animal swam into the specific area that used to contain the platform was recorded. These two measures reflect the animals memory for where the platform was located. The better the memory, the more often the animal should enter the exact location of the platform, and the more the animal should swim around in the quadrant in which the platform had been located.

III.C. Measuring Grip-Strength The peak force a mouse exerted by the forelimbs was measured using a grip strength meter (Columbus Instruments). The grip strength meter consists of a steel frame which

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supports a steel shaft. A push-pull strain gauge in the horizontal attitude is attached to the top of the steel shaft. A waffle style grip plate is attached to the end of the push-pull strain gauge. Each mouse was tested twice a day for 4 days before the treatment, 1 month after, and again after 2 months of treatment. Mice were grasped firmly at a point near the base of the tail and held above the grip plate. Rodents reflexively sprawl, extending the limbs and flexing the head and body upward. The mouse was lowered toward the grip plate until it firmly grasped the plate. The mouse was gently pulled away from the grip plate until it released. The tension force of the forelimbs and compression force of the hind limbs was measured in Kilogram units.

III.D. Assay for Cytochrome C Oxidase (Complex IV) After the completion of all testing on live mice, all mice were sacrificed, their brains and hearts were collected and were snap frozen in liquid nitrogen, and immediately stored at -80 ˚C. Brains and hearts were homogenized in ice-cold PBS/1mM EDTA/0.1 % triton-x100/antiproteases, aliquoted and stored at -80 ˚C. The homogenates were later used for further investigation. The activity of cytochrome c oxidase (complex IV) was measured by following the oxidation of reduced cytochrome c at 550 nm. Briefly, commercial cytochrome c from horse heart (Sigma, St. Louis, MO) is mostly oxidized. The substrate for complex IV is the reduced form of cytochrome c. Thus, reduced cytochrome c was prepared by incubating cytochrome c with excess ascorbic acid. Reduced cytochrome c was then separated from ascorbic acid on P-2 gel filtration resin (Bio-Rad, Hercules, CA) and the concentration was determined using the mM excitation coefficient of 19.6 mM-1cm-1 for the difference between reduced and oxidized cytochrome c. Reduced cytochrome c was used to assay complex IV activity in the brain and heart homogenates as described previously [49]. Briefly, the assay homogenate was potassium phosphate buffer (20 mM, pH 7) and n-dodecyl--D-maltoside (0.45 mM). Cytochrome c was added to the assay buffer and nonenzymatic oxidation was measured and subtracted from the oxidation rate following the addition of the homogenate (50-100 µg protein). The rate of complex IV activity was calculated and normalized to mg protein.

III.E. Assay for NQO1 NQO1 activity was measured as previously described [50] using DCPIP as an electron acceptor. The reaction buffer consisted of 25 mM Tris-HCl with 2 mM EDTA, pH 7.5. A known amount of the protein (100-150 µg) from the homogenate was added to the reaction buffer containing 200 µM NADPH. DCPIP was added at final concentration of 40 µM, and the reduction was monitored at room temperature using the decline in 600 nm absorbance and 700 nm as background. The inhibition by dicoumarol prepared in DMSO (final concentration 20 µM) as indication for the specificity of the reaction catalyzed by NQO1. The mM extinction coefficient 21 mM-1 cm-1 was used to calculate the activity of NQO1.


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III.F. Assay for Monoamine Oxidase (MAO) MAO was assayed using Amplex Red/monoamine oxidase/HRP assay kit from Molecular Probes (Invitrogen, Carlsbad, CA). The substrate for MAO was benzylamine, which upon oxidation produces H2O2. H2O2 is then used by HRP to oxidize Amplex red to resorufin. Fluorescence microplate reader was used to measure the production of resorufin using excitation at 560 ± 10 nm and fluorescence detection at 590 ± 10 nm. Total MAO was measured in this assay. We did not distinguish between the activity of MAO-A and MAO-B.

III.G. Quantification of MB in the Brain The steady state level of MB in the brain was quantified in the homogenates using LC/MS with electrospray ionization (ESI) [51], with modification. We used 1,9dimethylmethylene blue as an internal standard (instead of methylene violet [51], which was added to each extracted sample and to MB standard. Brain homogenate from MB-treated mice was extracted by acetonitrile, 0.1 µM 1,9-dimethylmethylene blue was added, and liquid chromatography was performed using a Shimadzu HPLC system (Shimadzu, Columbia, MA) and an Aquasil C18 50 x 2.1 mm column (Thermo, Torrance, CA). The column was operated at ambient temperature. The mobile phase was acetonitrile-methanol-ammonium formate (35:53:12 v/v) containing 20 mM ammonium formate at a flow rate of 0.2 ml/min. Each sample solution was injected via an autosampler at an injection volume of 10 L and eluted using an isocratic mobile phase from 0 to 20 min. Sample solutions were kept at 4 C in the autosampler before injection. The eluate was monitored using a Waters Micromass Quattro LC triple quadrupole mass spectrometer (Waters, Milford, MA) equipped with an electrospray ionization (ESI) source. Selected reaction monitoring (SRM) measurements were performed at 1.2 kV multiplier voltage. SRM transitions monitored in the positive ion mode were m/z 284.1 m/z 268.1 for methylene blue and m/z 312.25 m/z 296.3 for 1,9dimethylmethylene blue (internal standard). Masslynx and Quanlynx software (Waters, Milford, MA) were used for system control and data processing. The source temperature and capillary temperature were kept at 130 C and 350 C. The optimum cone voltage, extractor voltage, and spray voltage were set to 45 V, 3 V, and 3.5 kV, respectively.

III.H. Protein Assay The protein concentration of each brain homogenate was determined using the Bradford quantification reagent (Bio-Rad, Hercules, CA) and fat free BSA as standard. Each brain was homogenized in 5 ml homogenization buffer (ice-cold PBS/1mM EDTA/anti-proteases).

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III.I. Statistical Analyses Graphing, regression, and statistical analysis were performed using Prism 5.0 (GraphPad, San Diego, CA). For comparison between the groups one-way ANOVA or other tests were used. Significance was considered at an alpha level of p = 0.05.

IV. Results IV.A. the Effect of MB on Body Weight and Intake of Food and Water At the end of the three months of treatment with MB, no significant difference was found among the groups in body weight or water intake (Figure 1). Food intake, on the other hand, significantly decreased in MB-treated old mice. This effect of MB on food intake suggests that MB may work as a calorie restriction (CR) mimetic; an agent that could mimic the benefits of CR [52]. However, loss of body weight, which is a constant byproduct of CR, did not happen in MB-treated mice. Therefore, the effect of MB on food intake is possibly a result of an MB effect on appetite. MB enhances -oxidation and fat metabolism [34], which may contribute to this effect of MB. Although, we do not have at this point a definite explanation, it might be due to combination of the above listed factors in conjunction to MB‟s effect on mitochondria.

Figure 1 (Continued)


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Figure 1. The effect of Methylene blue on body weight and food and water intake in old mice. Old mice (22 months) were treated with MB in drinking water (250µM) for three months. Control groups of old and young mice were maintained on drinking water only until the end of the experiments (see Experimental Procedures). Body weight of each mouse was measured on weekly bases, while food and water intake were measured every week for each cage (3-5 mice per cage) and the average of daily intake for each mouse was calculated. Data presented are mean±sem (n=10 young, 10 old, and 9 old+MB). One-way ANOVA (Fisher's LSD Multiple Comparison Test) was used to compare the groups.

IV.B. MB Restores the age-Related Decline in Cognitive Function and in Grip Strength The final age-related decline in the spatial memory of old mice was about 30% (P<0.001), as demonstrated by the increase in the time (in seconds) that the old mice needed to find the location of the hidden platform (Figure 2 top). This increase in time to find the platform did not occur in the old mice treated with MB. When comparing the cognitive performance of old and young groups using the water maze test, the age-related decline in vision may contribute to the decline seen in the old group. In our experiment both groups of interest were old (MB-treated and control), suggesting that the vision limitation seems unlikely to explain our findings. After 6 days of training, the platform was removed on the 7th day and a single probe trial was given. The number of times the animals entered the area where the platform used to be (Figure 2 middle) and the distance the animal swam in the quadrant the platform used to be in (Figure 2 bottom) were recorded. The old mice entered the platform area less (P<0.05) and swam less in the platform quadrant (P<0.05) than either young animals or old animals treated with MB. There was no difference on these measures between MB treated animals and young animals.

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Figure 2. The effect of Methylene blue on the age-related decline in spatial memory in mice. Top- Old mice (22 months) were treated with MB in drinking water (250µM) for three months. Control groups of old and young mice were maintained on drinking water only until the end of the experiments. Spatial memory was measured using water-maze test as described in the Experimental Procedures. The time to platform increases in old mice not treated with MB. The time to platform is similar between the young mice old mice treated with MB. Data presented are mean±sem (n=10 young, 10 old, and 9 old+MB). One-way ANOVA (Fisher's LSD Multiple Comparison Test) was used to compare the groups. Middle- The effect of methylene blue on the age-related deficit in searching for the platform in the probe trial. The number of entries into to the area occupied by the platform on probe trials decreases in old mice not treated with MB. The number of entries into the platform area is similar between the young mice old mice treated with MB. Data presented are mean±sem (n=10 young, 10 old, and 9 old+MB). One-way ANOVA (Fisher's LSD Multiple Comparison Test) was used to compare the groups. Bottom- The effect of methylene blue on the age-related deficit in searching in the platform quadrant during the probe trial. The distance (in cm) swam in the quadrant the platform had been in on probe trials (B) decreases in old mice not treated with MB. The distance swam in the platform quadrant is similar between the young mice old mice treated with MB. Data presented are mean±sem (n=10 young, 10 old, and 9 old+MB). One-way ANOVA (Fisher's LSD Multiple Comparison Test) was used to compare the groups.


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We also tested the effect of MB on the grip strength as a measure for the possible effect of MB on muscle strength in general. The age-related decline in the grip strength applied by the front limb was about 20% (P<0.001). This decline did not occur in old mice treated with MB (Figure 3).

Figure 3. The effect of Methylene blue on the age-related decline in grip strength in mice. Old mice (22 months) were treated with MB in drinking water (250µM) for three months. Control groups of old and young mice were maintained on drinking water only until the end of the experiments. Grip strength was measured using water-maze test as described in the Experimental Procedures. The tension force of the forelimbs (in Kilogram units) is lower in old mice not treated with MB. The tension force is similar between the young mice old mice treated with MB. Data presented are mean±sem (n=10 young, 10 old, and 9 old+MB). One-way ANOVA (Fisher's LSD Multiple Comparison Test) was used to compare the groups.

The data presented in figures 2B through 3 do not distinguish between the possibilities that MB slows the decline in cognitive function with age or reverses it. The reason for this is that the cognitive function of the old mice, as compared to the young, was not established at the beginning of the treatment with MB in this experiment. The positive effect that MB has on old mice is obvious, however the exact mechanism of the effect of MB will be clarified in future studies.

IV.C. MB Crosses the Blood Brain Barrier The steady-state level of MB in the brain following treatment with MB is 128 nM (0.51 ± 0.25 pmols/mg protein) as measured by LC-MS-ESI [51]. As expected, no MB was detected in the brains of the control mice. Thus, although MB reaches the brain, the concentration is quite low. However, since it is within the 100 nM concentration range we found to be optimal for delaying cell senescence and protecting the mitochondria in cultured cells [17], it is reasonable to assume that MB is active in the brain at a nanomolar range concentration.

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IV.D. MB Reverses the age-Related Decline in Protein Content of the Brain Following brain homogenization, we were surprised to find that the protein content significantly declines with age. Interestingly, the age-related decline in protein content was prevented by MB. MB restores protein content in the brain to levels similar to young mouse brain (Figure 4, one way ANOVA). A 5-15 % decline in protein content in human brain has been reported previously [53, 54].

Figure 4. The effect of Methylene blue on the age-related decline in brain protein content in mice. Old mice (22 months) were treated with MB in drinking water (250µM) for three months. Control groups of old and young mice were maintained on drinking water only until the end of the experiments. Brain homogenates were prepared as described in the Experimental Procedures. Protein was measured using Bradford based assay. Fatty acid free BSA was used as standard. Oneway ANOVA (Tukey's Multiple Comparison Test) was used to compare the groups. Groups with different letters are significantly different. Data presented are mean±SD (n=10 young, 10 old, and 9 old+MB).

IV.F. MB Increases the Activities of Brain and Heart Mitochondrial Complex IV by 100 And 50 %, Respectively A previous in vitro study showed that MB enhances mitochondrial function and increases complex IV [17]. Thus, we examined the effect of MB cytochrome c oxidase (complex IV) in vivo. We found that the activity of complex IV in the brain of old mice treated with MB was doubled (Figure 5). We also measured the effect of MB on complex IV from the heart. Interestingly, MB significantly increased the activity of complex IV in the heart of old mice by 50% (Figure 6). These effects indicate that MB is active in tissues other than the brain.


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Figure 5. The effect of Methylene blue on the activity of complex IV in the brain of old mice. Old mice (23 months) were treated with MB in drinking water (250µM) for three months. Control groups of old and young mice were maintained on drinking water only until the end of the experiments. Brain homogenates were prepared and brain‟s complex IV activity was measured as described in the Experimental Procedures. One-way ANOVA (Newman-Keuls Multiple Comparison Test) was used to compare the groups. Groups with different letters are significantly different. Data presented are mean±sd (n=10 young, 10 old, and 9 old+MB).

Figure 6. The effect of Methylene blue on the activity of complex IV in the heart of old mice. Old mice (22 months) were treated with MB in drinking water (250µM) for three months. Control groups of old and young mice were maintained on drinking water only until the end of the experiments. Heart homogenates were prepared and heart‟s complex IV activity was measured as described in the Experimental Procedures. One-way ANOVA (Tukey's Multiple Comparison Test) was used to compare the groups. Groups with different letters are significantly different. Data presented are mean±sd (n=10 young, 10 old, and 9 old+MB).

The activity of complex IV in the brain homogenates did not change significantly in old vs. young mice. The effect of normal aging on complex IV decline is not conclusive [55-58]. However, the decline in complex IV in age-related disorders, such as Alzheimer‟s disease, has been established [59]. Thus, the induction of complex IV by MB may be useful for delaying the decline in complex IV in Alzheimer‟s disease which may offer clinical benefits for Alzheimer‟s patients.

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One possible explanation for the lack of effect of age on complex IV in our experiment could be that the old group was not old enough. Alternatively, a possible disconnects between physiologic age and chronic age may explain the lack of difference with age in complex IV activity. Such disconnect is not uncommon and could sometimes mask significant changes with age or interesting patterns of aging.

IV.G. MB Lowers the Activity of Monoamine Oxidase in the Brain of old Mice MB caused a significant decrease in the activity of MAO in the mouse brain (Figure 7). This decrease is likely due to direct inhibition by MB [60]. Interestingly, in our experiment, mice exhibit a 30 % age-related decline in the MAO in the brain, which was further enhanced by MB (Figure 7). In general, MAO activity in mice seems to decline at middle age and slightly increase after 25 months of age [61]. MAO activity in human brain increases with age [61-63]. The increase in MAO in the aging human brain may lower the bioactive amines (e.g., dopamine) and may increase the risk of age-related dementia [63, 64]. The inhibition of MAO by MB is consistent with previous findings [45].

Figure 7. The effect of Methylene blue on the activity of monoamine oxidases in the brain of old mice. Old mice (22 months) were treated with MB in drinking water (250µM) for three months. Control groups of old and young mice were maintained on drinking water only until the end of the experiments. Brain homogenates were prepared and brain‟s monoamine oxidase activity was measured using Amplex Red /HRP assay kit as described in the Experimental Procedures. Oneway ANOVA (Tukey's Multiple Comparison Test) was used to compare the groups. Groups with different letters are significantly different. Data presented are mean±sd (n=10 young, 10 old, and 9 old+MB).

IV.H. MB Did not Affect the Activity of NQO1 The activity of NQO1 (NAD (P)H quinone oxidoreductase) on the other hand was not affected by chronic treatment with MB, suggesting that it has no effect on NQO1 activity (Figure 8). NQO1 is a detoxification enzyme that functions to prevent one electron reduction of quinones resulting in the production of radical species. Thus, the preservation of NQO1


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indicates that MB likely does not interfere with the detoxification system in the cell. This finding is contrary to what we previously proposed [17], where we used a reporter gene system to study the effect of MB on NQO1. In the previous experiments we used HepG2 cells (transformed cells), which may explain, in part, the different responses to MB in the two experiments.

Figure 8. The effect of Methylene blue on the activity of NQO1 in the brain of old mice. Old mice (22 months) were treated with MB in drinking water (250µM) for three months. Control groups of old and young mice were maintained on drinking water only until the end of the experiments. Brain homogenates were prepared and brain‟s NQO1 activity was measured using Amplex Red /HRP assay kit as described in the Experimental Procedures. One-way ANOVA (Tukey's Multiple Comparison Test) was used to compare the groups. Groups with different letters are significantly different. Data presented are mean±sem (n=10 young, 10 old, and 9 old+MB).

V. Discussion We demonstrated that chronic treatment with MB improves spatial memory and muscle strength in old mice. In the water maze, not only did the young animals and MB-treated animals find the platform more quickly than old mice, they also swam more in the quadrant of the platform and entered the area the platform had been located in more often when the platform was removed in the probe trial. These differences in performance support our conclusion that the young and MB-treated animals had a better memory of where the platform had been located than the old animals. Previous research on the effects of MB on cognitive function in rodents have been limited to young animals, using short term (single day) treatment with MB [37, 38, 65]. Our findings suggest that low chronic dose of MB can have a positive impact on performance in a spatial memory task in old animals. In future studies, a memory task that is independent of motor ability should be used to better tease apart the decline in muscle strength and memory. MB‟s concentration in the brain at the end of the three month treatment was 0.51 ± 0.25 pmols/mg protein indicating that MB crosses the Blood Brain Barrier. The ability of MB to cross the Blood Brain Barrier following i.v. injection also has been demonstrated in previous studies [66]. These observations are encouraging since they indicate that a low chronic dose of MB administered in drinking water reaches the brain, enhances mitochondrial function, and protects against the age-related memory impairment in old mice. MB also prevents the

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decline in brain-protein content in old mice. A decrease of 5-15 % in protein content in the aging human brain (ages from 30 to 90 years old) has been previously described [53]. The activity of MAO, which increases with age in the human, was decreased by MB. The increase MAO with age in humans may interfere with brain concentrations of bioactive amines including serotonin and the catecholamines. Interestingly, MAO is also involved in producing hydrogen peroxide, which may further contribute to mitochondrial impairment with age. Thus, modulating the activity of MAO by MB may also contribute to the effect that treatment with MB has on the aging brain. The most intriguing finding of this study is that low chronic levels of MB increase the levels of complex IV in brain and heart. This increase in the activity of complex IV is consistent with the effect of MB on IMR90 cells in vitro [17], where the level of complex IV also increased. Complex IV is found in ≈5 fold excess over the other electron transport complexes of the mitochondria [67], which may indicate the physiological significance of excess complex IV. However, there is no definitive answer yet as to the physiologic significance of the excess in complex IV. The following discussion may provide some insights. Energy production by mitochondria relies on four electron transport complexes (ETC), which are complex I, II, III, and IV. Electron transfer through each one of the complexes starts at complex I, which catalyzes a two-electron oxidation of NADH, and continues until water is formed by complex IV (Scheme 2). Complex IV consumes more than 95% of the O2 that reaches the cell. The production of O2-free radicals is enhanced by the stalling of electrons upstream of complex IV; at complexes I and III [68, 69]. Complex I and complex III are two sites responsible for the production of free radicals by non-specific transfer of electron to O2 [70]. Thus, induction of complex IV by MB may play a key role in lowering the steady-state concentration of intracellular O2 and, as a result, lowering the production of oxidants by the mitochondria.

NADH  ETC I  Coenzyme Q  ETC III  cyt c  ETC IV (complex IV)  O2  H2O FADH2 ETC II Scheme 2: The electron transport chain (ETC) of the mitochondria. The four complexes are: complex I (ETC I), complex II (ETC II), complex III (ETC III), and complex IV (ETC IV) in addition to ATP synthase (i.e., complex V). The electron transfer through each of the ETCs starts at ETC I, which catalyzes two electrons by the oxidation of NADH and continues until water is formed on ETC IV. Coenzyme Q serves as low molecular weight electron carrier from ETCs I and II to III. Cytochrome c (cyt c) serves as electron carrier from ETC III to ETC IV.

In attempt to understand how MB induces complex IV, we used our experimental findings in conjunction with previous studies on MB and proposed a molecular mechanism to explain, in part, MB‟s effect on complex IV [17]. Briefly, we propose that MBH2 and MB serve as electron carriers between several dehydrogenases and heme-proteins (e.g., cytochrome c) (Scheme 1). Complex IV in turn recycles the reduced cytochrome c (Scheme


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2), which trigger the induction of additional complex IV. This mechanism is explained in more detail in [17]. We do not yet know the molecular mechanism that leads to improving cognitive function or muscle strength in old mice. We are proposing a possible mechanism based on emerging research from various studies. The ETC complexes are biochemically and physiologically connected and directed at energy production. High levels of complex IV correlate with neuronal metabolic activity [71] and with cognitive performance [72, 73]. Thus, we predict that the induction of complex IV by MB will increase the cellular energy charge. An increase in energy charge and the metabolic activity of neurons improves learning and memory retention. Furthermore, learning and memory retention require adequate neuroplasticity and neurogenesis. Recent research showed that neurogenesis occurs within the adult brain (e.g., dentate gyrus of the hippocampus) [74-77]. Interestingly, an age-related decline in neurogenesis of the adult brain [78-82] and muscle endogenous progenitor cells [83] has been demonstrated. This decline may be attributed, in part, to senescence of the progenitor cells or senescence of the somatic cells that are surrounding the progenitor cells (thus indirectly affecting the normal function of the endogenous progenitor cells) [83]. Since, MB is very effective in delaying cell senescence in vitro [17], it may retain its anti-senescence action also in vivo (as it retained its effect on mitochondria) and restore the function of neuronal progenitor cells. MB may improve the mitochondrial activity and energy metabolism in neuronal progenitor cells. Pharmacological approaches that are directed at inducing neurogenesis hold promise for preventing or delaying age-related disorders [77, 84-86]. Thus, MB could in part, improve memory and muscle function as part of such an approach. MB prevents the decline in complex IV, which is a key cytopathology of Alzheimer‟s disease (AD). Thus, by increasing the brain reserve of complex IV, we propose that MB could delay or slow the age-related decline in complex IV, thus preserving mitochondrial function, energy metabolism, and memory retention in AD. Due to its effect on heart complex IV, MB may improve physical activity and delay the onset of sarcopenia. Thus, MB could be used to improve the quality of life of the elderly. MB is a drug with an extended medical and safety record in humans; thus, FDA approval for its use in clinical trials in connection to aging and age-related disorders should not be denied on safety grounds (discussed in [17]).

Conclusions We proposed mitochondria-based approach to prevent or delay the onset of the agerelated degenerative disorders (e.g., Alzheimer, sarcopenia). This strategy is directed at enhancing mitochondrial activity using low chronic dose of MB to increase the level of complex IV. Energy deficiency and increase free radical production in Alzheimer and aging may be contributed by mitochondrial function. In vitro and in vivo MB exerts its effect at very low (nM) concentration, which in conjunction with its safety record in human further minimizes any risk of side effects of chronic exposure to MB. MB can also induce heme synthesis, thus, it may improve iron metabolism and preventing heme deficiency, which are key cytopathologies in Alzheimer.

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Acknowledgment The authors would like to thank the Bruce and Giovanna Ames Foundation and the American Federation for Aging Research (AFAR) for supporting this research. We are grateful to Prof. Gerald Litwack and Drs. William Phillips and William Zhering for commenting on the manuscript. We also would like to thank Gordon Wu, Tina Ghirarduzzi, Tegan Hall, and Lara Corkrey for technical assistance.

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Chapter 4

Receptor Specific Features of Excitotoxicity Induced Neurodegeneration Sergei M. Antonov* and Dmitrii A. Sibarov

Sechenov Institute of Evolutionary Physiology and Biochemistry RAS, Saint-Petersburg, Russia

Abstract Excitotoxicity is a term that describes the neuronal death caused by neurotoxic effects of glutamate, which is the most abundant excitatory neurotransmitter in the vertebrate central nervous system. Glutamate is well known to be involved in cognitive functions like learning and memory, but its excessive accumulation in extracellular space can lead to neuronal damages and eventual cell death via necrosis and apoptosis. As a result excitotoxicity contributes to pathogenesis of numerous neurodegenerative diseases. Both normal function and pathological action imply an activation of the same glutamate receptors particularly of NMDA- (N-methyl-D-aspartate), AMPA- (α-anino-3-hydroxyl5-methyl-4-isoxazole-propionate) and KA- (kainate) subtypes. Many achievements in the mechanisms of neurodegeneration were obtained using different experimental approaches on primary neuronal cultures. Double successive acridine orange and ethidium bromide staining combined with confocal microscopy offers fast, easy, sensitive and reproducible method by which necrosis and apoptosis can be recognized and quantified in a population of living neurons. Together with immunostaining they provide many research advantages and allow analysis of protein expression patterns. The growing quantity of evidence reveals the diversity of apoptosis cascades. Whereas our data show the same profiles of excitotoxicity for NMDA and KA, we found receptor subtype specific differences in neuronal death mechanisms. For example, apoptosis caused by prolonged NMDA receptors activation develops through the caspaseindependent cascades via release of apoptosis inducing factor (AIF) from mitochondria *

Corresponding author: [email protected], [email protected]

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Sergei M. Antonov and Dmitrii A. Sibarov and its direct action on nuclear chromatin. In contrast AMPA and KA receptors mediated apoptosis includes caspase-dependent pathway. On the basis of our data and literature the chapter will review the contemporary state of research concerning the aspects of excitotoxicity mechanisms discussed above.

Introduction Glutamate receptor (GluR) is the main subject of investigations, concerning excitotoxicity and neuroprotection. Functional disregulation of neuronal metabolism, resulted from overactivation of GluRs is thought to cause neuronal death and to underline a wide range of central nervous system (CNS) disorders like spinal cord and brain injuries, stroke, neurodegenerative diseases, etc. Loss of cerebral blood flow causes massive neuronal depolarization and Glu accumulation in extracellular space [Lipton, 1999]. The prolonged presence of Glu released from neurons and glial cells by non-quantum secretion [Antonov, Magazanik, 1988] and activation of ionotropic GluRs have extensive consequences for neuron functioning, starting with an increase in intracellular Ca2+ concentration, imbalance of transmembrane gradients of the main electrogenic ions (Na+ and K+), and an activation of various intracellular cascades, and ending with destruction of the plasma membrane or nuclear apparatus of neurons [Choi 1987, 1988; Jonston, 1994, Oiney, 1994; Schoepp, Sacaan, 1994; Hatanaka et al, 1996]. Massive cytoplasmic calcium ions accumulation is thought to be one of the most important activators of various cell death mechanisms. It‟s important to note, that a great body of facts concerning Glu stimulated neuronal disfunction were obtained in neuronal culture models. Cell cultures provide uniform cell composition and highly controllable extracellular environment which is favorable to study fine intracellular mechanisms. However, we must take into account that this model focuses on neurons alone and ignores tissue control. Other brain tissue cell types like astrocytes and oligodendrocytes can promote or prevent excitotoxic neuronal death. Actrocytes not only regulate neuronal ion balance, but can also maintain low extracellular Glu levels due to uptake with glial GLAST and GLT-1 transporters [Rothstein et al, 1994]. Glial cells express all kinds of ionotropic GluRs and respond to Glu stimulation with neurotrophic factors secretion like BDNF (brain-derived neurotrophic factor). Glu induced excitotoxic neuronal death involves activation of at least three types of receptors: NMDARs (selective agonist N-methyl-D-aspartate, NMDA), AMPARs (selective agonist α-anino-3-hydroxyl-5-methyl-4-isoxazole-propionate, AMPA) or KARs (selective agonist kainite, KA). Because KA is not strictly selective to KARs and can activate both KARs and AMPARs, below we mention these receptors together. NMDARs are highly permeable to Ca2+, as well as to Na+ and K+. An accumulation of free Ca2+ inside the cell triggers multiple predominantly Ca2+-dependent mechanisms of cell death usually ending as apoptosis [Khodorov, 2004]. Apoptosis, or programmed cell death, plays an enormous role in the development and formation of organs, as well as in the functioning of rapidly renewing tissues [Abrams et al, 1993; Jonston, 1994]. However, in nervous tissue, where there is virtually no regeneration, although there are now some data on de novo neuron formation from glial cell precursors [Skachkov et al, 2006], apoptosis is the key factor in the pathogenesis of many nervous system disorders, along with necrosis [Jonston, 1994].

Receptor Specific Features of Excitotoxicity Induced Neurodegeneration

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In general, AMPARs and KARs are permeable to Na+ and K+, and much less permeable to Ca2+ than NMDARs. The impermeability of AMPARs and KARs to Ca2+ is determined by the presence of GluR2 subunit [Burnashev et al, 1996; Dingledine et al, 1999]. GluR2 is expressed in all brain areas and neuronal subsets [Sun et al., 2005] except motor neurons and interneurons having comparatively low GluR2 expression [Magazanik et al, 1997], which makes them selectively vulnerable to AMPAR-mediated excitotoxicity. Prolonged activation of AMPARs and KARs elevates intracellular Na+ an contributes to cell swelling [Choi, 1987, 1988], which increases the probability of necrotic cell death. On this basis, we expect the distinctive features of NMDARs and AMPARs/KARs to underlie the different ratio of necrotic and apoptotic neuronal death while neurodegeneration caused by selective activation of particular receptor types using NMDA or KA.

Figure 1. Simplified diagram of caspase-dependent and caspase-independent pathways of apoptosis. In response to different stress types protein P53 becomes activated and induces Bax expression and translocation to mitochondrial membrane. Bax promotes opening of mitochondrial permeability transition pores (PTP). It allows AIF and Cytochrome-C to be released to cytoplasm. AIF represents caspase-independent pathway and can directly cause DNA fragmentation. Cyt-C (caspase-dependent pathway) activates caspases phosphorilation cascade ending at Caspase-3, causing DNA fragmentation. Proapoptotic Bax protein promotes Cyt-C and AIF exit to cytoplasm. Antiapoptotic protein Bcl-2 has an opposite function

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Sergei M. Antonov and Dmitrii A. Sibarov

Although the cell death during apoptosis and necrosis have clear morphological and biochemical differences [Cafforio et al, 1996; Philpott et al., 1996], most methods do not allow the simultaneous identification of apoptosis and necrosis in living tissue, are very laborious and require preliminary biochemical processing, fixation, or resuspension of the study material [Gavrieli et al, 1992]. The development of a vital kit for rapid analysis of the cellular composition of neurons in tissue cultures [Mironova et al, 2007] allows the automatic quantification of cell populations to be performed for the presence of apoptosis and necrosis. It greatly facilitates the study of neurodegenerative process dynamics. Its well known, that the development of apoptosis in various tissues can occur via two basic mechanisms (Figure 1): the caspase-dependent cascade and a cascade not involving caspases, i.e., via the direct action of apoptosis-inducing factor (AIF) on cell nuclei [Hong et al, 2004]. Inactive procaspases always exist in cytoplasm. Procaspases activation requires proteolytic splitting of proenzyme into two subunits and further cleavage of N-terminal ends [Ermshaw et al., 1999]. Subunits are then assemble into active oligomers. Initial procaspase proteolysis can be done by various proteases, including other caspases. Caspase independent mechanism can be triggered via hyperproduction of proapoptotic proteins like Bax and Bak, which can induce apoptotic cell death even in the presence of caspase inhibitors [Xiang et al, 1996, McCarthy et al, 1997]. An appearance of caspase 8, which activates procaspases 3, 6 and 7 is enough to start apoptosis. The key enzyme in the caspase-dependent pathway is caspase 3 (Cas-3), activation of which leads to irreversible destruction of nuclear DNA. Apoptosis can be reversed before an activation of Cas-3 with Cas-8. There are several proteins promoting and preventing caspases activity at this moment [Kidd, 1998; Adams, Cory, 1998; Reed et al, 1998; Huppertz et al 1999; Gross et al, 1999]. These proteins include inhibitors of apoptosis like A1, Bcl-2, Bcl-W, Bcl-XL, Brag-1, Mcl-1 and NR13, and proapoptotic proteins Bad, Bak, Bax, Bcl-XS, Bid, Bik, Bim, Hrk, Mtd. Most of these proteins belong to Bcl-2 family of proteins which is evolutionary conservative. For example in sponges Geodia cydomium and Suberites domuncula, homologous proteins are involved in morphogenesis process [Adams, Cory, 1998]. This raises the question about the contribution of different cell death mechanisms to apoptosis evoked by selective activation of different neuronal GluR subtypes. Identification of the receptor specificities of apoptosis pathways simplifies the search of intracellular targets for new neuroprotective agents. The goals of this work, performed on primary neuronal cultures from rat cortex were: to evaluate necrosis/apoptosis ratio during exposure to selective GluR agonists - NMDA and KA, as well as the Glu itself; to study potential neuroprotective properties of selective NMDAR, AMPAR and KAR antagonists, and to elucidate possible diversity of apoptotic cascades determining excitotoxicity triggered by excessive activation of GluR subtypes.

Methods Experiments were performed at room temperature (20-22oC) on primary cultures of neurons from embryonic rat brain cortex. All procedures using animals were in accordance to FELASA recommendations and were approved by the local Institutional Animal Care and Use Committee. The method used for preparing cultures has been described in detail


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previously [Antonov, Johnson, 1996; Mironova, Lukina, 2004]. Briefly, pregnant Wistar rats were sacrificed by CO2 inhalation 16 days after conception. Cortices were obtained by aseptic dissection, incubated for 30-40 min at 37oC in growth medium containing 83% minimum essential medium (MEM), 25 mM Hepes, 2 mM L-glutamine, 10% D-glucose plus 0.03% trypsin, and then dispersed mechanically by trituration with a fire-polished glass Pasteur pipettes. Cells were pelleted by light centrifugation (410 x g, 5 min at 25 oC), the supernatant discarded and the cell pellet resuspended in pre-warmed Hanks solution. For better washout of reagents this procedure was repeated. A plating suspension at a density of 130,000 cells per ml was prepared in growth medium. This density was optimal for the neuronal survival, the neuronal network formation and further experimental manipulations with the cultures. The suspension of cells was plated onto 15 mm diameter glass coverslips that had been coated with poly-D-lysine in 35 mm plastic Petri dishes. Cultures were kept at 37 oC in a humidified 5% CO2-containing atmosphere. The growth medium was refreshed twice a week. Cells were used for experiments after culturing 7 days in vitro (DIV). Directly before the experiments, coverslips with neuronal cultures were placed in the bathing salt solution. Since extracellular Mg2+, which blocks NMDAR channels [Antonov, Johnson, 1999], has neuroprotective effects on “young” neurons [Antonov et al, 2006], Mg2+-free bathing salt solution was used of the following composition: 140 mM NaCl, 2.8 mM KCl, 1.0 mM CaCl2, 10 mM Hepes; pH was adjusted to 7.2 – 7.4 with NaOH. To initiate excitotoxic insults 3 mM Glu, 30 µM NMDA and 30 µM KA were added to the bathing salt solution. In the case of NMDA in all experiments 30 µM glycine was coapplied, as a coagonist of NMDAR [Johnson, Ascher, 1987]. When effects of antagonists and modulators were studied the compounds were coapplied with the GluR agonists. Measurements of the proportion of dead cells among whole cell population were performed after 120 and 240 min treatment with the compounds. Cell viability was determined utilizing the vital fluorescence assay. Confocal images captured after 120 and 240 min of incubation with compounds were subjected for automatic cell counting after staining of all nuclei with acridine orange and dead cell nuclei with ethidium bromide. First, cells were treated with 0.001% acridine orange for 30 s. After complete washout of contaminating acridine orange cells were exposed to 0.002% ethidium bromide for 30 s. This procedure was applied directly before every measurement. As a result in confocal microscopy experiments the nuclei of live neurons, labeled with acridine orange, looked green and the nuclei of injured neurons, labeled with ethidium bromide, looked red (Figure 2, A) [Pulliam et al, 1998; Mironova et al, 2007]. In the absence of correlated pixels (Figure 2) the cell viability was estimated by the ratio of green pixels (the number) to the total number of lightened pixels (red plus green, Figure 2, B). If some population of neurons exhibited apoptotic transformations their nuclei looked yellow-orange, revealing the colocalization of fluorescence in green and red spectral regions (Figure 3). In this case the fractions of live, apoptotic and necrotic cells were calculated on the basis of correlation plot (Figure 3) as the ratio of green, yellow-orange and red pixels to the total number of lightened pixels (the sum of green, yellow-orange and red), correspondently. To improve the validity of cell viability measurements, 3 non-overlapping images from a single coverslip were pooled to calculate the mean value for this particular coverslip, which represented a single experimental measurement.

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Fluorescence images were captured using a Leica (Leica Microsystems, Heidelberg, Germany) TCS SL scanning laser confocal microscope (upright) equipped with argon laser of 50 mW (excitation wavelengths 458, 476, 488 and 514 nm, approximately 10 mW each). Cultures were viewed with a 40x water objective (HCX APO L 40x/0.80, Leica Microsystems, Heidelberg, Germany). To resolve fine details an additional electronic zoon with a factor of 1.5 –3.5 was used. Since ethidium bromide has a second peak of excitation, which is consistent with the excitation of acridine orange, both acridine orange and ethidium bromide could be visualized using the same laser line. For two-channel imaging of acridine orange and ethidium bromide, neuronal cultures were excited with 488 nm laser line, which could be varied between 0.1 – 10 mW by means of a neutral density filter. The emitted fluorescence was acquired at 500 – 560 nm (green region of spectrum, for acridine orange) and > 600 nm (red region of spectrum, for ethidium bromide) and collected simultaneously using separate photo multiplier tubes. Microscope settings were adjusted so that imaging conditions for both channels were kept equal and constant. To improve signal-to-noise ratio 6 scans (512 x 512 pixel array) were averaged at each optical section. The confocal images from both channels were merged using standard Leica software and program ImageJ (http://rsb.info.nih.gov). In order to quantify colocalized and non-colocalized fluorescence the correlation plot, which sorts values of given pixels in the first image as the x-coordinate and values of corresponding pixels in the second image as the y-coordinate, was generated for each of measurements. On the resulting image non-correlated pixels looked green and red and were attributed to live and necrotic neurons, correspondently. Correlated pixels looked yellow-orange and were attributed to nuclei of apoptotic neurons. Apoptotic proteins were identified immunochemically using mouse monoclonal antibodies to proapoptotic proteins P53 (dilution 1:400), AIF (dilution 1:1000), Cas-3 (dilution 1:100) and Bax (dilution 1:200). Monoclonal antibodies were visualized using secondary antibodies conjugated with the fluorochromes FITC, Phr, and Cy3, diluted 1:150. Images were recorded using a Leica TCS SL confocal scanning microscope. Fluorochromes were excited by light at a wavelength of 488 nm (for FITC), 488 nm (for Phr), and 514 nm (for Cy3). Emission was recorded in the green spectral region (emission peak at 525 nm) for FITC, in the red spectral region (emission peak at 620 nm) for Phr, and in the yellow spectral region (emission peak at 570 nm) for Cy3. All antibodies were obtained from Sigma. Before fixation, neurons on coverslips were treated with neurotoxic factors (Glu, NMDA or KA) separately or in combination with antagonists for 240 min. Cells were then fixed with 4% paraformaldehyde solution in PBS (phosphate-buffered saline) for 30 min. After fixation, cells were washed out twice with PBS (15 min x 2). Before treatment with BSA (bovine serum albumin, 2%), cells were incubated with Triton X-100 (0.2%) for 15 min, washed out with PBS, and exposed to monoclonal antibodies for 12 h at 4°C. After washing out to remove primary antibodies, fluorochrome-conjugated secondary antibodies were added. Reactions with secondary antibodies lasted 40 min at a room temperature of 23°C. Before recording of data, antibody-bound preparations were fixed on slides with Moviol glue to prevent fading of fluorochromes. All drugs were kept frozen in stock solutions at concentrations of 1 – 100 mM and were diluted to the required concentrations before use. All chemical reagents were obtained from Sigma Chemical Co. (St. Louis, MO, USA).


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Data were analyzed statistically using Excel, Origin 6.1, and SigmaPlot 8. All histograms and Table 1 show means ± standard errors of mean (s.e.m.). Student‟s two-tailed t-test and ANOVA were used for statistical comparisons.

Results Double sequential staining with acridine orange and ethidium bromide allowed cells dying by apoptosis or necrosis and living cells to be discriminated [Mironova et al, 2007]. In course of exposing of neuronal cultures to 3 mM Glu for 120 min green nuclei of live neurons and red nuclei of injured neurons were observed (Figure 2, A). The intensity correlation plot (Figure 2, B) reveals non-overlapping areas of red and green pixels. An absence of colocalization between green and red fluorescence points on necrotic type as a mechanism of neuronal death under these particular conditions. This data suggests that during 120 min excitotoxic insult cells die by necrosis. To induce apoptosis a longer treatment by Glu or its agonists is required.

Figure 2. Algorithm of recognition and automatic quantiﬁcation of live and dead neurons using the sequential acridine orange and ethidium bromide confocal microscopy assay. (A) Neurons after exposure of neuronal culture to 3 mM Glu during 120 min. Dead neurons are labeled with ethidium bromide and have red nuclei. Live neurons are labeled with acridine orange and have green nuclei. (B) The correlation plot for the image A. The absence of correlation between images obtained in green and red spectral regions demonstrates the lack of apoptotic nuclei. Dashed lines indicate thresholds, chosen empirically, that separates visible ﬂuorescence from dark pixels

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Figure 3. Distribution and automatic counting of living and dead cortical neurons in cultures treated with 3 mM glutamate for 240 min. Vital sequential staining with acridine orange and ethidium bromide using confocal microscopy is shown Three groups of nuclei can be distinguished: live neurons (example marked with green circle are visible only in green spectral region, left panel); neurons dying by necrosis (marked with red circle are visible only in red spectral region, intermediate panel), and apoptotic neurons (orange circle mark can be seen in both parts of the spectrum. The right image represents the result of merging red and green spectral regions). Frame size is 139 × 139 µm. The correlation plot shows correlated pixels or colocalization of fluorescence between the images recorded in the green and red spectral region. Correlating pixels are orange. The dotted lines show the empirically selected limits discriminating lighting-up pixels from non-lighting-up pixels. The proportion of live cells (37% for this experiment) was evaluated by the ratio of the number of green pixels to the total number of pixels lighting up.

Figure 3 shows images captured in the green and red spectral regions after exposure to 3 mM Glu for 240 min. The combined image shows the most typical situation, in which neurons can be discriminated into three classes on the basis of their nuclei. The nuclei of live neurons are visible only in the green part of the spectrum. They are detected by staining with and fluorescence of acridine orange. Yellow-orange nuclei on combined image (Figure 3) are seen in both the green and red areas and are detected because of the shift of acridine orange fluorescence towards the red spectral region as a result of nucleus acidification while apoptosis. [Mpoke, Wolfe, 1997]. Red nuclei are seen only in the red part of the spectrum, and are the nuclei of neurons with plasma membrane destruction, i.e., cells which have died by necrosis and which are detected by the fluorescence of ethidium bromide. Table 1. Comparison of the proportions of necrotic, apoptotic, and living neurons by activation of different types of glutamate receptors for 240 min. Values marked with * are significantly different from the control (p < 0.05, Student’s two-tailed t test) Treatment Control 30 µM NMDA 30 µM NMDA + 50 µM AP5 30 µM kainite 30 µM kainate + 30 µM CNQX 3 mM Glu 3 mM Glu + 30 µM CNQX 3 mM Glu + 50 µM AP5

Necrosis, % 3±1 15 ± 1* 3±1 16 ± 2* 1 ± 0.5 35 ± 8* 19 ± 6* 49 ± 13*

Apoptosis, % 15 ± 5 45 ± 9* 16 ± 4 52 ± 5* 16 ± 4 31 ± 3 16 ± 13 17 ± 3

Living cells, % 82 ± 6 40 ± 10* 81 ± 5 32 ± 6* 83 ± 4 34 ± 5* 65 ± 8 34 ± 6*

Number of experiments 4 5 4 5 4 4 4 4


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The proportion of neurons in each of three physiological states can be determined as ratios of the numbers of correlating pixels (lighting up in both parts of the spectrum) and noncorrelating pixels (lighting up in only one part of the spectrum) to the total number of pixels lighting up. In the experiment shown in Figure 3, 3 mM Glu induced the apoptotic processes in 39% of neurons; 24% died via necrosis and 37% of neurons remained live. Average data are shown in Table 1. Under the control conditions (incubation in the bathing salt solution for 240 min) only 3% of necrotic and 15% of apoptotic neurons were found. Significantly increased number of necrotic and apoptotic cells at the cost of decreased number of live neurons illustrates the strong neurotoxic action of Glu. Obviously, neurodegeneration triggered by excitotoxic effects of Glu has two components. Neuronal death by necrosis occurred rather quickly and was accompanied by delayed activation of apoptotic process. The first step in necrotic cell death is usually cell swelling. Multiple cell swellings or varicosities along dendrites were observed after treatment of neurons with 30 μМ NMDA. The dynamics of varicosities swelling can be seen on Figure 4. The process of cell swelling in response to NMDA application is possibly related to activity of volume-sensitive chloride channels (VSOR Cl-) [Inoue and Okada, 2007]. VSOR Cl- channels play a great role in cell volume regulation, but cause neuronal body and processes swelling after prolonged NMDA excitotoxic insult, which leads to necrotic cell death. Despite the data about the mechanisms of NMDA induced necrosis [Inoue and Okada, 2007], information about necrotic processes caused by KA is scanty.

Figure 4. Confocal microscopy images, demonstrating the dynamics of varicosities formation in neuronal cell culture continuously exposed to 30 μМ NMDA. Vital staining with ANEPPS. Arrows indicate the place of single varicosity growth

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Selective agonists of different Glu receptor subtypes can be utilizes to assess the particular receptor type that determines the major contribution to neurodegeneration induced by natural neurotransmitters. In order to evaluate the functional role of NMDARs and AMPAs/KARs in Glu induced excitotoxic stress, we used the approach described above. We have analyzed the effects of 3 mM Glu in combination with 50 µM AP5, a selective competitive antagonist of NMDARs, and 30 µM CNQX, a selective antagonist of AMPARs/KARs [Dingledine et al, 1999]. CNQX was found to provide highly significant protection of neurons against the toxic action of 3 mM Glu: the viability of neurons in the presence of CNQX increased, on average, from 34% to 65% (Figure 5). On the contrary, AP5 did not have any neuroprotective action suggesting that NMDARs are not involved in the transmission of 3 mM Glu excitotoxicity. This observation clearly demonstrates that an activation of AMPARs/KARs provide a dominant contribution to the neurotoxic effect of 3 mM Glu, which is consistent with data obtained using trypan blue as an indicator of necrosis [Mironova et al, 2006].

Figure 5. Effects of 240-min exposure of glutamate receptor agonists and antagonists on ratio of live, necrotic and apoptotic neurons in whole cellular population. (Glu) 3mM glutamate; (NMDA) 30 µM NMDA; (KA) 30 µM KA; (CNQX) 30 µM CNQX, (AP-5) 50 µM AP-5. * The data significantly differ from corresponding values, obtained in the absence of antagonists (p < 0.05, Student‟s two-tailed t test).

The ability to induce neurodegeneration by activation of AMPARs/KARs was supported by experiments using KA – a selective agonist of these receptors. After 240 min exposure to 30 µM KA 16% necrotic neurons and 52% of neurons dying by apoptosis ware observed (see Table 1, Figure 5). KA produced a statistically significant reduction in the proportion of live cells and increased the proportions of necrotic and apoptotic cells in comparison to the control (see Table 1). When KA was coapplied with CNQX, the neurotoxic effect was significantly weakened; the proportion of apoptotic and necrotic cells decreased, on average, to 16% and 1% respectively (Figure 5), and did not significantly differ from the control values (see Table 1).


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The role of NMDARs in induction of neurodegenerative processes in neuron cultures was evaluated by studying the neurotoxic action of 30 µM NMDA. All experiments with NMDA were performed in the presence of a saturating concentration of glycine (30 µM) which is a coagonist of NMDARs [Johnson, Ascher, 1987; Dingledine et al, 1990]. The data are presented on figure 5. Activation of NMDARs for 240 min induced neurodegeneration mostly via apoptosis (45%) rather than necrosis (15%). The data obtained in the presence of NMDA with respect of all necrosis, apoptosis and live neurons were significantly different from those of the control (see Table 1). NMDA induced neurodegeneration was completely prevented by an addition of AP5 (Figure 5). The ratio of apoptotic, necrotic, and live cells obtained after application of 30 µM NMDA combined with 50 µM AP5 did not significantly differ from the control (see Table 1). This demonstrates the neuroprotective properties of NMDAR antagonists. At this stage of our study we employed a simple procedure using vital fluorescence stains acridin orange and ethidium bromide. Recently it has been demonstrated the double sequential acridine orange and ethidium bromide staining combined with the confocal microscopy offers an express, fast, easy, sensitive and reproducible method by which necrosis, apoptosis and live neurons can be recognized and automatically quantified in a population of living cells [Mironova et al., 2007]. The utility of this assay has a broad experimental potential ranging from the analysis of dynamics and mechanisms of cell death to the pharmacological studies on a variety of live preparations. The data suggests that either NMDARs or AMPARs/KARs can mediate excitotoxicity. Excessive and selective activation of these Glu receptor types results in the development of neurodegeneration via both mechanisms, necrosis and apoptosis, although apoptosis is predominant. Overall, the neurotoxic action of KA was more pronounced than of NMDA, as the total number of necrotic and apoptotic neurons was greater when excitotoxic insults were induced by KA. Selective antagonists produced neuroprotective effects, preventing the neuronal death by necrosis as well as the induction of apoptotic processes. Long-lasting presence in the media of the natural neurotransmitter, Glu, which at concentrations used in our experiments, presumably, open all NMDARs, AMPARs and KARs, led to substantially more remarkable neurodegeneration in comparison with the selective agonists of particular receptor types (Figure 5). In case of Glu the necrotic component was noticeably larger, as the quantity of necrotic neurons was significantly greater when Glu was applied as a neurotoxic agent, than those obtained after NMDA or KA exposure (p<0.05 for both comparisons, Student‟s two-tailed t test). In consistence with previous studies [Mironova et al, 2006; Mironova et al, 2007] our experiments favor the conclusion that onset of necrosis requires much less prolonged Glu receptor agonists exposure then of apoptosis. As could be seen from Figure 2, 120 min agonist exposures caused rundown of live neuron because of necrosis. At this period of time no visible features of apoptosis were found. The development of apoptosis required a longer agonist treatment so, as a large portion of neurons exhibited apoptotic features after 240 min agonist exposures. In conclusion, Glu and selective agonists of Glu receptors are triggering neurodegeneration, which mechanistic pattern changes in time: at the beginning of excitotoxic insults neurons predominantly die via necrosis, to start apoptosis a longer period of time is required. The vital fluorescent assay applied to neuron cultures provides an estimate of integral pattern of apoptosis and can not provide any information about the particular intracellular cascades involved in this pathology process. The particular apoptotic pathways that

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participate the neurodegeneration evoked by different Glu receptor agonists (3 mM Glu, 30 µM NMDA and 30 µM KA) were analyzed in immunocytochemical experiments. We studied an expression patterns of two apoptotic proteins P53 and Bax and the key proteins of two basic apoptotic cascades AIF (apoptosis inducing factor) and Cas-3 (caspase 3). Neuron cultures were incubated in the bathing salt solution supplemented with one of the agonists for 240 min. Cells then were fixed and the proteins of interest were visualized using immune reactions with monoclonal antibodies. Each of the agonists induced an increase of the number of cells that were immunopositive to proapoptotic protein P53 (Figure 6, A). After exposure of neuronal cultures to any agonist the number of neurons expressing P53 was significantly greater than those under the control conditions (Figure 6, A), though the proportion of P53-positive cells exhibited no significant difference between agonists (p>0.05, ANOVA). P53 is known to be involved in apoptosis regardless of its particular cascades. This is a protein which is involved in the reparation when DNA damages are present, and P53 expression increases as the cell progresses along the apoptosis [Bates, Vousden, 1999; Miller et al, 2000]. In the case of our experiments P53 expression level in neuron cultures increased during excitotoxic stress caused by any agonist and could be taken as an integral measure of the apoptosis intensity. This is supported by an agreement in assessments of apoptosis either by P53 immune-positive cell count in fixed tissues and the vital fluorescence assay. For example, the proportion of apoptotic neurons among whole cellular population in course of treatment with 30 µM NMDA (240 min) were 45 ± 9% (n = 5) and 40 ± 10% (n = 4) obtained using the vital fluorescence assay and the reaction for P53, respectively, and did not differ significantly (p > 0.76, Student‟s two-tailed t test). The same experimental protocol as for P53 has been used to study another proapoptotic protein Bax, which demonstarated similar to P53 expression profile. Numerous Bax positive cells were found after treatment of neurons with 30 µM NMDA, 30 µM KA and 3 mM Glu (Figure 6, B). Glu was the most effective inductor of Bax expression, since in it‟s presence nearly 90% of neurons were Bax-positive. Selective activation of NMDARs or KARs was less effective, causing substantial increase of Bax-positive neurons up to 50% (Figure 6, B). It is well evaluated, that an elevation of Bax expression observed during apoptosis is independent of involved apoptotic cascade (caspase-dependent or caspase-independent) [Saikumar et al, 1998]. Bax translocation to mitochondrial membrane is critical for these processes. With this respect an estimation of P53 and Bax expression can serve as two reliable marks of apoptosis onset. These data suggest that any type of Glu receptors can mediate neurotoxic insult, that promotes Bax expression. Unlike P53 and Bax, AIF and Cas-3 are key molecules mediating caspase-independent (AIF) or caspase-dependent (Cas-3) apoptotic cascades. AIF or Cas-3 inmunostaining allows recognition of particular cascades involved in excitotoxicity. Under the control conditions AIF protein was present in the cytoplasm of most neurons and only a few (on average 6%) contained AIF in the nucleus (Figure 6, C). Treatment with 30 µM NMDA for 240 min produced a statistically significant increase in the proportion of cells containing AIF in the nucleus (p < 0.001, Figure 6, C). The effects of 30 µM KA and 3 mM Glu on AIF expression did not differ, but were significantly weaker than 30 µM NMDA induced increase of AIF expression. An elevation of Cas-3 expression depended on Glu mimetic used (Figure 6, D). The histogram in Figure 6, D summarizes the results obtained. The quantity of neurons exhibiting


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high nuclear content of Cas-3 during 30 µM NMDA exposure did not differ from the control level. The number of cells containing nuclear Cas-3 in the presence of 3 mM Glu and 30 µM KA were significantly greater than in the control (Figure 6, D).

Figure 6. Apoptotic proteins P53, Bax, AIF and Cas-3 expression in response to glutamate, NMDA and KA for 240 min exposure. (A) immunostaining for P53 after exposure to 30 µM KA; (B) immunostaining for Bax after exposure to 30 µM NMDA; (C) immunostaining for AIF after exposure to 30 µM; (D) immunostaining for Cas-3 after exposure to 30 µM kainate. Immunoreactive neurons are indicated by arrows. Histograms presented below the images show percentage of neurons immunopositive for P53, Bax, AIF and Cas-3. (Intact) incubation in the bathing salt solution; (NMDA) treatment with 30 µM NMDA; (KA) treatment with 30 µM kainate; (Glu) treatment with 3 mM glutamate. * The data differ significantly from the data obtained under the control condition (p < 0.05, Student‟s two-tailed t-test)

In fact, 30 µM NMDA was successful to increase the quantity of AIF-positive cells, but did not affect the expression of Cas-3. In opposite, 3 mM Glu and 30 µM KA, whose effects are realized by AMPARs and KARs, increased the number of Cas-3-positive cells, but did not affect AIF nuclear expression (Figure 6). In two sets of our experiments performed to evaluate the apoptosis (immunostaining for P53 and Bax in fixed culture and the vital fluorescence assay) regardless the methods NMDA and KA induced similar apoptosis intensity. Observed difference of expression pattern for Cas-3 and AIF in the case of NMDA and KA allows us to suspect that NMDARs-mediated apoptosis predominantly involves AIFdependent cascades, while AMPARs and KARs operate via the Cas-3-dependent apoptosis pathway.

Conclusion It is generally accepted that research to investigate the nature of processes and the extent of changes in cells and cellular structures under different physiological states, including pathogenesis, provides complex understanding of morphological and functional processes

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occurring in development, normal functioning, regeneration and neurodegeneration of CNS. A variety of stains [Noraberg, 1999; Patterson, 1979] and biochemical methods [Bergmeyer, Bernt, 1974; Koh, Choi, 1978; Uliasz, Hewett, 2000] have recently become available whose combination allows an assessment of the whole cellular cycle from the cell appearance till the death. Each of these approaches has its own advantages and disadvantages. Analysis of neurodegeneration processes in our studies was addressed by the recognition of neuron death using sequential staining with acridine orange and ethidium bromide. The characteristics, suitability for experiments on living tissues, effectiveness, and reliability of this method of identifying necrosis and apoptosis have recently been described in detail [Mironova et al, 2007]. The method is based on the ability of acridine orange to penetrate membranes and stain the nuclei of living cells; ethidium bromide can only detect cells with a disintegrated plasma membrane, staining their nuclei [Pulliam, 1998]. In addition, acridine orange allows apoptosis to be detected because of the difference in staining of apoptotic and living nuclei which has previously been used to identify apoptosis in cell populations of Drosophila embryos [Abrams et al, 1993; White et al, 1994], in Tetrahymena, and chick chondrocytes [Mpoke, Wolfe, 1997]. Many studies demonstrated that apoptosis is accompanied by acidification of cells [Gottlieb et al, 1995; Li, Eastman, 1995], that appears as a result of nuclear disintegration and is associated with decreases in pH due to fusion of nuclei with lysosomes [Mpoke, Wolfe, 1997]. The difference in staining of living and apoptotic nuclei results from a displacement of acridine orange emission to the red spectral region while acidification is occurring during apoptosis [Zelenin, 1966]. Experiments have demonstrated that prolonged exposure of neurons to the GluR agonists induces neurodegeneration, with substantial contributions being made by both apoptosis and necrosis. Unlike Glu, the neurotoxic actions of NMDA and KA mainly induce apoptosis rather than necrosis. This is not surprising because NMDA and KA are specific agonists of a particular receptor type, whereas Glu can simultaneously activate any Glu receptors [Gibb, Colquhoun, 1992]. Furthermore, the large contribution of necrosis to neurodegeneration evoked by 3 mM Glu can also be explained by the fact that it is a natural neurotransmitter and, therefore, has broad physiological effects which are not restricted by the activation of ionotropic GluRs only. In the continuous presence in extracellular media, Glu may interfere and interrupt the functioning of neuronal and glial Glu transporters, presynaptic structures by the interaction with metabotropic Glu receptors as well as induce an impairment of the transmembrane ionic gradients at the least for Na+, K+ and Ca2+ [Antonov, Magazanik, 1988; Antonov, 2001]. The Glu concentration producing the neurotoxic effects in our experiments on primary cortical neuron cultures of 7 DIV was rather high. This concentration was chosen since a lower one (1 mM) had no particular effect throughout 240 min experiment [Mironova et al, 2006]. This would appear to be associated with the receptor expression level, as the effective (in terms of excitotoxicity) Glu concentrations for neurons of 14 DIV were almost 100-orders of magnitude lower than for 7 DIV [Mironova et al, 2007]. These lead us to the conclusion that the endogenous agonist at high concentrations is more effective in the induction of necrosis than its synthetic analogs. The opposite situation was observed in relation to the promotion of apoptosis. Overall our study on rat cerebral cortical neurons of 7 DIV revealed two components of neurotoxic Glu action. One component was associated with the induction of necrotic cell death, while the other was associated with triggering of apoptotic mechanisms. Both components were clearly recognized for selective and natural agonists of Glu receptors rising


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up the possibility of separate characterization of their pharmacological and neurochemical properties. Neurodegeneration induced by NMDA and KA was completely abolished (to the control level) by the competitive NMDAR antagonist - AP5 and the competitive AMPAR and KAR antagonist – CNQX, respectively. This observation suggests that selective activation of any receptor type is sufficient to trigger apoptosis that is consistent with previously published data [Xiao et al, 2001; Wise-Faberowski et al, 2006]. This also demonstrates the potential of selective antagonists of Glu receptors revealing different mechanisms of action, including non-competitive inhibitors [Antonov, Johnson, 1996; Antonov et al, 1998], as neuroprotective agents that are able to prevent neuronal injuries and the development of apoptotic processes. The neurotoxic action of Glu was slightly reduced by CNQX, as a significant increase in neuronal viability was found in the presence of CNQX, although no significant decrease in necrosis and apoptosis was observed. At the same time, the effects of 3 mM Glu were completely resistant to AP5, a specific antagonist of NMDARs. These suggest that the neurotoxic effects on neurons of 7 DIV induced by 3 mM Glu are presumably mediated by an activation of non-NMDA receptors [Mironova et al, 2006]. We identified cells expressing proapoptotic peptides P53, Bax, AIF and Cas-3 using immunocytochemical methods. P53 is known to be a universal protein which is indirectly involved in all apoptotic mechanisms. In our experiments, P53 expression is the same when selective agonists or Glu were used, which is consistent with other data [Miller et al, 2000]. Under similar conditions, the number of neurons expressing P53 coincided well with the apoptosis estimates obtained with the vital fluorescence assay. Immunostaining for P53 is often utilized as universal marker for apoptosis, since an expression of this protein is proportional to the degree of cell genome damages [Bates, Vousden, 1999]. In the case of moderate genome damages, the cell division stops, DNA reparation occurs, and the cell survives. In conditions of extreme genome fragmentation, when DNA can no longer be repaired, receptor- and Cyt-C - dependent apoptotic cascades activate caspases. The first data concerning the involvement of caspases in the neuronal death were obtained from studies of their inhibitor – P53 – in cultures of substantia nigra neurons [Rabizadeh et al, 1993]. In these cells, P53 blocked caspases, which resulted in inhibition of apoptosis induced by hypoglycemia, Ca2+ excess and deprivation of neurotrophic factors. An activation of caspases is evidently one of the possible mechanisms of neuronal death in neurodegenerative conditions. P53 induces expression of proapoptotic Bax protein. In normal conditions small amounts of Bax exist in the cytoplasm. During apoptosis Bax can be translocated to mitochondrial membrane where it interacts with proteins of mithochondrial permeability transition pores (PTP), forcing them to open. Opened PTP are permeable to AIF and Cytochrome-C. An active involvement of Bax into Glu induced neuronal death has been demonstrated in experiments with Bax-knockout cortical neurons cultures [Dargusch et al., 2001]. Antibodies for Bax are now widely used in investigations of neurodegeneration [Zhang, Bhavnani, 2005; Raghupathi et al., 2003]. We also have some preliminary data that increased expression of Bax during apoptosis is accompanied with suppressed production of antiapoptotic Bcl-2 protein. The balance between pro- and antiapoptotic Bcl-2 family proteins it thought to be the key parameter in switching neuronal programmed cell death. An activation of ionotropic receptors (usually NMDARs) results in increased influx of 2+ Ca into cells, triggering proteolysis and degradation of cellular structures [Hatanaka et al,

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1996; Jonston, 1994]. This process is also accompanied by increased lipid peroxidation and subsequent development of oxidative stress [Tapia, 1992; Waters, 1995; Boldyrev, 2001]. Although an activation of Glu receptors is evidently accompanied with neuronal oxidative stress [Waters, 1995], there are nevertheless specific cascades triggering apoptosis whose involvement depends on activation of a given receptor type. This statement is supported by our experimental data on AIF and Cas-3 expression. The induction of apoptosis via the activation of NMDARs was not accompanied with increased Cas-3 expression, though overall assessment of apoptosis using P53 and the vital fluorescence assay gave high values. On the contrary the significant increase in the number of neurons expressing AIF was observed. Induction of apoptosis by KA and Glu led to the significant increase in Cas-3 expression as compared to the control, though AIF production was substantially lower than in the case of NMDA application. It can be suggested that hyperactivation of AMPARs and KARs, which have low Ca2+ conductance, is associated with impairments to energy metabolism and complex alterations of mitochondrial and sarcoplasmic reticulum functions [Khodorov, 2004]. Complex disregulation of mitochondrial membrane permeability is associated with the release of several apoptogenic factors: Cyt-C and procaspases 2, 3, and 9. Cyt-C catalyzes Cas-3 activation which determined caspase-dependent pathway of apoptosis. The increase in Cas-3 in the experiments with 3 mM Glu and 30 µM KA provide evidence that apoptosis was triggered via the caspase pathway. Another mechanism regulating apoptotic death occurs when NMDARs are activated. This mechanism involves the release of AIF, which is translocated from the mitochondrial membrane to the nucleus, inducing DNA degradation via the caspase-independent pathway [Yu et al., 2002]. It is supported by observed increase of nuclear AIF presence after 30 µM NMDA excitotoxic insults. The results described in this chapter are indirectly supported by data that NMDARs mediated apoptosis can not be blocked with caspase inhibitors, but is prevented in the case of Bcl-2 hyperexpression [Wang et al., 2004]. Thus, we can conclude that apoptosis induced by the NMDARs activation develops through the caspase-3 - independent pathway that involves direct AIF accumulation in nuclei. The AMPARs/KARs mediated apoptosis includes a caspase-3 – dependent mechanism. An existence of the receptor specific apoptosis cascades during excitotoxicity reveals new directions in selective therapy of neurodegenerative states. The work presented in this chapter is supported by RFBR grants 05-04-49789 and 08-0400423.

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Chapter 5

Mitochondrial Uncoupling Proteins – Therapeutic Targets in Neurodegeneration? Susana Cardoso, Cristina Carvalho, Sónia Correia, Renato X. Santos, Maria S. Santos and Paula I. Moreira* Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal

Abstract Uncoupling proteins (UCPs) are mitochondrial inner membrane proteins that uncouple electron transport from ATP production by dissipating protons across the inner membrane. UCP1 was the first uncoupling protein described and is present in brown adipose tissue being involved in the non-shivering thermogenesis. Subsequent studies demonstrated that neurons express at least three UCPs isoforms including the widely expressed UCP2 and the neuronspecific UCP4 and UCP5. UCPs control the mitochondrial membrane potential, free radicals production and calcium homeostasis and thereby influence neuronal function. Given that mitochondrial energy impairment and free radicals production are thought to be central players in neurodegeneration, recent data suggest that UCPs may have an important role in neuroprotection and neuromodulation. The function of neuronal UCPs and their impact on the central nervous system are attracting an increased interest as potential therapeutic targets in several disorders including neurodegenerative diseases. Here we will discuss the uncoupling process as an intrinsic mechanism of mitochondria physiology. The role of UCPs in healthy and pathological brain conditions will be also considered. Finally, we will discuss UCPs as potential therapeutic targets in stroke and neurodegenerative diseases.

Keywords: Brain, mitochondria, neurodegenerative diseases, stroke, UCPs

*

Corresponding author: Paula I. Moreira, Center for Neuroscience and Cell Biology, Faculty of MedicinePhysiology, University of Coimbra, 3000-354 Coimbra, Portugal. [email protected]/ pismoreira @ gmail.com

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Susana Cardoso, Cristina Carvalho, Sónia Correia et al.

I. Introduction Mitochondria are organelles located in the cytoplasm of all eukaryotic cells and involved in many processes essential for cells survival and function, including energy production, calcium homeostasis, redox control, and some metabolic and biosynthetic pathways [1]. Energy is generated as electrons are passed from donors at lower to acceptors at higher redox potential through various protein complexes. Coupled with electrons transport, protons are pumped from the matrix outward generating a potential difference across the inner membrane. The resulting potential energy produced by the proton gradient is used to drive phosphorylation of ADP to ATP [2]. Uncoupling proteins are a family of mitochondrial anion-carrier proteins located on the inner mitochondrial membrane, and their primary function is to leak protons from the intermembrane space into the mitochondrial matrix [3,4]. Through this process, the ATP synthesis is uncoupled from the electron transport, dissipating energy in the form of heat. The process of uncoupled respiration was first discovered in brown adipose tissue, where uncoupling is affected by UCP1, the most well-characterized isoform. In the last few years, studies brought evidence of the existence of other members of the UCP family that promote partial uncoupling of oxidation from phosphorylation. These proteins include UCP2, UCP3, UCP4, and UCP5 (also known as Brain Mitochondrial Carrier Protein 1, BMCP-1) that differ among each other in tissue distribution and regulation and may have distinctive physiological roles [3]. The specific role of UCPs has been widely discussed and although there is no general agreement, there is a strong conviction that these proteins may be involved in the defense against reactive oxygen species (ROS) therefore protecting or reversing oxidative damage. The first evidence came from a study made by Nègre-Salvayre and colleagues [5] revealing that the inhibition of UCP1 activates the formation of ROS in brown fat mitochondria. Subsequent data suggested that UCP activity may lead to an increase in proton conductance through the interaction with superoxide [6] or ROS products [7]. The alteration of mitochondrial energy metabolism leads to reduced ATP production, impaired calcium buffering, and generation of ROS. Indeed, up to 90% of intracellular ROS are generated in mitochondria during the oxidation of nicotinamide dinucleotide (NADH) and flavin adenine dinucleotide (FADH2) [8]. The generation of ROS is increasingly recognized as playing an important role in diabetes, obesity, ischemia/reperfusion, neurodegenerative disorders and aging where mitochondria are both sources and targets of these reactive species [9-11]. Therefore, it was suggested that an increased ROS production would lead to uncoupling that in turn would decrease ROS formation, leading to reduced oxidative damage [12]. Thus, this possible role of neuronal UCPs in neuroprotection and neuromodulation has been faced as an important way to control and limit the formation of free radicals in several disease and physiological processes. In this chapter we will begin by introducing the uncoupling process as an intrinsic mechanism of mitochondria. We will then discuss the role of neuronal UCPs in normal brain function. Finally, the role of UCPs in neurodegeneration and stroke will be discussed highlighting their interest as potential targets for therapeutic intervention in brain diseases.

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II. Mitochondrial Physiology and Uncoupling Mitochondria are intracellular organelles highly efficient in their ability to utilize oxygen (O2) and substrates to produce energy in the form of ATP [13]. The oxidative phosphorylation system (OXPHOS) system is located in the inner mitochondrial membrane and is composed by five respiratory chain complexes, NADH ubiquinone oxidoreductase (Complex I), succinate ubiquinone oxidoreductase (Complex II), ubiquinone-cytochrome c reductase (Complex III), cytochrome c oxidase (Complex IV) and ATP synthase (Complex V). There are two electron carriers, ubiquinone (coenzyme Q), located in the inner mitochondrial membrane and cytochrome c, located in the intermembrane space (Figure 1) [14]. Reducing equivalents produced in the Krebs cycle and in the β-oxidation pass through complexes I to IV and the energy generated by the electron transfer is used to pump protons from the mitochondrial matrix into the intermembrane space creating an electrochemical proton gradient used to drive complex V to generate ATP (Figure 1) [15]. The efficiency in use the formed H+ gradient is called coupling. In a perfectly coupled system, protons only enter the mitochondrial matrix through ATP synthase in the presence of ADP, this form of respiration is classified as state 3 (O2 is consumed only in the presence of substrate and ADP). But mitochondria can also use O2 even in the absence of ADP, which occurs when protons leak back into the matrix via a mechanism independent of ATP synthase that will uncouple respiration from oxidative phosphorylation. O2 consumption in the absence of ADP or in totally uncoupled mitochondria is designated to as state 4 of respiration [2]. H+ gradient can be consumed by many proteins, like members of the mitochondrial carrier family (ADP/ATP carrier and glutamate/aspartate carrier) and others like phosphate carrier and other carriers employing substrate-H+ symport [16]. Additionally, under normal conditions, a portion of the created H+ gradient is consumed by proton backflow to the matrix via non-protein membrane pores or protein/lipid interfaces [17]. These mechanisms allow H+ backflow to the matrix bypassing ATP synthase and thereby provoke protein-mediated respiration uncoupling [18]. Thus, uncoupling is an inherent part of mitochondrial physiology. The process of proton leak has been suggested to be involved in thermogenesis, regulation of energy metabolism or carbon fluxes, control of body mass, and attenuation of ROS production [19]. Mitochondrial uncoupling proteins (UCP1, UCP2, UCP3, UCP4, and BMCP-1) that are located into the inner mitochondrial membrane function as proton carriers and are responsible for basal proton leak (Figure 2), which in the rat accounts for 10-27% of resting O2 consumption, depending on the organ studied [19]. UCPs isoforms are activated by free fatty acids (FFAs), superoxide and inhibited by purine nucleotides [20], possessing some degree of homology among each other. UCP1 was first described in brown adipose tissue and is responsible for heat generation in the newborn and may be involved in the normal response to cold stress in the adult [21]. UCP2 is 59% identical to UCP1 and is widely expressed in spleen, lung, stomach, white adipose tissue and also in the brain [22]. UCP3 that is mainly expressed in skeletal muscle [23] possesses approximately 57% and 73% of similarity with UCP1 and UCP2, respectively. Finally, UCP4 [24] and BMCP-1 [25] are mainly expressed in the central nervous system being 34% and 30% identical to UCP1, respectively, and BMC1 is the only UCP found in the parenchymal cells of the liver [8]. All the UCPs share in common a tripartite structure that consists of three repeats of approximately 100 amino acids, each one containing two hydrophobic stretches that correspond to transmembrane alpha helices that

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span the phospholipids bilayer of the mitochondrial inner membrane. The two attached alpha helices are linked by a long hydrophilic loop, which is oriented toward the mitochondrial matrix side. UCPs have a monomer molecular weight of about 30 kDa with both the N- and the C-terminal ends oriented toward the cytosolic side of the inner mitochondrial membrane. The functional unit of UCPs is believed to be a homodimer formed by two identical subunits that contain 12 transmembrane helices [1,3,26].

Figure 1. Schematic representation of the mitochondrial electron transport chain (ETC). ETC is composed by four protein complexes (I-IV). Electrons from oxidative substrates are transported through ETC, by the two electron carriers, coenzyme Q that accepts electrons from complexes I and II and donate them to complex III, and cytochrome c that transfers electrons from complex III to complex IV leading to oxygen reduction to water. In this process, protons are pumped across the inner mitochondrial membrane to establish a proton motive force. The energy that is conserved in this proton gradient drives the synthesis of ATP via complex V (ATP synthase) as protons are transported back from the intermembrane space into the mitochondrial matrix. UQ: coenzyme Q; Cyt c: cytochrome c; complex I: NADH ubiquinone oxidoreductase; complex II: succinate ubiquinone oxidoreductase; complex III: ubiquinone-cytochrome c reductase; complex IV: cytochrome c oxidase; complex V: ATP synthase.

III. UCPs in the Brain- Effects on Neuronal Function Neuronal UCPs specific roles are still under intense debate. Although initially neuronal UCPs (UCP4 and BMCP-1) were not considered real uncoupling proteins, it is becoming more clear that these proteins perform important roles in neuronal function. For instance, using specific antibodies for UCP4 it was found that this protein is present in fetal murine brain tissues as early as embryonic days 12-14, which coincides with the beginning of neuronal differentiation [27]. Moreover, UCP4 content decreases as the mice get older being its highest levels found in the cortex, which suggest a role for UCP4 in neuronal cell differentiation [27]. Nevertheless, it is important to note that the tissue distribution of neuronal UCPs mRNA differs between rat and mouse [28]. In this section it will discussed some possible roles of neuronal UCPs in normal brain function.


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Thermogenesis Energy homeostasis is a highly regulated process and is central to the control of body weight. UCPs have been identified and implicated as potential regulators of adaptive thermoregulation, body composition, and metabolism [29]. Furthermore, temperature differences in various brain regions have been observed in rodents and more recently in humans, by invasive and non-invasive techniques [30-33]. UCP2 mRNA is expressed throughout the brain with some variability. A markedly intense expression is found in several parts of the brain including hypothalamus, thalamus, brainstem, cerebellum and the choroid plexus [22]. Indeed, evidence reveals a dorsoventral temperature gradient that coincides with the occurrence of UCP2 and increased proton leak [34]. This may suggest UCP2-induced mitochondrial uncoupling involvement in thermoregulation of the brain. The mRNA abundance of UCP4 and UCP5 is modulated by nutritional and temperature manipulations in a tissue-specific manner [29]. On cold exposure, brain UCP4 rise significantly and remains elevated from 1 to 24 h, whereas UCP5 mRNA in brain and liver increases at 1 h and remains elevated through, at least, 6 h [29]. Additionally, body temperature changes were preceded by a rise in UCP4 (in brain) and UCP5 (in brain and liver) expression [29]. Accordingly, cold temperature significantly increased UCP4 mRNA levels in rat hippocampal neurons [35]. Thus UCPs may play an important role in thermoregulatory processes induced by cold exposure. Nevertheless, it was recently demonstrated that in adult mice brain UCP4 highest levels occur in the cortex, which does not support the thermoregulatory function of UCP4 [27]. Further research is needed in order to clarify UCP4 involvement in thermogenesis regulation.

Calcium Mitochondria play important roles in the regulation of cellular calcium (Ca2+) dynamics in physiological situations, including neurotransmitters release, and in pathological states. Mitochondrial bioenergetics and redox state are also influenced by intracellular Ca 2+ levels being mitochondria capable of promoting Ca2+ uptake into the matrix that is driven by mitochondrial membrane potential (∆Ψm) [36]. Within mitochondrial matrix increased levels of Ca2+ can affect mitochondrial ROS release and subsequent oxidative stress. Mitochondrial dysfunction, resulting from the disruption of calcium homeostasis and the generation of ROS, is a central player in neuronal dysfunction and death following acute brain insults [37]. Uncoupling agents like 2,4-dinitrophenol (DNP) have been shown to reduce mitochondrial Ca2+ increases that occur after quinolinic acid-induced NMDA receptor activation [38]. Also in an animal model of traumatic injury, DNP treatment reduced by 30% the ability of mitochondria to sequester Ca2+ [39]. More recently, Chan and colleagues [40] demonstrated that human UCP4 expression in PC12 cells regulates mitochondrial Ca 2+ sequestration and entry but not the release from intracellular stores. Furthermore, UCP4 expression decreased the magnitude of sustained elevation in intracellular Ca2+ concentration after cellular Ca2+ depletion and inhibited mitochondrial Ca2+ overload and oxidative stress thereby preventing cell death (Figure 2) [40].

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Neuronal ROS Production A small proportion of the electrons flowing through complexes I and III react with O2 forming superoxide anion [41] that can be converted into hydrogen peroxide (H2O2) utilizing both manganese superoxide dismutase (MnSOD), which is located to the mitochondria and copper-zinc superoxide dismutase (Cu/ZnSOD) found in the cytosol. H2O2 is promptly converted to H2O via catalase and glutathione peroxidase, but has the potential to be converted to the highly reactive hydroxyl radical via the Fenton reaction, underlying ROS neurotoxicity [42]. Within the cells ROS may have a dual role, acting as beneficial or harmful species [15]. In response to certain stimuli cells produce low/moderate levels of ROS that have physiological functions intervening in several cellular signaling pathways, therefore acting as second messengers [15,43]. In opposite, excessive ROS formation will attack unsaturated fatty acids in membranes leading to lipid peroxidation and the production of 4hydroxynonenal (HNE) that conjugates to membrane proteins impairing their function [39]. Moreover, situations of increased oxidative stress and mitochondrial Ca2+ overload promote the opening of the permeability transition pore (PTP), a situation characterized by the mitochondrial proton motive force disruption. PTP opening will lead to the release of proapoptotic proteins like cytochrome c, which induce the caspase-mediated apoptosis [15]. In order to overcome the oxidative insult, cells possess a variety of enzymatic and nonenzymatic antioxidant defenses. However, if an imbalance between antioxidant defenses and ROS formation occurs, oxidative damage of cells will happen contributing to the development of neurodegenerative diseases [44]. Mitochondrial ROS production is closely linked to ∆Ψm such that hyperpolarization (high ∆Ψm) increases and promotes ROS production [45]. While long-term, complete uncoupling of mitochondria would be detrimental, it has been postulated that mild uncoupling could be beneficial since it causes a decrease in ROS production [45]. In rat kidney mitochondria, superoxide activates UCPs from the matrix side of the mitochondrial inner membrane [46] while the presence of mitochondrial-targeted antioxidants prevents superoxide-induced uncoupling [46]. Interestingly, uncoupling is not inhibited by extracellular SOD that cannot access mitochondrial matrix side, so demonstrating that superoxide generated at the matrix side did not need to be exported or cycle across the inner membrane to cause uncoupling [46]. Also, UCP3 knockout mice exhibit increased ROS in muscle [47]. Several lines of evidence demonstrate that brain regions that overexpress UCP2 present a decreased energy coupled efficiency and a reduced ∆Ψm [48]. The GDP-stimulated uncoupling that occurs in the presence of ubiquinone is abolished by SOD suggesting that UCPs are activated by superoxide [49]. Liu and colleagues [35] demonstrated that UCP4 have an important role in promoting neuronal survival. Indeed, the authors observed that PC12 cells overexpressing UCP4 present a reduction in mitochondrial oxidative phosphorylation and ROS production that result from the UCP4-induced metabolic shift associated with enhanced glucose uptake and glycolysis to compensate for reduced ATP mitochondrial production [35]. Thus, UCP4 activity makes neural cells less reliant on mitochondrial respiration for maintenance of energy levels. In order to establish the function of endogenous UCP4 in neurons, it was performed RNA interference (RNAi) to deplete UCP4 in cultured rat hippocampal neurons. It was shown that cells transfected with UCP4 RNAi present a


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decreased survival rate compared to cells transfected with scrambled RNAi [35]. These results demonstrate that endogenous UCP4 is required for hippocampal neurons survival. BMCP-1/UCP5 is present in the cortex, basal ganglia, substantia nigra, cerebellum, and spinal cord [50]. Data show that neuronal cell lines transfected with BMCP-1/UCP5 had higher state 4 of respiration and lower ∆Ψm, revealing greater mitochondrial uncoupling [50]. In addition, it was also observed a reduction in mitochondrial ROS production [50] (Figure 2). Moreover, the exposure of neurons overexpressing BMCP-1 to linoleic acid further enhanced state 4 of respiration while the addition of bovine serum albumin prevented this augmentation [50], supporting the notion of UCPs activation by FFAs. Accordingly, the reduction of dietary fat in immature animals rapidly reduces neuronal UCPs expression/activity leading to increased mitochondrial ROS production [51]. These changes decreased animals resistance to excitotoxic insults resulting in increased neuronal death [51], thus implicating a neuroprotective role for UCPs in neuronal injury.

Figure 2. Potential neuroprotective mechanism of neuronal UCPs. Mitochondrial uncoupling proteins (UCPs) function as an uncoupler by acting as a channel for proton entry into the matrix, which then dissipates the transmembrane potential (∆Ψm) generated by respiratory complexes I through IV. This reduces the proton motive force leading to the uncoupling of respiration from oxidative phosphorylation, dissipating energy in the form of heat. UCP-induced ∆Ψm reduction in neurons will reduce ROS produced in mitochondria and prevent mitochondrial calcium overload. UQ: coenzyme Q; Cyt c: cytochrome c; complex I: NADH ubiquinone oxidoreductase; complex II: succinate ubiquinone oxidoreductase; complex III: ubiquinone-cytochrome c reductase; complex IV: cytochrome c oxidase; complex V: ATP synthase

Due to UCPs role in the regulation of energy metabolism, their possible role in diabetes has been exploited. Diabetes, namely hyperglycemia, leads to an oversupply of electrons in the ETC that result in mitochondrial membrane hyperpolarization and ROS formation [42,52]. Although UCP3 expression was thought to occur mainly in the muscle, a recent finding shows that UCP3 is also normally present in dorsal root ganglion (DRG) neurons

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[53]. Moreover, UCP3 levels were decreased in DRG neurons isolated from streptozotocininduced diabetic animals while UCP3 overexpression in cultured DRG neurons was able to prevent glucose-induced mitochondrial hyperpolarization, ROS formation and programmed cell death induction [53]. Also, the human neuroblastoma cell line SH-SY5Y when exposed to high concentrations of glucose presented a down-regulation of UCP3 protein expression, and an increase in ∆Ψm and intracellular ROS [54]. In opposite, the addition of insulin-like growth factor (IGF-1), which positively regulates UCP3 expression [55,56], prevented the glucose-induced neurite degeneration and UCP3 down-regulation leading to ROS levels and ∆Ψm normalization [54].

IV. UCPs in Neurodegeneration and Stroke: Potential Therapeutic Targets The brain is extremely sensitive to oxidative damage due to its high O2 demand, its high content of oxidisable polyunsaturated fatty acids, the presence of redox-active metals and a low activity of antioxidant enzymes [10,14,57]. Since oxidative stress increases with age and mitochondria are both targets and sources of ROS, there is the assumption that mitochondria have a central role in aging and neurodegenerative disorders [58]. Indeed, despite the fact that neurodegenerative disorders have disparate clinical features, they are characterized by mitochondrial dysfunction and oxidative stress [59]. It has been demonstrated that human UCP2 targeted expression in mitochondria of adult fly neurons promotes an increase in state 4 of respiration, a decrease in ROS production and oxidative damage accompanied with an extension in life span without the compromise of fertility or physical activity [60]. Due to UCPs ability to regulate both mitochondrial metabolic efficiency and free radical generation, they are of special interest in stroke and neurodegenerative pathologies, including Alzheimer´s (AD) and Parkinson´s (PD) diseases and amyotrophic lateral sclerosis (ALS).

Alzheimer’s Disease Alzheimer‟s disease (AD) is a progressive age-dependent neurodegenerative disorder and the most common form of dementia, accounting for 50-70% of dementia cases. While less of 5% of AD cases are familial and associated with mutations in amyloid precursor protein (APP) and presenilins 1 and 2 (PS1 and PS2), the majority of AD cases are sporadic in origin and involve genetic and environmental factors that taken alone are not sufficient to develop the disease [61]. This neurodegenerative disease is characterized by progressive cognitive decline and the presence of Aβ plaques and tau neurofibrillary tangles [11,62]. Abnormal APP processing is believed to play a central role in AD. APP may be metabolized along two distintic pathways: the amyloidogenic and the non-amyloidogenic pathways. In the latter, APP is cleaved by an α-secretase producing non-amyloidogenic proteins. In the amyloidogenic pathway APP is cleaved by β- and γ-secretases producing the Aβ peptides [63]. It has been reported that Aβ oligomers and plaques are potent synaptotoxins, which block the proteasome function, inhibit mitochondrial activity, increase oxidative stress, and alter intracellular Ca2+ levels leading to synaptic dysfunction [64].


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Recent data from postmortem brain tissue from AD patients with different degrees of severity demonstrated that AD is associated with impairments in mitochondrial gene expression, namely in complex IV of the mitochondrial respiratory chain, increased levels of p53 gene expression and increased molecular indexes of oxidative stress, such as upregulation of nitric oxide synthase (NOS) and NADPH-oxidase (NOX). Additionally, the authors performed real time quantitative RT-PCR studies and found that in the brain, UCP4 and UCP5 expression is approximately 200 and 800-fold higher, respectively, than UCP 2 expression [65], thus suggesting a protective role of UCP4 and UCP5 against oxidative stress under normal circumstances. However, in AD brains UCPs gene expression decreased significantly relative to control brains and the mean levels tended to be lower in AD brains with higher grades of neurodegeneration [65]. So, the failure to maintain normal levels or increase the expression of UCPs may potentiate oxidative stress leading to progressive mitochondrial DNA damage and energy depletion. Data show increased levels of UCP4 expression in the cortex and hippocampus of mice undergoing dietary restriction [35,66]. Since dietary restriction has been shown to be neuroprotective in animal models of AD [67], it was suggested that neuronal UCPs may have a role against chronic neurodegenerative diseases [37,68].

Parkinson’s Disease PD is the second most common neurodegenerative disorder that begins by causing motor dysfunction but ultimately affects the mind and personality [62]. This disease is clinically characterized by progressive rigidity, bradykinesia and tremor and pathologically by the degeneration of pigmented neurons in the substantia nigra and by the presence of intraneuronal proteinaceous cytoplasmic inclusions that immunostain for α-synuclein and ubiquitin, designated Lewy Bodies [58,62]. It is well established that oxidative stress and mitochondrial dysfunction are associated with the degeneration of dopaminergic neurons in PD. The involvement of mitochondrial dysfunction in PD arose from the finding that 1methyl 4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP), a synthetic opiate, caused parkinsonism in drug addicted individuals [69]. MPTP is metabolized to MPP+ in glial cells and this metabolite inhibits the complex I of the mitochondrial respiratory chain [69]. Horvath and colleagues [70] have previously shown that CoQ therapy in a PD animal model induces mitochondrial uncoupling in the substantia nigra preventing MPTP-induced cell death. Moreover, it seems that this effect is mediated by UCP2 activation that precedes the CoQ prevention [70]. Later on, Andrews and co-workers [68] demonstrated that dopamine neurons sensitivity to MPTP is increased in UCP2 knockout mice, whereas UCP2 overexpression decreased MPTP-induced nigral dopamine cell loss by increasing mitochondrial uncoupling and decreasing ROS production [68]. Furthermore, electron microscopic analysis revealed that the substantia nigra from UCP2 knockout mice had significantly lower number of mitochondria compared with control [68]. Similarly, Conti and colleagues [71] showed that transgenic mice overexpressing UCP2 in catecholaminergic neurons present increased uncoupling of their mitochondria, a reduction in oxidative stress and retention of locomotor functions after MPTP exposition. These results suggest that UCP2 is an essential homeostatic

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protein regulating cell survival and vulnerability in the presence of harmful toxins. UCP2 role in PD was also explored in normal nigrostriatal dopamine function and previous studies show that mice lacking UCP2 exhibited reduced dopamine turnover in the striatum, reduced tyrosine hydroxylase immunoreactivity in the substantia nigra, striatum and nucleus accumbens and reduced dopamine transporter immunoreactivity in the substantia nigra [72]. Moreover, when evaluating those deficits in locomotor function, the authors observed that UCP2 knockout mice exhibited reduced total movement distance, movement velocity and increased rest time compared with wild type rats [72], giving the suggestion of UCP2 involvement in the maintenance of normal nigrostriatal dopamine neuronal function. Recently, the same authors reported that UCP2 mediates ghrelin-induced neuroprotection against dopaminergic cell loss in the substantia nigra after MPTP treatment through alterations in mitochondrial respiration, ROS production, and biogenesis [73]. Neuronal UCP5 role was investigated in a neuroblastoma cell line exposed to MPTP [74]. The authors observed that UCP5 knockdown increased caspase 3 levels and cell death. UCP5 knockdown also increased cytotoxicity induced by low doses of MPTP but had no effects in oxidative stress and membrane depolarization [74]. However, in the presence of high doses of MPTP, UCP5 knockdown exacerbated cytotoxicity, and increased oxidative stress and mitochondrial membrane polarization [74], demonstrating the importance of UCP5in oxidative stress-induced neurodegeneration.

Amyotrophic Lateral Sclerosis ALS is the most common adult-onset motor neuron disease [75] resulting in weakness, paralysis and subsequent death. Approximately 90% of the cases are sporadic and the remaining 10% are familial [58]. Mitochondrial and bioenergetic defects are widely implicated in ALS being reported alterations in mitochondrial structure, number and localization in motor neurons and skeletal muscle [76]. Additionally, the presence of mutant SOD1 within motor neuron causes alterations of the mitochondrial respiratory chain [77], namely in mitochondrial complexes II and IV [78]. Other studies show an involvement of ROS in the pathology of ALS [79]. Therefore, there is a strong notion that mitochondrial dysfunction may play a critical role in ALS pathology. Motor neurons have high energy demands, which make them particularly vulnerable to the adverse effects of mitochondrial impairment. When metabolically compromised, motor neurons become unable to maintain membrane potential, resulting in the opening of voltage dependent NMDA glutamate receptors and excessive calcium influx [80]. Indeed, it has been shown that glutamate levels are increased in the cerebrospinal fluid of half of ALS patients [81], this effect being associated with the loss of spinal motor neurons [82,83]. Neuronal UCPs are expressed in the mice spinal cord and their levels do not seem to be affected in SOD1 transgenic mice at different ages [84]. Nevertheless, UCP3 mRNA and protein levels are selectively increased in ALS skeletal muscle, both in animal models and in human biopsies [84]. On the other hand, other studies suggest that transient mitochondrial uncoupling can confer neuroprotection following spinal cord injuries [39]. For instance, rats administrated with DNP, a mitochondrial uncoupler, present less tissue loss, improved behavioral outcomes and a concomitant reduction in mitochondrial oxidative damage, Ca2+


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loading and dysfunction after spinal cord injury [39,85]. Further research is needed in order to clarify the role of UCPs in ALS.

Stroke Acute brain injury, caused by stroke and trauma, is a major cause of morbidity and mortality in the industrialized countries. During a stroke a drastic reduction in blood supply to neurons occurs resulting in cellular hypoxia and glucose deprivation, which impair oxidative phosphorylation and ATP formation [13]. In experimental models of stroke, ischemic neuronal damage includes excitotoxicity, Ca2+ influx and accumulation within mitochondria, superoxide production, and subsequent oxidative stress, p53 and Bax expression, PTP opening, cytochrome c release and caspases 9 and 3 activation [86]. Therefore, mitochondrial dysfunction appears as a common pathway in neuronal cell death in acute brain injury. Mitochondrial UCPs reduce mitochondrial ROS production by dissipating the hydrogen ion gradient across the inner mitochondrial membrane. Mattiason and co-workers [87] found that cortical neurons overexpressing UCP2 are protected against oxygen-glucose deprivation (OGD)-induced cell death. Furthermore, transgenic mouse overexpressing human UCP2 protein subjected to ischemic preconditioning are protected after a severe ischemic insult presenting enhanced neurological recovery [87]. The authors found that UCP2 promote a shift in hydrogen peroxide release from the mitochondrial matrix to the extramitochondrial space, where it can be degraded by some antioxidant enzymes [87]. Recently, Liu and colleagues [88] also demonstrated that ischemic preconditioning caused increased expression of UCP2 in rat hippocampus that conferred protection against ischemia/reperfusion injury. Treatment with SOD at the time of ischemia preconditioning attenuated the increase of UCP2 staining, therefore implying a role for superoxide-induced UCP2 expression [88]. Interestingly, another previous work in UCP2 knockout mice demonstrate that deficiency in UCP2 results in increased resistance to cerebral ischemia that is associated with reduced oxidative injury and an increase of the cerebral neuronal anti-oxidant state [89]. Moreover, authors show that UCP2 mRNA induction was temporally associated with changes of mitochondrial glutathione levels following ischemia in mice. The authors suggested that a chronic adaptation to the lack of UCP2 occurs, which may contribute to the reduction of ischemic injury and levels of lipid peroxidation in UCP2 knockout mice [89]. Therefore, interventions aimed to maintain mitochondrial homeostasis may provide new directions to prevent or attenuate acute central system injury [37]. It has been also reported an increased UCP2 and UCP5 expression in ischemic lesions in brain slice sections prepared from embolic stroke and multiple infarction brains [90]. Moreover, UCP5 expression in the lesions was higher in multiple infarction cases than in embolic stroke cases suggesting that UCP5 may respond to repetitive ischemic stresses or have a long-term effect [90]. So, under ischemic conditions brains may develop adaptive mechanisms involving an increased expression of UCP2 and UCP5. Although more studies are needed to elucidate the mechanisms underlying the protective roles of UCPs, the above-discussed studies show that UCPs are attracting an increased interest as potential therapeutic targets in several brain disorders.

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Conclusion Similar to UCP1, also neuronal UCPs (UCP2, UCP4 and BMCP-1) appear to modulate proton leak through the inner mitochondrial membrane decreasing the mitochondrial electrochemical potential. However, while the main UCP1 function is to produce heat to maintain body temperature, neuronal UCPs biological function seems to be different and include the control of ROS production. Neurodegenerative disorders are characterized by a progressive decline in neurological function and neuronal cell death and despite the fact that these disorders have disparate clinical features, they are characterized by mitochondrial dysfunction and oxidative stress. Research in neuronal UCPs is still an emerging field where future analyses of such proteins are needed. Nevertheless, the results obtained in experimental models indicate that their possible neuroprotector and neuromodulator role could be a promising avenue to develop better therapies to prevent or ameliorate stroke and neurodegenerative disorders.

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Chapter 6

Targeting Caspases in Neonatal Hypoxic-Ischemic Brain Injury and Traumatic Brain Injury Xin Wang*1, Rachna Pandya1, Jiemin Yao2,4, He Ma3 and Jianmin Li2 1

Laboratory of Neuroapoptosis Drug Discovery, Department of Neurosurgery, Brigham and Women‟s Hospital, Harvard Medical School, Boston, Massachusetts, USA 2 Department of Neurosurgery, Third Affiliated Hospital, 3 Department of Anesthesiology, Tumor Hospital, Guangxi Medical University, Nanning, Guangxi China 4 Department of Medicine-Infectious Disease, The University of Texas Health Science Center at San Antonio, San Antonio, Texas , USA

Abstract Mounting evidence implicates apoptosis in the pathogenesis of both acute and chronic neurological disorders. The caspase family of cysteine proteases plays a central role in the initiation and execution of neuronal apoptosis. So far the caspase family has been expanded to 18 cysteine protease members. About two decades of investigation involving the caspase family has produced a wealth of information. Studies indicate that targeting the caspase family can prevent neuronal cell death in neurological disorders. This chapter will discuss the role of the caspase family in experimental models of neonatal hypoxia-ischemia brain injury and traumatic brain injury in vivo and in vitro, as well as in human neonatal hypoxic-ischemic encephalopathy and traumatic brain injury. Given that elucidation of the roles of individual caspases could yield multiple points of possible therapeutic intervention, from the drug discovery and treatment perspective, the review will summarize what is currently known about the beneficial effects of targeting caspases using a variety of treatments against neonatal hypoxia-ischemia brain injury and * Address correspondence to correspondence author:Xin Wang, Ph.D., Brigham and Women‟s Hospital, Harvard Medical School, Department of Neurosurgery, Boston, Massachusetts 02115, USA, Phone: (617) 732-4186, Fax: (617) 732-6767, E-mail: [email protected]

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1. Introduction Caspases, a family of 18 enzymes, play a pivotal role in various neurological diseases of neonates and adults including neonatal hypoxic-ischemic encephalopathy (H-IE) and traumatic brain injury (TBI), respectively. In acute neurological diseases due to ischemia, necrosis is responsible for cell death in the core of the lesion; however, at the penumbra of the lesion apoptosis mediates cell death. Caspases are chronically activated in this area of ischemic/hypoxic lesion, eventually leading to cell death. Thus caspase inhibition is a possible approach to treatment of acute neurological diseases[1]. Caspases, first identified in the nematode Caenorhabditis elegans[2], are evolutionarily conserved in many multicellular organisms. Genes for Ced-3, ced-4, egl-1, and ced-9 are identified as loci controlling these enzymes[3]. In 1996 the nomenclature „Caspase‟ was coined based on their structure and function (as cysteine-dependent aspartate specific proteases), and numbers were given according to hierarchical discovery of each caspase. They are the central mediators in apoptosis or programmed cell death, through which the body regulates the number of cells in an organ[4]. Functionally caspases are zymogens containing an N-terminal prodomain. Their mature enzymes are heterotetramers of two subunits of ~20 kDa and two subunits of ~10 kDa each[5, 6]. The active enzyme is obtained via endoproteolytic cleavage and exists either as a dimer or monomer. These active enzymes, obtained through cleavage at the Asp 297 site, recognize tetrapeptide motifs and cleave their substrates on the carboxyl end of aspartate residues. All caspase recognition sequences contain Asp at -1 position. However, the specificity of the individual caspase is determined by positions -2, -3, and -4[7]. Caspases can be broadly classified as either effector caspases (3, 6, 7), which have short N-terminal domains and are activated by proteolysis of other caspases, or initiator caspases (1, 2, 4, 5, and 8-13), which have long N-terminal domains and are activated by nonproteolytic signaling molecules[4, 5]. Caspase-14, which has been found in cornifying epithelia such as skin[8], is the only purely non-apoptotic caspase with a role in cytokine maturation[1]. Eckhart et al. identifies and characterizes caspase-15 as a mammalian caspase with proapoptotic activity[9]. Eckhart et al. also demonstrates that caspase-16 is most similar in sequence to caspase-14, caspase-17 to caspase-3, and caspase-18 to caspase-8[10]. The cell death receptor pathway is activated by the ligation of Fas (CD 95/Apo1), the TNFR1 homologue, with the cognate ligand. The death inducing signal complex (DISC) is formed[11] by the DD (Death Domain) in the receptor as well as the adaptor molecule (FADD) and the DED (Death Effector Domain) in the adaptor molecule and procaspase-8, eventually leading to activation of caspase-8. Caspase-8 further activates effector caspases (3, 6, 7), which carry out limited proteolyis. The mitochondrial death pathway is activated by mitochondrial permeabilization leading to release of proapoptotic factors cytochrome c (cyto. c), Smac and apoptosis inducing factor (AIF)[12]. An apoptosome of procaspase-9, cyto. c, and Apaf-1 is formed eventually activating caspase-9. These in turn activate the effector caspases (3, 6, 7). Caspase-12 is located in the endoplasmic reticulum (ER) and is activated

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by ER stress, calcium influx, and accumulation of excessive proteins in the ER[13], leading to activation of the ER death pathway, which in turn acts on the effector caspases. Caspase-3 (32 kDa) is one of the primary protease executioners of apoptosis and is activated by intra-chain proteolytic cleavage, which generates a large subunit (17 kDa) and a small subunit (12 kDa). Typically, after apoptotic stimuli, the level of cleaved caspase-3 increases while that of procaspase-3 decreases. The effector caspases act on the protein kinases, other signal transduction proteins, DNA repair proteins, chromatin modifying proteins, and nuclear matrix proteins which eventually hails the death of the cell. Activation of CAD (caspase activated DNAse) via caspase-3 leads to DNA fragmentation. In addition, the effector caspases cleave a variety of molecules including poly ADP-ribose polymerase (PARP), protein kinase, spectrin, actin, and DNA-dependent protein kinase. Rip2 is the upstream modulator of pro-caspase-1 in the apoptotic cascade that acts on Bid downstream to execute cell death[14]. The final mechanism of caspase regulation is proteasome degradation and inhibitors of apoptosis proteins (IAPs) seem to parcipate in clearing the active caspases[15].

2. Targeting Caspases in Neonatal HypoxiaIschemia Brain Injury and Hypoxic-Ischemic Encephalopathy 2.1. Neonatal Hypoxia-Ischemia Brain Injury and Hypoxic-Ischemic Encephalopathy The incidence of hypoxic-ischemic encephalopathy (H-IE) is 1-8 cases per 1000 births in the United States. Perinatal asphyxia, stroke, and intraventricular hemorrhage are general causes of neonatal brain injury, with hypoxia-ischemia as the final common pathway. In severe neonatal hypoxia-ischemia brain injury (HIBI), the mortality rate is 25-50%. Those who survive have a substantial risk of permanent disability such as mental retardation, motor impairment, behavioral and cognitive disabilities, and seizures. Currently no treatment for neonatal HIBI is available, and only general supportive measures are used to prevent further morbidity. Hence there is an unmet need to discover novel therapeutics for H-IE. Various techniques reliably identify neurons rendered apoptotic by multiple mechanisms of brain injuries such as caspase assays[16], TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining[17], Flourojade B staining[16], DNA fragmentation assay[18], and Annexin V staining[19]. In vivo the Rice-Vanucci model demonstrates the effect of hypoxia-ischemia (H-I) injury on experimental animals. P7 day pups of different rat or mouse species are used, as their neural development simulates that of human neonates of 36-37 weeks gestation. Levine‟s procedure is used, consisting of unilateral carotid artery ligation, and the pups are made hypoxic with 8-10% of O2 and equilibrated N2 in a warm bath of 37°C for a specified period[20, 21]. The pups are sacrificed later to study the effects. Primary cerebellar granular neurons[22] and mouse primary cortical neuron (PCN)[23] are used as in vitro models. HIBI and H-IE can be diagnosed by various imaging modalities. In less experienced hands cranial ultrasonography, magnetic resonance imaging (MRI) techniques and pulse

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sequences such as T1 and T2 weighted images remains the modality of choice for neonatal imaging. However, in tertiary care centers, diffusion weighted imaging (DWI) and magnegtic resonance spectroscopy (MRS) are conventionally used[24]. Early use of positron emission tomography imaging in detecting brain injury due to perinatal and intrauterine insults can provide new insights in prognosis and instituting early therapy[25]. These imaging modalities in association with EEG help prognosticate the outcome of H-I injury in the neonate.

2.2. The Activation of Caspases in Neonatal HIBI and H-IE Studies have shown that neonatal HIBI is mediated by apoptosis[26-32] and that caspases are vital to mediate this injury. We and other researchers found that initiator caspase-8 may directly process and activate effector caspase-3, or indirectly activate caspase-3 through cleaving and activating the cytosolic BID, which promotes the release of cyto. c and feedback activation of caspase-9 and -3[14, 33]. The FAS-mediated cell death receptor pathway, leading to activation of caspase-8, plays an important role in H-I neurodegeneration[34]. Neuronal loss due to H-I injury is associated with the cleavage of initiator caspase-8 and 9[33]. Caspase-10 activation occurs primarily in the ischemia/reperfusion models[35]. Caspase-2[36] and caspase-3[37, 38] are activated extensively in the immature brain after H-I injury. Cleaved effectors caspase-7 and -3 are activated in neurons in the ipsilateral hippocampus after H-I injury[39]. Caspase-1 is an apical mediator of neuronal cell death, as shown by in vitro hypoxia and in vivo ischemia models[14]. Caspase-1 mRNA was increased in neonatal rats after HIBI with a time frame consistent with brain injury[40]. Caspase-1 converts pro-interleukin-1 (IL-1) to IL-1which mediates hypoxic ischemic brain damage in experimental animals and human neonates[41], and ICE deficient mice are resistant to mild to moderate HIBI. Post-mortem studies of neonatal brain in cases of pontosubicular necrosis have shown caspase-3 activation in human perinatal HIBI[42]. Caspase-12 is involved in apoptosis induced by ER stress[43] or Bax shuttling[44] in neonatal HIBI rats.

2.3. Targeting Caspases in Treatment of Neonatal HIBI and H-IE Neonatal HIBI is an evolving process that develops (after a certain delay) mainly due to neuronal apoptosis mediated by caspases. After the primary insult there is a complete recovery, and the secondary energy failure, which causes the lesion, occurs after about a delay of 6 to 48 hours. This presents a therapeutic window during which the neurodegeneration leading to H-IE could be prevented via a host of potential targets for intervention[45]. In vivo and in vitro studies have shown that inhibition of the caspase family prevents cell death in neurodegenerative diseases, and treatments targeting caspases may potentially play a neuroprotective role in neonatal HIBI[26] and in human perinatal H-IE. Herein, we review the current literature on a variety of treatments for neonatal HIBI and H-IE via caspase inhibition (Fig. 1 and Table I).


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Figure 1 Treatments targeting individual caspases in neonatal HIBI and TBI. Various treatments act on caspases in the cell death receptor pathway, mitochondrial death pathway and ER death pathway in neonatal HIBI and TBI. Treatments are labeled red for both HIBI and TBI, and Blue for HIBI and H-IE only, and green for TBI only.

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Table I. Treatments for HIBI and H-IE by inhibition of the caspases. Targeting caspases with different treatments in neonatal HIBI reduces brain tissue loss and reduces infarct volume in various brain areas. Different species of experimental animals are tabulated. CO: cortex, HC: hippocampus, TH: thalamus, ST: striatum, CrC: corpus callosum, CC: cerebral cortex, SN: subthalamic nuclei, IC: internal capsule, EC: external capsule, AN: amygdaloid nucleaus.


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Treatments Targeting te Mitochondrial Cell Death Pathway in HIBI and H-IE Since caspases play a major role in neuronal apoptosis, caspase inhibitors can effectively protect neurons from inappropriate apoptosis and help in prolonging cell survival. Indeed, caspase inhibition has proven efficacious in the treatment of neonatal HIBI and H-IE involving apoptosis. M826 is a selective caspase-3 inhibitor that can block almost all the activities of caspase-3 by intracerebroventricular injection[23]. A third generation dipeptidyl, broad spectrum pan-caspase inhibitor quinoline-Val-Asp(Ome)-CH2-O-phenoxy (Q-VDOPh) has a carboxy terminal phenoxy group conjugated to the amino acids valine and aspartate. It has proved to be neuroprotective on a gender specific basis (females are more protected than males), given either before or after ischemia[46]. It inhibits all the three pathways of caspase mediation (-3/9, 8/10 and 12[47, 48]) and reduces brain injury due to HI. BAF (boc-aspartyl(OMe)-fluoromethylketone), a pan-caspase inhibitor either alone or with hypothermia reduces apoptosis and protects against neuronal damage in HI models[49]. However, of a variety of tested strategies, hypothermia, is the only intervention that has translated to some clinical benefit in newborn babies[50]. Hypothermic medicine with either selective head cooling or systemic body cooling to 30-34°C rectal temperature has proven beneficial in neonatal HIBI in some way inhibiting the expression of caspases. The effect of hypothermia depends on timing of initiation, depth and duration of cooling after resuscitation. More pronounced protection is attained when cooling is started during ischemia. Intraischemic hypothermia[51] or post-ischemic hypothermia has proven to be of therapeutic value. The infarct size, extent of neuronal loss, and severity of brain injury are significantly reduced in the hypothermic group compared to the control group. Mild hypothermia (34°C) decreases expression of caspase-3 mRNA and lowers caspase-3 enzyme activity[52]. Prolonged hypothermia for about 72 hours post-H-I decreases caspase-3 in the parietal cortex and hippocampus[53]. Systemic hypothermia at 30°C beginning immediately post-H-I and lasting for 10 hours decreases caspase-2 and -3 in the cortex and dentate gyrus[36]. These results indicate that hypothermia may act at least partially through inhibition of the intrinsic pathway of caspase activation in the neonatal brain, thereby preventing apoptotic cell death. The library of the Neurodegeneration Drug Screening Consortium of 1,040 compounds assembled by the National Institute of Neurological Disorders and Stroke includes minocycline, doxycycline, nicotinamide, and melatonin. Minocycline, a semisynthetic derivative of tetracycline, is a FDA approved antibiotic used for decades in the treatment of various infectious diseases. We and other groups have reported that minocycline has proven effective in neurodegenerative diseases[1, 54-56]. If administered either immediately before or after H-I insult, it provides near complete neuroprotection in the neonatal brain by decreasing caspase-3 levels and thus preventing from neuronal injury due to apoptosis[57]. Doxycycline (another FDA approved tetracycline), given either before or immediately after H-I insult to experimental neonates, not only reduces caspase-3, but also reduces microgliosis, and hence prevents cell death. It reduces caspase-3 activation in a time-dependent manner. This prosurvival effect is seen in most vulnerable regions of the brain such as CA1, dentate gyrus, cortex and striatum[58]. Nicotinamide (vitamin B3), a precursor of NAD and a form of niacin, plays a vital role in cell survival and cell growth. Nicotinamide confers effective protection against HIBI in adult rats and has also proven effective in the neonatal rats[59]. Its proven pharmacological actions include reducing lipid peroxidation, reducing PARP[60], inhibiting apoptosis[60] and preventing ATP depletion[61]. It improves motor coordination and reduces the brain injury. Nicotinamide can

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also control the mitochondrial permeabilization and prevent cyto. c release, which reduces caspase-3 and -9 activity. Melatonin is a free radical scavenger and antioxidant. We have demonstrated that melatonin targets caspases in a number of neurological disorders including ischemic brain injury[62, 63]. It has been shown to play a role in the sleep-wake cycle and circadian rhythm[64]. It inhibits cyto. c release and loss of mitochondrial membrane potential[63]. When administered to pregnant spiny mice, melatonin reduces CNS inflammation and apoptosis in pups in a H-IE model[65]. Melatonin administered before and after H-I provides protection against brain injury as well as long-term improvement in behavioral asymmetry and learning deficits induced by H-I[66]. Because free radicals play an important role in mediation of HIBI, drugs that act as free radical scavengers and antioxidants can be neuroprotective. Edaravone, a free radical scavenger and antioxidant that also inhibits lipid peroxidation, is shown to improve motor functions in clinical trials of acute cerebral infarction. It is reported that edaravone also confers neuroprotection in H-I brain damage in newborn rats[67] through impeding apoptotic, necrotic, and mitochondrial mechanisms. This effect was dose- and time-dependent[68]. Nacetylcysteine (NAC) is a precursor of glutathione, a potent antioxidant, and a free radical scavenger. NAC is widely used as a mucolytic agent and as an antidote for paracetamol poisoning[69]. It is a precursor of glutathione, a potent antioxidant, and a free radical scavenger. Antenatal infection and H-I have a synergistic effect in neurodegeneration. NAC protects against lipopolysacharide-sensitised H-I and inhibits caspase-3 and -1 activation[70]. Caffeic acid phenethyl ester is another antioxidant, an inflammatory and antiviral agent with immunomodulatory functions. It inhibits neurotoxicity due to induced nitric oxide (NO) synthase and caspase-1 expression in vivo and in vitro, and it also prevents Ca2+-induced cytochrome c release and blocks caspase-3 activation in isolated brain mitochondria[71, 72]. NO plays a role in neuronal apoptosis by generating reactive nitrogen species, which causes nitration of lipids, DNA, and proteins, leading to neuronal cell damage[73]. Polyphenols have antioxidant properties[74]. Pomegranate polyphenols and resveratrol, when given to the dams, reduce activation of caspase-3 in the hippocampus of neonatal rats after H-I[75]. Another polyphenol amentoflavone reduces brain tissue loss due to H-I injury. It blocks caspase-3 activation in rats[76]. Pyrrolidine dithiocarbamate (PDTC) is an antioxidant, that confers neuroprotection and reduces cleaved caspase-3 expression in the PDTC-treated rats[77]. Though hydrogen gas neutralizes free radicals and reduces oxidative stress, its clinical use as a gas poses issues regarding safety and convenience. When saturated hydrogen saline is administrated intraperitoneally immediately and 8 hours post HI, it reduces the infarct ratio and improves long-term neurological and neurobehavioral function. It reduces the levels of malondialdehyde (a marker of oxidative damage), Iba-1, and caspase-3[78]. Hyperbaric oxygen preconditioning provides protection to various organs including the brain in neonatal H-IE[79]. It reduces infarct ratio and increases survival. Suppression of antiapoptotic pathways of caspase-3 and -9 activities play a role in this neuroprotection[79]. PAF (platelet-activating factor) is a lipid-mediator, released from various cells such as platelets, monocytes, macrophages, endothelial cells, and neutrophils[80]. PAF concentrations increase during ischemia and oxidative stress and have a proinflammatory effect[81], mediating neurotoxicity and neuronal degeneration. Thus PAF antagonist can be neuroprotective in HIBI. Indeed, the PAF antagonist ABT-491 reduces caspase-3 activity and TUNEL staining in experimental animals[82]. The use of NMDA receptor antagonist MK801 reduces caspase-3 activation as shown by western blot. Brain injury, caspase-3 activation,


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and DNA fragmentation are all reduced in the cerebral cortex showing that NMDA plays a role in apoptotic mechanisms in HIBI[83]. The role of neurotrophic factors in apoptosis during development is important. BDNF is a neurotrophic factor that blocks caspase-3 activation in neonatal HIBI. Intracerebroventricular injection of BDNF prevented activation of caspase-3 in vivo[84] and protected the brain from tissue loss. Insulin-like growth factor-1 (IGF-1) is another neurotrophic factor that plays an important role in neuronal cell survival. It reduces phosphorylation of Akt and GSK3after H-I injury, while caspase-3 and -9 activity is significantly reduced but caspase-8 or -1 activity is not affected[85]. Additionally, FGF 1 is neurotrophic factor. In transgenic rat expressing FGF-1, apoptosis is markedly reduced via decreased caspase-9, caspase-3 and its substrate PARP in rat pups and in PCNs[86]. It also blocks the H-I induced reduction of anti-apoptotic and survival promoting protein XIAP expression, thus providing neuroprotection[86]. Trapidil is an antiplatelet agent that acts as a phosphodiesterase inhibitor and a competitive inhibitor of platelet derieved growth factor (PDGF)[87]. Besides its biological effects in vasodilation and stimulating prostacyclin, trapidil plays a role in inhibiting neuronal apoptosis in a neonatal rat model of HIBI by noticeably reducing caspase-3 levels[87]. 2-, iminobiotin is an inhibitor of neuronal and inducible nitric oxide synthase. It is neuroprotecgtive by improving the cerebral energy state, decreasing vasogenic edema, and inhibiting neuronal apoptosis in experimental animals. It can be intravenously administered after H-I and is neuroprotective 24 hours after the insult[88]. Mixed lineage kinases are expressed in the neuronal cells and CEP-1347 is their semisynthetic inhibitor. Mitogen activated protein kinase activates mixed lineage kinases, leading to phosphorylation of c-jun, which in turn causes cyto. c release and activation of the caspases. CEP-1347 reduces neonatal brain damage and apoptosis, as shown by reduction in caspase-3 activity[17]. Hormone has been shown to be neuroprotective. Hexarelin is a peptide with a potent ability to stimulate growth hormone secretion. Hexarelin reduces caspase-3 activity in cerebral cortex of ipsilateral hemisphere by intracerebroventricular injection, and increases Akt and pGSK3 phosphorylation[89]. Erythropoietin (EPO) belongs to the cytokine superfamily and has traditionally been viewed as a hematopoiesis-regulating hormone. The receptors of EPO are present in central nervous system. It offers protection and reduces caspase-3 activation in neonatal mice after H-I injury[90]. Deferoxamine displays antioxidative actions, and EPO confers anti-apoptotic and anti-inflammatory effects, each has been shown to provide neuroprotection in neonatal rodent models of brain injury. Furthermore, intraperitoneal administration of deferoxamine and/or EPO, in rats reduces the number of cleaved caspase-3 positive cells[91]. Though 17 estradiol, a circulating hormone, attenuates neonatal HIBI, however caspase-dependent pathways play a little role[92]. Both extrinsic and intrinsic apoptotic pathways mediate the neuroprotective effects of Hsp70 overexpression in neonatal H-I[33]. The direct binding of Hsp70 to Apaf-1 may be one of the mechanism that reduces caspase-9 cleavage, thus reducing apoptosis. Apaf-1 interacting protein (AIP), containing N-terminal caspase recruiting domain, binds to Apaf-1 thereby inhibiting the formation of apoptosome and caspase-3 and -9 activation following HI. Transgenic mice overexpressing AIP (Tg-AIP mice) prevent H-I brain injury while intraperitoneal administration of TAT-AIP fusion protein confers neuroprotection and attenuates activation of caspase-3 and -9[93]. Bcl-xl, located mainly in the mitochondrial

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membrane, is an important anti-apoptotic molecule. A fusion protein with TAT (forming TAT-Bcl-xl) is protective against cell damage in the neonatal rat brain. This has been shown with the reduction of caspase-3 and -9 assays after intraperitoneal injection of the fusion protein in experimental animals[94]. On the other hand, mice deficient in interleukin-1 converting enzyme (ICE) are resistant to neonatal HIBI, and caspase-1 activity significantly contributes to the progression of neonatal HIBI[95]. Neonatal mice deficient in Atg7 gene show nearly complete protection from both H-I-induced caspase-3 activation and neuron death[39]. Endogenous BAX plays a role in regulating cell death in the CNS following neonatal H-I, Bax -/- mice had significantly decreased caspase-3 activation as compared to bax expressing mice following H-I[96]. Additionally, Bax-inhibiting peptide (BIP), a novel membrane-permeable peptide, which can bind Bax in the cytosol and inhibit its translocation to the mitochondria, significantly suppresses both the number of TUNEL-positive cells and the increase in caspases-3 and -9 activities induced by glutamate in cerebellar granule neurons, a cellular model of HIBI[22]. Studies have shown simvastatin to be neuroprotective in neonatal HIBI[97, 98] via inhibition of caspase-3, which is independent of calpain activation. Caspase-3 proteolytically cleaves many substrates including PARP, which is the biochemical hall mark of apoptosis. PARP cleavage is reduced in simvastatin-treated animals. Early necrotic death due to calpain activation was not inhibited, however at 48 hours cell death due to caspase-3 activity is inhibited. In addition, caspase-1 activity, IL-1 and ICAM-1 mRNA are also reduced due to simvastatin proving that its neuroprotective activity is due to inhibition of apoptotic cell death[99]. Treatments Targeting Cell Death Receptor Pathway in HIBI FLIP (FLICE-like inhibitory protein) acts as an endogenous cytoplasmic decoy for caspase-8 and provides neuroprotection[100]. Antioxidant status alters the expression of FLIP and caspase-8 simultaneously in opposite directions[101]. Animals overexpressing superoxide dismutase had excessive activation of pro-caspase-8, and activated caspase-8 leaded to apoptosis. On the other hand, animals overexpressing glutathione peroxidase have increased expression of FLIP which provides neuroprotection[101]. Treatments Targeting the ER Pathway in HIBI Administration of molecular hydrogen after HI insult reduces caspase-12 levels in a timedependent manner. This inhibition of caspase-12, leading to inhibition of downstream apoptotic mechanisms (such as caspase-3 activation), in turn provides neuroprotection by reducing the infarct volume in cortex and hippocampus[102]. Thus, these experimental studies of H-I models using various treatments show that targeting caspases in different ways can protect the neurons from neuronal degeneration and may prevent the mortality and morbidity associated with H-IE.


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3. Targeting Caspases in Traumatic Brain Injury 3.1. Traumatic Brain Injury The incidence of TBI is approximately one-sixth the total numbers of injuries. About seven million patients sustain a TBI annually worldwide due to traffic accidents, falls, assaults, or sports injuries[103]. In the United States, the estimated incidence of TBI is 100 per 100,000 persons, with 52,000 annual deaths. According to the World Health Organization, TBI will surpass many diseases as the major cause of death and functional disability by the year 2020[104]. Those sustain and survive a TBI are left with significant cognitive, behavioral, and communicative disabilities. Neuronal injury can be detected as soon as 10 minutes after TBI in cerebral cortex, thalamus, hippocampus, and other regions can be observed[105]. On the other hand, progressive gray and white matter atrophy and neuronal death can progress for one year following TBI[106, 107]. The pathophysiological mechanism of CNS injury in the acute phase and chronic nerve cell damage of TBI have been investigated by us and other groups[108-111]. It is generally believed, neuronal cell death plays an important pathophysiologic role in the cascade of CNS cell degeneration and associated neurologic deficits[106, 112]. Cell death in the CNS following TBI can take the forms of apoptosis and necrosis[113], and both caspase-dependent and independent apoptotic pathways have been identified in CNS trauma. Herein, we focus on the current interventional therapies targeting caspase-mediated apoptosis. A number of experimental animal models, organotypic cultures and cultured cell models of TBI have been developed to replicate the characteristics of human head injury, such as contusion, concussion, and/or diffuse axonal injury. In caspase-mediated TBI studies, animal models that have been used include controlled cortical injury (CCI)[114], lateral cortical contusion[115], and fluid-percussion (FP) brain injury[105, 116, 117]. Besides the ex vivo organotypic hippocampal cultures[118], cellular models include primary mouse[119] or rat[120] cultures of cerebral cortical neurons, neuronal-glial cultures[121], P2Y2R-1321N1 astrocytic cells[122], nerve growth factor differentiated PC12 cells[123], and septohippocampal cell cultures[124] are used. Several methods similar to those utilized in neonatal HIBI, have been used to detect apoptosis in TBI including caspase assay[117], TUNEL staining[125, 126], FJB staining[127], DNA fragmentation analysis[128], ApopTag assay and Wright staining[120]. Advanced techniques such as computed tomography scan[129] and MRI[130-132] have been used to identify damage induced by TBI. Furthermore, the more sensitive magnetic resonance DWI, diffusion tensor imaging (DTI), white matter fiber tractography (DTT), functional magnetic resonance imaging (fMRI), and MRS have been also used to investigate TBI[131-136].

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3.2. The Activation of Caspases in TBI The extrinsic pathway of apoptosis has been reported to be involved in TBI. Caspase-8 is expressed in cortical areas[117, 137] and thalamus[117], while it is shown to be activated in neurons, astrocytes, and oligodendrocytes[138]. The activation of caspase-8 contributes to caspase-3-mediated apoptotic cell death in experimental animals after TBI[138]. High levels of caspase-8 gene induction are observed after cortical impact in rats[139]. In addition, TNFR1 and TRAF1 are recruited to lipid rafts in rats after TBI. Subsequently, the signaling complex contains activated caspase-8, thus initiating cell apoptosis[140]. In a nerve growth factor-differentiated PC12 cell model of TBI, caspase-8 and -3 are activated upon stearic acid and palmitic acid apoptotic induction[123]. Moreover, using a mouse CCI model in vivo and primary cultures of cerebral cortical neurons in vitro, caspase-8 and -3 are activated while Fas is reported to retain its function as a death receptor after TBI. Furthermore, the interactions between Fas receptor and FADD, pro-caspase-8 and pro-caspase-10, and Fas-RIP-RAIDDcaspase-2 are apparent after CCI[119]. Our studies suggest that caspase-1 plays a key role in H-I-associated neuronal death[14]. Caspase-1 generates the pro-apoptotic tBid fragment, which plays a role in the release of mitochondrial apoptogenic factors cyto. c/Smac/AIF. Released cyto. c induces apoptosome assembly, resulting in caspase-9 and -3 activation[14]. Regarding models of TBI, caspase-1 is not only activated in the rat model of TBI[114], but also plays an important functional role in mediating neuronal cell death and dysfunction after TBI in mouse brain[141], while neuroprotection following TBI is achieved in a transgenic mouse expressing a dominant negative inhibitor of caspase-1[142]. In addition, caspase-1 mRNA content is increased in rats after FP-induced TBI[143]. Finally, in a rat FP injury model, TBI induces the activation of processing of caspase-1 and increases expression of caspase-11[144]. The intrinsic apoptotic pathway has been proposed as one mechanism of cell death after TBI. Indeed, the release of cyto. c from mitochondria and the activation of caspase-1 and then -3 in the injured cortex of a CCI rat model of TBI confirms the involvement of the mitochondrial death pathway[114]. Furthermore, activated caspase-2 has been shown to trigger mitochondrial apoptotic events by inducing conformational changes in Bax/Bak with subsequent release of mitochondrial apoptotic factors including cyto. c, AIF, and endonuclease G[145]. In addition, caspase-2 mRNA expression is increased in the cerebral cortex of rats after TBI[146]. Initiator caspase-9 is activated in rats after TBI[137, 139, 147]. After acute injury to mature brain, injury-induced cyto. c-specific cleavage of caspase-9 is reported to be followed by activation of caspase-3 correlated with marked Apaf-1 increases[148]. Moreover, initiator caspase-9 is predominantly expressed in cortical areas and in the thalamus while caspase-3 is expressed throughout the traumatized cerebral cortex and hippocampus[117]. Caspase-3 is induced by at least two major initiator pathways: a caspase8-mediated extrinsic pathway and a caspase-9-mediated intrinsic pathway. Activation of caspase-3 after TBI has been widely recognized[117, 139, 143, 147, 149-151]. Caspase-3specific spectrin breakdown products (SBDPs) are increased in cerebrospinal fluid after TBI in rats[152, 153]. Caspase-7, another apoptosis executioner, is generally believed to be present in only minute amounts in the brain (with highly restricted activity) or is completely absent. Larner, S. F. et al. demonstrate that caspase-7 is up-regulated and activated after TBI in rats[154].


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Furthermore, Larner, S. F. et al. also report increased expression and processing of caspase-12 following TBI in rats[155]. Therefore, the caspase-12-mediated ER death pathway may play a role in rat TBI pathology independent of the cell death receptor or mitochondria apoptotic pathway. In summary, the caspases are activated in the extrinsic and intrinsic death pathways and the ER death pathway following TBI in animal models in vivo and cultured cell models in vitro. Growing evidence demonstrates that caspases are activated in human TBI. After severe TBI, caspase-9 and caspase-3 are activated and cyto. c is released in the cerebrospinal fluid of patients[156-158]. Activated caspase-3 is also reported in specimens of human brain tissue with TBI[125, 159, 160], while caspase-3 is being investigated as a specific biomarker of proteolytic damage following TBI[161]. In addition, caspase-7 and its cleavage product, as well as the cleavage of caspase-1, are increased in human brain tissue[162, 163], caspase-1 is increased in the ventricular cerebrospinal fluid of TBI patients[164]. Through the analysis of brain tissue samples from adult patients with severe intracranial hypertension after TBI or post-mortem TBI brain samples and comparison with post-mortem control brain tissue samples, caspase-8 mRNA and protein are found to be increased in TBI adult patients while proteolysis of caspase-8 to 20-kDa fragments is seen only in severe TBI adult patients, and caspase-8 protein was predominately expressed in neurons[165]. In addition, Fas-procaspase-8 interaction is significantly increased in contused brain samples removed from severe TBI patients[119]. Taken together, experimental evidence demonstrates that the caspases in both intrinsic and extrinsic death pathways are activated in human TBI.

3.3. Targeting Caspases in Treatment of TBI The damage to the traumatized brain includes two phases: the initial irreversible primary phase being the injury itself, and the secondary phase, which begins at the time of injury and continues for days, weeks, or longer. Secondary brain injury represents a window of opportunity in which treatments with neuroprotective properties could be administered[166]. Determination of the specific expression profiles of caspases affected by injury is critical to develop targeted therapeutics for TBI. We review the current literature on treatments targeting caspases in TBI, and provide evidence supporting the therapeutic use of caspase inhibitors and other treatments in the setting of these conditions (Fig. 1 and Table II). Pan-caspase inhibitor BAF treatment reduces acute cell death in rats after TBI by inhibiting mitochondrial release of cyto. c, initiator caspase-2, and effector caspase-3[167]. In addition, post-CCI with intracerebroventricular injection of BAF in mice significantly reduces caspase-3 activation, suppresses caspase-cleaved APP and increases in Abeta, and improved histological outcome[168]. Administration of caspase-3 inhibitor z-DEVD-fmk (Nbenzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone), a specific tetrapeptide inhibitor of caspase-3, markedly reduces post-traumatic apoptosis and significantly improves neurological recovery following TBI[143]. Intracerebral administration of z-DEVD-fmk after TBI reduces caspase-3-like activity and DNA fragmentation in injured rat brain[169]. Given that glutamate toxicity in TBI causes cortical neuron death and dysfunction, in a cellular model of TBI, z-DEVD-fmk provides strong neuroprotection in PCNs following glutamate exposure[120]. In addition, z-DEVD-fmk, like pan-caspase inhibitor BAF, significantly

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attenuates apoptotic cell death in neuronal-glial cultures in vitro after mechanical injury by combined 3-nitropropionic acid and glucose deprivation treatment, whereas the caspase-1 selective inhibitor z-YVAD-fmk has no effect[121]. Inhibition of caspase-1 activation reduces trauma-mediated brain tissue injury, a conclusion is supported by several findings: 1) reduction of tissue injury and free radical production following TBI in the brain tissue of a transgenic mouse expressing a dominant negative inhibitor of caspase-1[142]; 2) pharmacological inhibition of caspase-1 by intracerebroventricular administration of the selective inhibitor of caspase-1, acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone (cmk) or the non-selective caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fmk confers neuroprotection[142]; 3) the pan-caspase inhibitor z-VAD-fmk (carbobenzoxy-valyl-alanylaspartyl-[O-methyl]-fmk) reduces cold injury-induced brain trauma in mice by preventing caspase-1 activation and DNA fragmentation[170]; 4) antibody therapy using anti-ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) neutralizing antibodies administered immediately after FP injury to injured rats reduces caspase-1 activation, IL-1processing, and cleavage of X-linked inhibitor of apoptosis protein, thus significantly decreasing contusion volume after TBI[144]. Taken together, the available evidence indicates that pharmacological inhibition of caspases by caspase inhibitors prevents cell death and improves functional outcome after TBI, offering an experimental rationale for the evaluation of effective pharmacological agents in human trauma patients. Hypothermia reduces the evolving secondary deterioration after TBI. Clinical trials have been performed in patients and different durations and temperatures have been studied in experimental animals[171]. Post-traumatic hypothermia significantly attenuates caspase-3 activity and cell death within the hippocampus following FP injury[172]. In another report, hypothermia promotes a rapid acute activation of caspase-3 in rats after TBI but suppresses the activation of caspase-3 at later time points[173]. In contrast, heat acclimation attenuates the activation of caspase-3 and provides sustained improvement in functional recovery and reduction in lesion volume in mice after TBI[174]. Intraperitoneal administration of minocycline, a derivative of the antibiotic tetracycline, before or after TBI in mice, improves neurological function and reduces tissue damage. Given that the activity of caspase-1 is increased in mice that underwent TBI, and this increase is significantly diminished in minocycline-treated mice, the neuroprotection of minocycline in TBI is suggested to take place through a caspase-1-dependent mechanism[141]. Minocycline can also block nitric oxide (NO)-induced neurotoxicity[175, 176] and inducible NO synthase up-regulation[176]. Lu et al. found that NO induces macrophage apoptosis, as shown by positive TUNEL staining and caspase-3 immunostaining after TBI[177]. Furthermore, the same group reported the neuroprotective effects of aminoguanidine, a selective inducible NO synthase inhibitor, after lateral FP brain injury in rats. In rats receiving prophylactic or postinjury treatment of aminoguanidine after TBI, the number of caspase-3 immunopositive neurons is reduced in the cerebrum[178]. Antioxidants and free radical scavengers have been reported to be neuroprotective in TBI. Selenium is an antioxidant with protective function in ROS-mediated apoptotic neural precursor cell death in vitro and in vivo in an experimental mouse model of TBI through attenuation of secondary pathological events. This action most likely results from its comprehensive effects in blocking caspase-3 and -9 activation, resulting in the maintenance of functional neurons and in inhibition of astrogliosis[179]. Cerebral contusions are one of the


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most frequent traumatic lesions and the most common indication for secondary surgical decompression. Hyperbaric oxygen therapy has been used in treatment of these lesions[180]. Table II. Treatments for TBI by Inhibition of the Caspases. The neuroprotective effects of treatments for TBI with targeting indicated caspases in different species and cell lines are tabulated.

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It decreases not only the number of TUNEL-positive cells[180] but also apoptosis in TBI via inhibition of the activation of caspase-9[181]. In addition, peroxynitrite can inhibit caspase-3mediated apoptosis in neurons following TBI in vitro and in vivo mostly because of its effect on cysteinyl oxidation of caspase-3[112]. The free radical scavenger S-PBN and MEK inhibitor U0126, both confer neuroprotection in rats after TBI. They attenuate the early activation of ERK and reduce activation of caspase-3 and subsequent DNA fragmentation[182]. The NMDA receptor plays an important role in the pathophysiological process of TBI[183, 184]. The protective effect of NMDA receptor antagonist MK-801 against TBI along with the inhibition of caspase-3 has been reported[183]. Macrophage colony stimulating factor (M-CSF) and its receptor are upregulated in the brain in an experimental model of TBI. NMDA induces caspase-3 activation and neuronal apoptosis in organotypic hippocampal cultures, whereas treatment with M-CSF (like caspase inhibitor z-VAD-fmk) protects hippocampal neurons from NMDA-induced caspase-3 activation and neuronal apoptosis[118]. The cyclin-dependent kinase inhibitor flavopiridol significantly inhibits the activation of caspase-3 in rats following TBI in vivo and in rat PCNs induced by etoposide in vitro[185]. Cyclooxygenase-2-specific inhibitor DFU (5,5-dimethyl-3(3-fluorophenyl)-4(4methylsulfonyl)phenyl-2(5)H)-furanone) enhances functional recovery and decreases the total number of activated caspase-3-immunoreactive cells in injured cortex and hippocampus in a lateral cortical contusion rat model[115]. Rosiglitazone is a potent agonist of peroxisome proliferator-activated receptor-gamma that confers neuroprotection in experiments models of focal ischemia, spinal cord injury and Parkinson‟s disease[186]. Furthermore, rosiglitazone-treated TBI mice also show significantly fewer TUNEL-positive apoptotic neurons and curtailed induction of caspase-3 and Bax compared to vehicle-treated controls[187]. Simvastatin not only provides neuroprotection in H-IE but also confers beneficial effects in TBI. These effects include activation of Akt, Forkhead transcription factor 1, and NFB signaling pathways, which suppress the activation of caspase-3 and apoptotic cell death, thereby leading to neuron function recovery of rats after TBI[188]. In addition, a ketogenic diet reduces cyto. c release and caspase-3 activation following TBI in juvenile rats[189]. A growing body of literature supports the benefit of hormone therapies for neuroprotection and improved cognitive recovery after TBI. The neuroprotective effect of estrogens, with significant reduction of active caspase-3, has been demonstrated in SpragueDawley male rats after TBI[190]. Furthermore, premarin, an estrogen sulfate, protects against cortical and hippocampal apoptosis after FP injury through mechanisms stimulating estrogen receptor-alpha and preventing caspase-3 activation in male rats as well[191]. Moreover, the neurosteroid progesterone and its metabolite allopregnanolone reduce the expression of the pro-caspase-3 and the activity of caspase-3 and confer anti-astrogliotic effects after TBI in adult male rats[192, 193]. In addition, the administration of progesterone improves morphologic and functional outcome and the marked attenuation of caspase-3 immunoreactivity after diffuse TBI in rats[194]. Studies also suggest that allopregnanolone appears to be more potent than progesterone in facilitating CNS repair after TBI[193]. EPO belongs to the cytokine superfamily and has traditionally been viewed as a hematopoiesisregulating hormone. It improves functional recovery, and reduces caspase-3 activation and neuronal apoptosis, as well as inflammation in a rodent model of TBI[195].


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Genetic deletion or overexpression affects the expression or activation of caspase(s) and functional outcome after TBI. Bid is a proapoptotic member of the Bcl-2 family that mediates cell death. Compared with normal animals, mice genetically deficient in Bid (Bid-/-) in a CCI model of TBI show the decreased early post-traumatic brain cell death and tissue damage, as well as decreased numbers of cells expressing cleaved caspase-3[196]. However, overexpression of Bcl-2, a major member of the Bcl-2 family, is only partially neuroprotective; there is no significant difference in the cleavage of caspase-3 or -9 in hippocampal samples from Bcl-2 transgenic or wild-type mice after TBI[197]. Transgenic expression of interleukin-6 (IL-6) in the CNS under the control of the glial fibrillary acidic protein (GFAP) gene promoter (GFAP-IL6 mice) affords neuroprotection against acute TBI associated with reduced oxidative stress, neurodegeneration, and apoptosis including reduced caspase-3 TUNEL staining[198]. In addition, neuronal survival has been found to correlate with increased expression of uncoupling protein 2 (UCP-2). Transgenic mice overexpressing UCP-2 show diminished brain damage after TBI while UCP-2 reduces cell death and inhibits the oxygen and glucose deprivation-induced caspase-3 activation in rat PCNs[199]. 1321N1 astrocytic cells expressing recombinant P2Y2 nucleotide receptors (P2Y2R-1321N1) are a well-established in vitro model of TBI. Activated caspase-9, but not caspase-8, is inhibited in P2Y2R-1321N1 astrocytic cells, suggesting the direct involvement of this nucleotide receptor in modulating cleaved caspase-9 after traumatic injury towards cell survival; while PD1693, a MKK3/6 inhibitor, inhibits trauma-induced death and abolishes the expression of cleaved caspase-9 in P2Y2R-1321N1 astrocytic cells[122]. Additionally, sAPPalpha (soluble amyloid precursor protein alpha), is a product of the non-amyloidogenic cleavage of an amyloid precursor protein. sAPPalpha reduces neuronal injury and improves functional outcome as well as significantly reduces the number of caspase-3 apoptotic cells in the hippocampus and cortex of rat brains following diffuse TBI[200]. We and other researchers have worked on caspase related stem cell therapy for neurological disorders[201-203], and there is increasing interest in the use of stem cells as a therapeutic tool in TBI. Direct intrathecal implantation of mesenchymal stromal cells in rat brain with TBI leads to enhanced neuroprotection via an NFB-mediated increase in IL-6 production and inhibition of caspase-3[201]. Moreover, treatment of TBI with human mesenchymal stem cells during the acute phase of rat brain injury can enhance neurological functional outcome, with increased levels of neurotrophic factors and decreased caspase3[203]. Interestingly, the pan-caspase inhibitor BAF reduces acute cell death in rats after TBI by inhibiting mitochondrial release of cyto. c and initiator caspases-2 and effector caspase-3, but not through caspase-8[167]. Moreover, even though caspase-8 is activated in a palmitic- or stearic acid-induced nerve growth factor differentiated PC12 cell model of TBI, blockade of caspase activity with the pan-caspase inhibitor z-VAD-fmk does not prevent cell death[123]. The evidence may imply that targeting caspase-8 in the cell death receptor pathway of TBI is not crucial. To date, there is no treatment for TBI that targets caspase-10 and -12.

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4. Perspective Because they are significant clinical problems, effective treatment strategies are urgently needed to cure H-IE and TBI based on their pathophysiological process. The synergistic mitochondrial death pathway, cell death receptor pathway, and ER death pathway could be coactivated to mediate neonatal HIBI and TBI. In future years, combination therapies targeting multiple caspases may become a viable strategy to enhance the beneficial effects of each. Such strategies will be employed to explore multimodal and neuroprotective therapies for neonatal HIBI and TBI using currently available compounds and treatment combinations (e.g. selective caspase-3 inhibitor as a “magic bullet” in combination with hypothermia). In addition, the development of more specific caspase inhibitor(s) should elucidate the action mechanism of caspases in the pathophysiology of neonatal HIBI and TBI, thus providing a new perspective in our understanding of the regulation of neuronal apoptotic cell death in the two neurological disorders. Mesenchymal stem cell therapy has provided beneficial effects for TBI[201, 203]. Furthermore, trials of stem cell therapies are ongoing, offering great promise as new treatment modalities for neonatal HIBI and H-IE[204-207]. Hopefully, stem cell therapy focused on caspase-mediated apoptosis will culminate in treatment stategies to reduce the risk of death or disability in infants with H-I encephalopathy and offer hope to those suffering from TBI.

Acknowledgments This work is supported by grants from the National Institutes of Health/National Institute of Neurological Disorders and Stroke (to X.W.) and the Muscular Dystrophy Association (to X.W.).

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Chapter 7

Alterations in N-Methyl-D-Aspartate (NMDA) Receptor Function and Potential Involvement in AnestheticInduced Neurodegeneration Cheng Wang*, Xuan Zhang, Fang Liu, Merle G. Paule and William Slikker Jr.

National Center for Toxicological Research, U.S. Food & Drug Administration, Jefferson, Arkansas

Abstract Advances in pediatric and obstetric surgery have resulted in an increase in the duration and complexity of anesthetic procedures. It is known that the most frequently used general anesthetics have either NMDA receptor blocking or γ-aminobutyric acid (GABA) receptor activating properties. It is also known that anesthetic agents can cause widespread and dose-dependent apoptotic neurodegeneration in the developing brain. Exposure of developing mammals to NMDA-type glutamate receptor antagonists affects the endogenous NMDA receptor system and enhances neuronal cell death. The NMDA receptor regulates a calcium channel and calcium influx that overwhelms the mitochondrial buffering capacity can result in increased production of reactive oxygen species (ROS) and cell death. Meanwhile, stimulation of immature GABA receptors is thought to be excitatory early in development but inhibitory in mature neurons. Stimulation of immature neurons by GABA agonists is thus thought to increase overall nervous system excitability and may contribute to NMDA receptor-associated increased excitability during early development. This increased excitability may contribute to abnormal neuronal cell death during development. *

Corresponding author: The Division of Neurotoxicology, HFT-132; National Center for Toxicological Research/FDA; 3900 NCTR Road.; Jefferson, AR 72079-0502 USA., Phone: (870) 543-7259, Fax: (870) 5437745, Email: [email protected]

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Cheng Wang, Xuan Zhang, Fang Liu et al. The type of excitotoxic insults that lead to neuronal apoptosis or necrosis are not adequately understood but surely depend upon animal species, the concentration of stressors, durations of exposures, the receptor subtypes activated and the stage of development or maturity of a particular cell type at the time of exposure. It has been proposed that prolonged blockade of the NMDA receptor in the developing brain by NMDA receptor antagonists such as the dissociative anesthetics ketamine or phencyclidine (PCP) causes a compensatory up-regulation of NMDA receptors. Neurons bearing these up-regulated receptors are subsequently more vulnerable to the excitotoxic effects of endogenous glutamate, because this up-regulation of NMDA receptors allows for the influx of toxic levels of intracellular Ca2+ under normal physiological conditions. Although many more studies will be necessary in order to develop adequate quantitative models to explain the relationships between altered NMDA receptor function and anesthetic-induced neurodegeneration, a general hypothesis has been constructed and tested in an interactive manner using carefully selected agents as defined by their pharmacological and physiological properties. The integrative and iterative evaluation of these kinds of models will lead to a better understanding of the potential neurotoxicity of NMDA antagonists and GABA agonists in the developing human.

Introduction Advances in pediatric and obstetric surgery have resulted in an increase in the duration and complexity of anesthetic procedures. It is known that the most frequently used general anesthetics have either N-methyl-D-aspartate receptors (NMDA) receptor blocking or γaminobutyric acid (GABA) receptor activating properties. It is also known that anesthetic agents can cause widespread and dose-dependent apoptotic neurodegeneration in the developing brain. Since the first report [1] that NMDA-receptor antagonist administration caused neurotoxicity in rats during early stage of central nerve system (CNS) development, more attention has been paid to the pediatric population exposed to anesthetics. There is evidence indicating that anesthetic drugs caused widespread and dose-dependent apoptosis in the developing rat brain [2-4], and the vulnerability of brains to neuronal effects of pediatric anesthetics is restricted to the period of rapid synaptogenesis, also known as the brain growth spurt [1]. The brain growth spurt occurs at different times relative to birth in different species. In rats and mice it happens in postnatal period. In human the brain growth spurt time extends from the sixth month of gestation to several years after birth. Thus, the brain growth spurt period lasts for several years, from pre to postnatal human development, during which immature CNS neurons are susceptible to drugs with N-methyl-D-aspartic acid (NMDA) antagonist or gamma amino butyric acid (GABA) mimetic properties [5]. Therefore, it is of interest that most of the currently used general anesthetic drugs have either NMDA receptor blocking property, such as ketamine or GABA receptor enhancing property, exemplified by midazolam and benzodiazepines. Activation of NMDA receptors mediates most of the excitatory neurotransmission in the central nervous system (CNS). Normal neuronal development, differentiation and outgrowth depend on stimulation of NMDA receptors. Since ketamine is widely used in clinical anesthesia, the reports by different groups on the neurodegenerative effect of NMDA receptor blockade in the developing rat brain raise concerns about the safety of ketamine and other anesthetics used for neonates. Exposure of developing mammals to NMDA-type glutamate

Alterations in N-Methyl-D-Aspartate (NMDA) Receptor Function and Potential… 157 receptor antagonists affects the endogenous NMDA receptor system and enhances neuronal cell death. The NMDA receptor regulates a calcium channel and calcium influx that overwhelms the mitochondrial buffering capacity can result in increased production of reactive oxygen species (ROS) and cell death. Meanwhile, stimulation of immature GABA receptors is thought to be excitatory early in development but inhibitory in mature neurons. Stimulation of immature neurons by GABA agonists is thus thought to increase overall nervous system excitability and may contribute to NMDA receptor-associated increased excitability during early development. This increased excitability may contribute to abnormal neuronal cell death during development. One of the intracellular transduction pathways that may contribute to anesthetic agent induced neurotoxicity involves N-methyl-D-aspartate receptors. NMDA receptors and the consequent Ca2+ influx caused by their activation are major events that may contribute to potential neuronal cell death. Therefore, long-term blockade of NMDA receptors by anesthetic agents during the synaptogenesis stage in developing brain can cause apoptotic degeneration of neurons.

Neurotransmission and Anesthetic Agents Glutamatergic Transmission and Potential Anesthetic-Induced Neurodegeneration during the Development The amino acid L-glutamate is generally recognized as the major excitatory neurotransmitter of the mammalian central nervous system (CNS) and glutamate receptors play a major role in fast excitatory synaptic transmission. Glutamate promotes neuronal migration, differentiation and plasticity during development and throughout life [6]. Malfunctions of the glutamate system can affect neuroplasticity and cause neuronal toxicity. In the case of anesthetic-induced neurodegeneration, many glutamate-regulated processes seem to be perturbed. Abnormal neuronal development, abnormal synaptic plasticity and neurodegeneration have been proposed as mechanisms that underlie anesthetic-induced neuronal cell loss. It is becoming clear that some of the most important functions of the nervous system, such as synaptic plasticity and synaptic formation, critically depend on the behavior of NMDA receptors, and that neurological damage caused by a variety of pathological states can result from exaggerated or inappropriate activation of NMDA receptors [7, 8]. The general anesthetics, such as ketamine, block subtypes of glutamate receptors: the Nmethyl-D-aspartate (NMDA) receptors. As a dissociative anesthetic, ketamine is commonly used to produce analgesia in children in emergency departments [9]. Clinically, ketamine‟s role in pediatric anesthesia is well established. However, recent studies have indicated that ketamine may cause dose-dependent, widespread apoptotic neurodegeneration in immature rat and monkey brains [1, 10, 11]. The window of vulnerability appears restricted to the phase of rapid synaptogenesis, also known as the brain growth spurt. Glutamatergic transmission is mediated by receptor families that are classed as ionotropic (iGluRs) or metabotropic (mGluRs). iGluRs are ligand-gated ion channels which can be subclassified into the following groups based upon their ligand binding properties: N-methyl-D-

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aspartate receptors (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPA), kainate receptors (KA) and, more recently, delta receptors. NMDA receptors appear to be heteromeric complexes [12]. NMDA-R1 (Grin1) subunits can form homo-oligomeric receptors that are functional, and the presence of this subunit is required to produce detectable NMDA-activated channel currents in vitro [13]. NMDA-R2 (Grin2A-D) subunits produce functional receptors only when co-expressed with NMDA-R1 [12], and coexpression of NMDA-R1 with NMDA-R2 subunits increases their responsiveness to NMDA and yields different functional properties [13]. Proposed mechanisms for the developmental neurotoxicity caused by anesthetic agents such as ketamine include a compensatory up-regulation of NMDA receptors and subsequent over-stimulation of the glutamatergic system by endogenous glutamate via this receptor upregulation [4, 10, 14]. This results in dysregulation of calcium signaling, oxidative stress [15], and activation of the NF-kB signaling pathway [14]. However, the exact molecular mechanisms underlying ketamine-induced apoptotic neuronal cell death remain elusive. To better understand the molecular pathogenesis of anesthetic-induced developmental neurotoxicity, systems biology approaches should be carried out to examine the changes in gene expression profiles in developing brains, which have been shown to display a high susceptibility to ketamine-induced apoptotic neuronal cell death.

Changes in Gene Expression Profiling and Anesthetic-Induced Neurodegeneration during the Development Consistent with previous morphological and biochemical findings on anesthetic agents, such as ketamine, our microarray study [16] identified disruptions in glutamate receptor signaling, synaptic long-term potentiation, PTEN (phosphatase and tensin homolog deleted on chromosome 10) signaling, pyrimidine metabolism, circadian rhythm signaling, etc. In addition, we also found remarkable changes in genes related to apoptosis. More specific and sharper differences did exist in that the Troponin T1 (Tnnt1) gene was significantly induced in the adult mouse brain [17], but it was not observed in our system. Conversely, while a significant up-regulation of the Grin1 (NR1) and Grin2 (NR2) genes was observed in our study, the previous report did not show similar results [17]. These similarities and differences most likely reflect the unique response patterns of adult and developing brains toward ketamine administration. Importantly, these data indicate that potential anesthetic-induced neurotoxicity may depend on the dose of anesthetic, the duration of exposure, the route of administration, the receptor subtype activated, and the stage of development or maturity at the time of exposure. The gene expression of the NMDA receptor subunit gene, Grin1 (NR1), was significantly up-regulated in ketamine-treated rat pups as detected in microarray experiments and subsequently confirmed with TaqMan analyses. The NMDA receptor NR1 subunit is widely distributed throughout the brain and is the fundamental subunit necessary for NMDA channel function. In the study [16], by using in situ hybridization techniques to detect the relative densities of NMDA receptor NR1 subunits, a potential parallel relationship between enhanced apoptosis and NMDA receptor expression levels was examined (Figure 1). Our in situ data provided direct evidence that repeated ketamine exposure results in a substantial increase in autoradiographic density (labeling) of NR1 subunit mRNA in the frontal cortex and

Alterations in N-Methyl-D-Aspartate (NMDA) Receptor Function and Potential… 159 hippocampus. These data indicate that ketamine-induced pathological change is closely associated with a remarkable up-regulation of NMDA NR1 subunit mRNA. It is possible that increased expression of Grin1 (NR1) was accompanied by an altered expression of other glutamate receptor subunits. Our microarray analyses and Q-PCR data [16] revealed an increase in Grin2a (NR2A; 1.5 fold) and Grin2c (NR2C; 1.7 fold), but no significant effects were observed in Grin2b (NR2B) or Grin2d (NR2D). Our findings are consistent with those of previous in situ hybridization and immuno-blotting data that demonstrated a compensatory up-regulation of NMDA-R1 and NMDA-R2 receptors following prolonged exposure to NMDA receptor antagonists [10, 18, 19].

In Situ Hybridization (Frontal Cortex) (NMDA Receptor NR1 Subunit mRNA)

I Control

Ketamine

II A

B

Ketamine (20 mg/kg x 6)

Control Shi et al., 2010

Figure 1. NMDA receptor NR1 subunit mRNA abundance in PND 7 rats. In situ hybridization was performed on rat brain sections (coronal) using a 35S-labeled oligonucleotide probe specific for the NMDA receptor NR1 subunit. Panel I shows a general view of NR1 in situ hybridization signals in frontal cortical areas from both control and ketamine-treated rats. Panel II illustrates that the autoradiographic density (labeling) for NR1 subunit mRNA was higher in ketamine-treated (20 mg/kg x 6 injections) rat brain frontal cortex (B) compared to control (A). Scale bar = 90 µm

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Cheng Wang, Xuan Zhang, Fang Liu et al. Table 1. Selective validation of the microarray results by Q-PCR Gene symbols Grin1 (NR1) Grin2a (NR2A) Grin2b (NR2B) Grin2c (NR2C) Grin2d (NR2D)

Fold-change (Q-PCR) 1.8* 1.5* 1.0 1.7* 1.2

* P<0.05, as compared to the control.

Fold-change (microarray) 1.5* 1.2 0.9 1.5* 1.1

TUNEL-Assay (PND-7 rat pups)

A

Control (10 µl saline/g, 6 injections)

B

Ketamine (20 mg/kg, 6 injections)

Figure 2. Ketamine-induced neurodegeneration in PND 7 rats assessed by TUNEL labeling. Representative photographs indicate that TUNEL-positive cells are more numerous in layers II and III of the frontal cortex in the ketamine treated rat brain (B). Only a few TUNEL-positive cells were observed in the control (saline treated) rat brain (A). Scale bar = 60 µm

NMDA receptor density has also been shown to increase in cultured cortical neurons after exposure to the NMDA receptor antagonists D-AP5, CGS-19755, and MK-801, but not after exposure to the AMPA/kainate receptor antagonist CNQX [20]. In our microarray study, no significant changes were detected in the gene expression patterns of AMPA or kainate receptors after repeated ketamine exposure. These findings support our previous pharmacological data showing that application of the non-NMDA receptor antagonist, CNQX, or nifedipine (an antagonist of the L-type voltage sensitive calcium channel) did not produce a significant protective effect against ketamine-induced neuronal apoptosis [4]. We hypothesize that continuous blockade of NMDA receptors by ketamine causes a compensatory up-regulation of NMDA receptors and this up-regulation makes neurons bearing these receptors more vulnerable, after ketamine withdrawal, to the excitotoxic effects of endogenous glutamate, because this up-regulation of NMDA receptors allows for the accumulation of toxic levels of intracellular calcium even under normal physiological conditions. In order to understand the underlying mechanism of anesthetic (e.g. ketamine)-induced neurodegeneration, brain tissues from the frontal cortical levels, where the most severe

Alterations in N-Methyl-D-Aspartate (NMDA) Receptor Function and Potential… 161 neuronal damage was expressed, were selected for RNA isolation and microarray analysis [16]. Consistent with the TUNEL assay and previous in vivo data (Figure 2), a total of 32 genes were found to be involved in apoptosis, and among them, 15 genes were up-regulated and 17 genes were down-regulated (Table 2) in animals exposed to 6 injections of 20 mg/kg ketamine, compared with the controls [16] . The apoptosis-related genes are a group of genes that has two distinct modes of operation: pro-apoptosis or anti-apoptosis. In response to various inducers such as stressful stimuli or sustained elevation of intracellular calcium levels, the ultimate fate of the brain cell is determined by the roles of these apoptosis-related genes in regulating the life/death cell balance. Table 2. Apoptosis-related genes identified by GOFFA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

Gene symbols Acvr1c Ahr Alms1 Amigo2 Atp7a Bnip3 Bub1b Cd24 Cdc2a Inhba Myc Ntf3 Pak7_predicted Pdia2_predicted Rasa1 Tnfrsf11b Unc5c Agt Alb Apoe Bag3 Cebpb Clu Cryab Gjb6 Hrk Igfbp3 Inpp5d Jun Mal Rassf5 Txnip

Gene names activin A receptor, type IC aryl hydrocarbon receptor Alstrom syndrome 1 adhesion molecule with Ig like domain 2 ATPase, Cu++ transporting, alpha polypeptide BCL-2/adenovirus E1B 19 kDa-interacting protein 3 budding uninhibited by benzimidazoles 1 homolog, beta CD24 antigen cell division cycle 2 homolog A (S. pombe) inhibin beta-A myelocytomatosis oncogene neurotrophin 3 p21 (CDKN1A)-activated kinase 7 (predicted) protein disulfide isomerase associated 2 (predicted) RAS p21 protein activator 1 tumor necrosis factor receptor superfamily, member 11b unc-5 homolog C (C. elegans) angiotensinogen (serpin peptidase inhibitor, clade A, member 8) Albumin apolipoprotein E Bcl-2-associated athanogene 3 CCAAT/enhancer binding protein (C/EBP), beta Clusterin crystallin, alpha B gap junction membrane channel protein beta 6 harakiri, BCL-2 interacting protein (contains only BH3 domain) insulin-like growth factor binding protein 3 inositol polyphosphate-5-phosphatase D Jun oncogene myelin and lymphocyte protein, T-cell differentiation protein Ras association (RalGDS/AF-6) domain family 5 thioredoxin interacting protein

Genes 1-17 were down-regulated and genes 18-32 were up-regulated.

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The mechanism(s) underlying the anesthetic (e.g. ketamine)-induced neuronal cell death have not been fully elucidated. However, our microarray data indicate that the majority (approximately two-thirds) of up-regulated genes were pro-apoptotic in nature including Agt, Clu, Gjb6, Hrk, Igfbp3, Inpp5d, Jun, Mal, Rassf5 and Txnip. Meanwhile, from these upregulated genes, expression of Alb, Apoe, and Cryab may provide inhibition of apoptosis [21]. It has been reported that silencing the expression of Bag3 (Bcl-2-associated athanogene 3) leads to reduced protein levels of Bcl-XL, Mcl-1 and Bcl-2 in colon cancer cells and increased apoptosis [22]. As a critical gene, Cebpb [CCAAT/enhancer binding protein (C/EBP)] acts as a major regulator of metabolic homeostasis and is involved in many cellular processes, such as differentiation, growth, immune responses, neoplastic growth, development of the reproductive system, and pro- and anti-growth pathways [23]. On the other hand, in genes that have been down-regulated (17 genes), about one-half are anti-apoptotic genes. The over-expression of Acvr1c has been shown to suppress the apoptotic effects and Amigo2 acts as an anti-apoptotic factor [24, 25]. Bnip3 encodes cellular proteins that interact with Bcl-2. In cortical cells, cyanide induces a rapid up-regulation of Bnip3 expression, followed by a caspase-dependent cell death [26]. Down-regulation of Cd24, Cdc2a, and disruption of the Rasa1 gene in early embryonic mice induce apoptosis of neuronal cells [27, 28]. In neocortical and hippocampal tissues, apoptotic effects can be demonstrated following Ahr activation [29] and this gene was found to be up-regulated in our studies. These observations may imply that the frontal cortex is the brain region most vulnerable to ketamine-induced neurotoxicity during development, and the neuronal survival in the early phases of the apoptotic cascades mostly depends on the balance between the proand anti-apoptotic factors of the apoptosis-related genes.

Gabaergic Neurotransmission and Potential Anesthetic-Induced Neurodegeneration during the Development Anesthetics that block the NMDA receptor (glutamate subtype) and/or positively modulate or gate the GABA(A) receptor have been associated with apoptotic neurodegeneration in the developing neonate [4, 11, 30, 31]. To induce or maintain a surgical plane of anesthesia, it is common practice in pediatric or obstetrical medicine to use a combination of agents from these two classes. The anesthetic gas nitrous oxide (N2O), an NMDA receptor antagonist, and isoflurane (ISO), a volatile anesthetic that acts on multiple receptors, including the postsynaptic GABA receptor, are most commonly used in surgical procedures for human infants. Therefore, this practice raises the concern that there is a risk of deleting immature neurons by increasing apoptotic cell death in the developing brain. In our previous study [32], to further clarify the brain damaging potential of N2O and ISO, PND-7 rat pups were exposed to clinically relevant doses of these anesthetics, either individually or in combination for 2, 4, 6 or 8 hours, and the neurotoxic effects were evaluated 6 hours after cessation of anesthesia. No significant increase in apoptotic neurodegeneration in layers II and III of the frontal cortex was detected, as indicated by caspase-3 immunostaining, silver staining and Fluoro-Jade C staining, in animals exposed to N2O (75 vol%) alone for up to 8 hours [32]. This was similar to findings from previous studies performed using the same model [11]. Meanwhile, it has also been reported that animals treated with isoflurane (0.75. 1.0, or 1.5%), which acts on multiple

Alterations in N-Methyl-D-Aspartate (NMDA) Receptor Function and Potential… 163 receptors including postsynaptic GABA receptors, exhibited dose-dependent neurodegeneration in the thalamic nuclei and parietal cortex [11]. However, our previous study demonstrated that application of isoflurane in a low dose (0.55 %; for 2, 4, 6 and 8 hours) showed no significant enhancement of apoptotic cell death in the frontal cortex compared with the corresponding control animals. This finding is very important because this concentration somewhat reflects the threshold dose of the neurotoxic effect of isoflurane [32]. In order to determine if a combination of NMDA antagonists and GABA agonists will prevent or enhance each other‟s effects, the combination of N2O (75%) with ISO (0.55%) has been applied. Co-administration of N2O and ISO is a frequently-used inhaled anesthetic combination to facilitate surgery in the neonate. Co-administration of N2O and ISO is a frequently-used inhaled anesthetic combination to facilitate surgery in the neonate. A striking enhancement in brain damage was noted [32] when N2O (75%) was combined with ISO (0.55%). Maximal neurodegeneration was observed after 6-8 hours, however, only a small amount of damage (not significant) was detected at 4 hours, and no significant effects at 2 hours. These findings were also consistent with previous studies performed using the same animal model [11]. The concordance between increased caspase-3 immunostaining, silver staining and Fluoro-Jade C staining, as well as a decreased BCL-XL/Bax ratio suggests this regimen produces cell death primarily through an apoptotic mechanism. In recent years there has been a great deal of interest in mechanisms of cell death, especially those that may be associated with the potential neurotoxic effects of anesthetics. Olney‟s group [33, 34] conducted a series of studies that led to the observation that during the developmental period of synaptogenesis, brief exposure to ethanol, which has both NMDA antagonist and GABAmimetic properties, can trigger enhanced apoptosis in the mammalian brain. The ability of ethanol to induce widespread apoptotic neurodegeneration throughout the forebrain during synaptogenesis provides a more likely explanation than has heretofore been available for the reduced brain mass and lifelong neurobehavioral disturbances associated with fetal alcohol spectrum disorder [35]. There is no doubt that the developing central nervous system is exquisitely sensitive to derangements in the internal milieu [36]. Accentuated neurodegenerative mechanisms in the immature brain increase neuronal susceptibility to various metabolic events or exposure to anesthetic agents [36]. Anesthetics and anticonvulsant drugs that block NMDA receptors or activate GABA(A) receptors consistently increase neuronal apoptosis in the neonatal brain [1, 5, 11, 34], suggesting that the physiological stimulation of the NMDA receptor is necessary for neuronal synaptogenesis differentiation and survival during development. Lack of NMDA receptor activation by glutamate decreases synaptogenesis and cell-cell interactions [37, 38]. Anesthetic agents directly suppress neuronal activation and also reduce extracellular concentrations of excitatory neurotransmitters [39], thereby reducing developmental inputs to immature neurons. Anesthetic agents suppress neuronal activity by as-yet unknown mechanisms. Similar to the proapoptotic effect of NMDA antagonists, vulnerability of the developing rat brain to GABA mimetics also changes with age [34], again raising the possibility that, depending upon the time of exposure [33], different patterns of neurodegeneration and potentially different neurobehavioral disturbances might occur. Meanwhile, immature GABA receptors are excitatory early in development and convert to an inhibitory role in mature neurons [40]. It can be postulated that prolonged supra-physiological stimulation of immature neurons by GABA agonists enhances the excitatory component in the action of GABA and may contribute to increased excitability during early development

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[41]. This increased excitability, along with NMDA antagonist-induced alteration of NMDA receptors, could lead to neuronal cell death. Consistent with previous reports [11], it appears that more profound neurodegeneration is induced if both NMDA and GABA(A) receptors are simultaneously altered, in that a more robust neurodegenerative response is seen after exposure to ethanol than to either an NMDA antagonist or GABAmimetic drug alone. Although this principle is corroborated by the present demonstration that combining a nontoxic concentration of the NMDA antagonist N2O with a GABAmimetic agent induced a pattern of neurodegeneration more pronounced than was induced by either drug category alone, more supportive data from additional agent combinations, such as ketamine and midazolam, and experimental models (in vitro and in vivo studies of nonhuman primate and rodent models) will be necessary to confirm this conclusion. In addition, the timing of the switch in the chloride reversal potential in various brain regions should be considered. At PND 7, many regions have either completed the switch or are well along in the process of switching. Thus, the matter of GABAergic excitation as a contributor to damage is complex and most likely region dependent.

Anesthetic-Induced Toxicity and Potential Protection Lines of evidence have indicated that activation of NMDA receptors caused neuronal cell death [1, 5, 34, 42-44]. Although neuronal apoptosis can be the final result of anestheticinduced toxicity, the pathways leading from mitochondrial dysfunction are not completely understood. It is increasingly apparent that mitochondria lie at the center of the cell death regulation process. Our previous data suggest the possible link between the formation of reactive oxygen species (ROS) and the regulation of either BCL-2 related genes or transcription factors [10, 14, 15, 45]. It was postulated that the continuous exposure of developing brains to general anesthetics may cause selective cell death by a mechanism that involves a dysregulation of NMDA receptor subunits [4, 14, 18, 31] accompanied by loss of mitochondrial membrane potential, alterations of calcium homeostasis and subsequent free radical formation. In addition, several factors may contribute to the topographic differences in anesthetic-induced neurodegeneration, such as animal species, developmental stage at the time of drug exposure, doses and duration of anesthesia exposure, etc. It is proposed that the administration of general anesthetics during critical developmental periods will result in a dose-related increase in neurotoxicity and loss of neurons [4, 10, 14, 30]. Polymers of α-2,8-linked sialic acid neural cell adhesion molecule (PSA-NCAM), a neuronal specific marker, is formed by the enzymatic transfer of large, negatively charged carbohydrate polymers of α-2,8-linked sialic acid to the fifth immunoglobulin domain of the NCAM molecule. To determine whether the expression levels of PSA-NCAM correlate with inhaled anesthetic-induced cell death, Western blot analysis for PSA-NCAM was performed [32]. An anesthetic combination (75% N2O + 0.55% ISO for 6 hours) caused a substantial increase in caspase-3, silver and Fluoro-Jade C staining, along with a concomitant decrease in PSA-NCAM staining. The decrease in PSA-NCAM corresponded to an approximately 45% decrease in PSA-NCAM immunoreactivity as assessed using an immunoblot assay. This decrease could be the direct result of neuronal loss induced by anesthetics, because this

Alterations in N-Methyl-D-Aspartate (NMDA) Receptor Function and Potential… 165 reduction of PSA-NCAM expression is consistent with the enhanced neurodegeneration as indicated by increased caspase-3, silver and Fluoro-Jade C staining positive neuronal cells. L-carnitine is a dietary supplement and has been reported to prevent apoptotic death [46]. L-carnitine is well known to exhibit membrane modulatory effects and thereby preserve cellular membrane integrity. The fact that co-administration of L-carnitine blocked cell death, as well as the loss of PSA-NCAM immunoreactivity, further indicates that the neuronal cell death induced by an anesthetic combination in developing rats is most likely apoptosis. Several reactive oxygen species, including nitric oxide and superoxide anion (O∙¯2), have been implicated in glutamate-induced neuronal death. However, little is known about the signaling pathway that mediates the postulated roles of peroxynitrite (ONOO). In previous in vitro studies, general anesthetics such as ketamine administration caused a significant upregulation of nitrotyrosine expression accompanied by enhanced neuronal apoptosis as indicated by cell death detection ELISA and decreased PSA-NCAM expression. Protein tyrosine nitration occurs during many neurodegenerative states and is an important marker of oxidative stress induced by peroxynitrite, and possibly other nitric oxide (NO)-derived oxidants. The toxicity of NO is linked to its ability to combine with superoxide anions (O-2) to form peroxynitrite, an oxidizing free radical that can cause DNA fragmentation and lipid oxidation [47, 48]. Recent findings show that peroxynitrite may act as a signaling molecule capable of up-regulating protein tyrosine phosphorylation, which plays an important role in the regulation of cell communication, proliferation, migration, differentiation and survival [49, 50]. In the brain, NO is produced by nNOS and is believed to be controlled by activation of NMDA receptors [51]. The finding that nNOS is connected to the NMDA receptor via a postsynaptic density protein (PSD95) indicates a direct link between NMDA receptor activation and nNOS stimulation [52]. The interaction of superoxide and NO, which results in the formation of peroxynitrite, can be prevented by targeting either superoxide or NO. The previous study [53] demonstrated that pharmacological manipulation of NOS by 7nitroindazole provides protection against ketamine-induced neuronal loss. Importantly, no protective effect was observed with cultures co-treated with ketamine and 7-nitroindazole, when 7-nitroindazole was omitted during ketamine washout. Primary neuronal cell cultures are thought to mainly contain neuronal NOS (nNOS). Therefore, 7-nitroindazole was used in this study since it has been reported to be a selective nNOS inhibitor. It has been demonstrated that pretreatment with 7-nitroindazole blocks methamphetamine (METH)induced dopaminergic neurotoxicity in mice [54] and selective nNOS knock-out mice are resistant to METH-induced dopaminergic neurotoxicity [55]. In addition to 7-nitroindazole, several other NOS inhibitors and iron porphyrin compounds have been utilized, such as FeTPPS and FeTMPyP, which efficiently degrade peroxynitrite to nitrate under physiological conditions. It appears, however, that 7-nitroindazole is the most selective nNOS inhibitor [5658]. The precise mechanisms by which NMDA regulates Bax (a proapoptotic protein) is unknown, but the prevention of this effect in vitro by the addition of catalase and superoxide dismutase [15], or in vivo by M40403 (superoxide dismutase mimetic) suggest the possible involvement of reactive oxygen species (ROS). Among the potential downstream regulators, the effect of ketamine on Bax and BCL-XL (an anti-apoptotic protein) was measured. Bax is a pore-forming cytoplasmic protein which translocates to the outer mitochondrial membrane,

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influencing its permeability and inducing cytochrome c release from the intermembrane space of the mitochondria into the cytosol, subsequently leading to cell death [59]. It is important to indicate that up-regulation of the NR1 subunit of the NMDA receptor is an important first step in the pathway to anesthetic-induced neurotoxicity. These up-regulated calcium channel receptors are vulnerable to endogenous glutamate concentrations within the tissues after ketamine washout. In our previous studies [4, 14] , to test whether the administration of antisense oligonucleotides (ODNs) targeted to the NR1 NMDA receptor subunit blocks steady state protein levels, an antisense ODN-targeting exon was used. Coadministration of antisense ODN specifically prevented NR1 up-regulation and blocked the reduction of PSA-NCAM expression induced by ketamine. Of particular interest to the data at hand are the possible mechanisms by which anesthetics such as ketamine could up-regulate NMDA receptors. Surprisingly, there is not an abundance of literature concerning this issue, but recently it has been demonstrated that the distal region of the NR1 promoter contains an active NF-kB site, which is developmentally regulated and appears to bind Sp3/Sp1 somewhat better than the NF-kB subunits [60]. The NMDA receptor NR1 subunit is widely distributed throughout the brain and is the fundamental subunit necessary for NMDA channel function. Our previous study [14] demonstrated that ketamine produced a significant up-regulation in NR1 protein expression. This result was consistent with literature demonstrating that treatment with NMDA antagonists produces an up-regulation of the NMDA receptor complex as measured by an increase in Bmax of NMDA receptor binding sites [20, 61]. The transcription factor NF-kB is known to respond to changes in the redox state of the cytoplasm and has been shown to translocate in response to NMDA-induced cellular stress [62]. NF-kB is normally sequestered in the cytoplasm, bound to the regulatory protein IkB kinase. The net result is the release of the NF-kB dimmer, which is then free to translocate into the nucleus. NF-kB translocation appears to be a necessary step in cell death induced by PCP [45], cyanide, and excitotoxic stimuli [63]. In our previous studies, ketamine produced a remarkable increase in translocation of NF-kB into the nucleus. The protection against cortical neuronal cell death and decreased PSA-NCAM by a peptide inhibitor of NF-kB translocation, SN-50, suggested that there was a causal relationship between these effects. There is evidence in the literature suggesting that the transcriptional regulation of target genes by NF-kB is tissue specific and possibly gene specific within a given cell type. The ability of SN-50 to prevent ketamine-induced cell death demonstrated that NF-kB is crucial to those processes. However, whether anesthetic-induced NF-kB translocation is specifically responsible for apoptotic or necrotic pathways observed in anesthetic studies is still unknown. Resolution of this question will require additional experiments.

Conclusion The integrative and iterative evaluation of the anesthetic models (in vivo & in vitro) lead to a better understanding of the potential neurotoxicity of NMDA antagonists and GABA agonists in the developing brains.

Alterations in N-Methyl-D-Aspartate (NMDA) Receptor Function and Potential… 167 For example, a total of 462 down-regulated genes and 357 up-regulated genes were identified after ketamine exposure in developing rat brain. Among them, 32 genes are associated with apoptotic pathways. Perturbations of NMDA-type glutamate and other receptor signaling were identified in our microarray analysis. Q-PCR assay confirmed that NMDA receptor genes including Grin1 (NR1), Grin2a (NR2A) and Grin2c (NR2C) were significantly up-regulated. The ketamine-induced up-regulation of NMDA receptor Grin1 (NR1) mRNA signaling was further confirmed by in situ hybridization. These observations, together with morphological evidence suggest that repeated anesthetic (e.g. ketamine) exposure causes compensatory up-regulation of NMDA receptors and subsequent overstimulation of the glutamatergic system triggering enhanced apoptosis in developing neurons, and that neuronal survival in the early phases of apoptotic cascades depends mostly on the balance between pro- and anti-apoptotic factors. Figure 3.

glutamate

IKK

Off

++ CaCa NR2

O22--

NR1 UP-regulated NMDA Receptor

M40403×

++ Ca Ca++

L-carnitine

++

C a ad rlo ove

IKK

NFKB I- KB

On

-A Proro-A cell death -A P Prnoti-A cc cell death e A me

room otocchhr t y ccy s aassees p s p ccaas

P NFKB

+ I- KB

SN50

P

KB p53genes

P-Gene A-Gene Modified from McInnis et al., 2002

nucleus

Figure 3. The cartoon illustrating the working model of anesthetic (e.g. ketamine)-induced neuronal cell death and potential protection mechanisms

In summary, Figure 3 illustrates a potential model of anesthetic (i.e. ketamine)-induced neuronal cell death in the developing brain. Excessive activation of up-regulated NMDA receptors results in a calcium overload that exceeds the buffering capacity of the mitochondria and interferes with electron transport in a manner that results in an elevated production of reactive oxygen species, and the dissociation of some transcription proteins, such as NF-kB, and their transport into the nucleus. In the nucleus these transcription factors bind to several DNA sequences of several known genes. The consequence of this binding is

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not completely understood, but the loss of the balance of pro- and anti-apoptotic genes is apparent; the diminished formation of anti-apoptotic heterodimers in favor of pro-apoptotic homodimers. These homodimers are thought to create mitochondrial membrane pores through which cytochrome c can leak into the cytoplasm where it can activate caspases that play a critical role in the ultimate demise of the neuron. As shown in above (text), several recent studies using antisense ODNs targeting specific NMDA receptor subunits, or blockers of oxidative stress such as L-carnitine, the superoxide dismutase mimetic, M40403, and the NOS inhibitor, 7-nitroindazole have indicated that reduction of oxidative stress may protect the developing animal from anesthetic-induced brain cell death.

Disclaimer This document has been reviewed in accordance with United States Food and Drug Administration (FDA) policy and approved for publication. Approval does not signify that the contents necessarily reflect the position or opinions of the FDA nor does mention of trade names or commercial products constitute endorsement or recommendation for use. The findings and conclusions in this report are those of the author(s) and do not necessarily represent the views of the FDA.

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Chapter 8

Genetics and Molecular Biology of Alzheimer's Disease and Frontotemporal Lobar Degeneration: Analogies and Differences Daniela Galimberti*, Chiara Fenoglio and Elio Scarpini

Elio Scarpini, Dept. of Neurological Sciences, "Dino Ferrari" Center, University of Milan, Fondazione Cà Granda, IRCCS Ospedale Maggiore Policlinico, Milan, Italy

Abstract Alzheimer‟s disease (AD) is the most common cause of dementia in the elderly, whereas Frontotemporal Lobar Degeneration (FTLD) is the most frequent neurodegenerative disorder with a presenile onset. The two major neuropathologic hallmarks of AD are extracellular Amyloid beta (A) plaques and intracellular neurofibrillary tangles (NFTs). Conversely, in FTLD the deposition of tau has been observed in a number of cases, but in several brains there is no deposition of tau but instead a positivity for ubiquitin. In some families these diseases are inherited in an autosomal dominant fashion. Genes responsible for familial AD include the Amyloid Precursor Protein (APP), Presenilin 1 (PS1) and Presenilin 2 (PS2). The majority of mutations in these genes are often associated with a very early onset (40-50 years of age). Regarding FTLD, the first mutations described are located in the Microtubule Associated Protein Tau gene (MAPT). Tau is a component of microtubules, which represent the internal support structures for the transport of nutrients, vesicles, mitochondria and chromosomes within the cell. Mutations in MAPT are associated with an early onset of the disease (40-50 years), and the clinical phenotype is consistent with *

Corresponding author: phone ++ 39.2.55033847, FAX ++ 39.2.50320430, E-MAIL: [email protected]

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Daniela Galimberti, Chiara Fenoglio and Elio Scarpini Frontotemporal Dementia (FTD). Recently, mutations in a second gene, named progranulin (GRN), have been identified in some families with FTLD. Progranulin is expressed in neurons and microglia and displays anti-inflammatory properties. Nevertheless, it can be cleaved into granulins which, conversely, show inflammatory properties. The pathology associated with these mutations is most frequently characterized by the immunostaining of TAR DNA Binding Protein 43 (TDP-43), which is a transcription factor. The clinical phenotype associated with GRN mutations is highly heterogeneous, including FTD, Progressive Aphasia, Corticobasal Syndrome, and AD. Age at disease onset is variable, ranging from 45 to 85 years of age. The majority of cases of AD and FTLD are however sporadic, and likely several genetic and environmental factors contribute to their development. Concerning AD, it is known that the presence of the 4 allele of the Apolipoprotein E gene is a susceptibility factor, increasing the risk of about 4 fold. A number of additional genetic factors, including cytokines, chemokines, Nitric Oxide Synthases, contribute to the susceptibility for the disease. Some of them also influence the risk to develop FTLD. In this chapter, current knowledge on molecular mechanisms at the basis of AD and FTLD, as well as the role of genetics, will be presented and discussed.

1. Alzheimer’s Disease and Frontotemporaal Lobar Degeneration Alzheimer‟s disease (AD) is the most common cause of dementia in the elderly, with a prevalence of 5% after 65 years of age. The disease was originally described by Alois Alzheimer and Gaetano Perusini in 1906, and it is clinically characterized by a progressive cognitive impairment, including impaired judgment, decision-making and orientation, often accompanied, in later stages, by psychobehavioural disturbances as well as language impairment. The two major neuropathologic hallmarks of AD are extracellular beta-amyloid (A) plaques and intracellular neurofibrillary tangles (NFTs). The production of A, which represents a crucial step in AD pathogenesis, is the result of the cleavage of a bigger precursor, named Amyloid precursor protein (APP), which is over-expressed in AD [1]. A forms highly insoluble and proteolysis resistant fibrils known as “senile plaques”. Neurofibrillary tangles are composed of the tau protein. In healthy controls, tau is a component of microtubules, which are the internal support structures for the transport of nutrients, vesicles, mitochondria and chromosomes within the cell. Microtubules also stabilize the growing axons, which are necessary for the development and growth of neurites [1]. In AD, tau protein is abnormally hyperphosphorylated and forms insoluble fibrils, which originate deposits within the cell. Frontotemporal Lobar Degeneration (FTLD) occurs most often in the presenile period, and age at onset is typically 45-65 years, with a mean in the 50s. Distinctive features in FTLD concern behaviour, including disinhibition, loss of social awareness, overeating and impulsiveness. Despite profound behavioural changes, memory is relatively spared [2]. Conversely to AD, which is more frequent in women, FTLD has an equal distribution among men and women. The current consensus criteria [3] identify three clinical syndromes: Frontotemporal Dementia (FTD), Progressive nonfluent Aphasia (PA) and Semantic Dementia (SD), which reflect the clinical heterogeneity of FTLD. Frontotemporal dementia is characterized by behavioural abnormalities, whereas PA is associated with progressive loss of

Genetics and Molecular Biology of Alzheimer's Disease and Frontotemporal Lobar… 175 speech, with hesitant, nonfluent speech output [4], and SD is associated with loss of knowledge about words and objects. This variability is determined by the relative involvement of the frontal and temporal lobes, as well as by the involvement of right and left hemispheres [5]. Despite the majority of AD and FTLD are sporadic and likely caused by the interaction between genetic and environmental factors, so far it was observed that clinically typical AD and FTLD can cluster in families and be inherited in an autosomal dominant fashion, suggesting a genetic cause.

2. Familial AD Autosomal dominant AD forms are characterized by mutations in three genes: APP [6], PSEN1 [7] and PSEN2 [8]. In 1987, a region of linkage with AD was reported on the long arm of chromosome 21, which encompassed a region harboring the APP gene, a compelling candidate for AD [9]. The gene is located at chromosome 21q21.22 and encodes for a transmembrane protein that is normally processed into amyloid fragments. In 1991, the first missense mutation in APP was reported [6]. Since then, 32 different mutations have been described in the APP gene in 89 families (http://molgen-www.uia.ac.be). All these mutations cause amino acid changes in putative sites for the cleavage of the protein, thus altering the APP processing, such that more pathological A42 is produced [10]. Interestingly, the chromosome 21, in which APP resides, is triplicated in Down syndrome and most of the cases manifest also AD by the age of 50. Post-mortem analyses of Down‟s patients who die young show diffuse intra-neuronal deposits of A, suggesting that its deposition is an early event in cognitive decline. The recent discovery of an extra copy of the APP gene in familial AD [11] provides further support that increased A production can cause the disease. The other two genes causing familial AD are Presenilin (PSEN)1 (14q24.3) and PSEN2 (1q31-q42). Presenilins represent a central component of -secretase, the enzyme responsible for originating A from the C-terminal fragment of the APP protein. Mutations in presenilins also alter APP cleavage, leading to an increased production of A42. So far, more than 178 mutations in PSEN1 have been identified and 14 additional mutations have been found in the homologous gene PSEN2 (http://molgen-www.uia.ac.be). Most variants in PSEN1 are missense mutations resulting in single amino-acid substitutions. Some are more complex, for example, small deletions or splice mutations. The most severe mutation in PSEN1 is a donor-acceptor splice mutation that causes a twoaminoacid substitution and an in-frame deletion of exon 9. However, the biochemical consequences of these mutations for -secretase assembly seem to be limited [12,13]. All these clinical mutations are likely to cause a specific gain of toxic function for PSEN1, determined by an increase of the ratio between Aβ42 and Aβ40 amyloid peptides, thus indicating that presenilins might modify the way in which -secretase cuts APP. Mutations in presenilins occur in the catalytic subunit of the protease responsible for determining the length of A peptides therefore generating toxic A fragments. However,

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presenilins have also non-proteolytic functions [14,15], the disruption of which might also contribute to familial AD pathogenesis. Despite several carriers develop the disease early (40-50 years of age) with a typical AD phenotype, in some cases patients carrying the same mutation develop signs and symptoms resembling FTD instead of AD [16]. In addition, other mutations are associated with myoclonus, seizures, bilateral spasticity, parkinsonian features or ataxia [17].

3. Familial FTLD Frontotemporal Lobar Degeneration is a heterogeneous disease characterized by a strong genetic component in its aetiology as up to 40% of patients report a family history of the disease in at least one extra family member [18]. In 1994 an autosomal dominantly inherited form of FTD with parkinsonism was linked to chromosome 17q21.2 [19]. Subsequently, other familial forms of FTD were found to be linked to the same region, resulting in the denomination “frontotemporal dementia and parkinsonism linked to chromosome 17” (FTDP-17) for this class of diseases. In 1998, MAPT gene on chromosome 17q21, which encodes the microtubule associated protein tau was described as the cause of the disease in these families [20-22] Currently, 44 different mutations in the MAPT gene have been described in totally 132 families (http://molgen-www.uia.ac.be). MAPT mutations are either non-synonimous or deletion, or silent mutations in the coding region, or intronic mutations located close to the splice-donor site of the intron after the alternatively spliced exon 10 [23]. Mutations are mainly clustered in exons 9-13, except for two recently identified mutations in exon 1 [24]. As regards possible effects on MAPT mutations, different mechanisms are involved, depending on the type and location of the mutation. Many of them disturb the normal splicing balance, producing altered ratios of the different isoforms. A number of mutations promote the aggregation of tau protein, whereas others enhance tau phosphorylation [25]. However, after the discovery for MAPT as causal gene for FTDP-17, there were still numerous families with autosomal dominant FTLD genetically linked to the same region of chr17q21 that contains MAPT but in which no pathogenic mutations had been identified, despite extensive analysis of this gene [26-28]. The neuropathological phenotype in these families was similar to the microvacuolar-type observed in a large proportion of idiopathic FTD cases with ubiquitin immunoreactive neuronal inclusions. Moreover, clinically, the disease in these families was consistent with diagnostic criteria for FTLD [3]. Sequence analysis of the whole MAPT region failed to find a mutation and tau protein appeared normal in these families [9] Moreover the minimal region containing the disease gene for this group of families was approximately 6.2 Mb in physical distance. This region defined by markers D17S1787 and D17S806 is particularly gene rich, containing around 180 genes. Collectively, these data strongly argued against MAPT and pointed to another gene. Systematic candidate gene sequencing of all remaining genes within the minimal candidate region was performed and after sequencing 80 genes, including those prioritized on known function, the first mutation in progranulin gene (GRN) was identified. It consists in a 4-bp insertion of CTGC between coding nucleotides 90 and 91, causing a frameshift and premature termination in progranulin (C31LfsX34) [30]. Tese results have been contemporarily replicated by Cruts et

Genetics and Molecular Biology of Alzheimer's Disease and Frontotemporal Lobar… 177 al. [31], who analyzed other families with a FTLD-U disease without MAPT pathology, finding a mutation five base pairs into the intron following the first non coding exon of the GRN gene (IVS0+5G-C). This is predicted to prevent splicing out of the intron 0, leading the mRNA to be retained within the nucleus and subjected to nuclear degradation [31]. At present there is no obvious mechanistic link between the mutations in MAPT and GRN, currently assuming that their proximity on chromosome 17 is simply a coincidence. Progranulin is known by several different names including granulin, acrogranin, epithelin precursor, proepithelin and prostate cancer (PC) cell derived growth factor [32]. The protein is encoded by a single gene on chromosome 17q21, which produces a 593 amino acid, cysteine rich protein with a predicted molecular weight of 68.5 kDa. The full-length protein is subjected to proteolysis by elastase and this process is regulated by a secretory leukocyte protease inhibitor (SLPI) [33]. Progranulin and the various granulin peptides are implicated in a range of biological functions including development, wound repair and inflammation by activating signaling cascades that control cell cycle progression and cell motility [32]. Excess progranulin appears to promote tumour formation and hence can act as a cell survival signal. Despite the increasing literature on the function of progranulin, its role in neuronal function and survival remains unclear. In the human brain, GRN is expressed in neurons but significantly is also highly expressed in activated microglia [30], with the result that GRN expression is increased in many neurodegenerative diseases. Since the original identification of null-mutations in FTLD in 2006, numerous novel mutations have been reported, spanning most exons, and to date more than 68 GRN mutations have been described (http://www.molgen.ua.ac.be/) Interestingly, the majority of mutations identified create functional null alleles, causing premature termination of the GRN coding sequence. This leads to the degradation of the mutant RNA by nonsense mediated decay, creating a null allele [30,31]. The presence of a null mutation causes a partial loss of functional progranulin protein, which in turn leads eventually to neurodegeneration (haploinsufficency mechanism), although how loss of GRN causes neuronal cell death remains unclear. Estimates of the frequency of GRN mutations in typical FTD patient populations suggests that they account for about 5-10% of all FTD cases, although numbers vary markedly depending on the nature of the populations considered [31,34,35]. Neuropathology analysis revealed that ubiquitin immunoreactive neuronal cytoplasmatic and intranuclear inclusions were present in all cases with FTDP-17, where pathological findings were available [36]. Furthermore, soon after the identification of mutations in GRN, biochemical analyses demonstrated that truncated and hyperphosphorylated isoforms of the TAR-DNA binding protein (TDP-43) are major components of the ubiquitin-positive inclusions in families with GRN mutations as well as in idiopathic FTD and a proportion of Amyotrophic Lateral sclerosis (ALS) cases [37]. TDP43 is a ubiquitously expressed and highly conserved nuclear protein that can act as a transcription repressor, an activator of exon skipping or a scaffold for nuclear bodies through interactions with survival motor neuron protein. Under pathological conditions, TDP-43 has been shown to relocate from the neuronal nucleus to the cytoplasm, a consequence of which may be the loss of TDP-43 nuclear functions [37]. The mechanism by which loss of progranulin leads to TDP-43 accumulation and whether this is necessary for neurodegeneration in this group of diseases is still to be clarified.

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In conclusion, the function of progranulin in the brain is currently unclear and why loss of this protein leads to a neurodegenerative diseases in mid-life remains to be established, and its possible role as regulator of a repair activity in the central nervous system, as it is well known to happen in periphery, remains a challenge for science. The gene encoding for TDP43, named TARDBP, has been extensively studied and a number of mutations found in its Cterminal glycine rich region. Unexpectedly, the clinical phenotype of carries was ALS, and aggregates made of TDP-43 have been described in brain and spinal cord of such patients (see [38] for review). A recently published collaborative study [39] analyzed GRN in a population of 434 patients with FTLD, including FTD, PA, SD, FTD/ALS, FTD/MND, CBD, PSP. Fifty eight variants were identified, including 24 pathogenic variants. The frequency of GRN mutations was 6.9% of all FTLD-spectrum cases, 21,4% of cases with a pathological diagnosis of FTLD-U, 16% of FILD-spectrum cases with a family history of a similar neurodegenerative disease, and 56.2 of cases of FTLD-U with a family history. Clinical information were available for 31 GRN mutation-positive patients from 28 different families. The most common clinical diagnosis was FTD (n=24); 3 patients were diagnosed with PA, 3 with AD and 1 with CBD. The majority of GRN mutations Introduced a premature termination codon, suggesting that their corresponding mRNA will be degraded through nonsense mediated decay, supporting the hypothesis that most GRN mutations create functional null allele [39]. Two additional genes have been shown to cause FTLD. In 1995 Brown et al. [40] reported linkage to the pericentromeric region of chromosome 3 in a large multigenerational family with FTLD from Denmark. Nevertheless, the aberrant gene in this family has only recently been identified [41]. It consists in a mutation of the splice acceptor site of exon 6 of CHMP2B (charged multivescicular body protein 2B), which is part of the endosomal ESCRTIII-complex. The change from G to C results in an alteration of the splice acceptor site of exon 6, causing aberrant mRNA splicing of this transcript, which leads to the insertion of 201 base pairs of the intron between exons 5 and 6. In addition, a further transcript was identified, resulting from the use of a cryptic splice site consisting of 10 base pairs from the 5‟ end of exon 6. Anyway, mutations in CHMP2B appear as a rare genetic cause of FTLD mainly due to their rare frequency of occurrence, showing moreover that the CHMP2B locus does not increase the risk for FTLD [42] lastly, the first evidence of linkage with chromosome 9q21-22 comes from a study carried out in families with Motor Neuron Disease (MND) and FTD [43]. Despite the evidence of linkage to chr9q21-22 in several additional FTD-MND families, the gene responsible for the disease in this locus has yet to be identified [44-46].

4. Sporadic AD Risk genes are likely to be numerous, displaying intricate patterns of interaction with each other as well as with non-genetic variables, and-unlike classical Mendelian (“simplex”) disorders- exhibit no simple mode of inheritance. Mainly due to this reason, the genetics of sporadic AD has been labeled “complex” [47]. The gene mainly related to the sporadic forms of AD is the Apolipoprotein E (APOE) [48], which is located at chromosome 19q13.32 and was initially identified by linkage analysis [49]. The relationship between APOE and AD has

Genetics and Molecular Biology of Alzheimer's Disease and Frontotemporal Lobar… 179 been confirmed in more than 100 studies conducted in different populations. The gene has three different alleles, APOE*2, APOE*3 and APOE*4. The APOE*4 allele is the variant associated with AD. Longitudinal studies in Caucasian populations have shown that carriers for one APOE*4 allele have a two-fold increase in the risk for AD [50]. The risk increases in homozygous for the APOE*4 allele, and this allelic variant is also associated with an earlier onset of the disease. Several linkage studies have been performed, giving rise to additional candidate susceptibility loci at chromosomes 1, 4, 6, 9, 10, 12 and 19. In particular, promising loci have been found at chromosome 9 and 10 [51,52]. Recently, a wide genome analysis identified variants at CLU (which encodes clusterin or ApoJ) on chromosome 8 and PICALM in chromosome 11 associated with AD [53]. Data on CLU were replicated in an independent study, which, in addition, demonstrated that CR1, encoding the complement component (3b/4b) receptor 1and locate on chromosome 1, is associated with AD [54]. Also, a large number of candidate genes studies have been performed in order to search a robust risk factor for the sporadic form of the disease. Several studies were mainly focused in genes clearly involved in the pathogenesis of AD such as genes encoding for inflammatory molecules or involved in the oxidative stress cascade, both considered major factors in AD pathology . One of the strongest evidence of the role played by genetic variants in inflammatory molecules to increase the risk of AD involves the Interleukin-1 (IL1) complex, which is located at chromosome 2q14-21 and includes IL1-, IL1-, and IL1R antagonist protein (IL-1Ra), all of which have significant polymorphisms found to be associated with AD in several case- controls studies carried out in different populations [55-57]. Several polymorphisms in IL-6, which is a potent inflammatory cytokine but has also regulatory functions, have been investigated as well. The IL6 gene is located at chromosome 7p21 and polymorphisms exist in the -174 promoter region and in the region of a variable number of tandem repeats (VNTR), which is located in the 3‟untraslated region. Both of them have been found associated with AD in case-controls studies [58,59]. Investigation of Tumor Necrosis Factor- (TNF) polymorphisms was initiated because genome screening suggested a putative association of AD with a region on chromosome 6p21.3, which lies within 20 centimorgans of the TNF gene. Furthermore, other polymorphisms located in the promoter region of TNF have been associated with autoimmune and inflammatory diseases [60]. As with TNF, investigations of the role of -2macroglobulin (A2M) were initiated as a result of screening studies of the genome. In this case, linkage was found in the region of chromosome 1p, where A2M and its low-density lipoprotein receptor are found. Blacker et al. [61] tested for association of polymorphisms with AD showing a strong involvement of this gene in AD. Moreover, polymorphisms in chemokines have been investigated with regard of susceptibility of AD. In particular, Monocyte Chemoattractant Protein-1 (MCP-1) and RANTES genes have been widely screened in different neurodegenerative diseases [62]. The distribution of the A-2518G variant was determined in different AD populations with concordant results [63,64] showing no evidence for association of this variant in AD compared with controls. Moreover, Fenoglio et al. [63] found a significant increase of MCP-1 serum levels in AD carrying at least one G polymorphic allele. Therefore, the A-2518G polymorphism does not seem to be a risk factor for the development of AD, but its presence correlates with higher levels of serum MCP-1.

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RANTES promoter polymorphism -403 A/G, found to be associated with several autoimmune diseases, was examined in AD population, failing to find significant differences between patients and controls [62].. CCR2 and CCR5 genes, encoding for the receptors of MCP-1 and RANTES respectively, have been also screened for association with AD. The most promising variants involve a conservative change of a valine with an isoleucine at codon 64 of CCR2 (CCR2-64I) and a 32-bp deletion in the coding region of CCR5 (CCR532), which leads to the expression of a non-functional receptor. A decreased frequency and an absence of homozygous for the polymorphism CCR2-64I were found in AD, thus suggesting a protective effect of the mutated allele on the occurrence of the disease [65]; conversely, no different distribution of the CCR532 deletion in patients compared with controls were shown [65,66]. Another chemokine recently tested for susceptibility with AD is IP-10. A mutation scanning of the gene coding region has been performed in AD patients searching for new variants. The analysis demonstrated the presence of two previously reported polymorphisms in exon 4 (G/C and T/C), which are in complete linkage disequilibrium, as well as a novel rare one in exon 2 (C/T). Subsequently these SNPs have been tested in a wide case-control study but no differences in haplotype frequencies were found [67]. Other genes under investigation are related to oxidative stress, a process closely involved in AD pathogenesis. In this regard, genes coding for the nitric oxide synthase (NOS) complex have been screened. The common polymorphism consisting in a T/C transition (T-786C) in NOS3, previously reported to be associated with vascular pathologies, has been tested in AD, but no significant differences with controls were found. Nevertheless, expression of NOS3 in PBMC either from patients or controls seems to be influenced by the presence of the C polymorphic allele, and is likely to be dose dependent, being mostly evident in homozygous for the polymorphic variant. The influence of the polymorphism on NOS3 expression rate supports the hypothesis of a beneficial effect exerted in AD by contributing to lower oxidative damage [68]. An additional variant in NOS3 gene has been extensively investigated in AD patients, although the results are still controversial. It is a common polymorphism consisting in a single base change (G894T), which results in an aminoacidic substitution at position 298 of NOS3 (Glu298Asp). Dahiyat et al. [69] determined the frequency of the Glu298Asp variant in a two-stage case-control study, showing that homozygous for the wild-type allele were more frequent in late onset AD. However, studies in other populations failed to replicate these results [70-73] More recently Guidi et al [74] correlated this variant with total plasma homocysteine (tHcy) levels in 97 patients and 23 controls, demonstrating that the Glu/Glu genotype is correlated with higher levels of tHcy, which represent a known risk factor for AD [75], and its frequency was increased in AD patients [74]. Thus the mechanism by which this genotype contributes to increase the risk in developing AD could be mediated by an increase of tHcy. However, NOS-1 is the isoform most abundantly expressed in the brain. Recent genetic analyses demonstrated that the double mutant genotype of the synonymous C276T polymorphism in exon 29 of the NOS1 gene represents a risk factor for the development of early onset AD [76], whereas the dinucleotide polymorphism in the 3‟UTR of NOS1 is not associated with AD [77]. To date, the promoter region of NOS1, located approximately 200 kb upstream of these polymorphism, has not been investigated for susceptibility to AD. Due

Genetics and Molecular Biology of Alzheimer's Disease and Frontotemporal Lobar… 181 to this reason and to further explore a possible association of NOS1 polymorphisms with AD, the distribution of a functional polymorphisms and a variable number of tandem repeats (VNTR) was analyzed very recently in a case-control study, which tested 184 AD patients as well as 144 healthy subjects [78]. The functional variant considered is located in exon 1c, which is one of the nine alternative first exons (named 1a-1i), resulting in NOS1 transcripts with different 5‟-untraslated regions [79]. Three SNPs have been identified in exon 1c, but only the G-84A variant displays a functional effect, as the A allele decreases the transcription levels by 30% in in-vitro models [80]. Regarding exon 1f, a variable number of tandem repeats (VNTR) polymorphism has been recently reported in its putative promoter region, termed NOS1 Ex1f-VNTR. This VNTR is highly polymorphic and consists of different numbers of dinucleotides (B-Q), which, according to their bimodal distribution, have been dichotomized in short (B-J) and long (K-Q) alleles for association studies. Both Ex1c G-84A and Ex1f-VNTR are associated with psychosis and prefrontal functioning in a population of patients with schizophrenia [81]. Notably, both Ex1c and Ex1f transcripts are found in the hippocampus and the frontal cerebral cortex, i.e. brain regions implicated in the pathogenesis of schizophrenia as well as AD. The presence of the short (S) allele of NOS1 Ex1f-VNTR represents a risk factor for the development of AD. The effect is cumulative, as in S/S carriers the risk is doubled. Most interestingly, the effect of this allele is likely to be gender specific, as it was found in females only. In addition, the S allele was shown to interact with the APOE*4 allele both in males and females, increasing the risk to develop AD by more than 10 fold [78]. Thus, NOS1 seems to be a risk factor for AD, but only in female population. This could be explained by a possible interaction with other genes or with additional environmental factors present in females but not males. Epidemiological data indicate that the prevalence of AD is increased in females compared with males. Therefore, it is conceivable that different factors contribute to the development of the pathology in females rather than males, including genetic ones.

5. Sporadic FTLD The best well-known risk factor for late onset SAD, Apo E4, has also been considered as a risk factor for sporadic FTLD. A number of studies suggested an association between FTLD and APOE*4 allele [82-87]. Other Authors however, did not replicate these data [88-90]. Recent findings demonstrated an association between the APOE*4 allele and FTLD in males, but not females [91], possibly explaining the discrepancies previously reported. An increased frequency of the APOE*4 allele was described in patients with SD compared to those with FTD and PA [89]. Concerning the APOE*2 allele in the development of FTLD, heterogeneous data have been obtained in different populations. Bernardi et al. [87] showed a protective effect of this allele towards FTLD, whereas other Authors failed to do so [87]. Despite these results, a recent meta-analysis comprising a total of 364 FTD patients and 2671 controls demonstrated an increased susceptibility to FTD in APOE*2 carriers [93]. Besides pathogenic mutations, several polymorphisms have been reported to date, both in MAPT and GRN. An association between Progressive Supranuclear Palsy (PSP) and a dinucleotide repeat polymorphism in the intron between MAPT exons 9 and 10 was described

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in 1997 [94]. The alleles at this locus carry 11 to 15 repeats. Subsequently, two common MAPT haplotypes, named H1 and H2, were identified [95]. They differ in nucleotide sequence and intron size, but are identical at the amino acid level. Homozygosity of the more common allele H1 predisposes to PSP and Corticobasal Degeneration (CBD), but not to AD or Pick Disease [95-96]. Regarding GRN, an association of a SNP located in the promoter and an increased risk to develop FTLD in patients who did not carry causal mutations has recent been demonstrated [97]. In addition, a known polymorphism in MCP-1 (A-2518G) has been shown to exert a protective effects towards the development of FTLD [98], whereas NOS3 G894T (Glu298Asp) and NOS1 C276T SNPs likely Increases the risk to develop FTLD [99-100].

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Genetics and Molecular Biology of Alzheimer's Disease and Frontotemporal Lobar… 187 [82] Reif, A; Herterich, S; Strobel, A; et al. A neuronal nitric oxide synthase (NOS-I) haplotype associated with schizophrenia modifies prefrontal cortex function. Mol Psychiatry, 2006, 11(3), 286-300. [83] Farrer, LA; Abraham, CR; Volicer, L; et al. Allele epsilon 4 of apolipoprotein E shows a dose effect on age at onset of Pick disease. Exp Neurol, 1995, 136, 162–170. [84] Gustafson, L; Abrahamson, M; Grubb, A; et al. Apolipoprotein-E genotyping in Alzheimer's disease and frontotemporal dementia. Dement Geriatr Cogn Disord, 1997, 8, 240–243. [85] Helisalmi, S; Linnaranta, K; Lehtovirta, M; et al. Apolipoprotein E polymorphism in patients with different neurodegenerative disorders. Neurosci Lett, 1996, 205, 61–64. [86] Stevens, M; van Duijn, CM; de Knijff P; et al. Apolipoprotein E gene and sporadic frontal lobe dementia. Neurology, 1997, 48, 1526–1529. [87] Fabre, SF; Forsell, C; Viitanen, M; et al. Clinic-based cases with frontotemporal dementia show increased cerebrospinal fluid tau and high apolipoprotein E epsilon4 frequency; but no tau gene mutations. Exp Neurol, 2001, 168, 413–418. [88] Bernardi, L; Maletta, RG; Tomaino, C; et al. The effects of APOE and tau gene variability on risk of frontotemporal dementia. Neurobiol Aging, 2006, 27(5), 702-709. [89] Geschwind, D; Karrim, J; Nelson, SF; Miller, B. The apolipoprotein E epsilon4 allele is not a significant risk factor for frontotemporal dementia. Ann Neurol, 1998, 44, 134138. [90] Short, RA; Graff-Radford, NR; Adamson, J; et al. Differences in tau and apolipoprotein E polymorphism frequencies in sporadic frontotemporal lobar degeneration syndromes. Arch Neurol, 2002, 59, 611–615. [91] Riemenschneider, M; Diehl, J; Muller, U; et al. Apolipoprotein E polymorphism in German patients with frontotemporal degeneration. J Neurol Neurosurg Psychiatry, 2002, 72, 639–641. [92] Srinivasan, R; Davidson, Y; Gibbons, L; et al. The apolipoprotein E epsilon4 allele selectively increases the risk of frontotemporal lobar degeneration in males. J Neurol Neurosurg Psychiatry, 2006, 77, 154-158. [93] Engelborghs, S; Dermaut, B; Goeman, J; et al. Prospective Belgian study of neurodegenerative and vascular dementia: APOE genotype effects. J Neurol Neurosurg Psychiatry, 2003, 74, 1148-1151. [94] Verpillat, P; Camuzat, A; Hannequin, D; et al. Apolipoprotein E gene in frontotemporal dementia: an association study and meta-analysis. Eur J Hum Genet, 2002, 10, 399– 405. [95] Conrad, C; Andreadis, A; Trojanowski, JQ; et al. Genetic evidence for the involvement of tau in progressive supranuclear palsy. Ann Neurol, 1997, 41(2), 277-281. [96] Baker, M; Litvan, I; Houlden, H; et al. Association of an extended haplotype in the tau gene with progressive supranuclear palsy. Hum Mol Genet, 1999, 8(4), 711-715. [97] Di Maria, E; Tabaton, M; Vigo, T; et al. Corticobasal degeneration shares a common genetic background with progressive supranuclear palsy. Ann Neurol, 2000, 47(3), 374377. [98] Galimberti, D; Fenoglio, C; Cortini, F; et al. GRN variability contributes to sporadic frontotemporal lobar degeneration. J Alzheimers Dis, 2010, 19(1), 171-7.

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[99] Galimberti, D; Venturelli, E; Villa, C; et al. MCP-1 A-2518G polymorphism: effect on susceptibility for frontotemporal lobar degeneration and on cerebrospinal fluid MCP-1 levels. J Alzheimers Dis., 2009, 17(1), 125-33. [100] Venturelli, E; Villa, C; Fenoglio, C; et al. The NOS3 G894T (Glu298Asp) polymorphism is a risk factor for frontotemporal lobar degeneration. Eur J Neurol., 2009, 16(1), 37-42. [101] Venturelli, E; Villa, C; Scarpini, E; et al. Neuronal nitric oxide synthase C276T polymorphism increases the risk for frontotemporal lobar degeneration. Eur J Neurol, 2008, 15(1), 77-81.


Chapter 9

The Cholinergic Neuron in Alzheimer’s Disease Christian Humpel* and Celine Ullrich

Laboratory of Psychiatry and Exp. Alzheimers Research, Department of Psychiatry and Psychotherapy, Innsbruck Medical University, Austria

Abstract Alzheimer´s disease (AD) is a chronic brain disorder characterized by cognitive decline, neuronal and synaptic loss, beta-amyloid-containing plaques, neurofibrillary tangles, inflammation and cerebrovascular damage. Numerous studies revealed that cholinergic neurons in the basal forebrain (septum, diagonal band of Broca, basal nucleus of Meynert) are affected in AD and a loss of acetylcholine directly correlates with memory dysfunction. (1) We will give an overview on the cholinergic neurons in the basal forebrain and discuss the role of the key enzyme choline acetyltransferase (ChAT). (2) We review the protective role of nerve growth factor (NGF) to support the cholinergic phenotype. (3) We demonstrate different in vitro and in vivo models, which are used to study cholinergic CNS neurons. (4) We reconsider if cholinergic neurons degenerate in AD or if cholinergic neurons only downregulate the key enzyme ChAT. (5) Finally, our review will summarize recent therapeutic strategies on augmenting cholinergic neurotransmission to improve or reverse cognitive deficits in AD. In summary our review focuses on the cholinergic CNS neurons and their role in AD. Alzheimer‟s disease is a severe and chronic degenerative disorder characterized by a progressive neurodegeneration, amyloid-containing plaques, neurofibrillary tangles, as well as cognitive dysfunction. Cholinergic neurons in the basal forebrain are located in six main central nuclei (Ch1-Ch6). The key enzymes for the cholinergic system, choline *

Corresponding author: Dr. Christian Humpel, Department of Psychiatry and Psychotherapy, Anichstr. 35, A-6020 Innsbruck, Austria, Phone: +43-512-504-23712; Fax: +43-512-504-23713, : christian.humpel @i-med.ac.at

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Christian Humpel and Celine Ullrich acetyltransferase (ChAT) and acetylcholinesterase (AChE) can be used for immunohistochemical staining and characterization of the system. Essential for the development and survival of cholinergic neurons in the basal forebrain is the nerve growth factor (NGF). The cholinergic neurotransmitter system in the basal forebrain is severely affected in AD and loss of the neurotransmitter acetylcholine directly correlates with cognitive dysfunction (Perry et al., 1981; Francis et al., 1985). Basic research of the neuropathologic hallmarks and treatment strategies in AD is a fundamental goal, due to immense costs of caring for patients with AD. The current review will highlight present knowledge of the cholinergic dysfunction in AD and will demonstrate different models, which are used to study AD, as well as possible therapeutic approaches.

The Cholinergic CNS Neuron The cholinergic system plays a crucial role in learning, memory and cognition as well as in the control of the cerebral blood flow, cortical plasticity, and sleep-wake cycle (Schliebs and Arendt, 2006; Mufson et al., 2008). Choline acetyltransferase (ChAT) is the key enzyme for the biosynthesis of acetylcholine (ACh) and is a good indicator for the functional activity of cholinergic neurons (Oda, 1999). ChAT mediates the reaction involving the transfer of an acetyl group from acetyl coenzyme A to choline at the synaptic endings of cholinergic neurons (Figure 1A). Two receptors are responsible for the action of acetylcholine, the nicotinic ACh receptors (nAChR) and the muscarinic ACh receptors (mAChR) (Pákáski and Kálmán, 2008). The action of acetylcholine is determined by the activity of the enzyme acetylcholinesterase (AChE), which is located presynaptically as well as postsynaptically and cleaves acetylcholine into choline and acetate (Figure 1A) (Mesulam et al., 2002). In addition, glial cells produce a second cholinesterase, butyrylcholinesterase (BChE), which has the potency to substitute for acetylcholinesterase (Mesulam et al. 2002). After the enzymatical cleavage of acetylcholine, choline is transported back from the synaptic cleft into the presynaptic terminal and recycled. The basal forebrain comprises six main central nuclei of cholinergic neurons (Ch1-Ch6), which extend their cholinergic fibers in specific pathways (Figure 1B) (Mesulam and van Hoesen, 1976; Mesulam et al., 1983; Mufson et al., 2003; Lucas-Meunier et al., 2003). Cholinergic neurons from the septum (Ch1) and the vertical limb of the diagonal band of Broca (Ch2) project only to the hippocampus, whereas pedunculopontinus nucleus (part of Ch5) and laterodorsal tegmental nucleus (Ch6) from the brainstem project to the thalamus (Mesulam et al., 1983; Mufson et al., 2003). Cholinergic nuclei from the lateral part of the horizontal limb of the diagonal band of Broca (Ch3) project to the olfactory bulb (Mesulam et al., 1983). The basal nucleus of Meynert (nBM) (Ch4) contains a population of large cholinergic neurons and their fiber pathway innervates the entire cortex and amygdala (Wenk, 1997; Lucas-Meunier et al., 2003; Schliebs and Arendt, 2006). The homologous structure of the basal nucleus of Meynert in human species conveys to the nucleus basalis magnocellularis in the rat basal forebrain (Wenk, 1997; Lucas-Meunier et al., 2003). In addition, cholinergic interneurons containing NADPH-diaphorase, GABA, calbindin and glutamate are found in diverse brain areas, such as e.g. the striatum (Mufson et al., 2003; Schliebs and Arendt, 2006; Lucas-Meunier et al., 2003).

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Figure 1. Scheme of a cholinergic nerve terminal (A). Choline acetyltransferase (ChAT), enzyme for the formation of acetylcholine (ACh), is anterogradely transported from the cell body to the presynaptic nerve terminal. ChAT mediates the reaction of acetyl-coA and choline into ACh. The vesicular acetylcholine transporter (VAChT) transports ACh into the synaptic vesicles. ACh is released into the synaptic cleft and binds to postsynaptical ACh receptors. Acetylcholinesterase (AChE), located in the presynaptic or postsynaptic membrane, terminates the reaction of ACh by enzymatically cleavage into choline and acetate. Choline is recycled and transported back into the presynaptic terminal by choline transporters. Metabolized acetate is taken up by glial cells. The postsynaptic cell (or adjacent glial cells) secrete the nerve growth factor (NGF), which diffuses to presynaptically located NGF receptors, TrkA or p75NTR. NGF stimulates the neuron as a "target-derived neurotrophic factor". Rat cholinergic central pathways and nuclei (B). Cholinergic neurons are located in six main central central nuclei (Ch1-Ch6), which project to different brain regions

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Nerve Growth Factor and Cholinergic Neurons Nerve growth factor (NGF) was the first discovered neurotrophin (Levi-Montalcini, 1987) and plays a key role in survival and phenotypic maintenance of cholinergic basal forebrain neurons (Salehi et al., 2004). NGF activates various intracellular signaling pathways via two membrane-bound receptors, the p75NTR (p75 neurotrophin receptor) and the TrkA (tyrosine receptor kinase-A) (Yuen et al., 1996; Schindowski et al., 2008; Yuen et al., 1996; Mufson et al., 2008; Schindowski et al., 2008). Binding of NGF to TrkA causes receptor dimerization, which is endocytosed at the synapse and retrogradely transported to the cell body (Rattray, 2001) (Figure 1A). NGF is synthesized as a precursor (proNGF), which is the dominant form of NGF in the central nervous system (CNS) (Fahnestock et al., 2004; Allen and Dawbarn, 2006). NGF is synthesized in the cortex and hippocampus and supports cholinergic neurons as a target-derived neurotrophic factor (Salehi et al., 2004; Allen and Dawbarn, 2006; Schindowski et al. 2008). The role of proNGF and mature NGF appears to be contradictory: while mature NGF maintains survival and function to certain neuronal populations, proNGF triggers cell death through p75NTR (Friedman, 2000). However, various studies suggests that proNGF binds TrkA and promotes neuronal survival and neurite outgrowth similar to mature NGF, but is probably less active than the mature NGF (Fahnestock et al., 2004; Rattenholl et al., 2001). Thus, it is indicated that NGF may elicit cell survival or cell death, depending on receptor and signaling pathways (Friedman, 2000). Nevertheless, in the absence of NGF, cholinergic neurons show cell shrinkage, reduction in fiber density and downregulation of transmitter-associated enzymes, e.g. ChAT or AChE, resulting in a decrease of cholinergic transmission (Svendsen et al., 1991; Weis et al., 2001; Humpel and Weis, 2002). Various studies demonstrated increased levels of NGF in the cortex and decreased levels of NGF in the basal forebrain in AD brains (Schindowski et al., 2008; Mufson et al., 2008; Allen and Dawbarn, 2006). A marked reduction of TrkA has been shown in the basal forebrain and in the cortex of AD brains (Schindowski et al., 2008; Mufson et al., 2003), thus supporting that a dysfunctional retrograde transport could lead to an accumulation of NGF in the cortex resulting in loss of trophic support for cholinergic neurons (Schindowski et al., 2008).

In Vitro and in Vivo Models to Study Cholinergic Neurons The implication of the cholinergic system in the pathology of AD is an important issue in AD research and the cholinergic phenotype can be studied in different in vitro and in vivo models. Dissociated primary cholinergic neurons, which were separated from diverse brain regions, are widely used to study the cholinergic phenotype. The effects of trophic interactions (Hefti et al., 1985; Wainer et al., 1991), toxic stimuli (Heaton et al., 1994) or pharmacological substances (Bailey and Lahiri, 2010, Bennett et al., 2009) on cholinergic neurons provide insights into the cholinergic system. The organotypic brain slice is an outstanding in vitro model to study neurons in an almost intact brain environment. Brain


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slices are able to maintain the survival of different cell types, the cytoarchitecture of the tissue, the connections between cells and neuronal properties (Duff et al., 2002). The organotypic brain slice model has been first introduced by Gähwiler and colleagues (Gähwiler and Hefti, 1984, 1997) and was modified by Stoppini et al. (1991). In slices, individual cells are in close contact and do not lose density-dependent regulatory mechanisms, three dimensional architecture as well as tissue specific transport and diffusion probabilities. Thus, slice cultures provide an easily accessible experimental model for studies of toxic, degenerative and developmental changes in the brain (Zimmer et al., 2000). The determination of cholinergic neurons is performed by immunohistochemical staining against the enzyme ChAT (Figure 2C) and the addition of NGF supports the survival of cholinergic neurons (Figure 2A,D) (Weis et al., 2001; Humpel and Weis, 2002). Single organotypic brain slices of the nBM or co-cultures of the nBM together with cortex have been used to study cholinergic neurons and the effects of aging (Marksteiner and Humpel, 2007), NGF withdrawal (Weis et al., 2001; Humpel and Weis, 2002) and other neuroprotective (Zassler et al., 2005) or neurotoxic (Zassler et al., 2003; Ullrich et al., 2009) substances. Several in vivo models have been used to determine the mechanisms of cholinergic dysfunction, cell death or recovery processes, such as injections of toxins (Smith, 1988; Waite et al., 1995; Nilsson et al., 1992; Weinstock, 1997; Hanin, 1992), genetically modified animals (Smeyne et al., 1994; Capsoni et al., 2006), fimbria-fornix transections (Hefti, 1986; Widmer et al., 1993) or radiofrequency (Dubois et al., 1985; Hepler et al., 1985). The development of cholinodeficient animal models is an important tool for studying the dysfunction of cholinergic neurons in the basal forebrain. The injection of ethylcholine aziridinium (AF64A), a toxin which has a structural similarity to choline, affects reference and working memory in rats and specifically disrupts cholinergic nerve terminals (Weinstock, 1997; Hanin, 1992). A selective lesion of cholinergic nBM neurons can be performed by injection of 192 IgG-saporin, which consists of the ribosome-inactivating protein saporin conjugated to 192 IgG, a monoclonal antibody to the rat p75NTR. This immunotoxin produces selective, dose-dependent and long-lasting cell death among p75NTR-positive cholinergic basal forebrain neurons and leads to an impaired performance in learning and memory tasks (Waite et al., 1995; Wenk, 1997; Schliebs and Arendt, 1998; Gu et al., 2000; Nilsson et al., 1992). Additionally, fimbria-fornix transections represent a well characterized method to study the effects of NGF on the survival of cholinergic neurons after axotomy (Hefti, 1986; Widmer et al., 1993). On the other hand, radiofrequency current is used to destroy neuronal perikarya and fibers and leads to lesions in the nBM (Dubois et al., 1985; Hepler et al., 1985). In the past decades, the development of transgenic animal models of AD provides an excellent opportunity to study the underlying neurobiological mechanisms of AD-related cognitive deficits. The establishment of mice lacking NGF producing cells can be characterized by severe loss of sensory and sympathetic neurons (Schliebs and Arendt, 1998; Winkler et al., 1998). Mice lacking TrkA receptors demonstrate a substantial decrease in AChE immunoreactive fibers in the cholinergic basal forebrain (Smeyne et al., 1994). In AD11 anti-NGF mice, which express transgenic antibodies neutralizing NGF, a progressive cholinergic neurodegeneration is seen, accompanied by intracellular neurofibrillary tangles, deposition of amyloid peptide and an impaired spatial memory (Capsoni et al., 2006).

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Figure 2. Cholinergic neurons in organotypic brain slices. Cholinergic neurons were incubated with (A, C) or without (B) 10 ng/ml nerve growth factor (NGF) for two weeks and stained immunohistochemically for the enzyme choline acetyltransferase (ChAT) using a chromogenic substrate. Note a marked neurodegeneration of cholinergic neurons when slices where incubated without NGF (B). Cholinergic ChAT-like immunoreactivity (green) is cytoplasmic as seen by nuclear DAPI (blue) co-staining and these neurons are embedded in a glutamine synthase positive (red) astroglial network (C). The number of cholinergic neurons remains stable when incubated for up to 6 weeks with NGF (filled squares), but declines markedly without NGF (filled circles). (D). The question (?) was if NGF may enhance the cholinergic ChAT+ neurons when added after 2-weeks to slices incubated without NGF. Our data show that the number of ChAT+ neurons did not increase, suggesting cell death of cholinergic neurons and not only down-regulation of ChAT

The Impairment of the Cholinergic System in AD A significant reduction of cholinergic basal forebrain neurons, as well as a substantial loss of the cholinergic innervation in the cerebral cortex has been observed in AD brains (Mufson et al., 2003; Mufson et al., 2008; Vogels et al., 1990; Mesulam, 2010). Furthermore, a progressive memory impairment and other cognitive dysfunctions characterize the clinically outcome of AD (McKhann et al., 1984). The concentrations of acetylcholine and ChAT are markedly reduced in the frontal, parietal, temporal, and visual cortices (Davies and Maloney, 1976; Bowen et al., 1976; Kásá et al., 1997) and in the hippocampus (Oda, 1999). Similarly, the AChE-positive fibers are significantly declined, most severe in the temporal lobes (Geula and Mesulam, 1989; Kásá et al., 1997). It has been shown, that a loss of 55% of the cortical


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cholinergic fibers was detected in AD brains (Geula and Mesulam, 1996). Furthermore, various studies demonstrated a decreased number of nicotinic and muscarinic (M2) ACh receptors in AD brains, in contrast to the maintenance of postsynaptic muscarinic (M1, M3) receptors (Francis et al., 1999; Mesulam, 2010). This is in line with a previous study by Drachman and Leavitt (1974), demonstrating that patients treated with the muscarinic antagonist scolopamine exhibits similar features to that seen in AD brains. This indicates for the first time that cortical cholinergic neurotransmission plays a crucial role in memory function and in AD pathology. On the contrary, the numbers of ChAT-positive cells in the putamen and the caudate nucleus are not affected in AD (Kásá et al., 1997; Mesulam, 2010), suggesting that this disorder does not affect the entire cholinergic basal forebrain system. The causes for cholinergic degeneration in AD brains are not fully clear, whereas various hypotheses exist. It has been demonstrated that cholinergic neurons in the Ch4 area are the most sensitive neurons to age-related neurofibrillary degeneration (Sassin et al., 2000) and that a decline in the number, size, or function of cholinergic neurons may be responsible for the cognitive impairments associated with aging and AD (Wenk, 1997). A correlation of the loss of cholinergic neurons of the basal forebrain and the extent of cognitive impairment revealed, that at least 30% of the cholinergic basal forebrain neurons need to be degenerated to see clinical symptoms (Schliebs and Arendt, 2006). Neuropathological studies described morphological alterations in cerebral capillaries, dysfunction of the neurovascular unit and ischemic infarcts in AD patients (Iadecola, 2004). Cholinergic neurons are closely associated with cerebral blood vessels of the blood-brain barrier (BBB) and tend to degenerate due to vascular dysregulation (Iadecola, 2004). Considering, Selkoe (2002) suggested that cholinergic hypofunction in AD brains is caused by synaptic dysfunction initiated by progressive accumulation of toxic beta-amyloid. Moreover, it is not known, if the cholinergic depletion is an early or late event in AD. On the one hand, Perry and colleagues (1981) demonstrated that ChAT activity is associated with early stages of AD pathology. Otherwise, it was reported that the loss of cortical cholinergic function and cholinergic markers correlated with severity of neuropathological lesions in AD (Davis et al., 1999). Cholinergic neurons are detected by immunohistochemical analysis for the key enzyme ChAT and a decline directly correlates with loss of cholinergic neurons (Perry et al., 1981; Francis et al., 1985; Schliebs and Arendt, 2006). However, the question remains if cholinergic neurons really die or only downregulate ChAT and cannot be visualized. It is well established that a fimbria-fornix transection leads to a shrunken morphology and a reduced number of ChAT-positive neurons (Naumann et al., 1992, 1994; Hefti, 1986). However, the number of ChAT-positive neurons increases after in vivo axotomy within 6 months (Naumann et al., 1994) suggesting the possibility of regeneration of transmitter synthesis within the cholinergic neurons. In vitro (Hefti et al., 1985) and in vivo (Naumann et al., 1992) studies demonstrated that NGF application results in reversal of ChAT expression and cell atrophy. Moreover, Widmer et al. (1993) demonstrated that ChAT expression declines in transected cholinergic neurons suggesting involvement of p75NTR after axotomy. Additionally, it was demonstrated that dissociated cholinergic neurons cultured with or without NGF showed that NGF enhanced cholinergic neuronal survival, but did not induce cellular ChAT activity (Hatanaka et al., 1988). In organotypic bran slices, axotomized cholinergic neurons cultured without exogenous NGF exhibited reduced and shrunken ChAT-positive neurons (Figure 2B). This reduction can be prevented by application of recombinant NGF (Figure 2A) (Zassler et al., 2005; Humpel and Weis, 2002). Furthermore, it has been demonstrated that NGF did not

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stimulate the cholinergic phenotype in organotypic brain slices of the nBM, but rescued the remaining cholinergic neurons from cell death (Humpel and Weis, 2002). Thus, this raises the question if the cholinergic neurons in AD brains undergo cell death or downregulate their ChAT activity, due to a disturbed NGF homeostasis. Whereas, it is not known if the cholinergic dysfunction is the primary characteristic in AD brains or if the deposition of beta-amyloid and neurofibrillary tangles promote the cholinergic depletion. A link between cholinergic dysfunction and amyloid precursor protein (APP) processing is demonstrated by co-localization studies of AChE with beta-amyloid deposits in AD brains (Mesulam, 1986). This was supported by cell culture studies, showing an upregulation of APP expression after NGF treatment (Villa et al., 2001). Moreover, only little is known regarding the role of the impaired cholinergic system in the phosphorylation of tau. Nonetheless, it has been shown that activation of mAChR decreased tau phosphorylation in various studies (Sadot et al., 1996; Fisher, 1997; Rubio et al., 2006).

Therapeutic Strategies in AD The cholinergic system has become an important target for therapeutic interventions, due to its obvious role in the pathogenesis of AD. Diverse therapeutic strategies are in the focus of intense research, such as the improvement of the cholinergic function in the basal forebrain, as well as the maintenance of the decline in memory and cognitive function. So far the most important therapeutic approach is to increase the availability of acetylcholine by inhibiting AChE and thus to enhance cholinergic neurotransmission (Blennow et al., 2006; Mufson et al., 2008). The efficacy of acetylcholinesterase inhibitors donezepil, rivastigmine, and galantamine has been extensively studied (Darreh-Shori and Soininen, 2010; Nordberg et al., 2009), but acetylcholinesterase inhibitors only temporarily mitigate some of the symptoms in AD. Side effects implicating the gastrointestinal system (e.g. nausea, vomiting, and diarrhoea, as well as fatigue or loss of appetite, limit their use (Mufson et al., 2008). Whereas, cholinesterase inhibitors only counteract AD-related deficiencies in cortical acetylcholine loss, but are not capable of slowing or reversing cholinergic neuronal loss. Alternatively, the cholinergic neurotransmission can be enhanced by activation of presynaptic nicotinic cholinergic receptors through appropriate nicotinic agonists (Oddo and LaFerla, 2006; Buckingham et al., 2009) or by the stimulation of muscarinic ACh receptors through the use of M1 agonists (Avery et al., 1997). Furthermore, recent studies reported a relationship between smoking, nicotinic cholinergic signaling and AD, suggesting that nicotinic treatments improve cognitive functions (Sabbagh et al., 2002; Levin et al., 2006). On the other hand, NGF delivery to the brain of patients appears to be an emerging potential therapeutic approach in AD. It is important to note, that NGF does not cross the BBB and must be delivered directly into the CNS. A continuous intracerebroventricular NGF injection has been tested in clinical trials with AD patients (Olson, 1993; Seiger, 1993; Eriksdotter Jönhagen et al., 1998; Winkler et al., 1998; Mufson et al., 2008). However, the NGF application was stopped because undesirable side-effects occurred, such as weight loss, back pain, pain from stimulation of dorsal root ganglion nociceptive neurons, sympathetic axon sprouting in the cerebral vasculature and Schwann cell hyperplasia (Eriksdotter


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Jönhagen et al., 1998; Tuszynski, 2007). An advanced method for NGF delivery into the AD brain could be gene therapy, where genetically modified cells which express e.g. NGF or ChAT are grafted into the brain (Tuszynski et al., 2005; Smith et al., 1999) or in combination with e.g. adeno-associated virus (AAV) vectors systems (Klein et al., 2000; Blesch et al., 2005; Mandel et al., 2006). Studies performed in aged monkeys using fibroblasts as vehicles of ex vivo NGF gene delivery demonstrated reversal of neuronal atrophy and restoration of cortical cholinergic inputs (Smith et al., 1999). In addition, studies were performed to deliver NGF, coupled to a transport protein across the BBB, such as e.g. transferrin-NGF coupled systems (Granholm et al., 1993; Begley, 2003) or the use of cell-based BBB-carrier systems, such as blood cells containing incorporated drugs (Begley, 2003). The intranasal route of administration of NGF could provide an alternative to ICV infusion and gene therapy (de Rosa et al., 2005; Covaceuszach et al., 2009). Furthermore, a recent in vitro study has shown that NGF loaded monocytes may migrate through a simple BBB and deliver NGF into the brain (Böttger et al., 2010). There will be a need to develop non-invasive delivery methods due to the large number of AD patients. In the case of NGF, this protein must be delivered early before degeneration of cholinergic neurons occurs and must be delivered continuously through life. In summary, cholinergic dysfunction plays an important role in AD and loss of acetylcholine in cortex and hippocampus correlates to cognitive decline. Therapeutic strategies are explored to enhance acetylcholine levels in the brain or to counteract cell death of cholinergic neurons.

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Chapter 10

Retinal Neurodegeneration Is an Early Event in the Pathogenesis of Diabetic Retinopathy: Therapeutic Implications Rafael Simó* and Cristina Hernández

Diabetes and Metabolism Reseach Unit, Institut de Recerca Hospital Universitari Vall D‟Hebron and CIBER for Diabetes and Associated Metabolic Diseases (CIBERDEM, Instituto de Salud Carlos III), Barcelona, Spain

Abstract Diabetic retinopathy (DR) remains the leading cause of blindness among workingage individuals in developed countries. Although tight control of both blood glucose levels and hypertension are essential to prevent or arrest progression of the disease, the recommended goals are difficult to achieve in many patients and, consequently, DR develops during the evolution of the disease. Therefore, new therapeutic strategies based on the understanding of the pathophysiological mechanisms of DR are needed. DR has been classically considered to be a microcirculatory disease of the retina due to the deleterious metabolic effects of hyperglycemia per se, and the metabolic pathways triggered by hyperglycemia. However, before any microcirculatory abnormalities can be detected in ophthalmolscopic examination, retinal neurodegeneration is already present. The two main features of retinal neurodegeneration are apoptosis and glial activation. Most of the information regarding retinal neurodegeneration has been obtained from rats with streptozotocin-induced diabetes (STZ-DM). Streptozotocin (STZ) is a potent neurotoxic agent and is able to produce neural degeneration. Therefore, neurodegeneration observed in rats with STZ-DM could be due to STZ itself rather than the metabolic pathways related to diabetes. However, the recent observation that both apoptosis and glial activation also occur in the retina of diabetic patients, even before any

*

Corresponding author: Diabetes and Metabolism Research Unit., Institut de Recerca Hospital Universitari Vall d‟Hebron., Pg. Vall d‟Hebron 119-129. 08035 Barcelona. Spain, Telephone: 34 934894172. FAX: 34 934894032, E-mail: [email protected]

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Rafael Simó and Cristina Hernández microvascular abnormality could be detected in ophthalmologic examination, reinforces the concept that neurodegeneration is a crucial pathogenic factor of DR. Neuroretinal damage produces functional abnormalities such as the loss of both chromatic discrimination and contrast sensitivity. These alterations can be detected by means of electrophysiological studies in diabetic patients with less than two years of diabetes duration, that is before microvacular lesions can be detected in ophthalmologic examination. In addition, neuroretinal degeneration subsequently initiates and/or activates several metabolic and signaling pathways which participate in the microangiopathic process, as well as in the disruption of the blood-retinal barrier (a crucial element in the pathogenesis of DR). Therefore, the study of the mechanisms that lead to neurodegeneration will be essential for identifying new therapeutic targets in the early stages of DR.

Introduction Diabetic retinopathy (DR) is the leading cause of blindness in working-age individuals in developed countries [1, 2]. The tight control of blood glucose levels and blood pressure are essential in preventing or arresting DR development. However, the therapeutic objectives are difficult to achieve and, in consequence, DR appears in a high proportion of patients. When DR appears, laser photocoagulation remains the main tool in the therapeutic armamentarium. The objective of laser photocoagulation is not to improve visual acuity but to stabilize DR, thus preventing severe visual loss. When laser photocoagulation is indicated in time, the risk of blindness is reduced by 90% in the following 5 years, and the loss of visual acuity is reduced by 50% in those patients with macular edema. However, timely indication is often passed and, therefore, the effectiveness of laser photocoagulation in current clinical practice is significantly lower. In addition, laser photocoagulation destroys a part of the healthy retina and, in consequence, side effects such as loss in visual acuity, impairment of either dark adaptation and colour vision, and visual field loss may appear. Vitreo-retinal surgery could be indicated in advanced stages of DR (ie., hemovitreous, retinal detachment). However, this therapeutic option requires a skilful team of ophthalmologists, is expensive, and fails in more than 30% of cases. With this scenario, it seems clear that new treatments based on the physiopathological knowledge of DR are needed. DR has been classically considered to be a microcirculatory disease of the retina due to the deletereous metabolic effects of hyperglycemia per se, and the metabolic pathways triggered by hyperglycemia (polyol pathway, hexosamine pathway, DAG-PKC pathway, advanced glycation end-products [AGEs] and oxidative stress). However, before any microcirculatory abnormalities can be detected under ophthalmoscopic examination, retinal neurodegeneration must be already present. In other words, retinal neurodegeneration is an early event in the pathogenesis of DR which antedates and participates in the microcirculatory abnormalities that occur in DR [3-6]. Therefore, the study of the mechanisms that lead to neurodegeneration will be essential for identifying new therapeutic targets in the early stages of DR. This is the main aim of the present project.

Retinal Neurodegeneration Is an Early Event in the Pathogenesis of Diabetic…

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Figure 1. Overexpression of GFAP in Müller cells (glial activation) in the retina from a representative diabetic donor (right panel) in comparison with non-diabetic donor (left panel). ONL: outer nuclear layer. INL: Inner nuclear layer GCL: Gnglionar cell layer

Neurodegeneration as an Early Event in the Pathogenesis of DR This concept was first introduced by Barber et al. [3]. These authors observed that one month after inducing diabetes in rats by using streptozotocin there was a high rate of apoptosis (TUNEL positive cells) in the neuroretina without a significant apoptosis in endothelial cells. In the same paper the authors compared the retinas from diabetic (n=2) vs. nondiabetic (n=3) donors and found a higher rate of apoptosis in the neuroretina from diabetic donors, even in the case of a diabetic donor without microvascular abnormalities. These findings have been further confirmed in experimental models. In addition, it has been demonstated that, apart from apoptosis, another of the features of retinal neurodegeneration is glial activation [4-8]. Our research group has been able to demonstrate that both apoptosis and glial activation also occur in the retina of diabetic patients (n=10) and precede microvascular abnormalities [9, 10] (Figure 1). In addition, these changes are also present in retinal explants cultured with a media with a high content of AGEs [11]. These findings suggest not only that neurodegeneration is an early event in the natural history of DR but also that it could play a crucial role in its pathogenesis. Neuroretinal damage produces functional abnormalities such as the loss of both chromatic discrimination and contrast sensitivity. These alterations can be detected by means of electrophysiological studies in diabetic patients with less than two years of diabetes duration, that is before microvacular lesions can be detected under ophthalmologic examination [6, 12, 13]. In addition, neuroretinal degeneration will initiate and/or activate several metabolic and signalling pathways that will participate in the microangiopathic process, as well as in the disruption of the blood-retinal barrier (a crucial element in the pathogenesis of DR). Nevertheless, these metabolic pathways remain to be characterized. The mechanisms involved in DR neurodegeneration are poorly understood. In addition, it is unknown which of the two primordial pathological elements (apoptotis or glial activation) is the first to appear and is, in consequence, the primary event. Nevertheless, it seems that these diabetes-induced changes occur in the early stages of DR and that they are closely related. P38 MAP kinase activation has been involved in the apoptotic retinal cell death that occurs in DR [6, 12, 13]. Apoptosis can be secondary to the classic mechanism related to caspase activation or can be due to a caspase-independent pathway. In this regard, it has been shown that high glucose concentrations can induce neuroretinal apoptosis without an increase

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in the caspase pathway. This caspase-independent pathway is at least in part mediated by the translocation of the apoptosis inducing factor (AIF) from the mitochondria to the nuclei [14]. Glial degeneration consists of astrocyte degeneration and the hypertrophy and hyperplasia of Müller cells. These activated or “gliotic” cells can be identified by means of immunohistochemical procedures as cells with prolongations similar to end-feeds that cross through all the retinal layers and reach the limiting inner membrane (Figure 1). However, although hypertrophic these cells are dysfunctional. In normal conditions Müller cells act as suppliers of retinal neurons and endothelial cells (which are the main constituents of the inner blood-retinal barrier). One of the primary functions of Müller cells is the absorption of fluid through potassium channels (mainly the Kir4.1), which allows transcellular water transport. Moreover, they play a key role in the uptake of potential neurotoxic metabolites such as glutamate and GABA [15-17]. Therefore, Müller cell dysfunction can precipitate neuroretinal apoptosis due to the accumulation of water (retinal edema) and neurotoxic metabolites. Moreover, glial cells are in close contact with endothelial cells of capillaries and, in consequence, glial degeneration could contribute to inner BRB disruption [18]. However, this possibility has still to be confirmed and the potential mechanisms involved are far from being elucidated.

Experimental Models to Study Retinal Neurodegeneration in the Setting of DR The experimental model currently used to study retinal neurodegeneration in DR is the rat with STZ-DM. In this model the presence of neural apoptosis and glial reaction has been detected one month after starting diabetes [3, 6-8]. In addition, electroretinographic abnormalities have been shown two weeks after inducing diabetes [19]. Retinal ganglion cells (RGCs) are the earliest cells affected and with the highest rate of apoptosis [20]. However, an elevated rate of apoptosis has also been observed in the outer nuclear layer (photoreceptors) and in the retinal pigment epithelium (RPE) [21, 22]. The mouse has been much less frequently used than the rat as an experimental model for the study of DR and retinal neurodegeneration. This is because it is more resistant to the STZ effect (mice need 3–5 doses of STZ to induce diabetes whereas in rats one dose is enough), it has a lower eye cup and presents higher resistance to the development of DR lesions. This relative protection to developing pathological lesions related to diabetes can be partly attributed to lower activity in the polyol pathway in comparison with rats [8, 23]. The interpretation of the results of retinal neurodegeneration in murine models with diabetes induced by STZ is hampered by the neurotoxic effect of STZ. It is worthy of mention that pathological changes to the brain after intraventricular injection of STZ are very similar to the neurodegeneration reported in DR [24, 25]. Therefore, neurodegeneration (apoptosis + glial activation) observed in rats with STZ-DM could be due to STZ itself rather than the metabolic pathways related to diabetes. For this reason, it would be advisable to use murine models with a spontaneous development of diabetes or at least experimental models in which diabetes has not been induced by a neurotoxic drug.


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Figure 2. Retinal neurodegeneration observed in a C57BL/KsJ-db/db diabetic mouse (right panel) at 4 weeks after starting diabetes in comparison with a C57BL/KsJ non-diabetic mouse (left panel)

Because of its great potential for genetic manipulation, the mouse offers a unique opportunity to study the molecular pathways involved in disease development. Among mice, C57BL/KsJ-db/db is the model that best reproduces the neurodegenerative features observed in patients with DR. C57BL/KsJ-db/db mice carry a mutation in the leptin receptor gene, and they are a model for obesity-induced type 2 diabetes. They develop hyperglycemia starting at 8 weeks of age as a result of excessive food consumption. It is noteworthy that they present an abundant expression of aldose-reductase in the retina (this is an important differential trait from other mouse models). Therefore C57BL/KsJ-db/db is a good model for investigating the underlying mechanisms of retinal neurodegeneration associated with diabetes and for testing new drugs. We have recently had the opportunity to verify retinal neurodegeneration in this model at 4 weeks after starting diabetes (Figure 2)

Therapeutic Candidates to be Explored As mentioned in the introduction, it is essential to find new drugs for DR treatment. In this regard, the results of two seminal studies: the FIELD study on DR [26] and the DIRECT programme [27, 28] have been recently published. These studies have demonstrated that fenofibrate (FIELD study) and candesartan (DIRECT programme) have beneficial effects in non-advanced DR independently of their primary actions (lipid lowering in the case of fenofibrate and lowering blood pressure in the case of candesartan). The mechanisms involved in these beneficial effects remain to be explored.

Fenofibrate It has been shown that fenofibrate exerts a neuroprotective action in a murine model of cerebral ischemia [29, 30], Parkinson‟s disease [31] and cerebral trauma [32]. Antioxidant, antiinflammatory and antiapoptotic mechanisms have been involved. However, there are no studies in DR.

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Angiotensin II receptor blockers (ARBs) Angiotensin and its receptors are overexpressed in the retina of diabetic patients and ARBs exert various pleiotropic effects within the retina (unrelated to blood pressure) that explain their therapeutic effectiveness in DR. Regarding neuroprotection it has been recently reported that candesartan (the ARB with the better diffusion across the blood-brain barrier) has a neuroprotective effect after brain focal ischemia [33, 34]. In addition, telmisartan and valsartan inhibit the synaptophysin degradation that exists in the retina of a murine model of DR [35]. Furthermore, valsartan is able to prolong the survival of astrocytes and reduce glial activation in the retina of rats with hypoxia-induced retinopathy [36]. Taken together it seems that neuroprotection could be a relevant mechanism involved in the beneficial effects of candesartan (and eventually other ARBs) in DR. In fact, it has been recently shown that losartan has neuroprotective effects in the retina of rats by re-establishing oxidative redox and the mitochondrial function [37]. Nevertheless, there are no specific studies on the neuroprotective effects of ARBs in patients with DR.

Other Candidates Apart from fenofibrate and ARBs there are other potential therapeutic candidates. Based on our background we have selected the following: Somatostatin Both neuroretina and retinal pigment epithelium produce somatostatin (SST), a peptide with antiangiogenic and neuromodulatory actions. These effects are mediated by its binding to 5 receptors which are also expressed in the retina. We have detected a marked deficit of SST (mainly due to SST-28) in the vitreous fluid of diabetic patients [38, 39]. In addition, we have also demonstrated that this deficit also exists in the retina at the early stages of DR and is associated with retinal neurodegeneration [9, 40]. Furthermore, intravitreal delivery of SST has been proposed as a new therapeutic approach for DR [41]. Recently, it has been shown that SST and SST analogues with selective high affinity to SST receptors 2 and 5 protect the retina from excitotoxicity induced by (RS)-alpha-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid hydrobromide in rats [42, 43]. However, to the best of our knowledge there are no studies evaluating the neuroprotective effect of SST in DR. Erythropoietin In previous studies we have demonstrated that in DR there is an increase in erythropoietin (Epo) production by the retina that is unrelated to hypoxia and occurs in the early stages of the disease [44-46]. Since Epo has neuroprotective properties, we have suggested that this increase detected in the retina of diabetic patients could be contemplated as a compensatory mechanism. In this regard, there is growing evidence that intravitreous or direct delivery of Epo to the retina can prevent retinal neurodegeneration and the breakdown of the bloodretinal barrier (BRB) [47, 48]. In addition, Epo protects retinal pigment epithelial cells from oxidative damage [49, 50]. Nevertheless, there are no data on this issue in murine models with spontaneous diabetes.


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Ascorbic acid We have recently compared by metabonomic analysis the metabolite profiles of vitreous humours of diabetic patients with proliferative diabetic retinopathy (PDR) and non-diabetic subjects. In this study we have detected a marked reduction of ascorbic acid levels in the vitreous fluid in patients with PDR (Invest Ophthalmol Vis Sci –first revision-). Acid ascorbic (AA), which is an essential substance in humans, acts as a cofactor in the enzymatic biosynthesis of collagen, catecholamines, and peptide neurohormones [51]. However, one of its most important functions is to act as an antioxidant and/or free radical scavenger [51-53]. Although most animals can synthesize vitamin C from glucose, humans can only acquire the vitamin from dietary sources because they lack gluconolactone oxidase, the enzyme required for AA biosynthesis. Vitamin C exists in two major forms. The charged form, ascorbic acid (AA), is taken into cells via sodium-dependent facilitated transport. The uncharged form, dehydroascorbate (DHA), enters cells via glucose transporters (GLUT) and is then converted back to AA within these cells. Retinal cells appear to be dependent on GLUT-1 transport of DHA rather than sodium-dependent AA uptake [54]. DHA uptake through facilitative glucose transport is competitively inhibited by D-glucose. In fact, the molecule of AA is very similar to D-glucose. Therefore, it could be postulated that chronic hyperglycemia of long-standing diabetes reduces DHA transport via GLUT1 at the blood retinal barrier (BRB). Exclusion of DHA from cells by hyperglycemia deprives the cells of the central antioxidant action of AA, thus favoring the accumulation of reactive oxygen species [55]. It should be noted that the retina is the only neural tissue that has a direct and frequent exposure to light, thus leading to free radical production due to photo-oxidation which becomes extremely toxic to retinal cells [56]. AA is present in the retina at a high concentration compared with its presence in other humans organs, and it is able to protect the retina against oxidative damage [51, 56, 57]. In fact, we have found 20 fold higher levels of AA in the vitreous fluid than in serum (unpublished results). Given that oxidative stress is a key factor in the pathogenesis of DR, the significant lower levels of AA detected in the vitreous fluid of PDR patients, point to this deficit as a crucial factor in determining DR development. AA is a required cofactor for several intracellular hydroxylases, including proline hydroxylase and dopamine hydroxilase. Therefore, AA deficit could also participate in the impairment of neuropetide production and, therefore, in neurodegeneration. In this regard, it has been reported that AA added to cultures of SH-SY5Y cells (cells derived from neuroblastoma) dramatically reduces the apoptosis induced by oxidative stress and decreases beta-amyloid protein production [58]. At present there are no published studies on the effectiveness of ascorbic acid as a neuroprotective agent in DR. In addition, it has been demonstrated that AA has antiangiogenic properties [59, 60] and, consequently, the lower levels that exist in diabetic patients can contribute to neovascularization, the hallmark of PDR. There are two main mechanisms which account for the lower levels of AA detected in the vitreous fluid of PDR patients: First, competitive inhibition mediated by hyperglycema in AA transport from systemic circulation to the retina; Second, AA consumption that exists in the diabetic retina in order to compensate the elevated degree of oxidative stress. Taken together, AA can be contemplated as a new therapeutic target. Prospective trials using diet supplements of vitamin C for preventing or arresting DR, and experimental studies addressed to increasing AA transport across BRB in the presence of hyperglycemia are needed.

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Conclusion The two most common forms of diabetes (type I and type II) are increasing at alarming rates in developed countries, and the complications associated with diabetes impose enormous burdens on health care systems. It should be emphasized that DR remains a leading cause of blindness in the working-age population and represents a major concern for patients with diabetes and for those who treat them, from both a quality of life and an economic standpoint. By identifying new targets for the detection and treatment of DR in its early stages, we will be able to reduce the healthcare costs associated with DR and will contribute towards improving the quality of life of diabetic patients. At present, DR is diagnosed by ophthalmoscopic examination in which we are looking for microvascular abnormalities (ie., mycroaneurisms, microhaemorrhages, hard exudades, neovessels). However, before any microcirculatory abnormalities can be detected by opthalmoscopic examination, retinal neurodegeneration is already present. Therefore, it is reasonable to hypothesize that retinal neurodegeneration is an early event in the pathogenesis of DR which precedes and mediates the microcirculatory abnormalities that occur in DR. Hence, the development of methods to explore the consequences of retinal neurodegeneration (i.e., electroretinograms, visual evoked potentials, optical coherence tomography [OCT]) will be essential not only for detecting the early stages of DR but also for testing new therapeutic agents. The effectiveness of current treatments of DR is limited, and they are currently indicated at too advanced stages of the disease. Therefore, novel strategies to detect, prevent and treat DR in its earliest stages are needed. The study of factors involved in neurodegeneration permit us to identify new therapeutic targets in the early stages of DR and, in consequence, to bring about improvements in clinical practice in the medium term. The combination of basic research focused on retinal neurodegeneration and clinical trials addressed to testing new neuroprotective agents in DR will open a new scenario aimed at reducing the burden and at improving the clinical outcome of this devastating complication of diabetes.

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Chapter 11

Molecular Imaging and Parkinson’s Disease Valentina Berti, Cristina Polito, Maria T. R. De Cristofaro and Alberto Pupi.

Clinical Pathophysiology Department, Nuclear Medicine Unit, University of Florence, Florence, Italy

Abstract Parkinson‟s disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic (DA) terminals in the striatum, resulting in functional changes in frontostriatal circuits. DA transporter imaging ([123I]FP-CIT SPECT imaging) and brain metabolic imaging 18F ([ ]FDG PET imaging) have been broadly employed to explore the biological substrate of PD, and together they could highlight the pathological processes occurring in early stages of PD. To evaluate the functional association between DA degeneration and cortical metabolism we performed both [123I]FP-CIT SPECT and [18F]FDG PET in the same PD sample; through a multiple regression analysis with SPM we explored the correlation between putaminal DA degeneration and cortical metabolic rate of glucose. In the putamen, which is the first and most affected striatal region in PD, the severity of dopaminergic impairment is directly related to cortical hypometabolism in premotor, dorsolateral prefrontal, anterior prefrontal and orbitofrontal cortices. [123I]FP-CIT SPECT and [18F]FDG PET allow to identify the early functional alterations in the frontostriatal circuits involved in PD.

Introduction Molecular imaging has been widely used for the assessment of several neurological disorders, and it has been demonstrated to provide useful complementary functional

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informations to anatomic imaging, such as specific receptor density, cerebral perfusion, cerebral metabolic rate of oxygen or cerebral metabolic rate of glucose. Functional neuroimaging, in particular with [18F]fluorodeoxyglucose ([18F]FDG) and positron emission tomography (PET), has revolutionized scientists‟ ability to accurately study cerebral glucose consumption of both normal and disordered brain. Most important, because of the intimate relationship between neural activity and brain metabolism, this technique can reveal the changes within local circuits of the brain at rest, thus highlighting the cerebral dysfunction closer to the pathology. This ability has provided an obvious opportunity to study several neurological disorders, such as neurodegenerative diseases.

Parkinson’s Disease The need for accurate and early diagnosis of neurodegenerative diseases motivated the use of molecular imaging techniques, which are able to show the presence of pathological alterations even before the condition is recognized clinically. Parkinson‟s disease (PD), as other neurodegenerative disorders, is characterized by a long preclinical period, during which potential therapeutic and restorative treatments could have a strong effect. For this reason, during last decades increasing attention has turned toward the application of neuroimaging techniques in PD. PD is a progressive neurodegenerative disorder, resulting from the progressive death of dopaminergic neurons in the nigrostriatal pathway, which is the best recognized neuropathological feature in PD and causes the alteration of cortico-striatopallidothalamocortical circuits [Wichmann T, et al. 2003]. The alterations in brain circuits occurring in PD result in motor disturbances, which begin only after a loss of approximately 70-80% of striatal dopamine- thus, there is a long latent period which precedes the development of clinical symptoms [Huang WS, et al. 2001]. Through the study of PD typical pathophysiological alterations, and therefore through the analysis of alterations occurring in striato-cortical circuits, it could be possible to obtain important informations about the pathways themselves and their functioning. N-ω-fluoropropyl-2β-carbomethoxy-3β-{4-iodophenyl}-nortropane ([123I]FP-CIT) single photon emission computed tomography (SPECT) has been used to estimate the loss of striatal dopaminergic terminals and it is the most widely used diagnostic technique to assess the integrity of the dopaminergic system in vivo [Booij J, et al. 2001; Spiegel J, et al. 2007]. The striatal radioactivity measured after administration of [123I]FP-CIT is a function of the quantity of dopaminergic terminals in the striatum. In patients with PD, decreased [123I]FPCIT uptake in the striatum has been reported by numerous studies, with the decrease being much more severe in the putamen than in the caudate nucleus. Moreover, PD patients showed an inverse correlation between the degree of motor deficit and [123I]FP-CIT uptake in the striatum, especially in the putamen [Marshall V, et al. 2001], and this correlation has been found even in early stages of disease [Berti V, et al. 2008]. Nigrostriatal dopaminergic neuronal loss in PD is, however, the cardinal but not the only neuro-functional step in the pathologic progression of the disease [Bezard E, et al. 2003; Braak H, et al. 2002; Obeso JA, et al. 2004]. PET imaging with FDG as been widely employed as a measure of local synaptic activity in the resting state and it can provide

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inferences regarding the status of neural pathways in PD patients. Indeed, the neuropathological processes can alter the functional connectivity across the entire brain in a disease-specific manner [Eckert T, et al. 2005] and [18F]FDG PET can highlight local changes in brain metabolism accompanying local changes in neural activity. [18F]FDG PET can improve diagnostic accuracy in the evaluation of patients with movement disorders, and recently it has been demonstrated that the expression of specific patterns of cortical hypometabolism could help in the differential diagnosis of parkinsonisms [Spetsieris PG, et al. 2009]. A number of reports described the expression of an abnormal PD related metabolic pattern, characterized by increased pallido-thalamic and pontine activity, associated with reductions in lateral frontal, paracentral, inferior parietal and parieto-occipital regions [Fukuda M, et al. 2001; Huang C, et al. 2007]. Besides, several studies showed the correlation between regional cortical hypometabolism and severity of motor impairment in PD, demonstrating an inverse correlation involving in particular anterior cingulate gyrus, orbitofrontal and occipitotemporal regions [Nagano-Saito A, et al. 2004]. However, even though both [123I]FP-CIT SPECT and [18F]FDG PET are able to identify the presence of PD pathophysiological alterations, taken apart they do not provide specific information about the neural circuits involved in the disorder. Only from the combination of the two neuroimaging techniques and thus from the relation of the impairment of nigrostriatal dopaminergic system with cerebral metabolic reductions, it could be possible to indirectly show the striato-cortical circuits in PD. Recently, our group evaluated the functional association between dopaminergic degeneration and cortical metabolism performing both [123I]FP-CIT SPECT and [18F]FDG PET in the same group of PD de novo patients; through a multiple regression analysis with Statistical Parametric Mapping (SPM) we explored the correlation between striatal dopaminergic degeneration and cortical metabolic rate of glucose (CMRglc), thus indirectly highlighting the alteration of striato-cortical circuits in PD [Berti V, et al. 2009].

Figure 1. Statistical parametric maps (SPMs) showing cortical regions with a direct association with putaminal dopaminergic impairment. In PD patients, the putaminal dopaminergic neuronal loss correlates with hypometabolism in premotor, dorsolateral prefrontal, anterior prefrontal and orbitofrontal cortices. From left to right side of figure: SPMs are displayed on the right and left lateral views of a 3D rendered standardized MRI

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In the putamen, which is the first and most affected striatal region in PD, the severity of dopaminergic impairment is directly related to cortical hypometabolism in premotor, dorsolateral prefrontal, anterior prefrontal and orbitofrontal cortices. Consistently with what is known about the organization of basal ganglia circuits [Albin R, et al. 1989; Obeso JA, et al. 2000], striatal dopaminergic impairment correlates with hypometabolism in several frontal areas. These findings are consistent with the pathophysiological changes occurring in the functional organization of the basal ganglia in PD, since the increased neuronal firing activity in the output nuclei of the basal ganglia characterizing PD leads to excessive inhibition of thalamocortical projections, which may be at the basis of reduced CMRglc in specific regions [Obeso JA, et al. 2008]. Besides, the correlation between striatal dopaminergic impairment and hypometabolism in frontal cortex emphasizes the functional inter-relationships between the neocortex and the striatum, consistently with the concept of fronto-striatal loops, which has been widely used to explain the functional organization of frontostriatal circuits. According to this model, this neural system includes five parallel but functionally distinct loops, including a “motor” loops and complex “non-motor” loops, which are involved in cognitive functions. At the striatum level, the motor loop is mostly centered on the putamen and the associative loop mostly on the caudate [Parent A, et al. 1995; Alexander GE, et al. 1986]. The functional correlation between dopaminergic loss in the putamen and cortical hypometabolism in the premotor cortex indirectly reflects the presence of the classical frontostriatal motor loop. However, the association between putaminal dopaminergic impairment and cortical hypometabolism is not confined to the frontal regions belonging to the motor loop, but it involves also dorsolateral, anterior prefrontal, and orbitofrontal regions, which are typically part of the associative and limbic loops [Alexander GE, et al. 1986]. These results can be explained by the parallel loss of dopaminergic terminals in putamen and in the caudate nucleus (even if to a minor extent in the latter), which is demonstrated by the correlation between putaminal and caudate nucleus dopaminergic impairment. Indeed, even in the early stages of the disease, the loss of dopaminergic projections in PD is not confined to the putamen but at the same time also occurs in the caudate nucleus and in ventral striatum (even if they are involved lately and to a lesser extent), which are the striatal relais of associative and limbic frontostriatal loops, therefore connected with dorsolateral prefrontal, anterior prefrontal and orbitofrontal cortices [Alexander GE, et al. 1986].

Conclusion In conclusion, through the study of the pathophysiological alterations occurring during a disease condition, it is possible to obtain important informations about specific brain circuits and their functioning. As an example, the double-tracer study of patients affected by PD, which, through the nigrostriatal neuronal loss affects striato-cortical brain circuits, indirectly highlithed the presence of specific brain circuits. Indeed, the correlation of dopaminergic impairment in specific striatal regions with cortical metabolism has shown pathways connecting the putamen and premotor and prefrontal cortex.

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Interestingly, the functional informations provided by molecular imaging are able to show and confirm several anatomical informations, such as anatomical connections between brain regions.

References Albin, R; Young, AB; Penny, JB. The functional anatomy of basal ganglia disorders. Trends Neurosci, 1989, 12, 366-75. Alexander, GE; DeLong, MR; Strick, PL. Parallel organization of functionally segregated circuits linking basal ganglia and cortex. Annu. Rev. Neurosci., 1986, 9, 357–381. Berti, V; Pupi, A; Ramat, S; Vanzi, E; De Cristofaro, MT; Pellicanò, G; Mungai, F; Marini, P; Sorbi, S. Clinical correlation of the binding potential with 123I-FP-CIT in de novo idiopathic Parkinson's disease patients. Eur J Nucl Med Mol Imaging, 2008, 35, 2220-6. Berti, V; Polito, C; Ramat, S; Vanzi, E; De Cristofaro, MT; Pellicanò, G; Mungai, F; Marini, P; Formiconi, AR; Sorbi, S; Pupi, A. Brain metabolic correlates of dopaminergic degeneration in de novo idiopathic Parkinson's disease. Eur J Nucl Med Mol Imaging, 2009, Sep 2. Bezard, E; Gross, CE; Brotchie, JM. Presymptomatic compensation in Parkinson‟s disease is not dopamine-mediated. Trends Neurosci., 2003, 26, 215–21. Booij, J; Speelman, JD; Horstink, MWIM; Wolters, EC. The clinical benefit of imaging striatal dopamine transporters with [123I]FP-CIT SPET in differentiating patients with presynaptic parkinsonism from those with other forms of parkinsonism. Eur J Nucl Med,, 2001, 28, 266–72. Braak, H; Del Tredici, K; Bratzke, H; Hamm-Clement, J; Sandmann-Keil, D; Rüb, U. Staging of the intracerebral inclusion body pathology associated with idiopathic Parkinson's disease (preclinical and clinical stages). J Neurol., 2002, 249(Suppl 3), III/1–5. Eckert, T; Eidelberg, D. Neuroimaging and therapeutics in movement disorders. NeuroRx, 2005, 2, 361–371. Fukuda, M; Edwards, C; Eidelberg, D. Functional brain networks in Parkinson‟s disease. Parkinsonism and Related Disorders, 2001, 8, 91–4. Huang, WS; Lin, SZ; Lin, JC; Wey, SP; Ting, G; Liu, RS. Evaluation of early-stage Parkinson's disease with 99mTc-TRODAT-1 imaging., J Nucl Med, 2001, 42, 1303-8. Huang, C; Tang, C; Feigin, A; Lesser, M; Ma, Y; Pourfar, M; et al. Changes in network activity with the progression of Parkinson‟s disease. Brain, 2007, 130, 1834–46. Marshall, V; Grosset, D. Role of dopamine transporter imaging in routine clinical practice. Mov Disord, 2003, 18, 1415–23. Nagano-Saito, A; Kato, T; Arahata, Y; Washimi, Y; Nakamura, A; Abe, Y; et al. Cognitiveand motor-related regions in Parkinson‟s disease: FDOPA and FDG PET studies. NeuroImage, 2004, 22, 553–61. Obeso, JA; Rodriguez-Oroz, MC; Rodriguez, M; Lanciego, JL; Artieda, J; Gonzao, N; et al. Pathophysiology of the basal ganglia in Parkinson‟s disease. Trens Neurosci, 2000, 2, S8–19.

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Obeso, JA; Rodriguez-Oroz, MC; Lanciego, JL; Rodriguez Diaz, M. How does Parkinson‟s disease begin? The role of compensatory mechanisms. Trends Neurosci., 2004, 27, 125– 7. Obeso, JA; Marin, C; Rodriguez-Oroz, C; Blesa, J; Benitez-Temino, B; Mena-Segovia, J; et al. The basal ganglia in Parkinson‟s disease: current concepts and unexplained observations. Ann Neurol, 2008, 64, S30–46. Parent, A; Hazrati, LN. Functional anatomy of the basal ganglia: I.The cortico-basal gangliathalamo-cortical loop. Brain. Res. Brain Res. Rev., 1995, 20, 91–127. Spetsieris, PG; Ma, Y; Dhawan, V; Eidelberg, D. Differential diagnosis of parkinsonian syndromes using PCA-based functional imaging features. Neuroimage, 2009, 45, 124152. Spiegel, J; Hellwig, G; Samnick, S; Jost, W; Mollers MO, Fassbender K, et al. Striatal FPCIT uptake differs in subtypes of early Parkinson‟s disease. J Neural Transm, 2007, 114, 331–5. Wichmann, T; DeLong, MR. Pathophysiology of Parkinson's disease: the MPTP primate model of the human disorder. Ann. N. Y. Acad. Sci., 2003, 991, 199–213.

Index

A Abraham, 185, 187 absorption, 206 acetylcholine, xi, 189, 190, 191, 194, 196, 197, 198, 200, 201 acetylcholinesterase, xi, 189, 190, 196, 199 acetylcholinesterase inhibitor, 196 acid, ix, 3, 30, 43, 68, 74, 82, 83, 111, 113, 120, 132, 136, 138, 141, 146, 147, 155, 156, 157, 164, 175, 177, 182, 202, 208, 209, 214 adamantane, 103 adaptation, 117, 204 adenine, 2, 4, 31, 38, 108 adenovirus, 161 adhesion, 161, 164 adipose, ix, 107, 108, 109, 119 adipose tissue, ix, 107, 108, 109, 119 ADP, 5, 6, 105, 108, 109, 127 advantages, ix, 87, 99 aetiology, 176 aggregation, 20, 46, 52, 54, 57, 60, 65, 82, 176 aging population, 80 agonist, 88, 95, 96, 97, 100, 140, 153 AIDS, 15, 18, 19, 21, 24, 28, 34, 36, 38, 39, 41, 42 albumin, 92, 113 alcohol use, 170 alcoholism, 83 alcohols, 55 allele, xi, 170, 174, 177, 178, 179, 180, 181, 182, 185, 187 ALS, viii, 3, 7, 8, 20, 45, 46, 53, 54, 55, 56, 57, 58, 60, 114, 116, 177, 178, 184 alters, 36, 82, 84, 134, 147, 148, 150, 186 amines, 76, 78, 83 amino acids, 109, 131 ammonium, 69 amygdala, 190

amyloid beta, 38, 39, 122 amyloid deposits, 196 amyotrophic lateral sclerosis, viii, 3, 8, 27, 29, 40, 45, 46, 56, 57, 58, 60, 114, 123, 124, 145, 154, 184 anatomy, 219, 220 anesthetics, ix, x, 155, 156, 157, 162, 163, 164, 165, 166 angiogenesis, 82, 213, 214 ANOVA, 70, 71, 72, 73, 74, 75, 76, 77, 92, 97 antibiotic, 46, 131, 138 antibody, 92, 138, 193, 199 anticonvulsant, 163 antidepressant, 65, 81 antigen, 161, 171 anti-inflammatory drugs, 55, 56 antioxidant, 2, 3, 7, 9, 11, 17, 19, 22, 23, 24, 27, 30, 33, 34, 37, 38, 42, 43, 47, 49, 60, 65, 112, 114, 117, 132, 138, 209 antisense, 58, 166, 168 antisense oligonucleotides, 166 apoptosis pathways, 89, 147 apoptotic mechanisms, 100, 133, 134, 144 appetite, 70, 196 Arabidopsis thaliana, 48, 58 architecture, 183, 193 argon, 91 arrest, xi, 9, 13, 33, 170, 203 artery, 83, 127 aryl hydrocarbon receptor, 161 ascorbic acid, 68, 209, 214 aseptic, 90 aspartate, ix, 87, 88, 102, 103, 109, 126, 131, 156, 157, 158, 168, 169, 171, 172 aspartic acid, 156 asphyxia, 127, 143, 145, 146 assessment, 80, 82, 99, 101, 215

222

Index

astrocytes, vii, 1, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 27, 29, 30, 31, 36, 38, 39, 40, 41, 42, 43, 50, 83, 88, 105, 136, 208 astrocytoma, 36 astrogliosis, 50, 57, 138 asymmetry, 132 asymptomatic, 36 ataxia, vii, 1, 8, 12, 30, 31, 43, 176 atherosclerosis, 24, 41, 43 ATP, ix, 5, 6, 78, 107, 108, 109, 110, 112, 113, 117, 123, 131 atrophy, 23, 135, 148, 195, 197 Austria, 189 autoimmune diseases, 180 autosomal dominant, x, 173, 175, 176, 182 autosomal recessive, 8 axons, 123, 150, 174

B back pain, 196 background noise, 55 bacteria, 6 barriers, 214 basal forebrain, xi, 189, 190, 192, 193, 194, 195, 196, 197, 199, 200, 202 basal ganglia, 113, 218, 219, 220 base pair, 177, 178 basic research, 210 BBB, 22, 23, 24, 195, 196 Bcl-2 proteins, 170 behaviors, 198 beneficial effect, 54, 125, 140, 142, 180, 207, 208 benzodiazepine, 152 biochemistry, 142 biological sciences, 46 biosynthesis, 30, 190, 209 biosynthetic pathways, 108 blindness, xi, 203, 204, 210 blood flow, 88, 190 blood pressure, 204, 207, 208 blood supply, 117 blood vessels, 195 blood-brain barrier, 22, 42, 154, 195, 197, 208 BMI, 13 body composition, 111 body weight, 70, 71, 111 bonds, 4, 5, 8, 18 brain contusion, 151, 152 brain damage, 128, 132, 133, 141, 143, 144, 145, 146, 147, 152, 154, 163 brain growth, 156, 157 brainstem, 111, 190

breakdown, 136, 149, 151, 208 budding, 161 Burkina Faso, 81 Butcher, 104

C Ca2+, x, 2, 4, 5, 6, 10, 19, 29, 88, 89, 100, 101, 103, 111, 112, 114, 116, 117, 132, 156, 157, 172 cadmium, 32 calcium, ix, 7, 16, 19, 21, 30, 38, 40, 48, 59, 60, 88, 103, 107, 108, 111, 113, 116, 121, 127, 155, 157, 158, 160, 161, 164, 166, 167 caloric restriction, 83, 122 calorie, 70 cancer, 8, 24, 30, 33, 42, 162, 170, 177 cancer cells, 162, 170 candidates, 208 capillary, 69 cardiovascular disease, 41 cartoon, 167 Caspase-8, 126, 136, 151 caspases, 89, 90, 100, 103, 117, 125, 126, 128, 129, 130, 131, 132, 133, 134, 137, 138, 139, 141, 142, 143, 150, 152, 168 catalysis, 4 cataract, 29, 50 catecholamines, 78, 209 category a, 164 CCR, 186 cDNA, 119, 182 cell body, 191, 192 cell culture, 55, 66, 95, 103, 105, 123, 135, 149, 165, 172, 196, 202 cell cycle, 9, 10, 13, 33, 84, 177 cell fusion, 37 cell line, 20, 36, 41, 48, 55, 59, 103, 113, 114, 116, 139, 202 cell lines, 113, 139 cell membranes, 22 cell metabolism, 8 cell signaling, 3, 27, 84 cell surface, 17 cellular signaling pathway, 112 central nervous system, vii, ix, 1, 35, 36, 39, 40, 87, 88, 107, 109, 118, 119, 120, 133, 145, 153, 156, 157, 163, 171, 178, 192, 200 cerebellar development, 33 cerebellum, 12, 25, 80, 111, 113 cerebral amyloid angiopathy, 182 cerebral blood flow, 88, 190 cerebral contusion, 151

Index cerebral cortex, 27, 42, 104, 130, 133, 135, 136, 181, 194 cerebral hypoxia, 144, 145 cerebrospinal fluid, 116, 123, 136, 137, 144, 149, 151, 187, 188, 198 cerebrum, 138 chaperones, 3, 4, 24, 43, 46, 48, 49, 54, 57, 58, 59 chemokine receptor, 186 chemokines, xi, 34, 151, 174, 179 chemoprevention, 43 child abuse, 151 cholinesterase, 82, 190, 196, 198, 200 cholinesterase inhibitors, 196, 198 choroid, 111 chromatography, 69 chromosome, 158, 175, 176, 177, 178, 179, 182, 183, 184, 185 chronic fatigue syndrome, 14, 34, 43 circadian rhythm, 132, 158 circulation, 209 class, 176 cleavage, 10, 16, 18, 89, 126, 127, 128, 133, 134, 136, 137, 138, 141, 144, 150, 151, 152, 174, 175, 190, 191 climate, 18 clinical diagnosis, 178 clinical symptoms, 195, 216 clinical syndrome, 174 clinical trials, 24, 79, 132, 196, 210 CNS, vii, viii, xi, 1, 2, 13, 15, 16, 18, 20, 21, 22, 23, 25, 26, 35, 50, 57, 80, 88, 99, 118, 132, 134, 135, 140, 141, 146, 156, 157, 189, 190, 192, 196, 199 CO2, 90 coding, 176, 177, 180 codon, 178, 180, 186 coenzyme, 109, 110, 113, 190 cognition, 190 cognitive ability, 64 cognitive deficit, xi, 153, 189, 193 cognitive deficits, xi, 153, 189, 193 cognitive dysfunction, xi, 189, 194 cognitive function, viii, ix, 63, 73, 77, 78, 80, 87, 196, 200, 218 cognitive impairment, 64, 174, 195, 212 cognitive performance, 71, 79 cognitive tasks, 64 coherence, 210 collagen, 209 colon, 150, 162, 170 colon cancer, 150, 162, 170 complexity, ix, 49, 52, 80, 149, 155, 156 complications, 42, 121, 210 composition, 83, 88, 89, 91, 111, 153, 169

223

compounds, 91, 131, 142, 165 computed tomography, 135, 216 computer software, 67 condensation, 144 conductance, 42, 101, 108, 121 connectivity, 217 consensus, 48, 174, 182 consumption, viii, 9, 63, 109, 207, 209, 216 contrast sensitivity, xii, 204, 205 control condition, 94, 97, 98 control group, 67, 131 controlled trials, 212 contusion, 135, 138, 140, 152, 153 convergence, 14, 34 conviction, 108 cooling, 131 coordination, 8, 131 corpus callosum, 130, 149, 150 correlation, xii, 34, 39, 91, 92, 93, 195, 215, 216, 217, 218, 219 cortex, 27, 42, 90, 104, 110, 111, 113, 115, 130, 131, 134, 136, 140, 141, 158, 159, 160, 162, 163, 170, 181, 187, 190, 192, 193, 194, 197, 218, 219 cortical neurons, 38, 93, 100, 101, 102, 103, 105, 117, 135, 136, 149, 150, 160, 169, 172 cost, 94 criticism, 55 crystallization, 66 CSF, 140, 200 cues, 25, 64 culture, 13, 17, 19, 22, 23, 33, 36, 50, 53, 55, 56, 66, 88, 93, 95, 99, 103, 104, 105, 123, 168, 169, 171, 196, 201, 211 cyanide, 65, 162, 166, 170 cycling, 8, 9, 13, 65 cyclooxygenase, 39 cystic fibrosis, 24 cystine, 17, 24, 38 cytoarchitecture, 37, 193 cytochrome, viii, 63, 65, 68, 74, 78, 83, 84, 105, 109, 110, 112, 113, 117, 126, 132, 145, 151, 153, 166, 168 cytokines, xi, 151, 174 cytoplasm, 5, 6, 7, 47, 89, 90, 98, 101, 108, 166, 168, 177 cytoskeleton, 30 cytotoxicity, 15, 116, 123, 143, 146

D damages, iv, ix, 5, 6, 7, 20, 87, 97, 100 decay, 177, 178 decomposition, 172

224

Index

defects, 14, 83, 116 deficiency, 10, 11, 12, 16, 21, 25, 31, 32, 65, 79, 83, 117, 123 deficit, 38, 72, 208, 209, 216 degenerate, xi, 189, 195 degradation, 4, 25, 40, 43, 101, 127, 177, 208 dementia, vii, x, 1, 2, 3, 19, 22, 28, 36, 37, 38, 39, 40, 41, 42, 65, 76, 83, 114, 173, 174, 176, 183, 184, 185, 186, 187, 197, 198 demyelination, 28 depolarization, 88, 116, 123 deposition, x, 173, 175, 193, 196, 200 deposits, 174, 175, 196 deprivation, 2, 101, 117, 138, 141 destruction, 88, 89, 94 detection, 69, 123, 165, 210 detoxification, 7, 8, 76 developed countries, xi, 203, 204, 210 developing brain, ix, x, 143, 155, 156, 157, 158, 162, 164, 166, 167, 168, 169, 170 developmental change, 193 diabetes, xii, 24, 43, 80, 81, 108, 113, 118, 121, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214 diabetic patients, xii, 203, 204, 205, 208, 209, 210, 213 diabetic retinopathy, vii, 209, 211, 212, 213 diagnosis, 54, 178, 200, 216, 217, 220 diagnostic criteria, 176, 182 diet, 67, 140, 153, 209 dietary fat, 113 differential diagnosis, 217 diffusion, 128, 135, 193, 208 dimerization, 192 direct action, ix, 88, 89 disability, 65, 127, 135, 142 disadvantages, 99 discrimination, xii, 204, 205 disease model, vii, 2, 198, 202 disease progression, 7, 15, 57 disequilibrium, 180 disorder, vii, x, xi, xii, 1, 8, 54, 114, 115, 163, 173, 189, 195, 215, 216, 217, 220 displacement, 99 disposition, 42 dissociation, 31, 167 disturbances, 163, 174, 198, 216 diversity, ix, 48, 87, 90, 169, 186 DNA, vii, x, 1, 3, 6, 8, 9, 10, 27, 29, 31, 32, 33, 36, 41, 50, 58, 59, 65, 82, 89, 90, 97, 100, 101, 103, 104, 115, 123, 127, 132, 133, 135, 137, 140, 143, 144, 147, 149, 152, 165, 167, 171, 174, 177 DNA damage, 10, 31, 32, 33, 97, 104, 115, 171

DNA lesions, 32 DNA repair, 8, 9, 10, 27, 127 DNA strand breaks, 31 donors, 14, 43, 108, 205 dopamine, 76, 115, 122, 123, 209, 216, 219 dopaminergic, xii, 29, 42, 115, 116, 123, 165, 172, 215, 216, 217, 218, 219 dosage, 19 Down syndrome, 175 down-regulation, 105, 114, 122, 194 drinking water, viii, 24, 63, 66, 67, 71, 72, 73, 74, 75, 76, 77 Drosophila, 48, 56, 57, 59, 61, 99, 102, 105 drug addict, 115 drug discovery, 125 drug resistance, 24, 43 drug treatment, 3 drugs, vii, 2, 22, 23, 24, 25, 26, 55, 56, 64, 92, 132, 156, 163, 197, 207, 212

E edema, 133, 149, 150, 204, 206, 211, 213 electroencephalogram, 151 electron, ix, 2, 4, 5, 6, 7, 9, 28, 65, 68, 76, 78, 83, 107, 108, 109, 110, 115, 121, 167 electrons, 3, 4, 5, 6, 7, 23, 78, 108, 110, 112, 113 ELISA, 165 elucidation, viii, 2, 125 embryogenesis, 102 emission, 92, 99, 216 encephalitis, 14, 34, 38, 41 encephalomyelitis, 42 encephalopathy, 15, 35, 81, 125, 126, 127, 142, 143, 154 encoding, 52, 178, 179, 180 endonuclease, 136 endothelial cells, vii, 2, 7, 15, 18, 20, 36, 37, 83, 132, 205, 206 endothelium, 19, 40, 211 energy consumption, 9 environmental conditions, 2 environmental factors, xi, 114, 174, 175, 181 enzymes, xi, 7, 81, 114, 117, 126, 189, 192, 198 epidemiology, 184 epithelia, 126 epithelial cells, 19, 208, 214 epithelium, 38, 206, 208, 211 erythropoietin, 147, 208, 213, 214 ESI, 69, 73 estrogen, 140, 153, 170 ethanol, 163, 164, 199 ethylene, 64, 81

Index eukaryotic cell, 4, 108 evil, 27 evoked potential, 210 excitability, x, 155, 157, 163 excitation, 68, 69, 91, 164 excitotoxicity, vii, ix, 21, 87, 88, 89, 90, 95, 96, 98, 100, 102, 117, 123, 152, 208 execution, 48, 53, 58, 125 exercise, 80 exons, 176, 177, 178, 181 experimental autoimmune encephalomyelitis, 42 exposure, x, 19, 20, 21, 46, 53, 65, 79, 90, 93, 94, 95, 96, 97, 98, 99, 111, 113, 137, 156, 158, 159, 160, 163, 164, 167, 168, 169, 209 extinction, 68

F FAD, 4, 28, 183 falciparum malaria, 81 family history, 176, 178 family members, 103 FAS, 128 fat, 69, 70, 108, 113 fatty acids, 65, 109, 112, 114, 149 FDA, 55, 79, 131, 155, 168 FDA approval, 79 fetal alcohol syndrome, 170 fever, 65, 82 fiber, 135, 190, 192, 199 fibers, 190, 193, 194, 199 fibroblast growth factor, 39 fibroblasts, viii, 10, 52, 63, 197 flavopiridol, 140 fluid, 116, 123, 135, 137, 148, 152, 187, 188, 198, 206, 208, 209, 213 fluorescence, 69, 91, 92, 93, 94, 96, 97, 99, 100, 101, 104 folate, 122 food intake, 70 forebrain, xi, 144, 163, 168, 169, 170, 171, 189, 190, 192, 193, 195, 196, 197, 199, 200, 202 fragments, 137, 175 free radicals, ix, 3, 23, 27, 78, 107, 108, 123, 132 frequencies, 180, 187 frontal cortex, 153, 158, 159, 160, 162, 170, 218 frontal lobe, 183, 187 functional changes, xii, 22, 215 functional imaging, 220 fusion, 18, 37, 99, 133

225

G gene expression, 21, 25, 41, 50, 51, 60, 61, 115, 158, 160, 186 gene promoter, 141 gene therapy, 197, 201 gene transfer, 197, 199 genes, x, 7, 8, 17, 29, 30, 34, 36, 48, 51, 52, 56, 58, 59, 60, 142, 149, 150, 158, 161, 162, 164, 166, 167, 173, 175, 176, 178, 179, 180, 181 genetic disease, 2, 8, 25 genetic factors, xi, 174 genetic linkage, 182 genetic mutations, 2 genetics, vii, xi, 174, 178 genome, 27, 100, 179 genomic instability, 9 genotype, 144, 180, 185, 187 gestation, 127, 156 glia, 15, 28, 30, 39 glial cells, 19, 36, 58, 88, 105, 115, 190, 191, 206 glioma, 33 glucose, xi, xii, 7, 10, 24, 39, 43, 54, 90, 112, 114, 117, 121, 138, 141, 203, 204, 205, 209, 211, 215, 216, 217 GLUT, 209 glutamate, viii, ix, x, 7, 17, 21, 38, 42, 56, 87, 93, 94, 97, 98, 102, 103, 104, 109, 116, 123, 134, 137, 143, 149, 155, 156, 157, 158, 159, 160, 162, 163, 165, 166, 167, 190, 202, 206 glutamate receptor antagonists, ix, 155, 157 glutathione, 3, 4, 17, 28, 29, 30, 39, 41, 42, 112, 117, 124, 132, 134 glycine, 91, 96, 103, 178, 184 glycogen, 49, 57, 147 glycolysis, 112 grades, 115 gray matter, 81, 148 growth factor, xi, 12, 18, 24, 32, 33, 39, 114, 122, 133, 161, 177, 184, 189, 190, 191, 192, 194, 198, 199, 200, 201, 202 growth hormone, 133 growth spurt, 156, 157

H HAART, 19, 21, 22 haplotypes, 182 head injury, 135, 148, 151, 152, 153 health care system, 210 heat shock protein, viii, 31, 45, 47, 56, 57, 58, 59, 60 heavy metals, 46

226

Index

hematopoietic stem cells, 10, 27 heme, viii, 63, 78, 79 hemisphere, 133 hemorrhage, 127 hepatic failure, 27 hepatitis, 34 hepatoma, 33 heterogeneity, 174 hippocampus, 79, 85, 103, 115, 117, 124, 128, 130, 131, 132, 134, 135, 136, 138, 140, 141, 150, 153, 159, 171, 181, 190, 192, 194, 197 histidine, 27 histochemistry, 119 histogram, 92, 98 HIV, vii, 1, 2, 3, 14, 15, 16, 18, 19, 20, 21, 22, 25, 28, 34, 35, 36, 37, 38, 39, 40, 41, 42 HIV/AIDS, 42 HIV-1, 14, 19, 21, 22, 34, 36, 37, 38, 39, 41, 42 HIV-1 proteins, 19, 38 HO-1, 8 homeostasis, ix, 2, 6, 9, 10, 11, 16, 19, 24, 25, 26, 32, 38, 43, 51, 107, 108, 111, 117, 121, 162, 164, 196 homocysteine, 180, 186 host, 6, 18, 19, 21, 25, 128, 184 human brain, 36, 74, 76, 77, 83, 120, 124, 137, 151, 177 human development, 156 human immunodeficiency virus, 28, 35, 36, 37, 38, 39, 40, 41, 43 hybridization, 50, 119, 158, 159, 167 hybridoma, 170 hydrogen, 3, 4, 78, 83, 112, 117, 118, 132, 134, 146 hydrogen gas, 132 hydrogen peroxide, 3, 4, 78, 83, 112, 117, 118 hydroperoxides, 3 hydroxyl, ix, 3, 4, 55, 87, 88, 112 hyperglycemia, xi, 113, 122, 203, 204, 207, 209 hyperplasia, 196, 206 hypersensitivity, 9 hypertension, xi, 137, 203, 213 hyperthermia, 50, 58, 172 hypertrophy, 206 hypoglycemia, 101 hypothermia, 131, 138, 142, 144, 145, 148, 152 hypothesis, x, 121, 156, 178, 180, 182, 198, 202 hypoxia, 104, 117, 125, 127, 128, 143, 144, 145, 146, 147, 148, 198, 208

I ICAM, 134 ice, 50, 68, 69

idiopathic, 176, 177, 219 image, 80, 92, 93, 94 image analysis, 80 images, 91, 93, 94, 95, 98, 128 imaging modalities, 127 imbalances, 52 immune reaction, 97 immune response, 150, 162, 185 immune system, 15 immunodeficiency, 8, 14, 24, 28, 35, 36, 37, 38, 39, 40, 41, 43 immunoglobulin, 164, 171, 202 immunomodulatory, 132 immunoreactivity, 116, 140, 164, 165, 194, 200, 213 impairments, viii, 13, 33, 64, 101, 115, 195, 199, 200 impulsiveness, 174 in situ hybridization, 50, 119, 148, 158, 159, 167 in vivo, xi, 13, 28, 33, 42, 50, 53, 58, 59, 60, 65, 66, 74, 79, 125, 128, 132, 133, 136, 137, 138, 140, 145, 152, 161, 164, 165, 166, 170, 172, 189, 192, 193, 195, 216 incidence, 20, 41, 127, 135, 210 induction, 18, 28, 30, 38, 39, 52, 53, 56, 58, 75, 78, 79, 84, 96, 100, 101, 114, 117, 136, 140, 148, 171 industrialized countries, 117 infants, 142, 145, 151, 162 infarction, 117, 120, 132 inferences, 217 inflammation, xi, 20, 38, 132, 140, 148, 153, 177, 189 inflammatory disease, 179 inflammatory responses, 60 inheritance, 178 inhibition, 12, 13, 18, 29, 32, 68, 76, 82, 83, 84, 101, 108, 126, 128, 130, 131, 134, 138, 140, 141, 142, 143, 147, 150, 152, 153, 154, 162, 209, 213, 218 inhibitor, 11, 12, 13, 17, 33, 49, 53, 81, 84, 100, 131, 133, 136, 137, 138, 140, 141, 142, 143, 145, 148, 161, 165, 166, 168, 171, 172, 177, 200 initiation, 6, 9, 10, 16, 39, 125, 131 injections, 159, 161, 193 inositol, 6, 161 insertion, 176, 178 insulin, 12, 33, 114, 119, 122, 161, 210 integration, 28 interference, 112 interneurons, 89, 190 intervention, viii, 43, 45, 55, 108, 128, 131 ion channels, 102, 149, 157 ionizing radiation, 9, 10, 32, 144 ions, 6, 38, 88, 102 IP-10, 180, 186 ipsilateral, 128, 133

Index Ireland, 146 iron, 4, 23, 27, 79, 165 irradiation, 81 ischemia, 81, 85, 108, 117, 124, 125, 126, 127, 128, 131, 132, 140, 143, 144, 145, 146, 147, 148, 207, 208 isolation, 161 isoleucine, 180 isomerization, 4, 5 Italy, 173, 215

J Jordan, 30, 35

K K+, 88, 89, 100, 104 kidney, 65, 81, 112, 121 kinase activity, 59 kinetics, 48 Krebs cycle, 109

L language impairment, 174 latency, 19, 43 lateral sclerosis, viii, 3, 8, 27, 29, 40, 45, 46, 54, 56, 57, 58, 59, 60, 114, 123, 124, 184 learning, ix, 79, 80, 87, 132, 152, 169, 190, 193, 200 left hemisphere, 175 leptin, 81, 207 lesions, xii, 32, 117, 124, 139, 150, 193, 195, 198, 204, 205, 206, 214 leukemia, vii, 1, 14, 15, 34, 35, 36, 37 lice, 23, 117, 201, 202 ligand, 5, 36, 126, 149, 157 lipid oxidation, 165 lipid peroxidation, 20, 21, 23, 101, 112, 117, 131, 132 lipids, 3, 132 liquid chromatography, 69 liver, 109, 111 localization, 30, 58, 116, 196, 200 locomotor, 115 locus, 178, 182, 184, 185 loss of appetite, 196 low-density lipoprotein, 179 LSD, 71, 72, 73 lumen, 2, 4, 5, 7 Luo, 29, 32, 84, 145, 213

227

lymphocytes, 15, 40, 56, 58 lymphoid, 8, 22 lymphoma, 32, 34 lysine, 49, 91

M machinery, 53 macrophages, 15, 21, 41, 132 macular degeneration, 29 magnetic resonance, 120, 127, 135, 149, 150, 152 magnetic resonance imaging, 127, 135, 149, 152 magnetic resonance spectroscopy, 120 majority, x, xi, 21, 25, 114, 162, 173, 174, 175, 177, 178, 185 malaria, 81, 83 mammalian brain, 163 manic-depressive psychosis, 81 manipulation, 49, 84, 120, 165, 207 MAP kinases, 39 markers, 10, 13, 23, 24, 37, 148, 176, 195, 198 Marx, 33 matrix, 108, 109, 110, 111, 112, 113, 117, 118, 121, 127 MCP, 179, 180, 182, 185, 188 MCP-1, 179, 180, 182, 185, 188 media, 96, 100, 205 mediation, 131, 132 MEK, 12, 13, 140 melatonin, 131, 132, 144, 146 membrane permeability, 101 membranes, 5, 6, 18, 22, 99, 112 memory, viii, ix, xi, 63, 64, 67, 71, 72, 77, 79, 80, 82, 84, 87, 152, 174, 189, 190, 193, 194, 196, 198, 199, 212 menadione, 83 mental health, 84 mental illness, 84 mental retardation, 127 mesenchymal stem cells, 141, 154 mesoderm, 183 messengers, 112 meta-analysis, 181, 187 metabolic pathways, xi, 203, 204, 205, 206 metabolic syndrome, 11, 32 metabolism, xii, 6, 8, 40, 64, 70, 79, 80, 82, 88, 101, 108, 109, 111, 113, 120, 121, 158, 215, 216, 217, 218 meter, 67 methamphetamine, 165, 172 methanol, 69 methemoglobinemia, 65 Mg2+, 91, 102

228

Index

microscope, 91, 92 microscopy, ix, 87, 91, 93, 95, 96, 104, 105 migration, 33, 154, 157, 165, 168, 171 mitochondria, viii, ix, x, 2, 5, 6, 7, 9, 10, 15, 16, 17, 19, 27, 28, 29, 63, 64, 65, 66, 70, 73, 78, 79, 83, 87, 103, 104, 107, 108, 109, 111, 112, 113, 114, 115, 117, 119, 120, 121, 132, 134, 136, 137, 164, 166, 167, 170, 173, 174, 206 mitochondrial DNA, 6, 29, 115 mitogen, 49 mitosis, 104 model system, 198 modeling, 22, 41 moderates, 81 modification, 11, 12, 49, 69 modifier gene, 29 molecular biology, vii molecular oxygen, 4, 5, 6 molecular weight, 42, 78, 110, 177 molecules, 3, 7, 24, 26, 47, 48, 49, 55, 81, 98, 102, 126, 127, 179 MOM, 214 monoclonal antibody, 193 monocyte chemoattractant protein, 185 morbidity, 117, 127, 134, 145 morphogenesis, 89 morphology, 148, 150, 195 morphometric, 150, 168, 199 mortality rate, 127 Moses, 149 motor neuron disease, 34, 35, 36, 116, 184 motor neurons, 56, 59, 60, 89, 116, 123 movement disorders, 217, 219 MRI, 127, 135, 150, 217 mRNA, 58, 105, 110, 111, 116, 117, 119, 128, 131, 134, 136, 137, 148, 149, 150, 158, 159, 167, 177, 178, 182 mtDNA, 5, 6, 10 multicellular organisms, 126 multiple regression, xii, 215, 217 multiple regression analysis, xii, 215, 217 multiple sclerosis, 3, 34, 150 multiplier, 69, 92 muscle strength, 64, 73, 77, 78 mutagenesis, 171 mutant, vii, 1, 15, 25, 26, 35, 36, 37, 43, 50, 54, 56, 58, 59, 60, 116, 177, 180 mutation, viii, 2, 15, 18, 25, 54, 175, 176, 177, 178, 180, 182, 183, 186, 207 myelin, 50, 161 myoblasts, 56 myoclonus, 176

N Na+, 88, 89, 100 NaCl, 91 NAD, 9, 76, 83, 84, 131, 145, 146 NADH, 78, 102, 108, 109, 110, 113 National Institutes of Health, 142 nausea, 196 necrosis, ix, x, 87, 88, 89, 90, 92, 93, 94, 95, 96, 99, 100, 104, 126, 128, 135, 145, 150, 156, 161, 171, 185, 202 nematode, 126 neocortex, 200, 218 neonates, 126, 127, 128, 131, 143, 156 neovascularization, 209 nerve, xi, 39, 53, 57, 59, 102, 135, 136, 141, 149, 156, 189, 190, 191, 193, 194, 198, 199, 200, 201, 202 nerve growth factor, xi, 39, 135, 136, 141, 149, 189, 190, 191, 194, 198, 199, 200, 201, 202 nervous system, vii, ix, x, 1, 2, 6, 34, 35, 36, 39, 40, 50, 87, 88, 102, 107, 109, 118, 119, 120, 155, 157, 163, 169, 171, 178, 192, 200 neural development, 127 neuroblastoma, 34, 39, 114, 116, 209, 214 neurodegenerative diseases, vii, ix, 2, 3, 7, 13, 22, 27, 30, 31, 40, 42, 55, 58, 59, 87, 88, 107, 112, 115, 121, 122, 128, 131, 142, 145, 146, 177, 178, 179, 182, 216 neurodegenerative disorders, 27, 56, 108, 114, 118, 187, 216 neurofibrillary tangles, x, xi, 114, 173, 174, 189, 193, 196 neurogenesis, 32, 33, 79, 84, 85 neuroimaging, 216, 217 neurological disease, vii, 1, 34, 126 neuromotor, 42 neuronal apoptosis, x, 38, 125, 128, 131, 132, 133, 140, 145, 147, 149, 150, 153, 156, 160, 163, 164, 165, 170 neuronal cells, 29, 59, 60, 133, 162, 165, 170, 213 neuropathy, 3, 20 neuroprotection, ix, 56, 88, 107, 108, 116, 122, 131, 132, 133, 134, 136, 137, 138, 140, 141, 143, 146, 147, 148, 153, 154, 198, 208, 212 neuroprotective agents, 89, 100, 210 neurotoxicity, x, 36, 40, 102, 104, 112, 122, 132, 138, 152, 156, 157, 158, 162, 164, 165, 166, 168, 169, 171, 172, 202, 213 neurotransmission, xi, 156, 189, 195, 196 neurotransmitter, viii, xi, 87, 96, 99, 157, 171, 190 neurotrophic factors, 88, 101, 133, 141, 154 neutrophils, 6, 103, 132

Index niacin, 131 nicotinamide, 2, 38, 108, 131 nicotine, 31 nigrostriatal, 116, 123, 216, 217, 218 nitrate, 65, 165 nitric oxide, 36, 39, 41, 65, 82, 115, 132, 133, 138, 146, 147, 148, 152, 165, 171, 172, 180, 186, 187, 188 nitric oxide synthase, 65, 115, 133, 147, 148, 172, 180, 186, 187, 188 nitrogen, 68, 132 nitrous oxide, 162 nitroxide, 43 NMDA receptors, ix, x, 87, 100, 102, 156, 157, 158, 160, 163, 164, 165, 166, 167, 168, 169, 171 N-methyl-D-aspartic acid, 156 noise, 55, 92 normal aging, 75, 83 Nrf2, 7, 8, 16, 17, 19, 22, 23, 27, 30, 32, 34, 39, 42 nuclei, xi, 89, 91, 92, 93, 94, 99, 101, 104, 130, 163, 189, 190, 191, 206, 218 nucleic acid, 83

O obesity, 108, 207 occipital regions, 217 oedema, 213 oligodendrocytes, vii, 2, 11, 15, 16, 50, 57, 88, 136 oligomerization, 82 oligomers, 89, 114 oncogenes, 33, 104 opportunities, 118 organ, 84, 109, 126 organelles, 2, 4, 52, 108, 109 organism, 46, 48, 52, 56 oxidation, 4, 6, 7, 9, 22, 23, 28, 40, 60, 61, 65, 68, 69, 70, 78, 82, 84, 108, 109, 123, 140, 165, 209 oxidation rate, 68 oxidative damage, 7, 19, 65, 82, 108, 112, 114, 116, 121, 132, 180, 208, 209, 214 oxygen, viii, x, 2, 4, 5, 6, 7, 9, 12, 27, 28, 34, 48, 57, 63, 82, 84, 108, 109, 110, 117, 119, 120, 121, 123, 132, 139, 141, 146, 147, 152, 154, 155, 157, 164, 165, 167, 209, 213, 216 oxygen consumption, viii, 63

P p53, 11, 16, 27, 32, 33, 36, 37, 115, 117 pain, 196 pancreas, 65

229

parallel, 17, 158, 218 paralysis, 43, 54, 116 parenchymal cell, 109 parietal cortex, 131, 163 parkinsonism, 115, 176, 183, 219 parvalbumin, 53 pathogenesis, ix, xii, 14, 20, 21, 27, 36, 54, 87, 88, 99, 125, 158, 174, 176, 179, 180, 181, 196, 204, 205, 209, 210 pathology, viii, x, 2, 13, 29, 45, 46, 54, 55, 97, 116, 118, 137, 174, 177, 179, 181, 185, 192, 195, 210, 216, 219 pathophysiology, 29, 142, 151 PBMC, 180 PCA, 220 PCP, x, 156, 166 PCR, 115, 159, 160, 167 PDGF, 133 peptidase, 161 peptides, 100, 114, 122, 175, 177 performance, 24, 42, 64, 71, 77, 79, 193 perfusion, 216 perinatal, 128, 143, 144, 145, 147, 149, 169 peripheral blood, 40 peripheral nervous system, 35 permeability, 5, 6, 10, 29, 90, 101, 112, 166, 211 permission, iv permit, 210 peroxidation, 20, 21, 23, 101, 112, 117 peroxide, 3, 4, 78, 83, 112, 117, 118 peroxynitrite, 140, 148, 165, 171, 172 PET, xii, 80, 215, 216, 217, 219 pH, 27, 68, 91, 99 pharmacological treatment, 55 pharmacology, 199 phencyclidine, x, 156, 169 phenotype, x, xi, 9, 12, 43, 55, 173, 176, 178, 183, 185, 189, 192, 196, 202 phospholipids, 110, 123 phosphorylation, 6, 7, 10, 11, 12, 13, 32, 46, 47, 48, 49, 53, 108, 109, 112, 113, 117, 133, 147, 153, 165, 171, 176, 196, 201 photographs, 160 physical activity, 79, 114 physiology, ix, 29, 55, 60, 80, 107, 109 plasma membrane, 4, 5, 7, 15, 18, 19, 52, 88, 94, 99 plasminogen, 43 plasticity, 122, 157, 190 platform, 64, 67, 71, 72, 77 point mutation, 15, 35 Poland, 36 polarization, 116 polymerase, 5, 6, 105, 127

230

Index

polymorphism, 179, 180, 181, 182, 185, 186, 187, 188 polymorphisms, 179, 180, 181, 185 polypeptide, 161 polyphenols, 132, 146 polyunsaturated fat, 114, 147 polyunsaturated fatty acids, 114, 147 positron, 128, 143, 216 positron emission tomography, 128, 143, 216 potassium, 68, 122, 202, 206 prefrontal cortex, 187, 218 prevention, 27, 42, 81, 84, 105, 115, 165, 212 primary cells, 65 primary function, 108, 206 primate, 18, 122, 164, 220 probability, 89 probe, 67, 71, 72, 77, 159 progesterone, 140, 153 prognosis, 128 progressive neurodegenerative disorder, 216 progressive supranuclear palsy, 187 project, 190, 191, 204 proliferation, 3, 4, 11, 12, 13, 14, 25, 32, 33, 41, 85, 152, 153, 154, 165, 170 promoter, 7, 49, 55, 166, 172, 179, 180, 182, 186 prophylactic, 81, 138 prostate cancer, 177 protease inhibitors, 19, 21, 41 proteases, 68, 69, 89, 104, 125, 126, 152 protective mechanisms, 145 protective role, xi, 30, 115, 117, 189 protein family, 102 protein folding, vii, 1, 2, 4, 8, 20, 28, 40, 43 protein kinases, 60, 127 protein misfolding, 20, 47 protein oxidation, 4 protein sequence, 50 protein structure, 40 protein synthesis, vii, 1, 46 proteolysis, 36, 89, 101, 126, 137, 150, 151, 174, 177 proteome, 24 protons, ix, 7, 107, 108, 109, 110 proto-oncogene, 33 psychiatric disorders, 65 psychosis, 81, 181 pumps, 6 pyloric stenosis, 186 pyrimidine, 158

Q quality of life, 65, 79, 210 quinolinic acid, 111, 120

quinone, 8, 76, 83 quinones, 76

R radiation, 8, 9, 10, 31, 32, 33, 46, 65 radical formation, 82, 164 radicals, ix, 3, 4, 23, 27, 29, 34, 78, 107, 108, 119, 123, 132, 146 radiosensitization, 82 RANTES, 179, 180, 185 reactants, 28 reactions, 4, 6, 8, 22, 97 reactive oxygen, x, 2, 12, 48, 57, 84, 108, 119, 120, 123, 155, 157, 164, 165, 167, 209 reactivity, 211 recognition, 60, 93, 98, 99, 104, 126, 172, 198 recovery processes, 193 recruiting, 133 rectal temperature, 131 red blood cells, 42 regeneration, 85, 88, 99, 195 regression, xii, 70, 212, 215, 217 regression analysis, xii, 215, 217 repair, 8, 9, 10, 13, 27, 29, 57, 64, 84, 140, 143, 177, 178, 184 reparation, 97, 100 replacement, 25 repression, 12, 13, 48 repressor, 11, 12, 177 reserves, 57 residues, 7, 8, 9, 27, 49, 102, 126 resistance, viii, 16, 24, 36, 43, 63, 113, 117, 120, 206 respiration, 6, 9, 11, 32, 108, 109, 112, 113, 114, 116, 120 respiratory syncytial virus, 14, 34 responsiveness, 158 retardation, 13 reticulum, vii, 1, 4, 27, 28, 29, 31, 32, 35, 38, 40, 41, 43, 54, 57, 101, 126, 143 retina, xi, 82, 203, 204, 205, 207, 208, 209, 210, 211, 213, 214 retinal detachment, 204 retinitis, 214 retinitis pigmentosa, 214 retinoblastoma, 58 retinopathy, vii, xi, 203, 204, 208, 209, 211, 212, 213 retirement, 80 retrovirus, vii, 1, 2, 14, 15, 19, 20, 21, 22, 23, 26, 28, 29, 34, 35, 37, 38, 39, 42, 43 retroviruses, vii, 1, 3, 14, 15, 18, 20, 40 rhythm, 158 ribose, 5, 6, 105, 127

Index RNA, 34, 50, 112, 161, 177, 186 RNAi, 112 rodents, 50, 56, 64, 67, 77, 111, 120 room temperature, 68, 90, 92 rosiglitazone, 140, 153

S sarcopenia, 65, 79 SARS, 37 SARS-CoV, 37 scavengers, 132, 138, 146 schizophrenia, 181, 187 sclerosis, viii, 3, 8, 27, 29, 34, 40, 45, 46, 54, 56, 57, 58, 59, 60, 114, 123, 124, 177, 184 screening, viii, 63, 179, 184 secretion, 8, 15, 24, 88, 133, 154 self-assembly, 59 senescence, viii, 4, 63, 64, 65, 66, 73, 79, 81 senile dementia, 83, 198 sensitivity, xii, 8, 9, 31, 115, 121, 204, 205 septic shock, 65 septum, xi, 189, 190 sequencing, 176 serine, 49 serotonin, 78, 83 serum, 81, 92, 113, 179, 185, 209 serum albumin, 92, 113 shock, viii, 31, 45, 46, 47, 48, 50, 51, 56, 57, 58, 59, 60, 61, 65 shrinkage, 192, 202 sialic acid, 164 siblings, 185 side effects, 65, 79, 204 signal transduction, vii, 1, 6, 9, 30, 47, 52, 127 signaling pathway, xii, 9, 14, 38, 46, 112, 140, 158, 165, 192, 204 signalling, 118, 198, 201, 205 signals, 6, 22, 26, 49, 148, 159 signal-to-noise ratio, 92 signs, 13, 15, 19, 64, 176, 198 silver, 162, 163, 164 Sinai, 28 siRNA, 10, 13 skeletal muscle, 80, 83, 109, 116 skin, 126 SLPI, 177, 184 smoking, 196, 201 SNP, 182 sodium, 57, 209 software, 67, 69, 92 spasticity, 176 spatial learning, 80

231

spatial memory, viii, 63, 64, 67, 71, 72, 77, 80, 84, 148, 193, 199 species, x, 2, 12, 27, 28, 48, 57, 58, 76, 84, 108, 112, 119, 120, 123, 127, 130, 132, 139, 155, 156, 157, 164, 165, 167, 190, 209 spectroscopy, 120, 128 speech, 175 spinal cord, 50, 56, 58, 88, 105, 113, 116, 140, 154, 178 spinal cord injury, 117, 140 sprouting, 196 stabilization, 42, 170 stem cells, 10, 25, 27, 32, 33, 43, 84, 85, 141, 154 stenosis, 186 stimulus, 50 stomach, 109 stressors, x, 156 striatum, xii, 82, 116, 130, 131, 190, 215, 216, 218 stroke, vii, ix, 88, 107, 108, 114, 117, 118, 124, 127, 145, 153, 212 stromal cells, 141, 154 structural changes, 148 substitutes, 105 substitution, 175, 180 substitutions, 175 substrates, 4, 10, 19, 103, 109, 110, 126, 134 Sun, 89, 105, 148 suppression, 12, 32, 152, 153 surveillance, 4 survival, xi, 7, 11, 13, 17, 24, 25, 26, 30, 37, 40, 42, 46, 52, 53, 54, 59, 91, 102, 108, 112, 116, 118, 122, 131, 132, 133, 141, 162, 163, 165, 167, 170, 177, 190, 192, 193, 195, 199, 208 survival rate, 113 susceptibility, xi, 150, 158, 163, 171, 174, 179, 180, 181, 184, 188 suspensions, 82 swelling, 54, 89, 94 symptoms, vii, 2, 176, 195, 196, 216 synapse, 192 synaptic plasticity, 122, 147, 157 synaptic transmission, 157 synaptic vesicles, 191 syndrome, 11, 14, 19, 26, 32, 34, 43, 161, 170, 175 synergistic effect, 132 synthesis, vii, viii, 1, 31, 46, 63, 79, 82, 108, 110, 122, 146, 171, 195

T T cell, 10, 15, 20, 22, 34, 35, 37 tandem repeats, 179, 181 tangles, x, xi, 114, 173, 174, 189, 193, 196

232

Index

tau, x, 13, 33, 114, 173, 174, 176, 183, 184, 187, 196, 201 TBI, 126, 129, 135, 136, 137, 138, 139, 140, 141, 142 technical assistance, 80 telangiectasia, vii, 1, 8, 30, 31, 32, 33, 43 temperature, 34, 35, 68, 69, 90, 92, 111, 118, 120 temporal lobe, 175, 194, 201 tension, 68, 73 terminals, xii, 193, 215, 216, 218 termination codon, 178 testing, viii, 63, 64, 67, 68, 207, 210 thalamus, 111, 130, 135, 136, 144, 190 therapeutic agents, 84, 200, 210 therapeutic approaches, xi, 22, 37, 190 therapeutic intervention, viii, 45, 108, 125, 196 therapeutic interventions, viii, 45, 196 therapeutic targets, vii, ix, xii, 107, 117, 204, 210 therapeutics, 127, 137, 182, 219 therapy, 21, 25, 26, 102, 115, 128, 138, 139, 141, 142, 147, 148, 152, 154, 197, 198, 201 thermoregulation, 111 tissue, ix, 4, 10, 41, 43, 64, 85, 88, 89, 107, 108, 109, 110, 111, 115, 116, 119, 130, 132, 133, 137, 138, 141, 144, 145, 150, 151, 166, 184, 193, 199, 201, 209 tissue plasminogen activator, 43 TNF, 17, 36, 179 toxicity, 41, 42, 54, 56, 58, 65, 83, 104, 123, 137, 157, 164, 165 toxicology, 146, 170 toxin, 193 training, 67, 71 transcription, x, 7, 17, 38, 47, 48, 50, 51, 55, 58, 59, 60, 61, 140, 164, 166, 167, 170, 174, 177, 181, 186 transcription factors, 50, 59, 164, 167 transcripts, 120, 181 transduction, vii, 1, 6, 9, 30, 47, 52, 56, 157 transection, 195, 200 transferrin, 197, 199 transformations, 91 translation, 4, 10, 52, 56, 186 translocation, 7, 17, 48, 49, 53, 58, 90, 98, 134, 143, 166, 169, 170, 206 transmission, 95, 157, 192 transplantation, 25 transport, ix, x, 2, 4, 5, 6, 9, 40, 65, 78, 83, 102, 107, 108, 110, 123, 167, 173, 174, 192, 193, 197, 201, 206, 209, 214 trauma, 117, 124, 135, 138, 141, 148, 149, 152, 153, 207

traumatic brain injury, 104, 125, 126, 148, 149, 150, 151, 152, 153, 154 tremor, 115 trial, 41, 67, 71, 72, 77, 81, 201, 212 triggers, 28, 48, 88, 150, 192, 202 tropism, 34 trypsin, 90 tumor, 32, 33, 43, 146, 148, 161, 185 tumor cells, 146 tumor necrosis factor, 148, 161, 185 tumorigenesis, 184 turnover, 4, 116 type 1 diabetes, 212 type 2 diabetes, 24, 43, 121, 207, 212 tyrosine, 18, 116, 165, 171, 192 tyrosine hydroxylase, 116

U UK, 34 ultrasonography, 127 UN, 38 underlying mechanisms, 207 uniform, 88 UV, 46, 65, 102, 214 UV light, 65, 214 UV radiation, 46

V vaccine, 34, 41 validation, 160 valine, 131, 180 vascular dementia, 187 vascular endothelial growth factor (VEGF), 24 vasculature, 196 vasodilation, 133 viral infection, viii, 2, 3, 27 virus infection, vii, 1, 37 viruses, 14, 15, 17, 37 vision, 71, 204 visual acuity, 204 visual field, 204 visualization, 103 vitamin B3, 131 vitamin C, 209 vomiting, 196 vulnerability, 30, 54, 60, 116, 123, 150, 156, 157, 163

Index

W wealth, 54, 125 weight loss, 196 western blot, 50, 132 white matter, 135, 145 wild type, 10, 116 withdrawal, 160, 193 workers, 115, 117

233

working memory, 193

Y yeast, 120

Z zinc, 112

Neurodegeneration_(1617611190)

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