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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.

NEUROSCIENCE RESEARCH PROGRESS Additional books in this series can be found on Nova‟s website under the Series tab. Additional E-books in this series can be found on Nova‟s website under the E-books tab.

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

ix

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

x


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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xi

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

xii


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


7

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.

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

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]

64

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

66

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


67

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

68


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.


69

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).

70


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)


71

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

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