Stephen C. Bondy - US grants

neurotoxins; synaptosomes;

Description: This project supports participation by eight U.S. scientists in a binational conference on molecular mechanisms underlying neural response to damage. The meeting is to be held at the National Institute of Mental Health and Neuro Sciences (NIMHANS) in Bangalore, India. It is co-organized by Dr. B.S. Sridhara Rama Rao, Head, Neurochemistry Department, Dr. G.N. Narayana Reddy, Director of NIMHANS, and Dr. Stephen Bondy of the University of California, Irvine. The focus will be on the molecular and cellular mechanisms underlying adaptive and regenerative events within the nervous system following physical or chemical damage. Effort will be made to integrate the specific presentations, by means of discussions and workshops, to make a synthesis of recent advances, to enhance the focus of definition of the molecular and cellular basis of neural repair mechanisms and to pinpoint precise areas for future research and especially joint research. Scope: This is the third in a series of Indo-U.S. neuroscience meetings held over the last five years, aimed at exchanges of information and discussions on recent advances in fundamental, mechanistic neurobiology. The theme of this meeting was recommended by the advisory panel of the previous meeting held in April 1987 in New Delhi. The U.S. and Indian organizers are very well suited to handle such a joint meeting. It is expected that significant exchanges of information will take place and that potentially good research collaboration would result.

The widespread use of alcohol results in serious societal problems, and a significant component of its abuse is damage to the integrity of the nervous system. The focus of this study is the evaluation of means by which such adverse effects might be attenuated. The concept underlying this research is that ethanol or its major metabolite, acetaldehyde, may disrupt membrane function by non- specific or by more selective damage. This insult may occur at the plasma membrane but may also involve the integrity and functioning of the endoplasnlic reticulum or mitochondrion. The ability of ethanol or acetaldehyde to disrupt cellular homeostasis will initially be studied by in vitro exposure of isolated synaptosomes to these agents. These studies will be supplemented later by studies of morphological fractions derived from ethanol or acetaldehyde-treated rats. Evaluation of cytotoxic events will be by use of several fluorescent probes which allow continuous monitoring of various indices of metabolic and structural integrity. Parameters include determination of transmembrane potentials across plasma and mitochondrial membranes, cytoplasmic pH and redox status, and microviscosity of the synaptosomal limiting membrane. In addition, free-radical induced oxidative activity will be assayed. Preliminary work has revealed that a metabolite of ethanol (acetaldehyde) can enhance generation of reactive oxygen species. Pretreatment with certain chemicals has been found to prevent synaptosomal deterioration induced by several xenobiotic agents. Tbe major putative protective materials to be studied are: a lipid soluble antioxidant vitamin, alpha-tocopherol; monosialoganglioside GM1 and an antagonist of n-methyl-d-aspartic acid (MK-801), the diamine product of ornithine decarboxylation (putrescine), and thiamine. The in vitro studies will be of value in further definition of the molecular lesions induced by ethanol, while the corresponding in vivo studies may contribute to the conceptual development of therapies for alcohol-related disease.

DESCRIPTION: (Applicant's Abstract) Aluminum has been long suspected to play a role in several neurological diseases associated with aging, but this linkage has never been unequivocally established. There is evidence that the potential of iron for enhancing free radical generation in nervous tissue is enhanced by aluminum, although this latter metal has no intrinsic pro-oxidant properties. The intent of this application is to illuminate the underlying molecular mechanisms by which such a potentiation may take place. The effect of chelation or protein sequestration of either metal upon their interactions will be examined. The biological substrate responsive to such oxidative stress induced by the combined metals, will be sought. The nature of the induced reactive oxygen species will be documented using both enzymic and electron spin resonance techniques. Subsequently, the biological relevance of aluminum-iron interactions will be investigated in animals exposed to these metals singly or in combination. The concentrations of these metals in various brain regions will be quantitated by atomic absorption. An attempt to correlate levels of aluminum and oxidative parameters in human post-mortem cerebral tissue from aged and Alzheimer's brains will also be made. In addition to study of parameters relating to oxidative damage to proteins, and membrane stability in dosed animals, and in treated cholinergic neuroblastoma cells, key proteins such as ubiquitin, beta-amyloid, and heat shock protein HSP 70, will be quantitated by immunological procedures. The final phase of the planned research is an attempted mitigation of changes induced in neural tissue in vitro and in vivo by these combined metals. This will involve dietary supplementation with antioxidant vitamins or the use of selective chelators. Results will illuminate the role that aluminum may play in potentiation of age-related neurological diseases.

DESCRIPTION: (Verbatim from the Applicant's Abstract) neurodegenerative diseases involve promotion of a pathological process by events associated with normal aging. Senescence is generally an essential concomitant of the progression of neurological disease and if this were retarded, the incidence of many such disorders would be significantly reduced. Prevention of deficits is more readily accomplished than attempts to compensate for impaired neurological function. This application posits that the cerebral mitochondrion is a susceptible target of age-related pro-oxidant events within the CSN, that the administration of exogenous factors may modify the velocity of these events and that this can influence other biological and behavioral consequences of aging. Maturational changes in mitochondrial function, structural integrity and mtDNA characteristics will be studied throughout the lifespan of C57BL/6 mice. Attempts to retard the onset of changes in mitochondrial parameters including gene deletions, membrane stability and levels of key enzymes, will be made by dietary supplementation with antioxidants, specifically targeted toward protection against free radicals produced by the reduced efficiency of the respiratory chain encountered with advancing age. Results will be substantiated with those obtained from mitochondria derived from neural cell lines exposed to low oxidant conditions for several generations. The role of both active oxygen and nitrogen species as contributory factors in these events and their potential modulation by antioxidants will be taken into account. The protective potential of antioxidants in combination, targeted toward several sources of oxidative events, will be evaluated. While the onset of neurological disease generally does not represent merely an acceleration of normal aging, there may be biological loci common to both. The identification and protection of such common targets will help in the development of agents that can reduce the incidence of neurodegenerative disorders.

DESCRIPTION: (Verbatim from the Applicant's Abstract) Aluminum has long been suspected to play a role in several neurological diseases associated with aging, but this linkage has never been unequivocally established. The relative inertness of aluminum salts has been cited as negating this possibility. Paradoxically, the investigator's recent findings suggest that this very inertness may provoke a neurotoxic response. In addition, although aluminum has no intrinsic pro-oxidant properties, they have evidence that the potential of transition metals for enhancing free radical generation in nervous tissue is enhanced by aluminum. These findings serve as the foundation for the following research objectives: (1) To define the precise chemical and molecular basis underlying aluminum's potentiation of transition-metal induced free radical activity within nervous tissue. The effect of aluminum on Fenton chemistry and oxidation state of several transition metals will be examined. (2) To locate the anatomical regions and cell types in which aluminum can effect tissue damage. (3) To define the sequence of the intracellular signaling cascades in doses animals and in human (neuronal and glial) cell lines responding to aluminum exposure. Second messenger pathways and consequent changes in transcription factors will be documented. Key proteins such as ubiquitin, cytokines, HSP 70, zinc finger proteins and NFkB will be quantified by immunological or gel shift procedures. Whether the properties of aluminum colloid can be intrinsically toxic or whether the toxicity of beta-amyloid can be enhanced will be determined. (4) To tract the effects of extended aluminum exposure upon genomic expression within neural tissues. This will include assay for deletions in mitochondrial DNA. (5) To explore possible pharmacological interventions that may prevent changes within neural tissues exposed to aluminum. This will involve dietary or growth media supplementation with antioxidant agents or the use of selective chelators. Together, these five objectives delineate a research strategy that will allow resolution of the seemingly contradictory evidence relating aluminum to neurological degeneration.

DESCRIPTION (Adapted from applicant's abstract): Free radical-based pathological processes are increasingly recognized as being a key component in the etiology of a wide variety of diseases associated with aging. A range of selective disorders are often superimposed on basal age-related events These include mental disorders, neurodegenerative disorders, cardiovascular diseases, cancer, and other less well-defined losses of cellular efficiency. In recent years, much investigation of oxidant-promoted cell damage has been conducted. Despite this growing amount of information concerning reactive oxygen and nitrogen species, there has been little effort made to integrate this body of knowledge across disciplines. The current application is designed to fill this gap by allowing comparison and discussion of results derived from several areas of medical investigation. The meeting is intended to serve as an integrative platform, and will be held in one of the world's finest conference centers, at the University of British Columbia, Vancouver, in July 2001 and the subsequent four years. objective of the meeting is to bring together participants from apparently unrelated fields to learn of each other's approaches to the study of this final common component of many age-related disorders. Such interactions will lead to the discovery of unexpected unifying concepts and set the groundwork for novel interactive approaches to clinical issues. It is intended to bring together leading authorities from differing fields, investigating various aspects of oxidative stress that underlies so many types of cell dysfunction. The colloquium will feature plenary lectures, The symposia, workshops, and poster presentations and will allow much time for informal discussion. The program is planned to bring together participants from diverse fields who would otherwise be unlikely to meet. This will allow the emergence of key analogies that can relate superficially diverse areas. By this means, such interaction is likely to accelerate the development of novel therapeutic strategies. The NIH support will foster the participation of international experts on free radical issues, and the meeting will be designed to assist in encouraging women and minority researchers to get involved in this exciting and rapidly emerging area. The fostering of the next generation of scientists by facilitating their participation is also a major intent of the meeting. The long-term support of this series of annual meetings will permit better planning for meeting organizers, attendees, students, and publishers.

DESCRIPTION (provided by applicant): Aging is a factor that exponentially increases the incidence of many diseases. Melatonin, a neuroendocrine agent, has been found to prevent the development or reverse some biochemical changes associated with brain aging. These changes include increases in oxidative and inflammatory events both of which are associated with senescence. The hypothesis underlying this application is that dietary melatonin may be of utility in retarding some of the undesirable aspects of brain aging. Arrest or reversal of trends associated with age will be evaluated by comparing patterns of mRNA and proteins from melatonin-treated mice with those from younger animals. Parallel studies using behavioral endpoints will reveal whether molecular changes detected, are reflected by altered cognition or motor strength. The degree of consonance between biological and behavioral changes may imply a causal relation. Changes in gene expression following treatment of aged mice with melatonin will be evaluated using array technology in combination with Northern blotting. Investigation of those genes associated with inflammatory or pro-oxidant events will be emphasized. The cytosolic pathways leading to genomic modulation will be sought. This will also involve the use of a cell line expressing melatonin receptors. Immunohistochemical identification of specific peptides from fixed tissue sections will allow a more precise localization of any changes in signaling pathways to be observed. Levels of proteins potentially affected by altered mRNA levels will be quantitated using ELISA and Western assays. The ability of melatonin to restore integrity of mitochondrial functioning and retard age-related mitochondrial DNA deletion will also be evaluated. The principal goal of this study is to contribute to the development of a rational nutritional strategy for delaying progression of brain aging.

[unreadable] DESCRIPTION (provided by applicant): Senescence is a multifactorial process, which appears to be associated with increases in both oxidative and inflammatory events. The ability to slow or reverse changes associated with brain aging by dietary supplementation will be investigated with a focus on these two processes. Following an initial survey of a broad range of agents, melatonin has been selected for detailed study. Changes in gene expression following treatment of aged mice with melatonin will be evaluated using array technology in combination with Northern blotting. Investigation of those genes associated with inflammatory or pro-oxidant events will be emphasized. The cytosolic pathways leading to genomic modulation will be sought. Levels of proteins potentially affected by altered mRNA levels will be quantitated using ELISA and Western assays. The ability of melatonin to restore integrity of mitochondrial functioning and retard age-related mitochondrial DNA deletion will be evaluated. Immunohistochemical identification of specific peptides from fixed tissue sections will allow a more precise localization of any changes observed. Arrest or reversal of trends associated with age will be evaluated by comparison of patterns of mRNA and proteins from dosed mice with those from younger animals. Parallel studies using behavioral endpoints will reveal whether molecular changes detected, are reflected by altered cognition or motor strength. The degree of consonance between biological and behavioral changes may imply a causal relation. Results will contribute to development of a rational strategy for delaying progression of brain aging. This is also likely to be relevant to slowing the onset of, and reducing the incidence of, several neurodegenerative diseases where age is a major co-factor. [unreadable] [unreadable]