Julie K. Andersen - US grants

neural degeneration; disease /disorder model; molecular genetics; transfection /expression vector; complementary DNA; gene expression; genetic recombination; glial fibrillary acidic protein; neurotoxins; genetically modified animals;

Superoxide and hydrogen peroxide are produced during normal metabolism. However, when produced in excess, the oxidant species can be converted by reaction with free iron to reactive hydroxyl radicals which can degrade DNA, proteins, and membrane lipids resulting in cellular degeneration. The brain is particularly susceptible to free-radical damage due to its unique cellular membrane composition, and its high levels of oxygen metabolism and free iron. Brain cells have developed several defense against these oxidant species including production of antioxidant enzymes and storage of iron in forms that will not catalyze formation of reactive radicals. Specifically, the antioxidant enzyme superoxide dismutase reduces superoxide to hydrogen peroxide, which is in turn reduced by either glutathione peroxidase or catalase to H20. Iron, in turn, is normally bound to ferritin, the major iron storage molecule in the brain. However, when balance in the brain is disturbed and free-radicals are allowed to accumulate, this can lead to iron- catalyzed production of hydroxyl radical and subsequent cell damage. It has been postulated that oxidative stress may play a role in the pathogenesis of neurodegenerative diseases like Parkinson Disease (PD) and cellular degeneration during normal aging. To examine the role of antiodixants in preventing neuronal cell damage, transgenic mice will be create which over-express the iron-binding protein ferritin, or the anti- oxidant enzymes glutathione peroxidase or catalase in neurons using neuron-specific promoters. Constructs will be evaluated for their ability to confer transgene activity on cultured cells by Northern analysis, immunocytochemistry, enzyme assays, and for sensitivity to iron, MPTP, 6-OHDA, and H202. Following injection into mouse embryos, genomic insertion of constructs will be confirmed by PCR and Southern blot analysis. Transgenic mice will be bred to create new lines and progeny examined for transgene expression in brain by Northern analysis, in situ hybridization, immunocytochemistry, and enzyme assays. Transgenic lines will be evaluated during normal aging for changes in lipid peroxidation by MDA accumulation, protein oxidation by glutamine synthetase activity, oxidative stress as assessed by GSH/GSSG ratios, and for cell loss or atrophy as measured by cell number and sizes in the substantia nigra using tyrosine hydroxylase immunocytochemistry compared with normal controls. Levels of striatal dopamine and its metabolite, DOPAC, tyrosine hydroxylase activity, 3H-dopamine binding, and dopaminergic cell numbers and sizes will be assessed in transgenic compared to controls following MPTP injection. Changes in brain morphology during development, and in protecting against the effects of oxidative stress in the brain, exploring the hypothesis that a genetic increase in one of these molecules may be involved in predisposition to or protection against PD or neuronal degeneration during aging.

A growing body of correlative evidence has implicated free radicals as a important factor in the pathology of both Parkinson's disease (PD) and normal brain aging. Dopaminergic nigrostriatal neurons, the predominant cell type lost in PD, are believed to be highly susceptible to free radical damage due to the propensity for dopamine to oxidize, producing elevated levels of reactive oxygen species. This problem is exacerbated by region-specific decreases in antioxidant defenses in the proximity of the substantia nigra. De-regulation of the glutathione system, a major component of the antioxidant defense system in the brain, has been strongly implicated as a causal factor in the resulting neuronal degeneration, but a direct role has yet to be definitively proved. We propose in this study to use genetic engineering to directly assess the involvement of the glutathione system in protecting the brain against neuronal damage due to either acute and chronic oxidative stress, and to test whether deregulation of this system can result in neurodegeneration like that seen in PD or during the normal aging process. This will be done using tissue culture and animal models in which levels of glutathione are altered by either over- or underexpression of glutamylcysteine synthase (GCS), the rate limiting enzyme in the synthesis of glutathione, or genetic over- or underexpression of glutathione peroxidase, the enzyme which acts with glutathione to de- toxicity reactive oxygen species. Such in vitro and in vivo systems should allow us to explore the hypothesis that genetic variations in levels of these molecules could be involved in predisposition to or protection against Parkinson's or neuronal degeneration during normal aging.

The goal of the proposed research is to explore the potential role of monoamine oxidase B (MAO-B) in neuronal degeneration associated both with normal aging and in neurodegenerative disorders such as Parkinson's and Alzheimer's disease. It has been hypothesized that due to its age-related increase in the brain coupled with its ability to produce reactive oxygen species (ROS) as a by-product of its enzymatic action, MAO-B could contribute to age-related neurodegeneration by eliciting increased oxidative stress and mitochondrial damage either on its own or possibly through its interaction with either endogenous and exogenous neurotoxic species. We plan to directly explore this hypothesis by creating genetically engineered PC12 cell lines with increased levels of MAO-B and transgenic animals in which levels of glial MAO-B can be inducibly increased in the adult animal. These models will be tested for effects of increased MAO-B activity on mitochondrial function and generation of oxidative stress in both the absence and presence of the Parkinsonian-inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP). The effect of elevation of brain MAO-B on microanatomical glial and neuronal changes associated with aging will also be assessed in adult animals. The transgenic lines proposed are superior to existing MAO-B transgenic lines previously created by ourselves in which MAO-B levels are altered not only in the adult but throughout development. Genetically controlled inducible delivery of the enzyme is more precise for examining the effects of MAO-B elevation in the adult and furthermore can be examined in a genetically homogeneous background in transgenic mouse lines, something that is often not possible in human studies. This work will complement on-going work in the laboratory exploring the role of free radicals in neurodegeneration during both aging and in neurodegenerative disease using mice with variant brain expression of molecules that are believed to be protective against free-radical damage including ferritin and glutathione/glutathione peroxidase.

Iron levels have been found by several investigators to be significantly increased in the substantia nigra of Parkinsonian patients vs. age-matched controls. This along with iron's ability to catalyze conversion of hydrogen peroxide produced as a by-product of dopamine metabolism to deleterious hydroxyl radical has lead to the hypothesis that iron may be involved in disease pathology via increased production of oxidative stress. Epidemiological studies performed to date have found no correlation between increased incidence of Parkinson's disease and either high dietary iron intake in the adult or prolonged occupational exposure to iron alone. However the role of early iron exposure on susceptibility to the disease has not yet been fully explored. What effects do neonatal iron exposure, genetic variations in iron metabolic factors, and increasing age have on susceptibility of dopaminergic nigral neurons to degeneration associated with Parkinson's disease? We plan to explore these questions by examining the effects of increased dietary iron on generation of oxidative stress and dopaminergic nigral cell degeneration in both the absence and presence of the Parkinsonian-inducing agent 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine (MPTP) in C57BI mice of various ages. In addition the combined effects of increased neonatal dietary iron and aging on susceptibility to MPTP will be examined in transgenic mouse lines recently constructed in our laboratory in which expression of the iron-binding protein ferritin has been up-regulated in dopaminergic nigral neurons. This will allow us to explore how combined variations in both dietary and endogenous metabolic factors in association with increasing age may predispose individuals for this disease and perhaps give us clues as to possible preventative interventions.

DESCRIPTION (From the Applicant's Abstract): A growing body of evidence has implicated oxidative stress as an important factor in the neuropathology associated with Parkinson's disease (PD). Dopaminergic nigrostriatal neurons, the predominant cell type lost in PD, are believed to be highly prone to free radical damage due to the propensity for dopamine to auto-oxidize and thereby produce elevated levels of hydrogen peroxide and catecholamine quinones. In vitro analysis suggests this reaction may be catalyzed by transition metals such as iron. Hydrogen peroxide formed during this process can either be converted by iron to form highly reactive and toxic hydroxyl radicals or removed through reduction by glutathione (GSH). GSH can also conjugate with quinones formed during dopamine oxidation preventing them from facilitating the release of iron from the iron-storage molecule ferritin. Alterations in both iron storage and glutathione (GSH) levels in the SN have been correlated with the neuronal degeneration accompanying PD but a direct causative role for either has yet to be definitively proved in vivo. We propose to use genetically engineered mouse lines generated in our laboratory as in vivo models to examine the role that alterations in GSH and free iron levels may play in the differential neurodegeneration of dopaminergic neurons of the substantia nigra in PD and how these two parameters may interact with one another to bring this about. This include the use of transgenic mice with altered SN expression levels of either ferrritin or glutamul cysteine synthetase (GCS), the rate limiting enzyme in GSH synthesis. We will use these in conjunction with the well-established MPTP toxicity model of PD to test whether chronic alterations in these molecules in vivo results in changes in neuronal loss associated with toxin treatment and/or aging and to delineate the specific biochemical processes responsible. Such in vivo systems should allow us not only to explore the mechanisms by which in vivo changes in these components may contribute to the neuropathology associated with PD but may also provide useful information for the design of future drug and genetic therapies for this disease.

DESCRIPTION (provided by applicant): Increased activity of the hydrogen peroxide-producing enzyme monoamine oxidase B (MAO-B) and decreased levels of the peroxide-scavenging compound glutathione (GSH) have both been postulated to contribute to the selective demise of dopaminergic neurons of the substantia nigra (SN) associated with Parkinson's disease (PD) via production of chronic oxidative stress in these cells. Oxidative stress may in turn impinge on mitochondrial function contributing to their subsequent neurodegeneration. Loss of mitochondria function in PD has been proposed to be the result of selective inhibition of mitochondrial complex I activity, however the specific cause or causes leading to its repression are unknown. We have recently demonstrated that subtle elevations in MAO-B or decreases in GSH in dopaminergic PC12 cells in vitro akin to that which occurs during brain aging or PD result in decreased mitochondriaI complex I activity. This appears to involve both direct oxidative damage to the complex itself as well as inhibition of the TCA enzyme alpha-ketoglutarate dehydrogenase (KDGH) which provides NADH as substrate to the complex. This in turn affects the capacity of the organelle to maintain function under stress conditions. As a logical extension of our published in vitro studies, we propose to analyze mitochondrial function and neurodegeneration in transgenic mouse lines created in our laboratory which possess inducibly increased levels of glial MAO-B mimicking those which occur during aging or decreases in GSH such as occurs in PD. This will allow us to assess the effects of this phenomenon on dopaminergic neurons of the SN which undergo degeneration in PD in the context of aging and stress. The ability to induce these changes in our transgenic models will allow us to examine the consequences of elevation in the adult animal bypassing any confounding developmental effects in a dosage-dependent and reversible manner. By looking throughout the lifespan, we will be able to assess the role that aging itself plays in this process. Information gleaned through these studies will allow us to test in future therapeutic strategies to minimize the effects of these alterations on mitochondrial function as it relates to PD and associated neurodegeneration.

Glutathione depletion is the earliest detectable biochemical event in the Parkinsonian substantia nigra (SN),[unreadable] occurring prior to selective loss of mitochondrial complex I (CI) activity associated with the disease. We have[unreadable] Dreviously demonstrated that down-regulation of total glutathione (GSH + GSSG) levels in cultured[unreadable] dopaminergic cell lines results in decreased mitochondrial function linked to a selective decrease in CI[unreadable] activity. Loss of CI activity following acute glutathione depletion appears to be due to[unreadable] reversible nitrosylation of protein subunits comprising this complex. The effects of prolonged chronic[unreadable] glutathione depletion on CI activity in dopaminergic cells, however, are unknown but may involve[unreadable] additional, irreversible oxidative events. A major goal of our project is to assess CI inhibition in our[unreadable] dopaminergic cell model at various times and levels of glutathione depletion in order to identify the limits of[unreadable] function vs. dysfunction including the thresholds for reversible vs. irreversible inhibition as well as the[unreadable] oxidant species and protein targets involved. Once such targets have been identified, we will assess the[unreadable] presence of similar alterations in a newly constructed dox-inducible antiGSH transgenic mouse model[unreadable] following titration of glutathione levels within SN dopaminergic neurons in vivo as a model for molecular[unreadable] events associated with Parkinson's disease.[unreadable] Acute reduction in glutathione in our cell model also results in inhibition of the GSSG reducing enzyme[unreadable] glutathione reductase (GluRd) and up-regulation of the enzyme g-glutamyl transpeptidase (GGT) which[unreadable] breaks down extracellular GSH to substrates which are transported back into the cell for glutathione[unreadable] synthesis. The former may contribute to the detrimental effects of glutathione depletion on mitochondrial[unreadable] function while the latter appears to be a compensatory event. We will also explore the molecular[unreadable] mechanisms involved in these molecular alterations following titration of glutathione pools including the[unreadable] oxidants and the enzyme targets involved and if/how these changes modify mitochondrial function. We[unreadable] will assess the effects of both transgenic GSSG Rd and GGT expression on CI activity and mitochondrial[unreadable] function in our dopaminergic cell model to see if they act to attenuate any detrimental mitochondrial effects.[unreadable]

We previously demonstrated that iron-mediated oxidative stress is causally involved in Parkinson's-related neurodegeneration in a well-established model of the disease, systemic MPTP administration. Iron's elevation in the Parkinsonian substantia nigra (SN) has been postulated to contribute to the selective dopaminergic neurodegeneration in this brain region associated with the disorder. The potential cause(s) of iron dysregulation in the diseased SN, however, is unknown. Iron levels are normally controlled by the physiological action of iron regulatory proteins (IRPs) which bind to iron-regulatory elements (IREs) in the RNAs of ferritin and the transferrin receptor (TfR) regulating their levels and thereby iron homeostasis. IRP1 binding is reported to be aberrantly sustained in the Parkinsonian SN in the face of elevated iron levels, a condition which would normally lead to its decrease. We have recently generated preliminary data suggesting that both increased neonatal dietary iron intake and decreases in SN levels of the thiol antioxidant glutathione result in aberrantly sustained IRP binding in the face of elevated iron levels. Previous studies have demonstrated that exposure of IRP1 to reactive oxygen species results in persistent non- physiological binding of the protein to IRE sequences. We hypothesize that: (1) increased iron and oxidative stress in the older SN as a consequence of increased neonatal dietary iron intake can result in sustained IRP1 binding and dysregulation of cellular iron homeostasis and (2) sustained depletion in dopaminergic SNglutathione levels can also result in persistent IRP1 binding and iron dysregulation. We propose to test these hypotheses in vivo by assessing the impact of: (1) neonatal iron feeding in a novel transgenic mouse line recently constructed in our laboratory in which levels of glutathione are increased in dopaminergic neurons of the SN and (2) glutathione depletion in a second recently constructed transgenic line in our laboratory in which dopaminergic SN glutathione levels are reduced. We plan to examine oxidative stress and iron homeostasis under both of these experimental conditions in comparison to the genesis of neurodegeneration. The long-terms goal of our studies will be to understand by what mechanism(s) iron dysregulation is occurring in the Parkinsonian SN which contributes to subsequent neurodegeneration associated with the disease. Through a better understanding of the possible intrinsic and extrinsic mechanisms by which iron dysregulation occurs in the Parkinsonian SN, our findings may aid in the development of novel therapies for the disease.

[unreadable] DESCRIPTION (provided by applicant): Age-related human diseases including cancer, Alzheimer's and Parkinson's disease show a clear correlation with mitochondrial dysfunction and reactive oxygen species (ROS). We propose to undertake a combined genetic, biochemical and bioenergetics approach to test the hypothesis that mitochondrial dysfunction and particularly mitochondrial reactive oxygen species (ROS) generation are causatively involved in age related disease. We propose to use three main manipulations of mitochondrial features-electron transport chain complexes, the glutathione pool and superoxide levels-and to measure outcomes models of these three age-related conditions. [unreadable] We believe that the proposed Program Project will make a significant contribution to our understanding of how mitochondrial function contributes to age-related disease with a view towards designing successful interventions. [unreadable] [unreadable] [unreadable]

NIH Roadmap Initiative tag

Glutathione depletion is the earliest detectablebiochemicalevent in the Parkinsonian substantia nigra (SN), occurring prior to selective loss of mitochondrial complex I (CI)activity associated with the disease. We have previously demonstrated that down-regulation of total glutathione (GSH + GSSG) levels in cultured dopaminergic cell lines results in decreased mitochondrial function linked to a selective decrease in CI activity (Jha et al.,2000). Loss of CI activity following acute glutathione depletion appears to be due to reversible nitrosylation of protein subunits comprisingthis complex.The effects of prolonged chronic glutathione depletion on CI activity in dopaminergic cells, however, are unknown but may involve additional, irreversible oxidative events. A major goal of our project is to assess CI inhibition in our dopaminergic cell model at various times and levels of glutathione depletion in order to identify the limitsof function vs. dysfunction including the thresholds for reversible vs. irreversible inhibition as well as the oxidant species and protein targets involved. Once such targets have been identified, we will assess the presence of similar alterations in a newly constructed dox-inducible antiGSH transgenic mouse model following titration of glutathione levels within SN dopaminergic neurons in vivo as a model for molecular events associated with Parkinson's disease. Acute reduction in glutathione in our cell model also results in inhibition of the GSSG reducing enzyme glutathione reductase (GluRd) and up-regulation of the enzyme g-glutamyl transpeptidase (GGT) which breaks down extracellular GSHto substrates which are transported back into the cell for glutathione synthesis. The former may contribute to the detrimental effects of glutathione depletion on mitochondrial function while the latter appears to be a compensatory event. We will also explore the molecular mechanisms involved in these molecular alterations following titration of glutathione pools including the oxidants and the enzyme targets involved and if/how these changes modify mitochondrial function. We will assess the effects of both transgenic GSSG Rd and GGTexpression on CI activity and mitochondrial function in our dopaminergic cell model to see if they act to attenuate any detrimental mitochondrial effects.

By providing management and organizational support services for all Projects and Cores, Administrative Core A will create an environment that encourages excellence and fosters interrelationships to ensure a successful multidisciplinary research approach. Included in these activities will be scheduled meetings every eight weeks among participants in the Program Project as well as an annual full-day retreat, both designed to encourage and facilitate interactions among participants in the Program Project. The Core will also manage the activities of an External Scientific Review Board whose purpose is to provide expert guidance and external review to the Program Project. They will also assist the Project and Core Leaders, both individually and as a group, to help foster new ideas, resolve technical issues and maintain project objectives. Progress reports, manuscript circulation and various mailings to the group will also be coordinated through this Core. An electronic bulletin board will disseminate information to all Program participants rapidly. Shared data will be posted in an Andersen PPG file on the Buck's server which has already been established for this purpose. The Core will liase with the Buck's IT Department to keep abreast of new technologies which will aid in dissemination of information amongst the various Program Project participants as well as other members of the Buck and outside scientific and lay communities as needed. The Administrative Core will in addition provide biostatistical assistance for the design and implementation of the proposed experiments in order to assure uniformity in statistical analyses of data generated by the various individual Projects and Cores as part of the Program Project. Finally, the Core will establish and be in charge of management of a tissue bank of materials from the various genetically engineered mouse lines to be shared by the various Projects as well as with other Buck and collaborators outside the Institute. This will be based on the individual tissue bank already established in the Andersen laboratory for catalogued storage of materials from their various genetically engineered mouse lines. In addition to the activities described above, the day-to-day administrative issues will be coordinated through this Core, including personnel, inventory, purchasing, maintenance and other administrative functions such as animal and isotope approvals. The Core will maintain financial records of expenditures and work with Institute finance staff to prepare monthly statements for all Project and Core leaders. The Core will also generate and submit all progress reports and grant renewals related to the Program Project.

DESCRIPTION (provided by applicant): Aging remains the single most important risk factor in human disease in North America and the idea that nutritional interventions can have profound effect on the onset of age-related disease is vitally important during a time of dramatic demographic change. Dietary restriction (DR), a reduction of nutrients in the diet, provides the most robust method of lifespan extension in species as diverse as yeast, worms, fruit flies and rodents. It has been shown in rodents that DR protects against a number of age related diseases including Huntington's, cancer, diabetes and other cardiovascular diseases. Given the universally protective effects of DR, investigating its molecular mechanisms will promote a greater understanding of the pathogenesis of various human age related diseases, which will in turn help advance the development of therapeutics for these disorders. We have identified the conserved nutrient sensing TOR pathway as a critical regulator of DR dependent lifespan changes. Using a combination of biochemical, genetic and genomic technologies we propose to understand the link between TOR, metabolism and aging. We hypothesize that modulation of mRNA translation by the TOR pathway leads to alterations in ATP generating pathways and mitochondrial function which mediates the lifespan effects of the TOR pathway. This proposal will examine the conservation of the effects of the TOR modulation on mitochondrial function between flies and human cells. Furthermore, using Drosophila we shall examine the cause and effect relationship between ATP generating pathways and lifespan. RELEVANCE: There is considerable interest in nutritional interventions that decrease the risk of disease and extend healthy lifespan. Since there is a high degree of genetic homology between humans and model organisms such as flies and worms, we believe it is timely to understand the dietary factors that regulate lifespan and metabolism in these simple organisms. Our findings will have a significant impact on helping uncover the role of nutrition in the etiology of a number of age-related diseases like cancer and diabetes.

DESCRIPTION (provided by applicant): Age-related human diseases including cancer, Alzheimer's and Parkinson's disease show a clear correlation with mitochondrial dysfunction and reactive oxygen species (ROS). We propose to undertake a combined genetic, biochemical and bioenergetics approach to test the hypothesis that mitochondrial dysfunction and particularly mitochondrial reactive oxygen species (ROS) generation are causatively involved in age related disease. We propose to use three main manipulations of mitochondrial features-electron transport chain complexes, the glutathione pool and superoxide levels-and to measure outcomes models of these three age-related conditions. We believe that the proposed Program Project will make a significant contribution to our understanding of how mitochondrial function contributes to age-related disease with a view towards designing successful interventions.

DESCRIPTION (provided by applicant): The Gordon Research Conference (GRC) on Oxidative Stress and Disease is designed to provide scientists from a wide range of disciplines with the latest research findings on the role of oxidative stress in a variety of disease processes with emphasis on shared mechanisms. This research conference has been held every 2 years since 1999;it is part of over 150 conferences that will be organized in 2011 by the GRC, an organization internationally known for the high- quality, cutting edge nature of its meetings. Although a comparatively young GRC conference, attendance at the meeting has grown steadily since its inception over ten years ago. The 2011 GRC on Oxidative Stress and Disease will be held March 14-18, 2011 at the Sheraton Four Point/Holiday Inn Express in Ventura, CA. The program for the meeting described in this application has been assembled around the theme of "Emerging research areas in the study of oxidative stress and disease" which have recently collectively changed the way in which we think about the impact of oxidative stress on cellular mechanisms involved in human disease. These will include the impact of oxidative stress on, for example, epigenetics, microRNAs, stem cells, and mitochondrial dynamics. Findings associated with these novel areas of research are in turn likely to affect the manner in which basic research is translated into interventions for oxidative stress-related disorders. This Gordon Research Conference will bring together leading international experts with a broad range of interests related to diverse aspects of oxidative stress and disease and will create a high quality scientific forum for discussion of the latest findings on basic mechanisms and their translational implementation into interventions aimed at novel disease therapies. In addition, for the first time in the history of this conference, a pre-conference Gordon-Kenan Research Seminar will be organized by junior investigators at the postdoctoral level. Activities during the Seminar will be oriented to junior investigators and are intended to: (1) provide them with the basic background on common mechanisms involved in oxidative stress and disease necessary to maximize their understanding of the science which will be discussed in the subsequent conference, (2) receive feedback on their ongoing research projects from experts in the field, and (3) facilitate their interaction with senior members of this scientific community to promote networking between junior and more senior researchers in the field. We fully anticipate that the scientific discussions, research talks, poster sessions, and other informal interactions between the participants of this conference will contribute to advancing our understanding of novel molecular mechanisms involved in oxidative stress- related human disease and will set the basis for the development of collaborative interventions aimed at promoting new therapeutic treatments for these disorders. PUBLIC HEALTH RELEVANCE: The Gordon Research Conference on Oxidative Stress and Disease together with the pre- meeting Gordon Kenan Research Seminar will bring together leaders in a variety of scientific disciplines relevant to the study of oxidative stress-related disorders and junior investigators constituting the future generation of researchers in this field. The scientific presentations, discussions and workshops during this conference are designed to expand our understanding of the mechanisms by which oxidative stress may contribute to disease processes. It is anticipated that the collegial and cooperative atmosphere that has traditionally characterized this conference will provide the perfect setting for the intellectual development and implementation of novel therapeutics for this set of related diseases.

DESCRIPTION (provided by applicant): Cellular senescence prevents the proliferation of mitotically-competent cells in the periphery in response to various stressors, including endogenous oxidative stress. Senescent cells express a 'senescence- associated secretory phenotype' or SASP, involving the secretion of pro-inflammatory factors that can cause degeneration of neighboring cells. While cell senescence in peripheral tissues has recently been shown to result in a number of age-related pathologies, whether it plays a causal role in brain aging and neurodegenerative disease is currently unknown. Preliminary data from our laboratory suggests that induction of astrocytic senescence may contributes significantly to neurodegeneration associated with Parkinson's disease (PD) and that this can be induced by candidate environmental toxicants previously linked to increased risk for PD. We propose to use two available environmental toxicant libraries to perform a small molecule screen in order to identify additional senescence-inducing agents, paying particular attention to bioavailable compounds previously associated with risk for PD. This will form the basis of future studies assessing the ability of environmental toxins identified in the screen to induce astrocytic senescence in vivo and its contribution to PD neuropathology. Based on this data, we plan to test the hypothesis that environmental agents may increase risk for PD in part via induction of astrocytic senescence that can in turn impact on PD-related neurodegeneration. If correct, our hypothesis has the potential to transform how we think about and treat PD and other related neurological disorders.

PROJECT SUMMARY The lysosome is considered a principle cellular reservoir for the safe storage of redox reactive iron. When lysosomal pH is disrupted, iron is released from the lysosome into the cytosol where it can be taken into the mitochondria via the membrane calcium uniporter. Once inside the mitochondria, the presence of elevations in redox reactive iron can result in increases in levels of mitochondrial oxidative stress, reductions in the mitochondrial membrane potential (MMP), and cell death. Lysosomal pH has recently been reported to be compromised in the context of mutations in the ATP13A2 gene resulting in an early onset form of Parkinson's disease (PD) known as Kufer-Rakeb syndrome. The ATP13A2 gene itself encodes a proton-pumping lysosomal ATPase whose loss of activity has been shown to disrupt both cellular iron homeostasis and mitochondrial function. We propose that ATP13A2 deficit results in a redistribution of chelatable iron which in turn predisposes neurons for increases in mitochondrial dysfunction and cell loss. This would constitute a novel function for ATP13A2 which could have important implications for mechanisms underlying the loss of cellular iron homeostasis associated with PD neuropathology.

DESCRIPTION (provided by applicant): We have discovered a series of chemical compounds that delay the onset of age-related pathology and extend lifespan. These compounds were identified in focused chemical screens and high throughput screens of both synthetic compounds and natural products. Here we propose to identify the mechanism of lifespan extension with a focus on vitamin D which we have shown maintains protein homeostasis and extends lifespan in C. elegans. This will uncover novel mechanisms for interventions in aging and age-related disease.

Project Summary/Abstract Alzheimer?s Disease (AD) is a massive economic and social burden and is one of the most feared diseases of late life. No treatments that halt or reverse the disease exist. There are several critical gaps in our current knowledge of the molecular and cellular mechanisms that drive AD. Critically, the proximal cause remains unknown. Our long-term goals are to discover the temporal order of changes that take place during disease progression to identify novel ways to treat the disease. Extensive studies of AD in many labs have revealed a complex picture of dysfunction in the brain. The characteristics of the disease result from neurons dying, but before neurodegeneration many molecular and metabolic changes are observed. One dominant hypothesis for AD has centered on one of these changes, the accumulation of neurotoxic protein species (Ab and tau). However, other features, such as an apparent ?energy crisis,? may accompany or even precede neurotoxic protein build-up. To determine the causes and the effects, we propose to study multiple systems affected by the disease simultaneously. We will employ a chemical tool which, based on strong preliminary evidence, induces autophagy, a cellular damage defense mechanism. This chemical (known as C1) modulates both the action of neurotoxic proteins and enhances mitochondrial function, making it a powerful tool to examine the interconnectedness of areas of dysfunction. We now propose four specific aims to advance our understanding of AD as follows: (1) determine the effects of the induction of autophagy by C1 in AD models in the invertebrate nematode worm C. elegans; the worm ages rapidly, allowing the disease to be modelled very efficiently in a contracted time period, (2) determine what energetic and metabolic dysfunction occurs and in what temporal order in AD C. elegans models, (3) determine the effect of the disease on protein abundance and protein aggregation, a hallmark of the disease; the effects of autophagy induction by C1 will also be tested in Aims 2 and 3, and (4) identify the global interplay between these biological systems to uncover the critical changes that occur during disease progression that drive the disorder. Collectively, our proposed research will impact AD research by identifying proximal causes of the disease and providing novel targets for pre-clinical and clinical science. If successful, this ?systems? level approach may be applied to other neurological diseases and other chronic conditions. !

PROJECT SUMMARY / ABSTRACT Losses in protein homeostasis associated with accumulation of damaged, misfolded and aggregated proteins is a characteristic feature of aging and many age-related neurodegenerative diseases. We hypothesize that this may in part be driven by age-related dysfunctions in autophagy which establishes a prodromal process resulting in decreased protein homeostasis and subsequent neurodegeneration. Growing evidence suggests that reduced activity of transcription factor EB (TFEB), a master regulator of autophagy and lysosomal biogenesis, could underlie many neurodegenerative diseases. Based on these findings, we conducted a chemical screen in a neuronal cell line for chemical compounds that induce TFEB. We identified a series of compounds that induce TFEB and its targets to levels far exceeding that produced by the classic TFEB inducer rapamycin. Our lead compound `C1' was tested across a wide range of proteotoxic disease models including in the nematode C. elegans, in in vitro human neuronal tauopathy models, and in an in vivo mouse model of Parkinson's disease (PD). In conjunction with elevations in autophagic flux, the compound was found to prevent the formation of neurotoxic proteins aggregates and enhanced mitochondrial function. Subsequent genetic and biochemical analysis shows that C1 induces TFEB by acting as a ?reverse agonist? of the nuclear hormone receptor DAF- 12/FXR, validated via the use of known modulators of DAF-12/FXR. Although FXR is best known for its ability to act in the liver and gut to maintain lipid homeostasis, it has recently been shown to be present in brain neurons although its role in here is currently unexplored. Our results highlight a novel previously uncharacterized role for FXR-TFEB signaling-mediated autophagy in age-associated neurodegenerative diseases. Based on these results, we hypothesize that neuronal FXR mechanistically acts to modulate levels of TFEB-mediated autophagy and as such constitutes a novel target for the treatment of age-related neurodegenerative diseases including Alzheimer's disease (AD). To test this hypothesis, we propose to determine whether: (1) FXR inhibition results in downstream TFEB signaling, triggering an increase in neuronal autophagy within neurons affected in AD and (2) prevents subsequent development of established AD-related pathologies. Proposed studies include analyses in both human iPSC-derived neurons and in brain tissues from an in vivo AD mouse model to interrogate features associated with human disease including progressive development of mitochondrial deficits, A? and tau neuropathology, losses in synapse integrity, and in mice, cognitive dysfunction.