Mel B. Feany, MD PhD - US grants

DESCRIPTION (Adapted from the application): Neurodegenerative diseases exact a massive toll on human and health care resources. They also pose the fundamental question of the mechanisms underlying selective degeneration of particular neuronal populations. Recent identification of the genes involved in several major neurodegenerative disorders, including Huntington's disease and Alzheimer's disease, represent major advances, but have not yet revealed how the encoded proteins produce cell death. Additional components of the neurodegenerative pathway must be identified. The applicants propose to use Drosophila as a model system to identify proteins required for neuronal degeneration. Both Huntington's disease and Alzheimer s disease are dominantly inherited neurodegenerative disorders most likely produced by toxic actions of the encoded gene products. Appropriate forms of both proteins will be expressed in Drosophila using the GAL4 system that facilitates transgene expression in a variety of defined tissue-specific and temporal patterns. The anatomic and behavioral abnormalities resulting from expression of the human transgenes will be characterized, and a phenotype suitable for generating second site suppressors and enhancers will be defined. Flies will then be mutagenized and genetic modifiers isolated. Mammalian homologues of these Drosophila modifiers will be human disease gene candidates and likely components of mammalian neurodegenerative pathways. The ability of many Drosophila proteins, including several discussed in the current application, to substitute functionally for their mammalian counterparts, suggests close analogies between the two systems. Even seemingly highly unique processes such as hindbrain compartmentalization and learning and memory show remarkable similarities. The basic cell biology of neurodegeneration should prove no exception. The applicant is an M.D./Ph.D. who will have completed a residency in anatomic pathology and subspecialty training in neuropathology prior to the proposed start date. She also holds a doctoral degree in neurobiology. The research will be carried out in a Drosophila laboratory group within the Harvard Medical School. A co-sponsor expert in human molecular genetics and neurodegenerative diseases has been selected to complement the primary laboratory's expertise in Drosophila molecular genetics and development.

DESCRIPTION (Provided by Applicant): Parkinson's disease is a common neurodegenerative syndrome characterized by loss of dopaminergic neurons in the substantia nigra, formation of filamentous intraneuronal inclusions (Lewy bodies), and an extra pyramidal movement disorder. Although several genes involved in familial Parkinson's disease have recently been identified, we still know very little about the molecular and biochemical events mediating neuronal dysfunction and death of dopaminergic neurons. To enable a comprehensive genetic analysis of Parkinson's disease, we have developed a Drosophila melanogaster model of the disorder. Expression of human a-synuclein in transgenic flies replicates the three cardinal manifestations of the human disease: adult-onset loss of dopaminergic neurons, filamentous intraneuronal inclusions containing a-synuclein, and progressive locomotor dysfunction. We now propose to exploit the genetic potential of the system by generating second site suppressors and enhancers of a-synuclein mediated neurodegeneration. A robust and titratable retinal phenotype suitable for genetic modification has been defined. Existing collections of well-defined mutant chromosomes will he assayed for their ability to modify the retinal phenotype. De novo mutations will also be generated and tested. Mutations that modify the retinal phenotype will be tested for their ability to alter dopaminergic neurodegeneration and inclusion formation. Modifiers of neurodegeneration and inclusion formation will be characterized molecularly. Mammalian homologues of these Drosophila modifiers will he human disease gene candidates and likely components of mammalian neurodegenerative pathways. We will also test the role of the ubiquitin/proteosome system, chaperones, and apoptosis in dopaminergic neurodegeneration using genetic methods. The role of the ubiquitin system and heat shock proteins will also be tested by looking for the presence of these proteins in Drosophila asynuclein aggregates. Ubiquitin co-localization studies will further address the relevance of the Drosophila system to human disease, because ubiquitination is a pervasive feature of human Lewy bodies. We can abolish inclusion formation in a-synuclein transgenic flies, and will determine if inclusions are required for neurotoxicity.

DESCRIPTION (provided by applicant): Alzheimer's disease is the most common neurodegenerative disease. Pathologically, Alzheimer's disease is characterized by neuronal loss in the context of amyloid plaques and neurofibrillary tangles. Amyloid plaques are composed of a small peptide, Abeta, which is formed from a larger precursor protein (APP). Neurofibrillary tangles are formed from abnormally phosphorylated and aggregated tau protein. Genetic evidence implicates abnormalities in APP processing in familial Alzheimer's disease. Mutations in the tau gene cause frontotemporal dementia. Despite identification of these critical genetic linkages, we still know very little about the molecular and biochemical events mediating neuronal dysfunction and death in Alzheimer's disease and frontotemporal dementia. To enable a comprehensive genetic analysis of these disorders, we have developed genetic models in Drosophila melanogaster. Expression of wild type and mutant human tau in transgenic flies truncates life span, and produces adult onset neurodegeneration associated with abnormally phosphorylated tau. These features replicate key manifestations of human neurodegenerative diseases associated with abnormal tau deposition. We now propose to exploit the genetic potential of the system by generating second site suppressors and enhancers of tau-induced neurodegeneration. Existing collections of well-defined mutant chromosomes will be assayed for their ability to alter the neurodegeneration. De novo mutations will also be generated and tested. Modifiers of neurodegeneration will be characterized molecularly. Mammalian homologues of these Drosophila modifiers will be human disease gene candidates and likely components of mammalian neurodegenerative pathways. We will also test the role of candidate modifiers, including chaperones and the ubiquitination/proteosome pathway in tau-induced neurodegeneration. In addition, we have created a genetic system for studying APP processing. The extracellular and transmembrane regions of APP were fused to green fluorescent protein (GFP) and a nuclear localization signal. Transgenic flies were created that show presenilin dependent nuclear localization of GFP. We will use our easily monitored, presenilin dependent cleavage assay to identify new proteins required for APP processing. These novel modifiers will represent candidate members of the presenilin complex, as well as upstream regulatory factors.

Tau is the major protein component of the characteristic intracellular protein aggregate of Alzheimer's disease and related disorders, the neurofibrillary tangle. Tau deposited in neurofibrillary tangles is markedly hyperphosphorylated, but the role of phosphorylation in influencing tau neurotoxicity has been difficult to dissect in conventional experimental systems. In preliminary experiments, we provide two lines of evidence supporting a critical role for phosphorylation in controlling tau toxicity in a transgenic Drosophila model of tauopathy. First, kinases and phosphatases comprise the major category of genetic modifiers identified in a forward genetic screen for modifiers of tau toxicty. These kinase and phosphatase modifiers include several enzymes known to phosphorylate or dephosphorylate tau in vitro, as well as novel kinases. Selective candidate testing also reveals that overexpression of the known tau kinases MARK, protein kinase A, and CDK5 enhances tau toxicity. Second, mutation of 14 proline-directed serine or threonine phosphorylation sites on tau, including the AT8, AT100, TG3, and PHF-1 sites, markedly attenuates tau toxicity. We now propose to investigate the role of proline-directed phosphorylation in determining tau neurotoxicity in detail. In Specific Aim 1 we will define the phosphorylation sites required for tau neurotoxicity by mutating sites individually or in small groups. These experiments will reveal if any individual phosphorylation site exerts a significant influence on tau toxicity, or if sites work primarily in concert. When the phosphorylation site(s) controlling tau toxicity have been defined, we will confirm the role of phosphorylation in determining toxicity by altering these site(s) to negatively charged amino acids to mimic phosphorylation. In Specific Aim 2 we will determine if the kinase modifiers we have identified through our genetic screens and candidate testing act on specific phosphorylation sites by determining if kinase modifiers are able to alter the toxicity of phosphorylation site mutant tau. Finally, in Specific Aim 3 we will investigate the role of our novel kinase modifiers in human neurodegenerative diseases characterized by deposition of abnormally phosphorylated and aggregated tau, including Alzheimer's disease, Pick's disease, and progressive supranuclear palsy.

[unreadable] DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by loss of motor neurons. Approximately 10% of familial ALS cases are caused by mutations in superoxide dismutase (SOD). Although expression of disease-linked mutant SOD in transgenic mice has led to the development of successful mouse models of the disorder, invertebrate models amenable to genetic analysis have not yet been described. To enable a comprehensive genetic analysis of the mechanisms underlying motor neuron death in ALS, we have developed a genetic model in Drosophila melanogaster. Expression of disease-associated mutant human SOD proteins in transgenic flies produces progressive locomotor dysfunction that ends in paralysis and truncates lifespan. Motor neurons in transgenic animals show degenerative changes and develop intracytoplasmic inclusions containing SOD. Transgenic animals expressing normal human SOD have no locomotor dysfunction or motor neuron degeneration. These features replicate key manifestations of ALS in patients. We now propose to exploit the genetic potential of the system by generating second site suppressors and enhancers of mutant SOD-induced neurodegeneration. A sensitive and robust flight assay will be used to select de novo modifiers in a forward genetic screen. Modifiers of neurodegeneration will be characterized molecularly. The effects of modifiers on locomotor function will be characterized, as will the ability of modifiers to influence motor neuron degeneration and inclusion formation. Mammalian homologues of these Drosophila modifiers will be familial ALS gene candidates and likely components of mammalian neurodegenerative pathways. We will also test the role of candidate pathways, including protein aggregation, oxidative stress and glutamate excitotoxicity in mutant SOD-induced neurodegeneration. SOD-containing protein aggregates will be characterized at the biochemical level and colocalization of ubiquitin and heat shock proteins to inclusions evaluated. Genetic manipulation of heat shock proteins and the ubiquitin/proteasome system will be performed. The presence of oxidative damage will be assayed by immunohistochemical analysis. Genetic studies will evaluate the functional importance of oxidative damage to motor neuron degeneration. The role of excitotoxicity will be explored with genetic manipulations, in particular by overexpression of glial glutamate transporters. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Age is the most important risk factor for developing neurodegenerative disorders such as Alzheimer's disease. However, the mechanisms controlling the special vulnerability of the aging nervous system to neurodegenerative stimuli remain undefined. We have previously documented a clear age-dependence of neurodegeneration in the Drosophila models of human neurodegenerative diseases that we have developed, including models relevant to Parkinson's disease and Alzheimer's disease. We will now use a powerful new regulated gene expression system to direct expression of toxic proteins related to human neurodegenerative diseases specifically to either the young or aged nervous system for defined periods of time. These experiments will allow us to test the hypothesis that the age-dependence of neurodegenerative diseases represents a selective vulnerability of older neurons. Alternatively, we may find that the increased prevalence of neurodegenerative diseases in older individuals represents the prolonged presence of a neurodegenerative stimulus within the long lived neuron. If we find that older neurons are indeed more vulnerable to toxic proteins related to Alzheimer's disease, Parkinson's disease and related disorders we will then be in the position to determine what feature of the aging nervous system endows selective vulnerability to these stimuli. There are well documented changes in the oxidative stress system and the ubiquitin/proteasome system and these systems have been implicated in the pathogenesis of neurodegenerative disorders. Genetic reagents that target these systems will thus first be used to determine the specific pathways that underlie the age-dependence of neurodegeneration. These studies have the potential to define the mechanisms underlying the specific vulnerability of the aged nervous system to neuronal death. Relevance: Neurodegenerative diseases like Alzheimer's disease and Parkinson's disease represent a devastating burden on our aging population, care takers and health care resources. These diseases are remarkable in that they preferentially target older individuals. The studies outlined in this proposal will determine if older neurons have undergone specific cellular changes that make them more vulnerable to neurodegeneration and will outline the pathways responsible for age-dependent vulnerability. Pathways critical for these vulnerabilities will provide important therapeutic targets. [unreadable] [unreadable] [unreadable]

Tau is the major protein component of the characteristic intracellular protein aggregate of Alzheimer's disease and related disorders, the neurofibrillary tangle. Tau deposited in neurofibrillary tangles is markedly hyperphosphorylated, but the role of phosphorylation in influencing tau neurotoxicity has been difficult to dissect in conventional experimental systems. In preliminary experiments, we provide two lines of evidence supporting a critical role for phosphorylation in controlling tau toxicity in a transgenic Drosophila model of tauopathy. First, kinases and phosphatases comprise the major category of genetic modifiers identified in a forward genetic screen for modifiers of tau toxicty. These kinase and phosphatase modifiers include several enzymes known to phosphorylate or dephosphorylate tau in vitro, as well as novel kinases. Selective candidate testing also reveals that overexpression of the known tau kinases MARK, protein kinase A, and CDK5 enhances tau toxicity. Second, mutation of 14 proline-directed serine or threonine phosphorylation sites on tau, including the AT8, AT100, TG3, and PHF-1 sites, markedly attenuates tau toxicity. We now propose to investigate the role of proline-directed phosphorylation in determining tau neurotoxicity in detail. In Specific Aim 1 we will define the phosphorylation sites required for tau neurotoxicity by mutating sites individually or in small groups. These experiments will reveal if any individual phosphorylation site exerts a significant influence on tau toxicity, or if sites work primarily in concert. When the phosphorylation site(s) controlling tau toxicity have been defined, we will confirm the role of phosphorylation in determining toxicity by altering these site(s) to negatively charged amino acids to mimic phosphorylation. In Specific Aim 2 we will determine if the kinase modifiers we have identified through our genetic screens and candidate testing act on specific phosphorylation sites by determining if kinase modifiers are able to alter the toxicity of phosphorylation site mutant tau. Finally, in Specific Aim 3 we will investigate the role of our novel kinase modifiers in human neurodegenerative diseases characterized by deposition of abnormally phosphorylated and aggregated tau, including Alzheimer's disease, Pick's disease, and progressive supranuclear palsy.

Affect; Alexander Disease; Alexander syndrome; Alzheimer; Alzheimer disease; Alzheimer sclerosis; Alzheimer syndrome; Alzheimer's; Alzheimer's Disease; Alzheimers Dementia; Alzheimers disease; Ammonia; Area; Astrocytes; Astrocytus; Astroglia; Astroprotein; Brain; Brain Diseases; Brain Disorders; Cells; Cellular Inclusions; Childhood; Class; Condition; Dementia, Alzheimer Type; Dementia, Primary Senile Degenerative; Dementia, Senile; Disease; Disorder; Drosophila; Drosophila genus; Drug Metabolic Detoxication; Encephalon; Encephalon Diseases; Encephalons; Factor XI Deficiency; Factor XI deficiency, congenital; Fiber; Fruit Fly, Drosophila; GFA-Protein; GFAP; Genes; Genetic; Genetic Alteration; Genetic Change; Genetic Screening; Genetic defect; Glia; Glial Cells; Glial Fibrillary Acid Protein; Glial Fibrillary Acidic Protein; Glial Intermediate Filament Protein; Hemophilia C; Idiopathic Parkinson Disease; Inclusion Bodies; Intermediate Filaments; Intracranial CNS Disorders; Intracranial Central Nervous System Disorders; Kolliker's reticulum; Lewy Body Parkinson Disease; Mammals, Mice; Metabolic Detoxication, Drug; Metabolic Detoxification, Drug; Metabolic Drug Detoxications; Metabolism of Toxic Agents; Mice; Modeling; Murine; Mus; Mutation; NRVS-SYS; Nerve Cells; Nerve Unit; Nervous System; Nervous System Diseases; Nervous System, Brain; Nervous system structure; Neural Cell; Neurocyte; Neuroglia; Neuroglial Cells; Neurologic; Neurologic Body System; Neurologic Disorders; Neurologic Organ System; Neurological; Neurological Disorders; Neurons; Non-neuronal cell; Overexpression; PTA Deficiency; Paralysis Agitans; Parkinson; Parkinson Disease; Parkinson's; Parkinson's disease; Parkinsons disease; Pathology; Phenotype; Position; Positioning Attribute; Primary Parkinsonism; Primary Senile Degenerative Dementia; Process; Protein Overexpression; Research; Retina; Retinal Degeneration; Role; Rosenthal Syndrome; Seizures; Testing; Toxic effect; Toxicities; Work; dementia of the Alzheimer type; demyelinogenic leukodystrophy; detoxification; disease/disorder; dysmyelinating; dysmyelination; dysmyelinogenic leukodystrophy; experiment; experimental research; experimental study; fibrinoid degeneration of astrocytes; fibrinoid leukodystrophy; fruit fly; gain of function; genome mutation; insoluble aggregate; macrocephaly with feeblemindedness and encephalopathy with peculiar deposits; megalencephaly with hyaline panneuropathy; mutant; nerve cement; nervous system disorder; neurological disease; neuronal; neurotransmitter uptake; overexpress; pediatric; primary degenerative dementia; protein aggregate; research study; retina degeneration; retinal degenerative; senile dementia of the Alzheimer type; social role; substantia alba; white matter

DESCRIPTION (provided by applicant): The symptoms experienced by patients with neurodegenerative disorders like Parkinson's disease, Alzheimer's disease and other less common conditions are quite distinct. The basis for the characteristic clinical manifestations of each of the disorders is dysfunction and death of different neuronal cell populations. However, despite many important molecular genetic, pathological and biochemical advances in our understanding of neurodegenerative diseases, the basis for neuronal cell type-specificity remains a fundamental mystery. We will take a genetic approach to the problem of specific degeneration of subsets of postmitotic neurons. Our approach will be to perform unbiased forward genetic screens in Drosophila to outline pathways responsible for cell type-specific neurodegeneration in differentiated adult neurons. We will outline pathways that maintain viability of subsets of neurons both in otherwise normal neurons and in neurons expressing toxic proteins implicated in Alzheimer's and Parkinson's disease. Importantly, we have previously demonstrated relevant neuronal cell type-specific degeneration in our Parkinson's and Alzheimer's disease models. Human homologs of candidates developed in the Drosophila system will then be examined for anatomic localization to vulnerable neurons in human tissue. In the longer term, the cell type-specific pathways of neuronal vulnerability identified in the current proposal will provide attractive therapeutic targets in Alzheimer's disease, Parkinson's disease and related neurodegenerative disorders. PUBLIC HEALTH RELEVANCE: As doctors and family members alike know, patients with Alzheimer's disease often experience loss of memory and other cognitive problems while Parkinson's disease is usually manifest by tremor, slowness of movement and rigidity. Although we know that these characteristic disease symptoms reflect loss of specific neurons in the brains of these patients, we have very little insight into why specific subsets of neurons are lost in particular diseases. Our studies seek to determine the mechanisms that underlie specific loss of identified neurons as part of a longer term effort to devise effective treatments for these devastating disorders.

DESCRIPTION (provided by applicant): Lysosomal storage diseases are a group of multisystem disorders characterized by the common pathology of abnormal accumulation of storage material in lysosomes. Lysosomal storage diseases often affect the central nervous system and cause significant neurological morbidity. Central nervous system manifestations have been particularly difficult to treat with currently available therapies. We propose here to screen directly for central nervous system-penetrant molecules that reduce abnormal lysosomal storage. We have previously characterized Drosophila lacking the lysosomal enzyme cathepsin D and have shown that they represent a robust model of the abnormal lysosomal storage characteristic of neuronal ceroid lipofuscinosis cause by mutations in the cathepsin D gene in patients and in ovine and murine models. We now propose carrying out an in vivo drug screen in our model to identify small molecules that can reduce or eliminate abnormal lysosomal storage material. Molecules emerging from our screen will not only have the ability to modulate lysosomal function in a fashion that promotes clearance of stored material, but will also have the ability to penetrate a functioning in vivo blood brain barrier. Thus, these drugs will represent attractive therapeutic drug candidates in neuronal lipofuscinoses, and possibly other lysosomal storage diseases as well. These molecules may also provide useful research tools to probe lysosomal function in normal and abnormal physiological conditions. Finally, given the emerging evidence that the autophagosomal/lysosomal system is altered in other neurodegenerative diseases, including Alzheimer's disease, the molecules we identify may also have therapeutic relevance to more common neurodegenerative disorders as well. PUBLIC HEALTH RELEVANCE: Lysosomal storage diseases are devastating disorders that cause severe disability and early death in affected children. We will use a fast, cheap in vivo model system to identify compounds that reduce the lysosomal storage thought to result in death of neuronal death and dysfunction in these diseases. Results from our system will provide important candidate therapeutics and may also help us understand how abnormal neuronal storage leads to cell death.

DESCRIPTION (provided by applicant): No highly effective treatment is currently available for Alzheimer's disease. To develop a suitable in vivo model for drug screening, and to investigate the basic pathogenesis of Alzheimer's disease and related disorders, we have created models of the disorder based on expression of human tau and Abeta in the fruit fly Drosophila. Our models recapitulate key features of the human disorders. Specifically, when we express human tau in Drosophila we observe shortened lifespan, behavioral abnormalities and accumulation of abnormally phosphorylated tau protein. Unbiased forward genetic screens have revealed conserved basic pathological mechanisms, including the importance of abnormal phosphorylation of tau, oxidative stress, reactivation of cell cycle in postmitotic neurons, and abnormalities of the actin cytoskeleton. We now propose using our model of Alzheimer's disease and related tauopathies to identify drugs that can ameliorate neurotoxicity in vivo. The small size and short lifespan of fruit flies allows screening of a relatively large number of compounds in intact animals. We will test the ability of 2,000 compounds (Spectrum Collection) to reduce toxicity of tau in our model. Approximately one half of the compounds we will test are USDA approved drugs. Many of the other compounds are natural products with pre-approval clinical history. Many of the drugs we will test have proven ability to reach the brain. We anticipate that the well- characterized nature of the compounds will facilitate translation of these therapeutic compounds to testing in vertebrate animal models and to eventual use in the clinic.

DESCRIPTION (provided by applicant): Alzheimer's disease is the most common neurodegenerative disorder and is characterized pathologically by the intraneuronal deposition of abnormally phosphorylated and aggregated tau protein and by the formation of extracellular amyloid plaques. Abnormal deposition of tau into neurofibrillary tangles is also the primary pathologic feature of a group of less common disorders, collectively termed the tauopathies. To define the molecular mechanisms controlling tau-induced neurodegeneration we (Dr. Gamblin) have performed extensive biochemical characterization of tau variants, including splicing isoforms and mutants linked to the familial tauopathy frontotemporal dementia and parkinsonism linked to chromosome 17. Importantly, these biochemical studies have defined tau variants with altered aggregation and microtubule binding properties. In parallel we (Dr. Feany) have created Drosophila models of tauopathy. Our models recapitulate key features of the human diseases, including age-dependent neurodegeneration, abnormal tau phosphorylation, aggregation of tau into fibrillary tangle-like inclusions, and early death. We will now combine our strengths in biochemistry and in vivo tauopathy modeling to define the species of tau that cause cellular and organismal toxicity in tauopathies.

DESCRIPTION (provided by applicant): Alzheimer's disease is the most common neurodegenerative disorder and is characterized pathologically by the intraneuronal deposition of abnormally phosphorylated and aggregated tau protein and by the formation of extracellular amyloid plaques. Abnormal deposition of tau into neurofibrillary tangles is also the primary pathologic feature of a group of less common disorders, collectively termed the tauopathies. To define the molecular mechanisms controlling tau-induced neurodegeneration we and others have modeled tauopathies in the simple and powerful genetic model organism Drosophila. Genetic, biochemical and cell biological experiments in Drosophila have provided important clues regarding the pathogenesis of tauopathies. However, the unbiased forward genetic screens providing the bases for these studies, while valuable, have to date remained incomplete. Here we propose to use newly created and powerful whole- genome transgenic RNAi collections to perform comprehensive genetic analysis of tau neurotoxicity in vivo. We will complement these studies by with a state of the art transcriptomics in human Alzheimer's disease neurons. These studies will for the first time provide a comprehensive analysis of mechanisms controlling tau toxicity to postmitotitc neurons and should identify many new high-value therapeutic targets. Our studies will be particularly important as more and more data emerges from genome wide associated studies showing genetic influences on Alzheimer's disease and related tauopathies, but with little clear evidence as to the mechanism of action of these newly identified gene products in neurodegenerative disease pathogenesis. PUBLIC HEALTH RELEVANCE: The proposed studies will combine the strengths of fruit flies as a fast, cheap model system to identify causal factors in tau neurotoxicity with state-of-the-art molecular analysis of tissue from patients with Alzheimer's disease to outline mechanisms controlling cell dysfunction and death in neurodegeneration. These studies will help us design better therapies for Alzheimer's disease and related neurodegenerative disorders.

DESCRIPTION (provided by applicant): Mitochondria are central regulators of cellular bioenergetics. Reflecting the critical role for mitochondria in metabolism, energy production, and production of reactive oxygen species, a wide range of human diseases have been linked to mitochondrial dysfunction. Included in these, genetic oxidative phosphorylation disorders represent the most common group of inborn errors of metabolism. Isolated complex I deficiency is the most frequent inherited oxidative phosphorylation disorder and leads to a variety of severe metabolic diseases. Patients with complex I deficiency have a range of clinical presentations that reflect particularly involvement of the brain, heart and skeletal muscle. Leigh's disease, a fatal encephalomyopathy, is the most common clinical syndrome. Isolated complex I deficiency usually leads to death within the first two years of life and there is no effective treatment. Although a substantial amount is know regarding the structure and biochemical function of complex I, the mechanisms leading to cellular dysfunction and death in diseases associated with complex I deficiency are much less well understood. A paucity of animal models has contributed to the slow progress in understanding the pathogenesis of complex I deficiency. To address these issues and allow for a detailed genetic analysis of complex I deficiency, we have modeled the disorder in the simple genetic model organism Drosophila. Results of preliminary genetic modifier analyses lead us to propose a novel hypothesis to explain complex I pathogenesis: accumulation of excess reducing equivalents leading to reductive stress. We will now test the role of reductive stress in complex I deficiency using a combination of genetics and biochemistry. We will first perform a genetic dissection of the enzymatic pathways leading to the production and metabolism of NADH, a critical substrate of complex I. We will then use biochemical assays to measure directly the levels of reductive equivalents in animals with altered complex I function, and in our complex I model in the context of genetically modified backgrounds. If successful, our studies will validate a novel hypothesis regarding the pathogenesis of complex I deficiency and thus set the stage for development of new therapeutic approaches.

This project will harness the power of Drosophila genetics to complete a genome-wide analysis of the mechanisms controlling toxicity of AxD-linked mutant human GFAP to glia. New, comprehensive collections of transgenic RNAi lines will be crossed to the Drosophila model of AxD. Rigorous validation studies will confirm the identities of modifier genes. Candidate genes and pathways will then be prioritized for detailed mechanistic analysis in close collaboration with other members of the Program Project. We will also confirm and extend preliminary data we have developed implicating NO as a secreted signal that promotes neuronal death. Specifically, we will use a new expression system we have developed to test the hypothesis that glia release NO, which then activates cell death pathways in neurons through cGMP-dependent pathways. These studies are expected to significantly enhance our understanding of the mechanisms by which glia promote nervous system dysfunction in AxD specifically, and in neurological diseases more generally.

Alzheimer's disease is the most common neurodegenerative disorder and is characterized clinically by cognitive dysfunction and pathologically by the formation of extracellular amyloid plaques and intraneuronal deposition of aggregated tau into neurofibrillary tangles. Although rare forms of the disorder are caused by highly penetrant mutations in autosomal dominant genes, the pathogenesis of more common forms of the disease remains incompletely understood. A thorough understanding of the basic mechanisms driving loss of neuronal integrity during aging would provide a crucial underpinning for efforts focused on identifying the pathways mediating neurodegeneration in Alzheimer's disease and related disorders. Thus, to provide a comprehensive knowledge of mechanisms driving brain degeneration in higher eukaryotes we will take advantage of the power of forward genetics in Drosophila to outline, in an unbiased fashion, mechanisms controlling preservation of neuronal function during aging. Then, to relate our findings to human disease directly we will integrate, using a systems biology approach, networks derived from eQTL analysis and RNA sequencing data from a unique and high-quality resource of laser captured temporal neurons from patients with Alzheimer's disease and carefully age- and sex-matched control patients without neurological disease. We will further discover pathways relevant to disease by performing an integrated metabolomics and phosphoproteomic analysis in Drosophila models relevant to Alzheimer's disease, namely human tau and Aß transgenic animals. The resulting networks, including previously undiscovered, or hidden, nodes will be tested for their causal relationship to neurodegeneration in Drosophila in well-characterized models of tau and Aß neurotoxicity. Our studies will thus discover on a genome scale novel mechanisms driving neurodegeneration and will provide a census of those mechanisms most likely to underlie cell death in Alzheimer's disease and related disorders. The genes and pathways we discover can then be examined in mechanistic detail in the appropriate mammalian models.

Parkinson's disease is the most common neurodegenerative movement disorder and is characterized pathologically by the intraneuronal deposition of abnormally phosphorylated and aggregated ?-synuclein protein. Abnormal deposition of ?-synuclein into neuronal and glial aggregates is also the primary pathologic feature of a group of collectively even more common disorders, termed the ?-synucleinopathies. To define the molecular mechanisms controlling ?-synuclein induced neurodegeneration we and others have modeled ?-synucleinopathies in the simple and powerful genetic model organism Drosophila. Genetic, biochemical and cell biological experiments in Drosophila have provided important clues regarding the pathogenesis of ?-synucleinopathies. However, the unbiased forward genetic screens providing the bases for these studies, while valuable, have to date remained incomplete. Here we propose to use a newly created and powerful Drosophila model of ?-synucleinopathies to perform a comprehensive genetic analysis of ?-synuclein neurotoxicity in vivo. These studies will for the first time provide a broad analysis of mechanisms controlling ?-synuclein toxicity to postmitotic neurons and should identify many new high-value therapeutic targets. Our studies will be particularly important as more and more data emerges from genome wide associated studies showing genetic influences on Parkinson's disease and related ?-synucleinopathies, but with little clear evidence as to the mechanism of action of these newly identified gene products in neurodegenerative disease pathogenesis.

Alzheimer's disease is the most common neurodegenerative disorder and is characterized clinically by cognitive dysfunction and pathologically by the formation of extracellular amyloid plaques and intraneuronal deposition of aggregated tau into neurofibrillary tangles. Aging is arguably the most important risk factor predisposing to the development of Alzheimer's disease and significant evidence implicates protein homeostasis, or proteostasis, failure as a key mechanism underlying age-related disease risk. However, the mechanistic basis for impaired proteostasis with advancing age remains incompletely understood. Recently work in model organisms has demonstrated cross talk between peripheral tissue proteostasis and misfolding and abnormal aggregation of proteins in the central nervous system, but the molecular and cellular mechanisms contributing to tissue-level proteostasis regulation are largely unknown. Here we capitalize on a recent unbiased forward genetic screen in Drosophila, which implicated multiple novel cellular pathways in age-related proteostasis failure and neurodegeneration. We have further determined that a number of novel proteostasis identified in the screen act in a non-cell autonomous fashion to regulate brain proteostasis. Based on these results we will expand our studies to define, using powerful model organism genetics, the range of pathways in peripheral tissues capable of altering brain proteostasis. We will further determine if peripheral manipulation of proteostasis through genetic manipulation of these pathways influences neurodegeneration in experimentally tractable Drosophila models related to Alzheimer's disease, namely tau and Aß transgenic flies. Finally, we will analyze tissue from aging mice, as well as from Alzheimer's disease patients and controls, to ensure that the insights we develop from our powerful, but simple, model organism are relevant to the disease itself. These studies will ultimately expand the array of molecular and cellular targets available for therapy development in Alzheimer's disease and related disorders.

Alzheimer's disease is the most common neurodegenerative disorder and is characterized clinically by cognitive dysfunction. Classic neuropathological features of the disease include the formation of extracellular amyloid plaques, intraneuronal deposition of abnormally phosphorylated and aggregated tau protein into neurofibrillary tangles, and gliosis. Glial pathology has generally been considered a secondary, or reactive, change. However, recent advances in understanding normal and pathological glial biology have instead suggested that glia may play an active role in neurological disorders, including Alzheimer's disease. Here we take a genetic approach to define proteins and pathways mediating the influence of glia on Alzheimer's-associated neurodegeneration. Taking advantage of the advanced molecular and genetic tools, short lifespan, and conserved glial biology in Drosophila we will identify glial proteins and pathways that can influence tau neurotoxicity in aging adult brains. In proof of principle studies we validate a novel system for studying non-cell autonomous neurodegeneration in tauopathy. In addition, based on the observation that many genes implicated in Alzheimer's disease through genome wide genetic association studies are expressed predominantly or substantially in glial cells, we will test the effect of upregulating and downregulating these genes in fly glia on tau-induced neurotoxicity. These studies will develop a novel methodology for studying the effect of glia on the neurodegeneration associated with Alzheimer's disease and will ultimately expand the array of molecular and cellular targets relevant for therapy development in this common and devastating disorder.

Parkinson's disease is the most common movement disorder and is characterized clinically by tremor and bradykinesia, as well as by cognitive decline in more widespread forms of the disease. Classic neuropathological features of Parkinson's disease include intraneuronal Lewy bodies formed by intraneuronal deposition of abnormally phosphorylated and aggregated ?-synuclein protein, as well as gliosis. Glial pathology has generally been considered a secondary, or reactive, change. However, recent advances in understanding normal and pathological glial biology have instead suggested that glia may play an active role in neurological disorders, including Parkinson's disease. Here we take a genetic approach to define proteins and pathways mediating the influence of glia on Parkinson's-associated neurodegeneration. Taking advantage of the advanced molecular and genetic tools, short lifespan, and conserved glial biology in Drosophila we will identify glial proteins and pathways that can influence ?-synuclein neurotoxicity in aging adult brains. In proof of principle studies, we will validate a novel system for studying non-cell autonomous neurodegeneration in ?-synucleinopathy. In addition, based on the observation that many genes implicated in Parkinson's disease through genome wide genetic association studies are expressed predominantly or substantially in glial cells, we will test the effect of upregulating and downregulating these genes in glia on ?-synuclein induced neurotoxicity. These studies will develop a novel methodology for studying the effect of glia on the neurodegeneration associated with Parkinson's disease and will ultimately expand the array of molecular and cellular targets relevant for therapy development in this common and devastating disorder.