Marie-Francoise Chesselet - US grants

DESCRIPTION (provided by applicant): The striatum is involved in pervasive motor and psychiatric disorders such as Parkinsons's and Huntington's disease, schizophrenia, dystonia, Tourette syndrome, and attention deficit and obsessive compulsive disorders. It has been recently recognized that a subpopulation of fast-spiking GABAergic interneurons is a critical element of striatal circuitry. These interneurons provide the major source of inhibition within the striatum. They powerfully inhibit large numbers of striatal efferent neurons and mediate their feed forward inhibition by cortical inputs. These GABAergic interneurons have unique molecular properties among striatal neurons, in particular a high level of expression of GAD67, parvalbumin, and the shaw-like potassium channel KV3.1. In addition, they are coupled electrotonically by GAP junctions made of connexin-36. They are regulated by nigrostriatal dopaminergic neurons, and depend on BDNF for their development. We will use a multidisciplinary approach combining molecular, morphological and electrophysiological techniques in genetically altered mice to elucidate the role of connexin-36, Kv3.1, dopamine and BDNF in the regulation of the molecular and functional properties of these fast-spiking GABAergic interneurons. We will measure Parvalbumin, GAD67, Kv3.1, connexin-36, and their mRNA by immunohistochemistry and single cell semi-quantitative in situ hybridization histochemistry, respectively. Changes in electrophysioiogical properties (action potential characteristics, spike trains, electrotonic coupling and cortical inputs) of the striatal fast-spiking GABAergic interneurons will be analyzed in striatal slices. These experiments will be done in mice lacking intrinsic molecules that may be critical for the functional properties of the fast-spiking GABAergic interneurons, specifically connexin-36 and Kv3.1, and in mice with alteration in extrinsic factors known to alter the function of these interneurons, specifically dopamine and Brain Derived Growth Factor (BDNF). These studies will provide basic information on the mechanisms involved in the maturation of a crucial population of striatal GABAerglc neurons and provide new cues for striatal pathologies.

DESCRIPTION: The striatum (caudate-putamen) a brain area involved in the control of movement and cognitive processes receives a massive excitatory input from the cerebral cortex. During the previous funding period, we obtained evidence that cortical lesions in adult rats produce long term changes in striatal gene expression and little compensatory axonal sprouting. The research described in this application is concerned with the hypothesis that greater neuronal plasticity exists in response to cortical lesions in the immature than in the adult striatum. We will use quantitative approaches to examine the extent and morphological characteristics of the innervation of the dorsolateral striatum by afferent from the contralateral cortex labelled by injection of anterograde tracers in rats. This will be done during normal development, and after lesions of the frontoparietal cortex induced by superficial thermocoagulation of pial blood vessels in pups. Reactive synaptogenesis in the denervated dorsolateral striatum will be measured by electron microscopy. In order to identify factors that may be critical for corticostriatal plasticity, we will examine the pattern of expression of growth factors and adhesion molecules known to play a role in neurite outgrowth in vitro and in vivo, but whose role in the striatum during normal postnatal development and after cortical lesions is unknown. Specifically, quantitative immunohistochemistry and in situ hybridization histochemistry will be used to examine the expression of basic fibroblast growth factor (bFGF), neurotrophins (BDNF, NT3), and the highly polysialilated neural cell adhesion molecule (PSA-NCAM). The results will be analyzed in relation to the development of the corticostriatal pathway, and the induction of compensatory axonal sprouting after lesions. Finally, we will determine the effects of cortical lesions induced at different times during postnatal development on the expression of mRNAs encoding glutamic acid decarboxylase (GAD), the enzyme of GABA synthesis, and neuropeptides present in striatal efferent neurons, which have been shown to be altered after cortical lesions made in adults. These experiments will provide new insights into the molecular mechanisms underlying neuronal plasticity in the striatum during postnatal development and in response to early postnatal cortical injury. This will provide the rational for new approaches to treat striatal dysfunction resulting from cortical alterations occurring in the young or in the adult as a result of neurodcgenarative diseases or brain injury.

Functionally, the basal ganglia consists of the striatum, the pallidum and the substantia nigra. These nuclei are a target for drugs used in the treatment of number of neuropsychiatric disorders, and are implicated in chronic neuronal dysfunctions such as Parkinson's disease, Huntington's chorea and tardive dyskinesia. GABA, an inhibitory neurotransmitter synthesized by glutamic acid decarboxylase (GAD) is the major neurotransmitter in these nuclei. We have shown that levels of GAD and GAD mRNA are modified in neurons of the basal ganglia after lesions of cortical and dopaminergic afferents, and chronic administration of antipsychotics. These observations suggest that GAD gene expression is regulated in GABA- ergic neurons in response to alteration of their activity and reveal novel information on the long term effects of lesions and pharmacological treatments on discrete populations of basal ganglia neurons. The research proposed in this application will extend this analysis to the role of other key neurotransmitter systems of the basal ganglia in regulating GAD gene expression in these brain regions. Three major sets of neurons will be examined: the subthalamic nucleus, the serotonergic projection from midbrain raphes, and intrastriatal cholinergic neurons. An additional goal will be to compare the regulation of genes encoding two distinct isoforms of GAD (Molecular weight 65,000 and 67,000 daltons) and of the corresponding immunoreactive proteins. For mRNAs, the main method of approach will be in situ hybridization histochemistry and quantitative autoradiography at the single-cell level in topographically identified neurons. The levels of GAD mRNA will be compared to those of each GAD isoform by quantitative immunohistochemistry with monospecific antibodies. Results will be compared with changes in the level of enkephalin and substance P mRNAs, neuropeptides co-localized with GABA in distinct subsets of striatal efferent neurons. The results of these experiments will provide new insights into the molecular regulation of GABA-ergic neurotransmission in the brain. In addition, by providing unique information on the effect of drugs used in the treatment of psychiatric illness on subpopulations of GABA-ergic neurons in the basal ganglia, the results will help to identify new directions in the treatment of psychiatric symptoms in diseases of the basal ganglia, and help in the development of antipsychotic drugs with less extrapyramidal side effects.

DESCRIPTION (provided by applicant): Since 1998, the Training Program in Neural Repair at UCLA has enabled a collaborative effort of Faculty with broad expertise in neural repair to provide in depth training in this expanding area of Neuroscience. This application requests support to pursue this program with expanded emphasis on training in skills that are made necessary by the evolution of our field. Our trainees will be schooled not only in the basic principles of neural development and response to injury but also in teamwork, innovative technical approaches, and the challenges of translating this basic understanding into benefits for patients. The training program will continue to draw on the unique strength of a group of faculty actively engaged in basic and clinical research on various aspects of Neural Repair at UCLA. Mentors for the program include established and junior investigators with expertise in stem cell differentiation, cell death and neuroprotection, neural development, plasticity and restoration of function after injury to the central nervous system. They include basic and clinical scientists, many of whom bridge the gap between the laboratory and advances in therapies for neurodegenerative diseases and brain injury. All have vigorous research programs and an active commitment to graduate and post-doctoral education. Graduate students in the training program obtain their degree in the Interdepartmental Graduate Program in Neuroscience or one of the ACCESS biomedical graduate programs at UCLA. The curriculum includes training in broad areas of cellular, molecular and system neuroscience, specialized courses in Neural Repair, weekly meetings with other trainees and faculty, seminars from renowned investigators in the field, and exposure to clinical research linked to advances in the field of neural repair. The trainees are encouraged to explore areas at the junction of multiple fields, to use multiple technical approaches, and to engage in collaborations between laboratories. Our goal is to train a cadre of young investigators that are fully prepared for the changing culture of science while retaining a solid background in their main area of expertise. This training approach will benefit from a history of fruitful collaborations and interactions among the mentors in the program and will continue to produce a cadre of young investigators with the ability to respond to the challenges of reducing the burden of disability resulting from degeneration and disruption of the central nervous system.

The UCLA Center for the Study of Parkinson's Disease will use an integrated multidisciplinary approach to elucidate the effects of nigrostriatal lesions and treatments of Parkinson's disease on the molecular and cellular characteristics of the subthalamic nucleus. This region of the basal ganglia has recently emerged as an important focus for the development of novel therapeutic strategies for the disease. The goal of the Center will be to identify new molecular targets for non-invasive pharmacological treatments for Parkinson's disease. The techniques of molecular neuroanatomy, slice electrophysiology, in vivo microdialysis and behavioral analysis will be used in four animal models: I) rats with nigrostriatal lesions; 2) lesioned rats treated with L-DOPA; 3) lesioned rats with chronic deep brain stimulation of the subthalamic nucleus; 4) lesioned rats with implanted OABA producing cells in the subthalamic nucleus. Key findings will be examined with molecular anatomical techniques in post-mortem human brain. Research in the Center will be supported by an Administrative and Communication core. In addition, an Animals and Neuropathology (Core B), Molecular Biology (Core C), and Neuroengineering (Core D) cores will provide standardized research tools for all projects and develop new cutting edge technology to enhance research in the Center. Core B will provide standardized surgical, behavioral, histological and neurochemical procedures for all the animal models examined in the Center, and will collect well characterized brains form patients with Parkinson's disease for study of the subthalamic nucleus. Core C will provide all projects with GABA-producing cells for in vivo transplantation, and will identify changes in gene expression with DNA microarray technology. Core D will develop and manufacture deep brain stimulation probes for rats and develop miniaturized probes for measuring neurotransmitter release. Interactions between the Center and clinical investigators in the Movement Disorder Program at UCLA will provide an ideal conduit for the rapid translation of research findings into clinical applications. The Center will provide a dynamic training environment that will expand the research capabilities of scientists at all career levels and their trainees. The Center will facilitate the participation of new investigators across the UCLA campus in research on Parkinson's disease and will reinforce the existing interactions between basic and clinical research on Parkinson's disease at UCLA.

Functionally, the basal ganglia consists of the striatum, the pallidum and the substantia nigra. These nuclei are a target for drugs used in the treatment of number of neuropsychiatric disorders, and are implicated in chronic neuronal dysfunctions such as Parkinson's disease, Huntington's chorea and tardive dyskinesia. GABA, an inhibitory neurotransmitter synthesized by glutamic acid decarboxylase (GAD) is the major neurotransmitter in these nuclei. We have shown that levels of GAD and GAD mRNA are modified in neurons of the basal ganglia after lesions of cortical and dopaminergic afferents, and chronic administration of antipsychotics. These observations suggest that GAD gene expression is regulated in GABA- ergic neurons in response to alteration of their activity and reveal novel information on the long term effects of lesions and pharmacological treatments on discrete populations of basal ganglia neurons. The research proposed in this application will extend this analysis to the role of other key neurotransmitter systems of the basal ganglia in regulating GAD gene expression in these brain regions. Three major sets of neurons will be examined: the subthalamic nucleus, the serotonergic projection from midbrain raphes, and intrastriatal cholinergic neurons. An additional goal will be to compare the regulation of genes encoding two distinct isoforms of GAD (Molecular weight 65,000 and 67,000 daltons) and of the corresponding immunoreactive proteins. For mRNAs, the main method of approach will be in situ hybridization histochemistry and quantitative autoradiography at the single-cell level in topographically identified neurons. The levels of GAD mRNA will be compared to those of each GAD isoform by quantitative immunohistochemistry with monospecific antibodies. Results will be compared with changes in the level of enkephalin and substance P mRNAs, neuropeptides co-localized with GABA in distinct subsets of striatal efferent neurons. The results of these experiments will provide new insights into the molecular regulation of GABA-ergic neurotransmission in the brain. In addition, by providing unique information on the effect of drugs used in the treatment of psychiatric illness on subpopulations of GABA-ergic neurons in the basal ganglia, the results will help to identify new directions in the treatment of psychiatric symptoms in diseases of the basal ganglia, and help in the development of antipsychotic drugs with less extrapyramidal side effects.

The Administrative and Communication Core will be responsible for the scientific and fiscal management of the Center. In addition to fiscal management, a major role of the core will be to insure the constant communication between investigators in the Center and the functioning of the cores. The administrative core will be involved in disseminating information within the Center, organizing regular meetings of the investigators and their trainees, and promoting communication between the Center and other clinical and basic investigators in Parkinson's disease in the US and internationally. The core will be responsible for all educational activities of the Center.

The core will provide standardized surgical procedures, behavioral, histological and neurochemical analysis to all projects. Specifically, the core will provide rats with 6-hydroxydopamine-induced lesions of the nigrostriatal pathway, with continuous L-DOPA delivery by osmotic minipumps, with implanted GABA-producing cells and with implanted deep brain stimulators of the subthalamic nucleus, and their respective controls. The core will provide behavioral analysis to assess motor behavior in untreated and treated rats with nigrostriatal lesions and their controls. The core will provide histological analysis of lesions, cannula and electrode placement, transplant location and size, and of morphological integrity of the subthalamic nucleus. The core will provide neurochemical analysis of dopamine depletion. In addition, the core will collect and perform neuropathological examinations of post-mortem human brains of patients with Parkinson's disease and controls. This tissue will be used in project 1 and will be made available to other investigators outside the Center.

Project l of the UCLA Center for the Study of Parkinson's Disease will use quantitative molecular and anatomical techniques to address the central hypothesis of the Center: loss of nigrostriatal neurons, as well as both pharmacological and surgical treatments of Parkinson's disease, alter the molecular and cellular characteristics o neurons of the subthalamic nucleus. Levels and pattern of expression of mRNAs encoding subtypes/subunits of glutamatergic. OABAergic and dopaminergic receptors will be examined in the subthalamic nucleus and its target areas with Polymerase Chain Reaction (PCR) and in situ hybridization histochemistry. This analysis will be extended to genes involved in signal transduction and regulation of these receptors after identification of alterations in the expression of candidate genes with DNA microarray technology in Core C. The following animal models will be generated in Core B and examined in this project: l) rats with nigrostriatal lesions; 2) rats with lesions treated with L-DOPA; 3) rats with lesions and chronic deep brain stimulation of the subthalamic nucleus; 4) rats with lesions and transplants of GABA-producing cells in the subthalamic nucleus. Salient findings will be examined in post-mortem human brain collected as part of CoreB. The data obtained in this project will provide the molecular informatioji necessary for the interpretation of in vitro electrophysioiogical experiments performed in project 2 and in vivo microdialysis and behavioral experiments performed in project 3 on the same animal models.

DESCRIPTION (Verbatim from the Applicant's Abstract): Anatomical plasticity, for example the sprouting of spared axons after a lesion, is likely to play a critical role in functional recovery after brain injury or neurodegeneration. However the mechanisms of anatomical plasticity in the adult brain remain poorly understood. Recent experimental results demonstrated a lesion-specific axonal sprouting from the homotypic contralateral cortex in the dorsolateral (motor) striatum of adult rats after lesions of the sensorimotor cortex. This axonal sprouting was observed after lesions induced by thermocoagulation of pial blood vessels, which produce a local ischemia in the cortex, but not after aspiration lesions. The goal of the present study is to determine the cellular and molecular mechanisms involved in this sprouting of corticospinal axons. We will take advantage of the fact that comparable cortical lesions made either by thermocoagulation or by aspiration have different effects on axonal sprouting in the striatum. This will allow us to identify those cellular and molecular mechanisms that are specifically associated with the robust anatomical plasticity seen after ischemic lesions. Recent data from our laboratory have shown that both lesions induce markedly different changes in neuronal activity and patterns of gene expression in the contralateral cortex. In a first set of experiments, we will further identify genes that are likely to be critical for the axonal sprouting by comparing patterns of mRNA expression after the two types of lesions. This will be done first by large scale screening with a DNA micoarray method, followed by confirmation with RT-PCR and mapping at the cellular level with in situ hybridization histochemistry and immunohistochemistry. In a second set of experiments we will block the changes in cellular activity in the cortex contralateral to the lesion to elucidate its role in 1) the molecular changes observed after the lesion 2) axonal sprouting. This multidisciplinary approach will allow us to identify the contribution of key molecular and cellular effects to axonal sprouting in the motor striatum. The results will help to understand the mechanisms responsible for differences in anatomical plasticity after various types of brain injury in the adult. This has relevance for the treatment of stroke and neurodegenerative diseases, two major health concern and leading causes of disability.

The Center for Gene-Environment studies in Parkinson disease at UCLA (UCLA-CGEP) will bridge three major NIH and VA-supported awards in Parkinson's disease (PD) and one NIH-sponsored study of Huntington's disease. The central hypothesis of the proposed UCLA-CGEP is that gene and environmental toxins combine to increase the risk for PD in susceptible individuals through an interplay between pesticides and mechanisms regulating dopamine homeostasis. We postulate that critical factors in this interaction are oxidative stress and resulting alterations in proteasomal function. Project I "Environmental toxins and genes that influence dopamine in Drosophila and humans" will examine interindividual variability of dopamine vesicular transporter (VMAT) expression due to promoter variants in two human populations in parallel with a reporter gene assay. These populations will be genotyped for functional VMAT2 variants and association analyses of gene-environment interactions and pesticide exposures collected in the parent grant will be conducted. In addition, Drosophila genetics will be used to determine how the expression of VMAT affects dopamine-mediated toxicity and identify genes that modulate VMAT function, which will then be examined in the human population for their relevance to increased risk of PD. Project II "Interaction between pesticides and genetic alterations in dopamine homeostasis in mice" will test the hypothesis that pesticides and genetic variations in combination increase the vulnerability of dopaminergic neurons, and that one of the mechanisms involved is oxidative stress. Genetically engineered mice with a reduction in expression of VMAT or the cytoplasmic dopamine transporters, and mice with altered expression of alpha-synuclein and parkin, two proteins known to cause familial PD, will be examined. Behavior and quantitative anatomy will be used to assess the effect of pesticides on dopaminergic neurons in these genetically altered mice. Histology, gene expression profiling, in vivo neurochemistry and slice electrophysiology will be used to examine the role of oxidative stress in this interaction. Project III, "Pesticides and Proteasomal Dysfunction: genetic susceptibility in cellular models" will test the hypothesis that proteasomal dysfunction is central to the deleterious effects of the combined environmental and genetic insults. Cell lines, primary neuronal cultures from genetically altered mice, and human lymphoblasts will be examined.

genetics; model

DESCRIPTION (provided by applicant): New evidence indicates that distinct mutations cause familial Parkinson's disease (PD) by mechanisms that may also operate in sporadic PD. These new data point to the importance of cell dysfunction preceding cell death and to the involvement of non-dopaminergic neurons in the disease. Accordingly, identifying mechanisms of cellular dysfunction that are common to multiple causes of PD may offer new therapeutic targets to halt or reverse the course of the disease. This renewal application for the UCLA UDALL Parkinson Disease Center of Excellence focuses on studies of progression of dysfunction, in complementary models expressing PD-causing mutations, and in a well characterized patient population. The center consists of 5 projects supported by an administrative core and a mouse genetics core. In the first three projects we propose to continue coordinated multidisciplinary work supported by the current award to characterize the progression of motor and non-motor behavioral anomalies and neuropathology (project 1), anomalies of neurotransmitter release (project 2) and of synaptic function (project 3) in genetic mouse models of PD, including novel models based on BAG technology. These projects will be complemented by the addition of cellular models (project 4) to analyze the mechanisms of cellular dysfunction leading to the phenotypes observed in the mouse. Studying progression of dysfunction will also be the focus of the new patient oriented component of the Center. In this project (project 5), we will conduct clinical longitudinal studies of disease phenotype after diagnosis, including psychiatric and cognitive co-morbidities. This will be coupled to the development and validation of an improved health-related quality of life assessment tool. These patient oriented studies will provide crucial clinical data for future analyses of genetic material from the same patients and for the translation of our basic research efforts into improved patient care. To identify the cellular alterations leading to neurodegeneration in PD, the UCLA UDALL Parkinson Disease Center of Excellence will focus on early manifestations of the disease occurring before the onset of motor symptoms and their progression. Integrating experimental models and clinical studies, the goal of our center is to understand the mechanisms of these cellular dysfunctions in order to spur the development of therapeutic strategies able to stop the disease process. Project 1 Title: Progression of Behavioral and Pathologic Defects Preceding DA Cell Death in Mouse Models of PD PI: Marie-Francoise Chesselet, MD, PhD DESCRIPTION (provided by applicant): New evidence indicates that distinct mutations cause familial Parkinson's disease (PD) by mechanisms that may also operate in sporadic PD. During the current funding period we have identified cellular dysfunction without cell death in mice expressing various mutations known to cause PD in humans. In this renewal application we will test the hypothesis that common mechanisms of dysfunction may be induced by distinct mutations and offer therapeutic targets for treatment at early stages of the disease, before further cell death causes irreversible damage. Project 1. 2. and 3 form the continuation of the current award and will continue to use a multidisciplinary approach to uncover the mechanisms of neuronal dysfunction in existing and novel mouse models. In specific aim 1 of project 1 we will determine the progression of behavioral deficits and neuropathology in already available mice overexpressing alpha-synuclein under different promoters and parkin KO mice. We will use a battery of sensitive motor tests we have developed to assess the motor phenotype of the mice, and will extend this analysis to non-motor behaviors because related symptoms can have a major impact on patient quality of life, as examined in project 5 of the Center. We will use immunohistochemistry to determine the progression of alpha-synuclein pathology and glial activation throughout multiple brain regions known to be affected in PD, and to detect anomalies in the expression of proteins involved in neurotransmitter release and examined in project 4 of the Center. We will use ligand binding and molecular approaches to identify dysregulation of dopaminergic transmission that will be further examined with neurochemical approaches in project 2 and with electrophysiology in project 3. Finally, eventual cell loss in brain regions that are affected in PD (locus coeruleus, ventral medulla, nigrostriatal dopaminergic neurons) will be assessed in older animals with unbiased stereology. In specific aim 2, we will extend this analysis to novel mouse models generated in the Mouse Genetics Core with state of the art BAG technology. These include a mouse model expressing a parkin mutation shown to cause the loss of dopaminergic neurons in Drosophila. These mice will be characterized with the same methods described above, before being further analyzed in projects 2 and 3. Studies in project 1 will parallel longitudinal clinical studies of project 5 that examine disease progression in PD patients, including psychiatric and cognitive comorbidity. They will provide critical information on the time course of the deficits and new models for projects 2 and 3 and test in an in vivo mammalian system the hypotheses generated in cellular models in project 4 of the Center.

The Administrative Core will support the infrastructure of the Center as described in detail in "Organizational and Administrative Structure " in the Overview of this application. It will enable the smooth functioning of the Center by assisting the Center Director with the daily management of the Center. The Core will organize the meetings of the Steering committee, Center Investigators, and Internal and External Advisory Boards. The Core will assist the Center Director in reviewing progress and preparing reports. The Core will provide logistical support for the Center educational activities. The Core will aid in the dissemination of scientific information to Center investigators and to the scientific community, and provide logistical support for collaborations with other Centers and attendance to the annual Udall Center meeting. The Core will play an essential role in facilitating regular interactions between the Center and patient support and advocacy groups.

New evidence indicates that distinct mutations cause familial Parkinson's disease (PD) by mechanisms that may also operate in sporadic PD. These new data point to the importance of cell dysfunction preceding cell death and to the involvement of non-dopaminergic neurons in the disease. Accordingly, identifying mechanisms of cellular dysfunction that are common to multiple causes of PD may offer new therapeutic targets to halt or reverse the course of the disease. This renewal application for the UCLA UDALL Parkinson Disease Center of Excellence focuses on studies of progression of dysfunction, in complementary models expressing PD-causing mutations, and in a well characterized patient population. The center consists of 5 projects supported by an administrative core and a mouse genetics core. In the first three projects we propose to continue coordinated multidisciplinary work supported by the current award to characterize the progression of motor and non-motor behavioral anomalies and neuropathology (project 1), anomalies of neurotransmitter release (project 2) and of synaptic function (project 3) in genetic mouse models of PD, including novel models based on BAG technology. These projects will be complemented by the addition of cellular models (project 4) to analyze the mechanisms of cellular dysfunction leading to the phenotypes observed in the mouse. Studying progression of dysfunction will also be the focus of the new patient oriented component of the Center. In this project (project 5), we will conduct clinical longitudinal studies of disease phenotype after diagnosis, including psychiatric and cognitive co-morbidities. This will be coupled to the development and validation of an improved health-related quality of life assessment tool. These patient oriented studies will provide crucial clinical data for future analyses of genetic material from the same patients and for the translation of our basic research efforts into improved patient care. To identify the cellular alterations leading to neurodegeneration in PD, the UCLA UDALL Parkinson Disease Center of Excellence will focus on early manifestations of the disease occurring before the onset of motor symptoms and their progression. Integrating experimental models and clinical studies, the goal of our center is to understand the mechanisms of these cellular dysfunctions in order to spur the development of therapeutic strategies able to stop the disease process.

The Research Development Core will provide seed money for pilot projects to attract new investigators to the itudy of Gene-Environment interactions in PD, foster collaborations between investigators from different disciplines, such as epidemiology, neurology, toxicology, genetic, and basic neuroscience, and provide the possibility to test innovative ideas and gather preliminary data for larger scale studies supported by new grants. Applications will be solicited-in person and through broad-mailing to UCLA investigators that may contribute novel expertise to the study of PD. Proposals will be reviewed anonymously and funding decisions made by the Steering Committee of the Center. Applicants and recipients of pilot funds will be invited to all activities of the Center (monthly meetings, annual symposium, External Advisory Board visits) and be required to present their project and progress on these occasions. Long term assessment of the success of the research core will include yearly updates on progress, publications, collaborations and grant support.

The goal of the UCLA-CGEP is to investigate the hypothesis that the cellular mechanisms of action identified for Putative Environmental Toxicants (PETs) contribute to a significant increase in PD risk;this project will focus on investigations in rodents. Our group identified specific cellular mechanisms that are affected by PETs: the proteasome, microtubule integrity, and aldehyde dehydrogenase detoxification. Together with altered VMAT function/expression that influences dopamine balance, these pathways-may affect the vulnerability of DA neurons to neurodegeneration. We propose to use our complementary expertise with molecular, neurochemical, electrophysiological, and viral gene delivery approaches to determine whether these cellular mechanisms are affected by PETs in vivo in rodent brains and identify additional molecular and functional alterations induced by PETs that can contribute to increased vulnerability of DA neurons. Using PET treatment regimens that lead to mild dysfunction of DA neurons we will determine whether PETs alter levels of K48 and K63 ubiquination in tissue and increase levels of mitochondrial aldehyde dehydrogenase substrates in DA neurons. We will identify new genes involved in the cellular mechanism of actions of PETs by assessing transcriptome changes induced by PETs in nigrostriatal DA neurons isolated by laser capture microdissection, and use slice electrophysiology to identify the mechanism of action of PETs on the cellular properties of DA neurons. Finally, we will assess the role of regulation of DA cytoplasmic levels in modulating PET toxicity by examining the effects of virally or pharmacologically induced alterations of VMAT in rats treated with PETs. These in vivo animal experiments will provide a link between molecular mechanisms identified in projects 1 and 2 and the toxicity of compounds found by our group (Project 4) to increase PD risk in humans. In turn, the transcriptome and electropysiological analyses in Project 3 may point to new cellular pathways to be investigated in genetic studies in Drosophila (Project 2) and in humans (Project 4).

The major theme of the UCLA CENTER FOR GENE ENVIRONMENT IN PARKINSON DISEASE (PD) is to identify novel mechanisms of pathogenesis for sporadic PD by understanding the primary cellular mechanisms by which agricultural pesticides that we and others have demonstrated to be risk factors for PD. produce dysfunction and death of dopamine neurons in cellular and animal models, and lead to PD in humans. Our group has begun to identify an association between exposure to specific pesticides and an increased risk for PD in an exceptionally well-characterized patient cohort in the agricultural region of California Central Valley. In parallel experiments, we have discovered that several of these pesticides affect specific cellular pathways potentially involved in PD. In particular, we have evidence that several agricultural pesticides associated with an increased risk of PD interfere with the ubiquitin-proteasome system (UPS). In addition to their effects on the proteasome, these pesticides to varying extent interfere with microtubule assembly, and/or inhibit aldehyde dehydrogenase, a key detoxification enzyme. The central hypothesis to be tested in the Center is that disruption of these particular cellular mechanisms by some agricultural pesticides is responsible for their ability to increase the risk of PD. Four integrated projects will combine human-based studies in a unique epidemiological cohort (project 4) with basic research studies in cellular (project 1), Drosophila (project 2) and rodent (project 3) models. We expect that the results of our studies will identify novel molecular pathways involved in neurodegeneration in PD, and specific therapeutic targets to stop or reverse the course of the disease. In addition, a better understanding of the potential neurotoxicity of widely used pesticides will have implications for their use in the environment to protect the health of workers and the general population, who are exposed to environmental pesticides in or near agricultural settings.