Gary W. Miller - US grants

DESCRIPTION: Parkinson's disease (PD) is characterized by a selective loss of nigrostriatal dopamine neurons and dopamine innervation to the striatum. Results of epidemiological studies suggest that pesticide exposure is a risk factor for PD, and levels of organochlorine pesticides in post-mortem brain tissue have been shown to be associated with diagnosis of PD. The specific targeting of the SNc dopamine neurons in MPTP toxicity and idiopathic PD has been proposed to be mediated by the plasma membrane dopamine transporter (DAT), which acts as the molecular gateway for MPP+, the toxic metabolite of MPTP. Inside the cell, MPP+ can inhibit mitochondrial respiration and lead to free radical production, or be sequestered into vesicles by the vesicular monoamine transporter (VMAT2). The principal investigator has shown that in vivo exposure to organochlorine pesticides increases dopamine transport via an upregulation of DAT in mouse striatum. He hypothesizes that: 1) exposure to organochlorine pesticides such as heptachlor alters normal dopaminergic function, and 2) organochlorine pesticide-induced alterations to the nigrostriatal dopamine system increase susceptibility to dopaminergic toxins. Cell lines expressing both DAT and VMAT2, transgenic mice expressing different levels of DAT and/or VMAT2, and techniques to assess dopamine function will be used to test these ideas. There are five specific aims. Aim 1 will determine the effects of organochlorine pesticides on plasma membrane and vesicular uptake of dopamine and on DAT and VMAT2 expression in stable cell lines. Aim 2 will characterize mRNA and protein expression of DAT, VMAT2, TH, Dl and D2 receptors in the brain, focusing on the nigrostriatal system of heptachlor treated mice. Aim 3 will determine whether heptachlor alters real time dopamine release and uptake using fast scan cyclic voltammetry in vitro, and whether heptachlor alters extracellular dopamine levels in vivo using microdialysis in freely moving animals. Aim 4 will determine whether organochlorine pesticides augment the toxicity of PP+ in stable cell lines expressing DAT and/or VMAT2. Finally aim 5 will determine whether exposure to organochlorine pesticides increases the subsequent toxicity of MPTP in wild type and DAT-VMAT transgenic mice. The proposed experiments should advance our understanding of the effects of pesticides on dopamine signal transduction and ultimately the onset of PD and other disorders in which dopamine is involved. In addition, these studies could aid in the development of strategies aimed at attenuating the adverse effects of pesticides on the dopamine system.

DESCRIPTION (provided by applicant): Developmental Organochlorine Exposure and Neurodegeneration Parkinson's disease (PD) is characterized by the selective death of nigrostriatal dopamine neurons. The resultant loss of dopamine input to the striatum leads to hallmark motor symptoms of the disease. Epidemiological studies have revealed pesticide exposure as a risk factor for PD and post-mortem studies have demonstrated that organochlorine insecticides are elevated in PD brains. Our lab has previously shown that exposure of adult mice to organochlorine insecticides alters the expression and function of the dopamine transporter (DAT), a key regulator of dopamine neurotransmission and a gateway for dopaminergic toxins. We now have evidence that exposure to these compounds during development increases DAT expression in young mice. The purpose of this study is to examine the effects of organochlorine insecticides on the development of the nigrostriatal dopamine system and to determine if such exposures predispose the animals to parkinsonism. Specific Aim 1. Gestational and lactational exposure to organochlorine insecticides disrupts the development of the nigrostriatal dopaminergic system. In this aim, we will test the hypothesis that developmental exposure to organochlorine insecticides disrupts nigrostriatal dopamine system gene expression in young and adult mice. Real-time PCR, laser capture microdissection, behavioral testing, neurochemistry, and immunochemistry will be performed to determine the effects of the insecticides on the dopamine system. Specific Aim 2. Gestational and lactational exposure to organochlorine insecticides increases susceptibility of dopamine neurons to MPTP. This aim will test the hypothesis that permanent alterations in the expression of key components of dopaminergic neurons by developmental exposure to organochlorines renders these animals more susceptible to the parkinsonism-inducing toxin MPTP. After developmental exposure to insecticides, adult mice will be exposed to MPTP and analyzed as in Aim 1. Completion of these specific aims will provide crucial information regarding the effects of developmental exposure of pesticides on the integrity and vulnerability of the nigrostriatal dopamine system.

DESCRIPTION (provided by applicant) The primary purpose of this broadly based multidisciplinary toxicology and environmental health sciences training program is to prepare trainees for biomedical research careers in schools of medicine, public health, and pharmacy, and in governmental or industrial laboratories with a sound knowledge in environmental health and toxicology. The areas of research emphasis represented by the 22 training faculty include neurotoxicity and neurodegenerative disease, mechanisms of cell death, oxidative DNA damage and repair, and environmental assessment and epidemiology. The 22 members of the training faculty have an established record of success in research training and are currently funded by a total of over $10,000,000 in federal grants with a third of that being from NIEHS. Several scientists from the Centers for Disease Control, including the Agency for Toxic Substances and Diseases Registry, also participate in the training of students through guest lectures and collaborative research. The most important component of training is laboratory research, first as a series of three research rotations, then in the dissertation laboratory. This training is complemented by core courses in toxicology, biochemistry, pharmacology, cell biology, molecular biology, and biostatistics, and advanced courses in toxicology specialty areas, seminar courses, and journal clubs. The program is designed to support six students and three postdoctoral fellows each year from a total eligible population of about 24 students and 30 postdoctoral fellows. The student who completes the pre-doctoral Training Program will have acquired broad familiarity with toxicology and environmental health, knowledge in depth in the area of dissertation research, the ability to search, read critically and report on the literature of the biomedical sciences, mastery of a variety of laboratory techniques useful in modern biomedical research, skill in planning and executing a research project, ability to write clear, accurate scientific reports for publication, and ability to present effectively the results of research. The postdoctoral fellow who completes the Training Program will have gained extensive experience in cutting edge toxicology research, and should have submitted an individual NRSA application to gain grant writing experience to aid in the transition into an independent research position. BACKGROUND This is the first competitive renewal for this Training Program submitted from Emory University. The program is administratively positioned within the Molecular Pharmacology Program (MSP) within the Division of Biological and Biomedical Sciences (DBBS). The goal of the program is to produce broadly trained, competitive scientists oriented towards productive careers related to the toxicological and environmental health sciences. The program is directed by Dr. Gary Miller and co-directed by Dr. Dean Jones. During the first four years of funding, the program has recruited its first classes of pre-doctoral students (total of 12) and postdoctoral fellows (total of 8), expanded the number of participating faculty mentors from 13 to 20, developed a toxicology curriculum within MSP and began the process of establishing the procedures by which students are selected and mentored.

DESCRIPTION (provided by applicant) The ability to several manufactured nanomaterials to induce oxidative stress has been suggested to be the most appropriate means of assessing the potential toxicity of manufactured nanomaterials. Since oxidative stress is a common pathogenic mechanism in numerous diseases, including various neurodegenerative diseases, it is possible that the various nanomaterials may contribute to the disease process. We have shown that the redox state (dynamic balance between reduced and oxidized components) of neurons (in vitro and in vivo) can be spatially resolved by subcellular compartment. Neurotoxicants can preferentially oxidize cytoplasmic, mitochondrial, or nuclear redox components, such as thioredoxin or GSH. We hypothesize that the overall toxicity of nanomaterials will correspond to their ability to induce oxidative stress in distinct subcellular compartments and that these measures will provide a superior means of assessing their potential toxicity. We propose a series of in vitro and in vivo experiments aimed at determining the subcellular redox state of a cellular population known to be especially vulnerable to oxidative injury, namely, the dopamine neurons in the substantia nigra pars compacta. Aim 1. To determine the ability of the manufactured nanomaterials fullerene (C60), fullerol (C60(OH)22-24), manganese oxide (MnO2), titanium dioxide (TiO2), magnetic iron oxide (FeO4), and nanoscale zero valent iron (n-ZVI) to preferentially oxidize sucellular redox components. In this aim, we will examine the ability of suspended nanoparticles to induce oxidative stress in cell cultures of neuronal origin in the absence and presence of an oxidative challenge (6-OHDA). In addition, we will assess the physico-chemical properties of the nanomaterials prior to and after exposure to the cellular model. Aim 2. To determine the ability of the manufactured nanomaterials "manganese oxide (MnO2), titanium dioxide (TiO2), magnetic iron oxide (FeO4), and nanoscale zero valent iron (n-ZVI) to induce oxidative stress in dopaminergic brain regions. This aim will examine the ability of nanomaterials to alter subcellular redox state and induce oxidative damage in dopaminergic brain regions and determine the physico-chemical state of the nanomaterials prior to administration and in the brain tissue of exposed animals. Aim 3. To determine the ability of bioavailable antioxidants to attenuate the oxidative stress induced by manufactured nanomaterials. In this aim N-acetyl cysteine and alpha tocopherol will be tested for their ability to attenuate oxidative stress in in vitro and in vivo settings. Completion of these aims will provide novel information on the ability of nanomaterials to induce oxidative stress with subcellular spatial resolution and determine if their physico-chemical state is altered after exposure to the biological system.

Parkinson Disease; programs; Research

ABSTRACT: ADMINISTRATIVE CORE The Administrative Core serves critical leadership, organizational, and identity roles for the HERCULES Center. During the first three years of the award the Administrative Core has performed the originally proposed duties and taken on additional unanticipated initiatives. We have reviewed the key responsibilities of the Administrative Core and designed an updated structure that will maximize effectiveness, meet these new developments, and remain flexible for future growth. Our Executive Committee receives regular input from an External Advisory Board, an Internal Advisory Board, and the Stakeholder Advisory Board. Using the platform of the exposome, our goal is to advance environmental health sciences at our home institutions and the larger scientific community and this goal remains in the forefront of all HERCULES decisions. The Administrative Core is the key driver of this goal and will pursue the following aims: Specific Aim 1. To provide strategic vision for the HERCULES Center. Specific Aim 2. To promote the mission of HERCULES within the home institutions. Specific Aim 3. To effectively manage center resources. The Administrative Core will work with the Facility Cores and with institutional finance officers to maximize the delivery of core resources. Specific Aim 4. To oversee the recruitment, appointment, evaluation, termination, training, and development of Center members. Specific Aim 5. To facilitate interactions with HERCULES partners. Specific Aim 6. To disseminate knowledge gained from HERCULES activities. By aggressively pursuing the above aims, the Administrative Core will assure that HERCULES remains on its path of excellence by providing a combination of inspirational vision, scientific oversight, fiscal management, resource allocation, and critical appraisal of activities.

As detailed in the preliminary data, we have demonstrated that mishandling of dopamine via reduced VMAT2 expression causes a progressive loss of dopamine terminals and cell bodies in the substantia nigra, synuclein aggregation, and L-DOPA responsive behavioral deficits, making the VMAT2 deficient mice a unique progressive model of PD. WE have shown that several environmental toxicants alter VMAT2 function. We hypothesize that the reduced ability to store dopamine causes oxidative stress in the presynaptic dopamine neuron, damage to cellular constituents, and the death of the neuron. Thus, the sxidative damage that occurs following improper storage of dopamine may underlie the disease process. We have recently shown that the redox state (dynamic balance between reduced and oxidized components) of neurons can be spatially resolved by subcellular compartment in that compounds used in animal models of Parkinson's disease can preferentially oxidize cytoplasmic, mitochondria!, or nuclear redox components, such as thioredoxin (Trx) or glutathione (GSH). Thus, the perturbation of redox state by altered dopamine homeostasis may be a key mediator of toxicity. Given the critical role of VMAT2, a-synuclein, and redox state the dopamine system, the goal of this project is to identify the interactions among these three components in order to better understand the underlying pathogenic processes that mediate cellular damage n Parkinson's disease. Hypothesis: Altered compartmentalization of dopamine disrupts the redox balance of the neuron resulting in enhanced vulnerability of the dopamine neuron Aim 1. To determine the role of asynuclein in regulation of the localization and function of VMAT2. We have preliminary evidence of a direct regulatory interaction of VMAT2 and a-synuclein. In this aim we will determine the nature of this molecular and functional interaction using site-directed mutagenesis, confocal microscopy, and vesicular uptake assays. Aim 2. t To determine if a-synuclein expression alters the vulnerability of dopamine-mediated injury due to reduced VMAT2 expression. By harvesting midbrain dopamine neurons from animals with differential expression of VMAT2 and a-synuclein, we will examine the effects of increased cytoplasmic dopamine and a-synuclein on dopamine-mediated cell death. Aim 3. To determine the effects of reduced or increased expression of VMAT2 on vulnerability to exogenous dopamine or MPTP. Aim 4: To expand our current kinetic-dynamic model of dopamine metabolism to encompass targets of environmental toxicants. We will extend our model to account for the oxidative stress that occurs with mitochondria! dysfunction and the impact of a-synculein on key components of the dopamine system. Completion of the above specific aims will improve our mechanistic understanding of how environmental and genetic factors disrupt proper storage of dopamine and contribute to the pathogenesis of Parkinson's disease

DESCRIPTION (provided by applicant) Parkinson's disease (PD) involves pathological loss of neurons. The long-term objective of this research is to understand how environmental and genetic neurotoxic agents interact to signal and regulate the survival/apoptosis machinery in PD pathogenesis. Mitochondrial dysfunction has been proposed as a key mechanism that mediates demise of dopaminergic neurons in PD. However, the detailed molecular mechanisms by which PD relevant environmental toxicants affect mitochondria! transcription and activity remain unknown. Recently published findings from this lab highlight the key role of nuclear transcription factor myocyte enhancer factor 2 (MEF2) in neuronal survival. Unpublished studies have revealed unexpected presence and function of MEF2 in mitochondria. Based on this, this proposal seeks to explore the role of mitochondrial MEF2 in mediating and integrating the toxic signals of PD relevant environmental toxicants in the degeneration of dopamine neurons. The specific aims are to: 1). Determine the role of mitochondrial MEF2 in regulating transcription of mitochondrial genome in dopaminergic neurons;2). Study the regulation of mitochondrial MEF2 by PD relevant environmental toxicants in mitochondrial dysfunction and neuronal death in cellular models;and 3). Establish the regulation and function of mitochondrial MEF2 in toxicant-induced animal models of PD. To accomplish Specific Aims 1-3 the role of MEF2 in mitochondrial gene transcription will be established in dopaminergic neuronal cell line SN4741 and primary neurons and a group of model toxicants including MPP+(metabolite of MPTP) and rotenone will be tested in cellular and rodent models to investigate whether de-regulation of mitochondrial MEF2 mediates the toxic effects of these toxins. Moreover, an attempt will be to extend these findings to PD patients by correlating the levels and activity of mitochondrial MEF2 with the disease. A combination of morphological, biochemical, functional and genetic methods will be employed in the proposed study. These studies will allow an assessment of whether or not targeting mitochondrial MEF2 underlies environmental toxicant-induced apoptosis of dopamine neurons. The novel insight gained from this study will demonstrate how environmental toxicants may disrupt mitochondrial function, providing a molecular explanation for the loss of dopamine neurons that may relevant to both sporadic and familial PD and a potential therapeutic target.

Parkinson's disease is a progressive neurodegenerative disorder that is characterized by the loss of dopamine neurons in the substanfia nigra pars compacta, resulfing in severe motor symptoms, which include a tremor at rest, rigidity, bradykinesia, akinesia and postural instability. In addition to motor deficits, there are a variety of non-motor symptoms associated with Parkinson's disease, including olfactory deficits, problems with gastrointesfinal mofility, sleep disturbances, sympathefic denervafion, anxiety, and depression. One of the promising areas of Parkinson's disease therapeufics is the administrafion of growth factors, such as glial cell derived neurotrophic factor (GDNF) or brain-derived neurotrophic factor (BDNF). Unfortunately, trials with GDNF and BDNF have not been successful, which is due, in part, to drug delivery and pharmacokinefic limitations. In collaboration with the Ye laboratory at Emory University, we have identified a class of orally active TrkB agonists that appear to recapitulate many of the actions of BDNF. Here we propose to test these novel compounds for their ability to restore funcfion in several animal models of Parkinson's disease. Based on preliminary evidence, we hypothesize that our novel orally-active TrkB agonists will restore function in these animal models. To test this hypothesis we propose to determine the ability of the novel TrkB agonists to restore funcfion in the MPTP mouse model (aim 1), to determine the anfiparkinsonian efficacy of the TrkB agonists in a chronic and progressive animal model of the disease that displays motor and non-motor deficits (VMAT2 LO mice, aim 2), and to determine the ability of these compounds to activate TrkB and alleviate symptoms in a non-human primate model of PD. Complefion of these specific aims will help us to determine the potential utility of selecfive and orally-acfive TrkB agonists as a therapeufic intervenfion for Parkinson's disease and posifion us to apply for further support from the NINDS Translafional Research Program.

Research Development Core Emory Parkinson's Disease Collaborative Environmental Research Center (PD-CERC). The Emory PD-CERC Research Development Core will provide support for initial development of research projects that are in line with the goals of the Center. Investigators not currently affiliated with the PD-CERC (key personnel) will be invited to submit proposals to develop novel ideas that will improve our understanding of environmental factors in neurodegeneration, primarily Parkinson's disease. The Research Develop Core provides a mechanism for enriching the PD-CERC team by providing support for the initial development of novel lines of investigation by non-CNS investigators that complement the ongoing CMS research projects. The PD-CERC will fund 3 pilots annually ($25,0007year each). Expectations of Awardees Pilot awardees will be expected to participate in the Emory PD-CERC seminar series and to present at the beginning and end of their funding period. At the time of award, each awardee is asked to give a brief presentation on their goals for the project. This provides an opportunity for Center investigators to offer advice and expertise on various aspects of the study. At the end of the funding period (within 3 months) the awardees will present at the PD-CERC Seminar. The awardees are also strongly encouraged to present their research at our CND Data club at some time during the project year. CND Data Club is a raw data driven seminar series that does not allow extensive background presentations. The idea is to challenge data quality and interpretation prior to the submission of results. Other Research Development Activities We will also use the auspices of the Research Development Core to foster career development of trainees within the Project Leaders'laboratories. Through a series of rotating lab meetings, we will provide input and advice regarding applications for fellowships and K-awards. This is something we have also done as part of our CND Data Club, but we have decided to develop a specialized forum for this mentorship. Each month the project leaders'labs will have a joint lab meeting with one trainee presenting ideas for career development. For example, a trainee may present and discuss potential specific aims for an NRSA application or for an R21 or K award for more senior trainees. Postdoctoral fellows will also be encouraged to take advantage of the career development resources provided by Emory Office of Postdoctoral Studies. There are several institutional training grants available to trainees of the PD-CERC and pilot awardee laboratories, including the NIEHS Toxicology Training Grant (Dr. Miller and Dr. Jones, Co-Directors), and the NINDS Translational Research in Neurology Training Grant (Dr. Levey, Director).

The HERCULES Center at Emory is based upon the emerging scientific interest in exposome-related approaches and the substantial research base in the Environmental Health Sciences (EHS) on campus. The other components of HERCULES address critical infrastructure needs that are essential for growth of our program. The Pilot Project Program perfectly complements the overall theme of HERCULES and provides an opportunity to broadly promote Environmental Health Science research at Emory University, while specifically advancing the concept of the exposome within the field.

The Career Development Core of the HERCULES Center will promote recruitment of new faculty in environmental health sciences into the Center and foster professional development of faculty in the Center. The goals of the program will be integrally aligned with those of the rest of the Center, promoting interdisciplinary partnerships, translational research, and growth of environmental health sciences at Emory. As described in the HERCULES Logic Model presented in the Strategic Vision section, the program will: Recruit new center investigators and ensure their integration into the Center: Internal to Emory: by targeting existing Emory faculty with skills that fill gaps in the Center and providing support to work with the Center External to Emory: by supplementing recruitment packages to bring key new environmental health faculty to Emory University in areas of priority to the Center, leveraging existing searches Augment existing support services fostering career development of Center's junior faculty through: Formal mentoring Work-in-progress and Grants-in-preparation presentations for critique and feedback Grant-writing consultation service Professional networking Monitor and promote progress of the Center's mid-level and senior investigators Promote cross-training of all ranks of faculty in emerging areas of environmental health sciences to facilitate new collaborations and entry of established scientists into environmental health research Enhance diversity in Center membership and environmental health research at Emory University

The scientific theme of the Emory HERCULES PSO (Health and Exposome Research Center: Understanding Lifetime Exposures) is to use exposome-related concepts and approaches to improve human health. Community engagement in the emerging exposome framework will be key to the effective translation of exposome discoveries into improved human health. The novelty of the exposome concept provides an excellent opportunity for community input at an early stage of the work. It also ensures that community perspectives are integrated into the exposome concepts as they evolve. The Community Outreach and Engagement Core (COEC) will build upon existing partnerships to promote dialogue among local communities, HERCULES scientists, and health-care providers. The COEC will provide the needed mechanisms to ensure that community voices inform the HERCULES scientists of local environmental health concerns, with a particular emphasis on neighborhoods disproportionately affected by environmental exposures. In turn, exposome concepts and research findings will be discussed with local communities and health-care providers in a format that is both relevant and accessible. The COEC will enhance community capacity to address local environmental health issues through consultation services and mini-grant programs. Importantly, the COEC will support HERCULES scientists and trainees as they engage community partners in exposome-based research. An evaluation plan will be implemented for continuous quality improvement of COEC approaches and its activities.

The Systems Biology Core, directed by Eberhard Voit, is composed of an educational component and a service component. The goal of the core is to educate HERCULES investigators on how systems biology approaches can help generate data that can be used in higher-level exposome models. The service component will provide bioinformatic, biostatistical, and systems biology support for all HERCULES investigators. The Integrated Health Sciences Facility Core, directed by Dean Jones, will provide three central functions within the Center, support of clinical research services, personalized exposure surveillance using a top-down high-resolution metabolomics method and targeted exposure analysis using mass spectrometry, immunoassay and chemical sensor methods. The core will facilitate translational and clinical studies by providing access and expertise with several well-characterized human/clinical populations. The IHSFC will act at the interface of the exposome and clinical and public health practice.

DESCRIPTION (provided by applicant) The vision of the HERCULES P30 (Health and Exposome Research Center: Understanding Lifetime Exposures) is for the exposome to gain equal footing to the genome in clinical and public health settings. The investigators believe that the pursuit of this simple and unifying visin will stimulate discovery, promote collaboration, and enhance communication in Environmental Health Sciences at Emory University. The investigators plan to use the P30 mechanism to create an intellectual climate that fosters innovation, collaboration, and progress. Specifically, they plan to enhance collaborative research at Emory, including increasing extramural funding, serving the local and regional community as a source for information on environmental issues, and to make an impact on the national and global level through cutting-edge research. To do this the investigators propose the following specific aims that align with their theme of using exposome-related concepts and approaches to improve human health: Specific Aim 1: Provide greater access to exposome-related approaches (systems biology, metabolomics, high throughput toxicology, spatial and temporal statistical models) through cores, pilot funding, and research forums. Specific Aim 2: Make major contributions towards the operational definition of the exposome (promoting a culture of team science, pilot funding, pursuing exposome-related research). Specific Aim 3: Provide career development activities around innovative and emerging concepts and approaches related to exposome. Specific Aim 4: Enhance and expand existing relationships with community partners to facilitate communication of the importance of environmental factors in disease using exposome principles. Specific Aim 5: Provide infrastructure and resources to facilitate rapid translation of novel scientific findings ito the development of novel sustainability, prevention or treatment strategies in humans. The organization of the Center is depicted in the HERCULES Logic Model in the strategic vision section. The Executive Committee will be guided by these aims and use the Logic Model to monitor progress of the Center.

The mission of the IHFSC Is to facilitate translation and integration of basic science research on environmental exposures into population and personalized health applications, with an emphasis on the impact of exposures on complex biological systems that change dynamically over time. This mission is driven by the overall theme of HERCULES to use exposome-based concepts and approaches to improve human health. A key aspect of the ISHFC is to operationalize exposome-related research and is primarily designed to provide access to, tools and research approaches for clinical and epidemiology research in environmental health. The definitions and implications of the exposome in environmental health sciences remain at a relatively early stage of development, both nationally and internationally. The IHFSC leadership (Jones, Barr, Ziegler) are active in workshops, symposia, and society activities promoting development of exposome-related research. As the IHFSC pursues its mission within HERCULES (Fig G.1) to help operationalize exposome-related research, they will work closely with faculty affiliated with: 1) the Systems Biology Core to enhance utilization and development of exposome-related tools, 2) the Career Development Program to enhance training opportunities in key exposure science technologies and environmental health approaches, 3) the Community Outreach and Engagement Core (COEC) and the Administrative Core to enhance global understanding of the exposome-related concepts and research and their importance in human health and environmental sustainability, and 4) the Pilot Project Program to facilitate translation and integration of exposome-related research tools and concepts into novel and competitive new research projects. This will be done by the IHFSC through 3 major components, universal exposure surveillance (Jones), targeted exposure analyses (Barr), and clinical research services (Ziegler) (Fig G. 1). This builds upon existing bench-to-bedside research in environmental health sciences, anchored by Dr Barr's expert capabilities in exposure science, Dr Jones's universal exposure surveillance method [1], and Dr Ziegler clinical research expertise and links to resources that capitalize on access to well-characterized patients and control subjects. The Core leaders have established local, national and international research leadership in exposure science, quantitative biology, nutrition, and clinical and translational research, have been active in development of exposome-related research and are well- positioned to lead efforts to enhance and facilitate the NIEHS mission in environmental health.

DESCRIPTION (provided by applicant) Environmental health science at Emory University has grown substantially over the past decade. It is no coincidence that this growth parallels the T32 Training Program from the NIEHS. As the investigators enter their 10th year of T32 funding they can document marked success in training, mentoring, and placement of our trainees. Of 34 trainees who have completed their training (either completion of Ph.D. or an Emory postdoctoral fellowship), five are in academic tenure track positions, 12 are in academic non-tenure track positions including postdoctoral fellows still in training, three serve in industry positions, two are in government, ad one is no longer in workforce. The trainees have received numerous awards, including individual NRSAs and K99/R00s. The training faculty has expanded from 19 to 40 with NIEHS grant funding more than doubling. The investigators NIEHS P30 Core Center was just awarded in May, 2013, which will provide trainees with access to expertise in systems biology, bioinformatics, metabolomics, analytical chemistry, and community engagement. The School of Public Health recently completed construction of a new building that greatly increased laboratory and classroom space for many of the T32 investigators. The investigators also successfully launched the new doctoral program in Environmental Health Sciences in 2011 (Dr. Sarnat, Director). This Program provides advanced training in environmental health science from the molecular level to the population level. Postdoctoral trainees have come from several departments on campus, play critical roles in faculty research, and participate in the professional development activities available on campus (Dr. Caudle, Director). Trainees have access to excellent RCR and professionalization workshops. The investigators have been involved in a multi-tiered strategy aimed at the recruitment and retention of under-represented minorities (URM) trainees (over 25% of their trainees are URM). In its first nine years, the Emory NIEHS T32 has provided outstanding training opportunities for 44 junior investigators in the areas of environmental health sciences and toxicology. The investigators request six pre-doctoral and three postdoctoral slots. They propose to continue their Program with innovative coursework, outstanding research training, trainee-focused career development, and a commitment to diversity.

DESCRIPTION (provided by applicant): A major feature of Parkinson's disease (PD) is the loss of dopamine-producing neurons in the substantia nigra pars compacta and the concomitant loss of dopamine in the striatum. While multiple processes likely contribute to the loss of dopamine neurons, we argue that disruption of the proper storage and release of dopamine from vesicles plays a key role. This theory is supported by the literature, which demonstrates that improper storage of dopamine causes the formation of oxidative dopamine byproducts and toxic aldehydes that damages neurons. Evidence from genetic and toxicological studies of PD demonstrates that many insults converge on the dopamine vesicle to exert their deleterious effects. We hypothesize that vesicular function mediates dopamine neuron toxicity, such that increasing vesicle function will enhance dopamine storage and release and confer resistance to neurotoxicants, while decreasing vesicle function will have the opposite effect. Our laboratory has generated and characterized two unique mouse lines that allow us to test this hypothesis: a bacterial artificial chromosome-based transgenic mouse that overexpresses VMAT2 (VMAT2-HI) and a genetic knockout of the synaptic vesicle glycoprotein 2C (SV2C-KO), which was recently implicated in Parkinson's disease pathogenesis. Here, we provide preliminary data that clearly demonstrate that increased vesicular function is beneficial to the dopamine system and that SV2C is a novel regulator of dopamine uptake and release. Together, these findings provide the basis for the following aims. Aim 1: To determine the effects of variable VMAT2 or SV2C expression on vesicular dopamine storage and release dynamics. We will determine how these genetic manipulations alter vesicular dynamics, including the readily releasable pool. Aim 2: To determine if altered vesicular storage and release alters vulnerability of dopamine neurons in a model of PD. We determine the effect of reduced SV2C or increased VMAT2 on MPTP toxicity in aged mice, a classical and well-established model. Aim 3: To determine if altered vesicular storage and release of dopamine influences vulnerability to the PD-related toxicity of polychlorinated biphenyls (PCBs). We expect that our mice will show an inverse correlation between vesicle function and PCB-induced. In addition to the novel mouse lines generated in our laboratory we will use a suite of cutting-edge techniques in our studies, including optogenetic stimulation, fast-scan cyclic voltammetry, in vivo microdialysis, and CLARITY. Completion of these aims will establish a pivotal concept in neuroprotection: increasing vesicle function improves neurotransmitter signaling and opposes dopamine neuron toxicity. This suggests that modulation of synaptic vesicle function represents an innovative and unexplored opportunity for addressing dopamine neuron dysfunction and provides a foundation for new therapeutic approaches.

The Biological Response Resource (BRR) will examined the interactions of exogenous and endogenous chemicals with our biological molecules as they are necessary for there to be a biological effect. One of the most important and practical reasons to include the biological response is that the vast majority of exposures are transient. The chemicals underlying exposures that occurred weeks, months, or years ago are long gone from the body. However, these past chemical exposures often leave molecular fingerprints of their presence. Whether this be damaged DNA, epigenetic modification, or protein adduct formation, the biological fingerprints may be able to provide information of these transient exposures of the past. Without the specific inclusion of the biological impact of exposures in the working definition of the exposome we are left with no way to assess prior exposures at a biological level. The overall goal of the Biological Response Resource BRR is to provide expertise and access to a suite of validated measures of biological responses. In order to offer maximal flexibility to potential users, our BRR is offering a wide array of assays that range from genomic and epigenomic offerings to measures of redox status, cytokine production, and pathway analysis. The goal will be pursued through the following objectives: Objective 1. To educate investigators on the benefits of biological assays available and assist in study design and sample optimization. Objective 2. To provide state-of-the-art measurements that assess the biological effects of, and susceptibility to, exposures on the human genome. Objective 3. To provide access to a variety of other indices of biological response, such as comprehensive profiling of redox state, multiplexed cytokine assessment, and microbiome characterization. Objective 4. To collaborate with the Untargeted Research Resource to identify biological responses that can be measured as part of the untargeted (metabolomics) analysis. Objective 5. To interface with the Developmental Research Resource and the Targeted Research Resource to identify biological markers which are highly associated with specific exposures. The Biological Response Resource will work with CHEAR clients, NEALE colleagues, and the entire CHEAR network to develop reliable and reproducible indices of environmentally-induced perturbations of biological systems.

PROJECT SUMMARY: HERCULES The vision of the HERCULES P30 is to demonstrably advance the role of environmental health sciences in clinical and public health settings using the platform of the exposome. Healthcare and biomedical research have become increasingly genome-centric. While much of this is due to the impressive achievements in genomics, which have consistently outpaced gains in environmental health, it is our contention that a more persuasive case needs to be made for environmental factors. Science and intuition support the idea that the environment plays just as large of a role as genetics for the majority of diseases. The exposome, which embraces a strategy and scale similar to genomic research, is poised to elevate the environment in discussions of health and disease. We will continue to grow and enhance the environmental health science research portfolio at Emory through cutting-edge technologies and innovative data solutions. We will build upon the superb relationships we have built with the local community and continue to push the mission of NIEHS on campus and across the scientific landscape. Based on the extraordinary progress over our first three years, we propose to retain our theme to use exposome-related concepts and approaches to improve human health. This simple and unifying vision will continue to stimulate discovery, promote collaboration, and enhance communication through the following Specific Aims: Specific Aim 1. To marshal physical and intellectual resources to support exposome-related approaches (high-resolution metabolomics, analytical chemistry, systems biology, machine learning, bioinformatics, high-throughput toxicology, and spatial and temporal statistical models) through cores, pilot funding, mentoring, and research forums. Specific Aim 2. To make major contributions towards exposome and environmental health science research. Specific Aim 3. To provide career development activities around innovative and emerging concepts and approaches related to the exposome. Specific Aim 4. To enhance and expand existing relationships with community partners to resolve environmental health issues in the community using exposome principles. Specific Aim 5. To provide infrastructure and resources to facilitate rapid translation of novel scientific findings into the development of prevention and treatment strategies in humans. Pursuit of HERCULES' aims will advance environmental health sciences within our institutions and in the scientific community.

Project Summary Human evolution has created complex metabolism systems to transform and eliminate potentially harmful chemicals to which we are exposed. Available evidence indicates that these systems generate a million or more different chemical metabolites, most of which are completely uncharacterized. Widespread use of mass spectrometry-based metabolomics methods shows that many unidentified mass spectral features are significantly associated with human diseases. Substantial epidemiological research implicates environmental contributions to many disease processes, and we believe that many of the unidentified mass spectral features are metabolites of environmental chemicals. We have an established and successful human exposome research center focused on improving the understanding of environmental contributions to disease. The present proposal is to build upon this foundation to develop powerful new chemical identification tools that can be scaled to identify hundreds of thousands of foreign chemical metabolites in the human body. We have assembled an exposome research team of analytical scientists with expertise in mass spectrometry, xenobiotic metabolism, computational chemistry and robotic methods, to develop and test new chemical identification tools to identify hundreds of thousands of foreign chemical metabolites. Our approach relies upon expertise in 1) computational chemistry to predict possible xenobiotic metabolites, respective adduct forms and ion dissociation patterns in mass spectrometry, 2) use of enzymatic and cellular xenobiotic biotransformation systems, which allows creation of multi-well panels containing specific biotransformation systems to generate xenobiotic metabolites, 3) ion fragmentation mass spectrometry and NMR spectroscopy methods to confirm chemical identities and 4) expertise with robotic systems which can be used to scale the approach to identify hundreds of thousands of metabolites of environmental chemicals. An Administrative Core will maintain an organizational structure and coordinate activities between the Experimental Core and the Computational Core, NIH and the Stakeholder Engagement and Program Coordination Center (SEPCC). The Experimental Core will develop and provide compound identification capability with ultra-high-resolution mass spectrometry support. The Computational Core will develop a predicted xenobiotic metabolite database to support metabolite identification. The Administrative Core will maintain interactions with HERCULES Exposome Research Center and support interactions with prospective Core users. Milestones are established to monitor progress toward goals to establish tools for compound identification that can be scaled to identify hundreds of thousands of foreign chemical metabolites. The results will catalyze metabolomics research by providing new ways to identify unknown metabolites of environmental chemicals, and also support identification of a broader range of metabolites of drugs, food, microbiome, dietary supplements and commercial products.

ABSTRACT For 25 years, we have investigated the genetic bases of familial and sporadic Alzheimer?s disease (AD) among Caribbean Hispanics in the US and in the Dominican Republic, contributing samples and data to the Alzheimer?s Disease Genetics Consortium and the Alzheimer?s Disease Sequencing Project. For this new proposal, we intend to integrate the genetic analyses of AD in Caribbean Hispanics with deep molecular phenotyping: epigenomics, transcriptomics, proteomics and metabolomics using whole blood, plasma, cerebrospinal fluid and brain tissue where possible. Multi-omics data generated in this minority population will be used to understand the effects of gene variants on disease, clarify the affected proteins and pathways that underlie AD and for comparison to results obtained from similar studies in white, non-Hispanic populations such as Accelerated Medicines Partnership ? Alzheimer?s Disease. Compared with non-Hispanics, we have shown that Caribbean Hispanics are three times more likely to develop AD by age 75 years. If they have family members with AD their risk is five times higher. From the study entitled, ?Estudio Familiar Investigar Genetica de Alzheimer? (EFIGA, RF1AG01543) we have clinical information and biological samples in 701 families multiply affected by AD, including 5,932 individuals of which 304 families (43%) have three or more affected relatives and 1,874 individuals with sporadic AD and 2,739 healthy controls. We have begun recruiting members of the Caribbean Hispanic Religious Orders aged 60 years and older who consent to blood sampling for DNA and plasma, lumbar puncture for cerebrospinal fluid and autopsy at death. Our systematic genetic analyses in CH have yielded putative variants in ABCA7, BIN1, CD2AP, CLU, CR1, EPHA1, MS4A4A/MS4A6A, PICALM, SORL1, PINX1 and SRCAP, each of which has been replicated. To covert these genetic findings into meaningful applications to AD we now want to focus our efforts on the relationships between the genetic variants and epigenomics, transcriptomics, proteomics, metabolomics. From the existing cohorts of genetically characterized individuals we will assemble a new multi-omics cohort. This multi-omics cohort of 1,000 individuals will identify systems-level alterations in AD and will provide insight into the mechanisms underlying genetic variants, assist in identifying disease pathways, putative protein-protein interactions and downstream metabolites that can be used to inform preclinical work, develop biomarkers and eventually therapeutic targets for drug discovery. The overarching goal is to use a genetic variant-centered, environmentally inclusive integration of multiple omics layers to identify specific causal genes and investigate how they may perturb pathways leading to disease.

Project Summary Advanced training in environmental health and data science: molecules to populations. This application represents the consolidation of three NIEHS T32 training grants at Columbia University into a unified training program designed to address critical needs in the field of environmental health sciences. We propose a program with 18 predoctoral students and 8 postdoctoral scholars. Our mentoring team has substantial funding from NIEHS (>$13,000,000 per year) and other agencies (>$40,000,0000 per year) that provides a wealth of opportunities for original research by both pre- and postdoctoral trainees. Our predoctoral trainees will participate in: 1) a core curriculum in environmental health sciences (using a life course approach to study molecular mechanisms of disease, epidemiologic methods, health effects of climate change, and the exposome) 2) a core curriculum in data sciences, 3) specialized coursework to support dissertation research, 4) research rotations, 5) small interdisciplinary training groups, and 6) dissertation research. Although our trainees will continue to take traditional didactic coursework, the addition of small interdisciplinary training groups, workshops, and boot camps creates a facile platform that can rapidly evolve to enable student exposure to cutting-edge methods that address future needs in the field. Through a collaboration with the Columbia University Data Science Institute (DSI) we propose a highly innovative training program for our postdoctoral trainees. One of the leading data science programs in the world, the Columbia DSI will provide complementary training and support for our fellows, including participation in their existing data science postdoctoral fellows program based in computer science and engineering. Fellows will acquire advanced data science skills to complement their environmental health science research (the primary focus on their training). In their second year, these fellows will enhance their leadership skills by facilitating our workshops, bootcamps, and mini-courses (machine learning, data visualization, network science) for the predoctoral trainees. Thus, the postdoctoral trainees will acquire a skillset that prepares them to apply advanced data science approaches to environmental health in the laboratory and in the classroom. Moreover, each postdoctoral fellow will lead (with an assigned faculty mentor) a small interdisciplinary training group of predoctoral trainees, providing an ongoing forum for interaction and collaboration between the pre- and postdoctoral trainees and enhancing their skills in guiding team science.

PROJECT SUMMARY In 2018, the NIH Strategic Plan for Data Science identified a number of goals and cross-cutting themes to address in order to maximize the value of data generated through NIH-funded efforts. This included the enhancement of data sharing, access, and interoperability of NIH-supported data resources. One key barrier to achieving this goal is the lack of biomedical scientists with the ability to apply data science techniques to maximize the usability of the data and metadata produced by their research. Our NIEHS-supported training grant (T32 ES007322) provides a single, unified training program for 18 predoctoral students and 8 postdoctoral fellows within the environmental health sciences. Our program is designed to ensure trainees acquire skills in advanced data analytics to complement their primary training in environmental epidemiology, climate science, molecular mechanisms of disease, and the exposome. The integration of additional training in making diverse epidemiologic, toxicological and clinical data findable, accessible, interoperable and reusable (FAIR) and ready for use with artificial intelligence and machine learning (AI/ML) is a natural progression for our multi-disciplinary training program. We also benefit from the co-location of two other NIH-funded training grants, in nursing informatics and neuroscience, with activities training biomedical researchers in data science. We aim to leverage our collective expertise to develop a multidisciplinary curriculum that enables our trainees to develop the competencies and skills needed to make diverse biomedical data FAIR and AI/ML-ready. This curriculum will be designed to be flexible and module-based so it can be implemented in-full, as part of existing training seminars, or as stand-alone bootcamps, depending upon the needs of individual training programs. Our novel curriculum will combine didactic seminars, guided discussions and hands-on training activities to develop competencies and skills in use of data standards, the FAIR principles and AI/ML-readiness. This module-based curriculum will be centered on core foundational concepts, such as ontologies, common data elements and metadata annotation. To construct these modules, we will draw upon expertise from faculty both internal and external to Columbia University from within the fields of semantic science, information science, environmental health data science, and computer science. We will consult with educational professionals who will advise on evidence-based curricular design and provide independent evaluation of our curriculum and training activities using both quantitative and qualitative measures. Following successful evaluation, we propose to incorporate the developed curriculum and training activities into multiple existing training programs. Recorded lectures, discussion guides and training materials will be made available within a shared resource library. Formalizing supplementary training in the FAIR principles and ML/AI-readiness across our multiple training programs will accelerate the achievement of research training aims and develop a cadre of scientists poised to advance biomedical research through the application of data science.

PROJECT SUMMARY/ABSTRACT The National Academy of Sciences describes the barriers that scientists experience on the path from mentored to independent research as the single most critical threat to the future of biomedical sciences in the United States (US). Women and individuals from marginalized groups face steeper obstacles, particularly in technology-intensive fields such as omic sciences. Despite NIH?s separate grant reviews and paylines, the mean age at which investigators receive their first R01 has continued to climb to an all-time high of 46 years, extending a 35-year negative trend. In parallel, the likelihood of PhD graduates becoming professors has dropped significantly in the last decades, putting the future of biomedical research in the US at risk. To address these challenges, we propose the 'Career MODE' program: Careers through Mentoring and training in Omics and Data for Early-stage investigators to empower diverse cohorts of young researchers across the US?i.e., postdocs in the final years of mentored training and faculty members within two years of their first appointment?to establish independent, successful careers in omics and data sciences. Omics? ranging from genomics to the microbiome?have revolutionized biomedical sciences. As they have become part of any biomedical field, the scarcity of a workforce within each discipline with appropriate omic training has emerged as a critical challenge. We seek to develop an 11-month program to provide intensive training in omics and data science and specialized mentoring from a nationwide, diverse network of 70+ mentors. Career MODE will also feature training in professional skills, including leadership and team management, grant writing, mentoring, goal setting and strategy development, communication and teaching, scientific rigor, transparency, reproducibility, responsible conduct of research, and health equity. By leveraging its nationwide group of mentors and trainees, Career MODE will also offer a structured approach to enhance collaborations and networking, help create teams for breakthrough research projects and K99/R00 and R01-type grant proposals, further career development, and help secure tenure-track positions and faculty promotions. Career MODE will feature hybrid (virtual and in-person) coursework and learning activities (boot camps, roundtables, panels, symposia, observerships), a robust evaluation of each objective, and will generate and disseminate extensive educational material on omics and data science. The program will do outreach to the most promising, motivated, and diverse junior investigators?a group traditionally with limited resources? to create empowered cohorts of scientists to pursue independence through advanced knowledge and tools in omics and data sciences applicable to all biomedical research fields. Thus, our program aligns with NIGMS goals and provides a high return for investment. We will also develop strategies to advance equity and access in omics research and promote Career MODE to academic institutions, sponsors, and stakeholders to reach financial sustainability and self-support beyond the five years of R25 funding.