Tomas R. Guilarte - US grants

The analysis of vitamin B in human milk and colostrum is greatly complicated by the lack of modern methodologies available as well as the large number of B vitamers present in these fluids. This project will develop a radiometric microbiological assay for vitamin B-6 and niacin to complete the development of similar assay procedures for thiamin, biotin, and pantothenic acid in these fluids. Specifically, the project will: 1) apply already developed RMA for vitamin B-6 and niacin for the anlaysis of these vitamins in human colostrum and milk. 2) complete the development and validation of RMA's for thiamin, biotin, and pantothenic acid and apply the RMA's in the analysis of these vitamins in human colostrum and milk.

Vitamin B-6 is an essential factor in infant nutrition in that it plays a vital role in the development and normal functioning of the central nervous system. Recent studies have shown that based on biochemical measurements, a state of deficiency or increased requirement exists in pregnant women on self-selected diets. Maternal vitamin B-6 status is known to have a direct effect on the vitamin B-6 content of the milk. Investigators have shown that low levels of maternal B-6 intakes does not maintain milk B-6 levels by mobilization of body stores. Therefore, mothers with low or deficient B-6 intakes prior and during gestation and lactation will affect the B-6 nutritional status of their infants with potential deleterious effects on central nervous system development. An association between maternal B-6 intakes and neurological disabilities of the infant at birth has been suggested by several investigators. Our preliminary studies have shown that marked alterations in the ontogenic development of several neurotransmitter systems occurs as a result of perinatal vitamin B-6 deprivation and that these effects occur more rapidly than previously known. The proposed study will investigate the effect of maternal vitamin B-6 nutrition during gestation and lactation on the neurochemical and behavioral development of the offspring. The use of various levels of maternal B-6 intake will make possible the study of functional relationships between B-6 levels and its neurochemical effects at different ages. These measures will be correlated with biochemical indicators of B-6 nutritional status in maternal blood. Furthermore, we propose studies to determine the role of 3-hydroxykynurenine in epileptic seizures associated with vitamin B-6 deficient neonatal rats.

Epidemiological studies during the last decade have described an association between chronic early life lead (Pb2+) exposure (CELLE) and mental disorders later in life, for example schizophrenia (SZ). There is scientific evidence that some of these mental health problems are the result of alterations in the function of the N- methyl-D-aspartate receptors (NMDAR) complex. The NMDAR is important for brain development, synaptic plasticity, learning and memory; and, Pb2+ is a potent inhibitor of the NMDAR. During the last 20 years, the long-term goals of the research funded by this grant has been to elucidate the molecular and cellular mechanisms by which CELLE affects brain development and impairs synaptic plasticity and cognitive function via NMDAR inhibition. Our current studies are defining molecular and cellular changes resulting from CELLE that resembles those associated with SZ. For example, CELLE results in the loss of parvalbumin-positive GABAergic interneurons (PVGI) in the frontal cortex and hippocampus of adolescent rats, neuropathology that is hallmark of SZ and is duplicated by NMDAR antagonist animal models of SZ. We also found that CELLE results in a hyperactive subcortical dopaminergic system and increases D2-dopamine receptor levels in the striatum, two features present in SZ subjects. The goal of the proposed studies is to determine the developmental trajectory of the PVGI loss produce by CELLE and the downstream effects on gamma oscillations (?-osc) and ultimately on cognitive function. We are focusing on PVGI because they are fast- spiking GABAergic inhibitory neurons that synchronize pyramidal cell firing, giving rise to ?-osc that are critical for cognitive function and PVGI development depends on NMDAR function. The proposed ?-osc studies will provide a neural network link to the PVGI loss and behavioral and cognitive function deficits in adolescent and young adult Pb2+-exposed rats. We also propose to test D-serine as a pharmacological therapy that may mitigate the behavioral and cognitive function deficits resulting from early life Pb2+ intoxication. At the present time there is no pharmacological intervention that mitigates the behavioral and learning deficits resulting from Pb2+ intoxication in chidren.

A recent report by a National Academy of Science expert panel entitled "Neurotoxicity: Identifying and controlling poisons of the nervous system indicates that there are thousand of chemicals in use today whose toxicity to the central nervous system (CNS) are not known. This is due, at least in part, to the lack of sensitive and general biomarkers to assess CNS neurotoxicity. Neuronal damaged caused by a number of insults has been shown to result in dose-related and region specific increases in glial fibrillary acidic protein (GFAP), an astrocyte specific protein. As a result, GFAP has been proposed as a biomarker of toxicity. The development of methods to assess GFAP levels in brain tissue represent important advances on the field of CNS biomarkers, but there are limitations. A major limitation of these methods is that they can not be applied for in vivo assessment of neurotoxicity. Thus, the development of a biomarker that can detect generalized neuronal damage, it's quantitative, provides topographical information of CNS damage, and can be applied for in vivo studies would be advantageous. We believe that the measurement of radiolabeled PK11195 to peripheral benzodiazepine (BDZ) receptors may be such a biomarker. The proposed studies will use quantitative methods to measure peripheral BDZ receptors as a biomarker of neurotoxicity. We propose to validate the use of in vitro [3H]-PKI 1195 and [125I]-PK11195 autoradiography to measure peripheral BDZ receptors in the brain of animals exposed to prototypical neurotoxins. The method is quantitative and provides excellent topographical information of brain damage. The proposed method will be compared in the same animals with GFAP levels, classical histological methods, or biochemical methods when appropriate. The second approach is the in vivo measurement of [125-I]-PK11195 radioactivity in the living mouse brain using a commercially available single photon radiation detection system (i.e., probe). This method is quantitative and is an important aspect of the proposed work since it may extend the use of the biomarker to study human populations. These approaches are feasible and will provide new and important information on the and important information on the development of a biomarker of CNS neurotoxicity.

DESCRIPTION (provided by applicant): The approved use of manganese (Mn) as an additive to gasoline is likely to result in increased accumulation of this metal in the environment. Chronic exposure to low-levels of Mn may pose a health risk to humans, but it is not known to what extent this may occur. A small number of studies describe acute neurological symptoms in humans after occupational exposure to Mn which resemble those observed in Parkinson?s disease. However, neither the mechanism of action nor the neurological consequences of chronic, low-level exposure to Mn is known. The objective of this proposal is to describe behavioral, in vivo neurochemical, and neuropathological changes that occur as a result of chronic exposure to low levels of Mn. The findings from the proposed studies will be fundamental in understanding the mechanism(s) of chronic, low-level Mn neurotoxicity. Moreover, these data will identify sensitive markers for the early detection of Mn neurotoxicity that can be used in vivo in humans. We propose that Mn neurotoxicity be studied in the non-human primate, Macaca mulatta (rhesus monkey), by assessing behavioral and in vivo brain chemistry using a prospective study design. Serial brain imaging will be performed by positron emission tomography (PET), single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI) and spectroscopy (MRS) techniques, throughout Mn exposure. The type of PET/SPECT scan performed will depend on the molecular endpoint to be examined. The MRI scans will show the regional brain distribution of Mn, as well as provide an anatomical template of the PET/SPECT scan images from each monkey brain. The MRS studies will provide brain metabolic changes resulting from Mn exposure. Following the conclusion of the behavioral and brain imaging studies, neuropathological techniques will be performed on the post-mortem tissue to fully characterize and define the changes found by the brain imaging studies and to assess other neuronal systems which may also be affected. This proposal merges the disciplines of behavioral toxicology, brain imaging, neuroscience and neurotoxicology in an innovative research proposal directly relevant to assessing human health risk to Mn exposure. Our long-term objective is to describe the neurotoxicity associated with exposure to chronic, low level Mn exposure and establish the basis for its mechanism of action. The proposed research using state-of the-art techniques will provide the best possible data with which to make human risk assessment and regulatory decisions about Mn in gasoline. The proposed studies will help us understand potential interactions between exposure to environmental agents and neurodegenerative disease.

[unreadable] DESCRIPTION (provided by applicant): Human exposure to anthropogenic or naturally occurring chemicals contributes to the incidence of neurological disease. To estimate and minimize the human risk of neurological disease from chemical exposure, it is important to identify whether specific chemicals, or classes of chemicals, produce neurotoxicity. Biomarkers of neurotoxicity permit evaluation of exposure to an agent (i.e., dose) and the vulnerability of specific brain structures and cell populations, such as neurons and glial cells, to damage (i.e., effect). A useful approach to assess neurotoxicity is to identify marker proteins of neuronal or glial origin that are sensitive to change as a result of neurotoxic insult. Our approach to the development of a biomarker of neurotoxicity focuses on the peripheral benzodiazepine receptor (PBR), a glia-specific protein. The rationale for this strategy is that reactive gliosis is the earliest and most widespread response of the nervous system to injury. Quantification of a widespread response is needed as a generic biomarker when there is a paucity of knowledge about neuronal targets that may be damaged by a specific chemical. Although the PBR has now been used extensively by us and others as a biomarker of neurotoxicity in the adult brain, it has never been tested in the developing brain. Thus, the specific aims of this proposal are: (1) apply the PBR as a biomarker of neurotoxicity in developing animals; (2) to continue the application of the PBR as an in vivo biomarker of neurotoxicity using small animal imaging; and (3) to determine if the pharmacological activation of the PBR can prevent damage and/or promote recovery from chemical-induced brain injury. A novel aspect of the proposed work is the use of the PBR as an in vivo biomarker of neurotoxicity using state-of-the-art small animal brain imaging techniques. To the best of our knowledge, this is the first validation of a biomarker of neurotoxicity that will permit the study of the living brain in small animals following environmentally-relevant exposures to neurotoxicants. The validation of this technology may serve as a first- tier screening method in neurotoxicity testing of chemicals. Lastly, emerging evidence suggests that the pharmacological activation of the PBR may have neuroprotective effects. We will test this hypothesis in an animal model of demyelination. These studies may have important implications in the development of a novel therapeutic strategy for the treatment of brain injury. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Childhood lead (Pb2+) intoxication remains a public health problem of global proportion. A number of studies over several decades have consistently shown that one of the most prominent effects of Pb2+ in children is to decrease their capacity to learn with devastating effects on cognitive and intellectual development. The consequences of childhood Pb2+ intoxication on the intellectual capacity of pediatric populations and society as a whole is incalculable as the world is more and more dominated by knowledge- based economy. Recent human studies have also shown that previous Pb2+ exposure is associated with longitudinal declines in cognitive function and loss of brain volume in aging individuals. Therefore, Pb2+ exposure in early life has immediate and long-term consequences to human neurological health. The long-term goal of the proposed studies is to understand the behavioral, cellular and molecular bases of Pb2+-induced deficits in cognitive function in developing animals. The ultimate goal is to define molecular mechanisms by which Pb2+ exposure impairs intellectual development in order to devise therapeutic strategies that may be beneficial to Pb2+ intoxicated children. The proposed studies will delineate mechanism(s) by which Pb2+ affects NMDA receptor mediated processes in developing synapses. This will be performed in primary culture of hippocampal neurons. We will also examine the cellular (neurogenesis) and molecular (NMDA receptor) bases of how environmental enrichment and voluntary exercise may reverse the learning deficits of Pb2+ exposed developing animals. Finally, we will begin to assess the neurological consequences of combined developmental Pb2+ exposure and stress. We will examine their combined effects within the context of learning and underlying neurobiological substrates. Previous studies have only examined the effects of Pb2+ on the developing brain without taking into consideration the context in which Pb2+ intoxication occurs. Therefore, the proposed studies will provide a greater relevance to the human condition. Relevance: Scientific evidence has demonstrated that changes in the way that central nervous system synapses develop or deficits in synaptic proteins, forms the bases for many neurological, psychiatric and neurodegenerative diseases. The proposed studies will help delineate molecular mechanisms by which Pb2+ intoxication alters synapse development. These studies will provide the basis for the testing of therapeutic strategies such as environmental enrichment and voluntary exercise on reversing the well-characterized learning deficits in Pb2+ intoxicated animals. Pediatric populations that are most likely to experience Pb2+ intoxication are the same ones that also experience stressful, low-socioeconomic conditions. Therefore, one of the stated goals of our project is to examine the relationship of Pb2+ exposure and stress on neurodevelopment. The proposed studies will have significant implications to public health policy as well as therapeutic interventions for Pb2+ intoxicated children. PUBLIC HEALTH RELEVANCE: The long-term goal of the proposed studies is to understand the behavioral, cellular and molecular basis of lead (Pb2+)-induced deficits in cognitive function in developing animals. The ultimate goal is to examine the detrimental effects of Pb2+ on cellular and molecular processes that depend upon activation of the NMDA receptor and how stress may influence such interactions, and to devise targeted therapeutic strategies to reverse these deficits. A major advantage of this unique team of multidisciplinary collaborators is that we will examine the effects of Pb2+ at the behavioral, systems, cellular and molecular levels in animals that are intoxicated with environmentally relevant levels of Pb2+.

DESCRIPTION (provided by applicant) This revised application is the second competitive renewal of the investigators' Program in Interdisciplinary Training in Environmental Health Sciences (EHS) at Columbia University. Since its inception in 1997, the EHS doctoral program has trained 26 Ph.D. pre-doctoral candidates, 13 of whom have been supported by this training grant. This interdisciplinary program combines public health with basic biomedical science skills and trains students to solve complex problems in three areas of environmental health: cancer, respiratory disease, and neurological disease, within two tracks: Molecular Toxicology and Molecular Epidemiology. The investigators' primary goal is to develop the next generation of independent, academic researchers in environmental health sciences. The Ph.D. program has thrived in a rich, collaborative and collegiate atmosphere that supplements classroom instruction and research with training in writing, public speaking, and teaching. The training faculty consists of 21 well-funded investigators, who collectively have substantial experience in mentoring both pre-doctoral and postdoctoral trainees. These investigators conduct research related to one or more of the Program's focus areas and are highly experienced in its collaborative, interdisciplinary aspects. The training program benefits greatly from its integration with numerous other environmental health activities within the Department, the School, and across the University. These include: the Center for Environmental Health in Northern Manhattan (CEHNM), the Columbia Center for Children's Environmental Health (CCCEH), the Superfund Research Program (CU-SRP) and the Columbia Earth Institute. This training grant, along with Departmental and University support, provides the core funding used to recruit Ph.D. candidates into the Program. By the end of their second year, trainees are typically supported on his/her mentor's R01 grant or on an individual fellowship. This frees up training slots to enable the recruitment of new candidates. Of the nine current students (which includes two underrepresented minorities), six are either currently supported or have been supported by the training grant. Presently, this training grant supports three doctoral candidates but because of the growth of the Department's program faculty, the renovation of its infrastructure, and the increasing number of high quality applicants, the investigators feel that the time is right to extend training grant support to an additional student. Therefore, this application is requesting support for four students so that the doctoral program can grow along with other aspects of the Department. The success of this program is exemplified by the outstanding success of the graduates in finding excellent post-doctoral positions; earlier graduates now hold faculty positions, while several are in senior positions in both industry and related governmental agencies. The renewal of this training grant will assure the continued success of this Program. Public Health Relevance: This is a revised application for continued support, through a training grant, to develop the next generation of researches in the field of environmental health sciences, with a focus on the critical areas of cancer, respiratory disease, neurological disease. A dedicated faculty with an outstanding track record in both research and training will provide the necessary instruction for the investigators' cohort of pre-doctoral students.

DESCRIPTION (provided by applicant): Manganese (Mn) is an essential metal for human health, but exposure to excess levels can cause neurological disease. Emerging evidence suggests that long-term exposures to low levels of anthropogenic or environmental sources of Mn may have detrimental effects on human neurological health. Automobile combustion of gasoline containing methylcyclopentadienyl manganese tricarbonyl (MMT) has the potential to significantly increase Mn exposures to human populations where this fuel additive is used. However, there is a paucity of knowledge on the neurological health effects of chronic exposures to low-levels of Mn. While moderate to high levels of Mn exposure are associated with motor abnormalities and cognitive dysfunction as well as basal ganglia dopaminergic dysfunction in humans and non-human primates. The extent to which lower levels of Mn exposure may target specific cognitive domains and alter brain chemistry is not known. The studies described in this application are the result of a unique and productive on-going collaboration of scientists with expertise in behavioral neuroscience, molecular imaging, molecular and cellular neuroscience, neurotoxicology, neurochemistry and neuropathology applying the latest state-of-the-art behavioral and neuroimaging modalities to understand the behavioral dysfunction and underlying molecular and cellular mechanisms of Mn-induced neurological disease in the living non-human primate brain. Our most recent evidence suggests that chronic low level Mn exposure in non-human primates produces in vivo neurochemical changes resembling those in schizophrenia patients. Further, exposure to Mn produces a cellular stress response and diffuse amyloid-2 plaques in the frontal cortex resembling those in the aging brain and in Alzheimer's disease. These preliminary findings provide a putative link of exposure to an environmental toxicant (Mn) and neurochemical and neuropathological changes associated with mental and neurological diseases. The proposed studies will expand on these findings and provide the most comprehensive assessment to date on the neurological consequences of chronic Mn exposure on the non-human primate brain. The knowledge gained will help set future public health policies and guidelines to limit Mn exposures in occupational settings and to the general population. Relevance: The work that we describe in this proposal will define the behavioral, in vivo neurochemistry and neuropathological effects at increasingly lower levels of chronic Mn exposure in non-human primates. They represent the most comprehensive assessment to date on the neurological consequences of chronic exposure to levels of Mn that segments of the population are likely to encounter in their environment. These studies have already provided novel findings and the knowledge gained will help set future public health policies and guidelines to limit Mn exposures in occupational settings and to the general population. PUBLIC HEALTH RELEVANCE: The long-term goal of the proposed studies is to determine the behavioral, neuroimaging and neuropathological effects of chronic exposure to increasingly lower concentrations of manganese (Mn) in order to identify the level of cumulative exposure that produces the earliest changes in behavior and brain chemistry. The ultimate goal is to determine the extent to which chronic exposure to environmentally- or occupationally- relevant levels of Mn contributes to human neurological disease. A major advantage of our unique collaboration is that behavioral and in vivo brain chemistry changes are monitored prospectively and in parallel with Mn exposure in non-human primates.

DESCRIPTION (provided by applicant): The severe nervous system developmental risks of early life exposure to lead (Pb2+) are well known. It is now becoming increasingly clear that much lower concentrations of Pb2+ can produce significant detrimental effects in children, heightening the need to understand the properties and extent of Pb2+ actions on the brain. Our laboratory has recently discovered that in vitro exposure to very low levels of Pb2+ produces long-term impairments in presynaptic transmitter release in cultured hippocampal neurons, and that these actions mimic those observed in animals lacking the trophic factor brain-derived neurotrophic factor (BDNF). We propose studies in hippocampal slices from rats exposed to low levels of Pb2+ during development to utilize 1) state-of- the-art two-photon imaging methods to assess long-term effects on presynaptic Ca2+ influx and vesicular transmitter release in intact synapses, and 2) whole-cell patch-clamp recording from CA1 pyramidal neurons to characterize the long-term effects of low level Pb2+ exposure on postsynaptic N-methyl-D-aspartate receptor (NMDAR)-gated currents. Our working hypothesis is that early Pb2+ exposure produces impairments in NMDAR function that leads to reduced BDNF synthesis and release and subsequent impairments of presynaptic transmission that are critical to normal cognitive function. One manipulation known to increase BDNF levels and release is an enriched environment. To test our hypothesis and identify potential methods of protecting the brain from developmental damage from Pb2+, we propose to 1) characterize the effects of low [Pb2+] exposure on BDNF gene expression, promoter methylation and TrkB receptor activation, and 2) test the ability of an enriched environment, exogenous intraventricular BDNF infusion or administration of the TrkB agonist 7,8-dihydroxyflavone to prevent Pb2+-induced long-term damage to NMDAR function and transmitter release. The research program we propose addresses the critical question of whether early developmental exposure to low-levels of Pb2+ than previously thought have long-term detrimental effects on brain function. These studies will provide novel information about Pb2+ effects on both presynaptic and postsynaptic mechanisms critical to cognition and memory storage. Further, we will examine novel therapeutic manipulations that elevate BDNF release and TrKB receptor activation in order to protect against the long-term detrimental effects of Pb2+ exposure.

DESCRIPTION (provided by applicant): The long-term goal of this research is to continue the validation and application of the Translocator Protein 18 kDa (TSPO), previously called the peripheral benzodiazepine receptor as a biomarker of neurotoxicity, neuroinflammation and neurodegeneration. While TSPO is being used in preclinical and clinical studies to detect and monitor brain injury and inflammation, the function of TSPO in microglia and astrocytes, the glial cell types that express and upregulate TSPO levels is not currently known. The proposed studies have several inter-related goals. First, we have discovered that in primary microglia, TSPO may be associated with NADPH oxidase (NOX) and mechanistic studies proposed in Specific Aim 1 are planned to extend this finding and to further understand the TSPO/NOX interaction. In Specific Aim 2 we propose studies to understand the function of TSPO in astrocytes. While the function of TSPO in microglia may be associated with the innate immune response, we hypothesize that the function of TSPO in astrocytes may be associated with the synthesis of neurosteroids. An understanding of the function of TSPO in microglia and astrocytes will assist in devising therapeutic approaches for mitigating neuroinflammation in neurodegenerative disease. Finally in Specific Aim 3 we proposed studies to validate and apply TSPO as a biomarker of neurotoxicity in the fetal and early postnatal brain. While most studies to date have used brain tissue from adult animals or humans, we have evidence that TSPO may serve as a biomarker of brain injury in the fetal brain. These studies will use a novel TSPO-GFP mouse model that will significantly facilitate the detection of brain injury in vivo and ex vivo. Te development of novel methods to screen the neurotoxic potential of chemicals is of high priority as articulated by Dr. Francis Collins, the present director of the National Institutes of Health. Thus, the proposed studies have significant translational implications to the human condition. The proposed studies will use a variety of methodologies ranging from cell culture to imaging and are in line with research goals of the National Institute of Environmental Health Sciences and the National Toxicology Program.

Project Summary/Abstract The FIU-RCMI Recruitment Core will focus on hiring established health disparities investigators who have existing track records of independent research support by NIH R-series, P-series, K-series and/or U-series awards, or other federal or non-federal awards. These new hires will bring their own programs of research to FIU, and will serve as mentors to early career and minority health disparities investigators affiliated with the FIU-RCMI. The Recruitment Core will develop and implement (a) search and screen outreach and recruitment strategies that ensure health disparities investigators from underrepresented backgrounds are part of the pool of applicants, and (b) policies and procedures for allocating up to $100,000 direct costs per year for up to two years for each newly hired health disparities investigators to augment institutional support. For the FIU-RCMI to be successful in its research, training, and community engagement goals, we need a transdisciplinary team of experienced, collaborative-minded, and dedicated health disparities experts. In particular, experienced investigators from affected racial/ethnic minorities are integral to identify novel and culturally relevant solutions, and effectively partner with affected communities. Florida International University (FIU) has a number accomplished investigators currently conducting community-based research on health disparities. However, our research, training, and community engagement endeavors in health disparities, substance abuse, and HIV will benefit greatly from the hiring of additional established investigators whose work complements and expands FIU?s current capacity and expertise.

4.2. Project Summary (Abstract): The long-term goal of the proposed research is to understand the role of the cholinergic system in manganese (Mn)-induced neurological dysfunction. Today, millions of welders, smelters, and miners in the United States (US) and throughout the world are chronically exposed to Mn- containing fumes, aerosols, and particles on a regular basis. Furthermore, drinking water with naturally high Mn concentrations is now recognized as an important source of chronic Mn exposure to large segments of the population in the US and globally. Therefore, the number of humans that are potentially exposed to neurotoxic levels of Mn worldwide are much larger than previously recognized, making it a public health problem of global proportion. Exposure to contemporary levels of Mn results in impairments in working memory and executive function and produces deficits in fine motor control and postural stability. These neurological effects of chronic Mn exposure are likely to have a pathophysiology that involves multiple neuronal systems. Previous studies from our laboratory have shown that chronic exposure to moderate levels of Mn in non-human primates produces dysfunction of nigrostriatal dopaminergic (DAergic) neurons by inhibiting striatal dopamine release. We now find a marked loss of striatal cholinergic interneurons (ChI) and these findings challenge the current dogma of Mn-induced pathophysiology from a solely DAergic perspective to one in which there is disruption of the DAergic-Cholinergic balance in the basal ganglia. Cholinergic neurons are important in the physiology of cognition, emotion, compulsive behavior, locomotion, and gait, domains that are affected in Mn-induced neurological dysfunction. Here, we also provide initial evidence that chronic Mn exposure in non-human primates results in an apparent basal forebrain cholinergic neuron loss or injury similar to what is found in Alzheimer's disease and other neurodegenerative disorders. Thus, we propose to rigorously characterize the effect of chronic Mn exposure on choline acetyltransferase (ChAT)-positive cholinergic neurons in the caudate/putamen/nucleus accumbens as well as in the basal forebrain and pedunculopontine nucleus in the non-human primate brain (specific aim 1). These studies will use rigorous unbiased stereological cell counting and soma size determination methods. We will also determine the effect of chronic Mn exposure on vesicular acetylcholine transporter (vAChT) in cholinergic axon terminals and varicosities (specific aim 2) to assess if chronic Mn exposure produces cholinergic neuron axonopathy. Finally, we will examine the role of neurotrophic factors on the Mn-induced loss of cholinergic neurons (specific aim 3) by measuring concentrations of Brain-Derived Neurotrophic Factor and Nerve Growth Factor in relevant brain regions. The proposed studies will provide a more precise mechanistic understanding of Mn-induced pathophysiology that can lead to the development of cholinergic- and/or neurotrophic factor- based therapies for the treatment of Mn-induced neurological dysfunction.

Project Summary (Abstract): The long-term goal of this research is to understand the function(s) of Translocator Protein 18 kDa (TSPO) in glial cells, specifically in microglia. TSPO is a glial stress response protein that we have previously validated as a biomarker of brain injury and inflammation and it is currently used in preclinical and clinical Positron Emission Tomography (PET) imaging studies throughout the world. TSPO is a sensitive biomarker that is able to detect brain injury and neuroinflammation in a number of human neurodegenerative and mental conditions as well as in neurodegeneration induced by exposures to environmental chemicals. Furthermore, we are currently using TSPO as a biomarker of neurotoxicity to screen the neurotoxicity of chemicals for which there is currently no information on their potential to damage the brain. Despite the widespread use of TSPO in clinical and preclinical studies, there is a paucity of knowledge on the function(s) of TSPO in glial cells (microglia and astrocytes), the cell types that upregulate TSPO under neuropathological conditions. This proposal is aimed at understanding a novel interaction that we have discovered between TSPO and NADPH Oxidase (NOX2) in microglia that may provide important insights on modulation of brain reactive oxygen species (ROS) production and neuroinflammation. We present rigorously performed studies demonstrating the TSPO-NOX2 interaction and its modulation by microglia activation. There are three specific aims in the proposed research. Specific aim 1 will examine the function of TSPO in primary microglia generated from wildtype and global TSPO knockout (TSPO-gKO) as well as microglia-specific conditional TSPO (mTSPO-cKO) mice. Specific aim 2 will examine the subcellular localization and functional significance of the TSPO-NOX2 interaction in microglia. Finally, specific aim 3 will examine the neurological effects of TSPO deletion (global and microglia-specific) at the whole animal level. Combined, the proposed studies will generate new information on the function of TSPO in microglia. We will use state-of-the-art molecular and cellular techniques to understand the function of TSPO in microglia. A precise understanding of this novel TSPO-NOX2 interaction will provide new insights for devising therapeutics strategies for neurodegenerative conditions that involve neuroinflammation since NOX2 is involved in microglia-mediated neurodegeneration. This research topic is consistent with strategic objective 1 in advancing environmental health sciences basic biological research which is an integral part of the National Institute of Environmental Health Sciences strategic plan 2018-2023: Advancing Environmental Health Science, Improving Health 2.0.