Pamela J. Lein - US grants

DESCRIPTION: Dendrites represent the primary site of sysnapse formation in the vertebrate nervous system, and the size of the dendritic arbor restricts the number of synaptic inputs a neuron receives. Therefore, characterizing the mechanisms that control dendritic growth may provide insights as to how synaptic contacts are established and maintained not only during normal development and remodeling, but also in pathological states. Osteogenic protein -1 1 (OP-1) has recently been identified as a unique trophic factor that selectively induces dendrite formation in cultured embryonic sympathetic neurons. To establish the relevance of these in vitro observations to neuronal morphogenesis in situ, it will be necessary to determine if the temporal and spatial distribution of OP-1 is coincident with dendritic growth and to identify the cell type(s) that produce OP-1. Immunocytochemistry, immunoprecipitation, in situ hybridization, and RNase protection analyses of sympathetic ganglia, nerves, and target tissues will be employed to accomplish these objectives. Since preliminary data suggest that both local and target cells may provide OP-1 to sympathetic neurons, regulation of neuronal access to OP-1 via retrograde transport will be assessed. Finding that retrograde transport is a major means of delivering OP-1 to neurons has significant implications regarding mechanisms of dendritic atrophy in axonal lesioning or degeneration.

DESCRIPTION (provided by applicant) There is increasing concern that perinatal exposure to organophosphate acetylcholinesterase (AChE) inhibitors (OPs) may cause cognitive problems in children. This concern is based on recent reports of widespread exposure of the public to OPs, and on animal studies indicating that the developing nervous system is more sensitive than the mature nervous system to the neurotoxic effects of OPs. It is reported that OPs cause developmental neurotoxicity at doses significantly below those that inhibit the catalytic activity of AChE. These data have been widely interpreted to mean that OPs target molecules other than AChE. However, recent evidence demonstrating that AChE functions to promote axonal growth in the developing but not mature neurons suggest an alternative explanation: OPs target AChE, but the mechanism of developmental neurotoxicity involves disruption of the morphogenic function of AChE not inhibition of its catalytic activity. Interference with the axon- promoting activity of AChE represents a biologically plausible mechanism for explaining the functional deficits observed in animals exposed perinatally to OPs. The goal of this proposal is to test the hypothesis that OPs disrupt axonal growth by interfering with the morphogenic activity of AChE. The specific aims are to: (1) Validate dorsal root ganglion (DRG) neurons as a model system for mechanistic studies of OP-induced axonal dysmor- phogenesis; (2) Determine if AChE mediates OP effects on axonal growth; and (3) Determine if OPs alter axonal and dendritic morphogenesis in primary cultures of hippocampal and cortical neurons. Neurons cultured from AChE wildtype (+/+) and AChE null (-/-) mice will be exposed to varying levels of chlorpyrifos (CPF) or its metabolites CPF-oxon (CPFO) and 3,5,6-trichloro-2-pyridnol (TCP). The number, length, and branching patterns of axons and dendrites will be quantified using Metamorph imaging software following immunocytochemical localization of axon- or dendrite- specific antigens. AChE enzymatic activity will be measured using a standard colorimetric assay; cell viability, by assessing protein and DNA synthesis and uptake of vital dyes. Direct interactions between OPs and AChE will be assessed by determining if OPs inhibit adhesion of AChE coated microspheres to tissue sections of developing cerebral cortex from and by autoradiography of proteins immunoprecipitated by AChE antibody from cultured DRG neurons exposed to radiolabeled CPF. These data will not only provide mechanistic insight into the developmental neurotoxicity of OPs, but also suggest a biological basis for the increased vulnerability of the developing nervous system. Impacts of these studies include the reduction of uncertainty in risk assessment, the development and evaluation of biomarkers for OP toxicity in children, and the design of in vitro assays for screening the developmental neurotoxicity of other OPs of concern.

DESCRIPTION (provided by applicant): There is increasing evidence that perinatal exposure to relatively low levels of polychlorinated biphenyls (PCBs) may cause cognitive and behavioral problems in children. However, assessment of this risk is complicated by large gaps in our understanding of critical exposure periods and mechanisms of PCB developmental neurotoxicity. Filling these gaps is dependent upon the development and characterization of appropriate model systems. In light of the human data, it is surprising that most experimental studies have employed model systems that analyze the effects of developmental PCB exposure on cognition in adult rather than juvenile animals. Therefore, the first specific aim of this application is to determine the feasibility of using juvenile rats to examine the effect of PCBs on cognitive function. The investigators will use the Morris swim task to test spatial learning and memory in rats beginning on postnatal day 24 (P24). To identify critical exposure periods, cross-fostering studies will be used to generate animals exposed to Aroclor 1254 (10 or 100 ppm in the maternal diet) during gestation only or lactation only. Another group of rats will be exposed continuously throughout gestation and lactation. The second specific aim is to generate pilot data regarding the hypothesis that PCBs impair cognitive function by disrupting dendritic growth. Dendritic morphology is a primary determinant of neuronal connectivity; disruption of dendritic growth or maturation is associated with neural deficits of various etiologies. Developing neurons use specific environmental cues to regulate the growth of dendrites in time and space. Several cues known to regulate dendritic morphogenesis in central neurons - thyroid hormone, cholinergic activity, and estrogen - are also known or suspected targets of PCBs. These observations suggest that PCBs may impair cognition via effects on dendritic growth. To assess the feasibility of this hypothesis, the investigators will determine if PCBs induce changes in dendritic morphology that correlate with cognitive deficits. Dendritic morphology will be analyzed in the brains of animals immediately following the conclusion of behavioral studies. Various morphometric techniques will be used including immunohistochemical localization and western blot analyses of MAP2 and PSD95, and image analysis of dendritic growth in individual neurons labeled with the Golgi-Cox stain. The long-term objectives of these studies are to delineate the cell and molecular mechanisms of PCB developmental neurotoxicity.

DESCRIPTION (provided by applicant): Inflammation is a common etiological factor in the pathogenesis of diverse neurological diseases; however, mechanisms by which inflammation causes neural dysfunction are not well understood. We hypothesize that the pro-inflammatory gamma interferon (IFNgamma) contributes to inflammation-associated neuropathology by acting directly on neurons to induce dendrite retraction and synapse loss. This hypothesis is derived from observations that (1) dendritic atrophy and elevated IFNgamma are coincidental in inflammatory reactions caused by viral infection, trauma or ischemia, in chronic inflammatory or viral diseases such as multiple sclerosis or HIV and in neurodegenerative diseases such as Alzheimer's and Parkinson's disease; and (2) dendritic atrophy has been linked to neural dysfunction. We have recently shown that IFNgamma causes dendritic retraction and synaptic loss in cultured peripheral and central neurons in the absence of adverse effects on cell viability or axons. What is not known is whether IFNgamma elicits the same response in vivo, and if IFNgamma generated by peripheral inflammatory events can initiate dystrophic signals that are propagated back to the soma. The significance of the latter is suggested by observations that many neuropathological inflammatory lesions are largely restricted to white matter or target tissues. Using neurons of the superior cervical ganglion (SCG) as a model system, we will address the following specific aims: 1) Determine if systemic administration of lFNgamma is sufficient to cause dendrite retraction and synapse loss in vivo; 2) Assess whether IFNgamma signaling from distal axons is sufficient to cause dendritic retraction in neurons grown in compartmented chambers; and 3) Determine if peripheral inflammation causes dendritic retraction in vivo and if IFNgamma is necessary for this effect. Aim 3 will be accomplished by quantifying dendritic arborization in SCG neurons that innervate airways in wildtype, IFNgamma-/- and IFNgamma Ralpha -/- mice following airway infection with Sendai parainfluenza virus. SCG neurons that project to airways will be identified by retrograde labeling with Fast Blue, and dendritic arbors will be visualized using DiI labeling. Major histocompatibility complex-I and nitric oxide synthase expression will be monitored in SCG as biomarkers of exposure to pharmacologically active concentrations of IFNgamma. These studies will provide significant insights regarding the effect of pro-inflammatory cytokines on neural circuitry, and identify a novel mechanism for conveying information about peripheral injury or inflammation to distal brain loci.

DESCRIPTION (provided by applicant): Dendrites are important determinants of neuronal connectivity and the importance of precisely regulating dendritic morphology is suggested by observations that aberrant dendritic growth is associated with neurodevelopmental and neurodegenerative disorders. Currently very little is known about the molecular mechanisms that regulate dendritic morphogenesis. Our goal is to test the central hypothesis that dendritic growth is controlled by regulation of specific genes. We have developed an in vitro model that is uniquely suited to rigorously testing this hypothesis. Rat sympathetic neurons cultured under defined conditions extend a single functional axon, but fail to form dendrites. However, bone morphogenetic proteins (BMPs) trigger these neurons to extend multiple dendrites without altering axon growth or survival. The size of the BMP-induced dendritic arbor is comparable to that of sympathetic neurons in vivo, suggesting that BMPs regulate the full complement of genes necessary for dendritic growth. We propose that isolation of genes differentially expressed in naive vs. BMP-treated sympathetic neurons will enable us to identify candidate genes involved in regulation of primary dendritogenesis. The specific aims of this proposal are to: (1) Identify genes that are differentially regulated in cultured sympathetic neurons during BMP-induced dendritic growth using oligonucleotide microarrays and determine the temporal expression pattern of these genes using quantitative RT-PCR; and (2) Evaluate the functional significance of BMP-regulated genes in dendritic growth by determining if overexpression of BMP-activated genes induces dendritic growth in sympathetic neurons grown in the absence of BMPs, and conversely, if overexpression of down-regulated genes blocks BMP-induced dendritic growth. The relevance of this proposal to the R21 mechanism is that it will generate pilot data establishing the feasibility of a new avenue of research into molecular mechanisms of dendritic growth. It also involves high risk experiments that could lead to a significant breakthrough in understanding of an important question in developmental neurobiology. Since dendritic dysgenesis is associated with neurological disease, defining the molecular mechanisms that regulate dendritic growth will impact our understanding of basic pathogenic mechanisms in the nervous system and provide opportunities for novel preventive/therapeutic approaches.

DESCRIPTION (provided by applicant): Polychlorinated biphenyls (PCBs), and in particular neuroactive non-dioxin-like PCBs, remain a significant children's health concern because of their persistence and inadvertent production by various industrial processes that continue to contaminate food and indoor air, especially in schools across the United States. PCBs have been identified as probable environmental risk factors for neurodevelopmental disorders (NDD), which affect 1 in 10 children born in the United States. Most NDD have complex etiologies that likely involve multiple genetic loci interacting with exposures to environmental factors during critical periods of neurodevelopment. While many genes have been associated with increased risk of NDD, there remains a huge gap in understanding how multiple genes interact to modify neurodevelopment and even less clarity as to how genetic susceptibilities interact with environmental factors to amplify NDD risk. We will address these questions by testing the hypothesis that exposure during critical window(s) of neurodevelopment to a mixture of PCBs found in mothers at risk for having a child with NDD will potently disrupt neuronal connectivity via ryanodine receptor (RyR)-dependent mechanisms and the net outcome will be influenced by heritable mutations that alter the fidelity of Ca2+ signals essential for activity-dependent dendriic growth. This hypothesis derives from data generated during the previous funding cycle demonstrating sensitization of the ryanodine receptor (RyR) by PCB 95 activates calcium-dependent signaling pathways that promote dendritic arborization, and increased dendritic arborization is associated with impaired cognitive behavior in weanling mice exposed to PCB 95 in the maternal diet. The studies described in this application will use a PCB mixture that is relevant to human NDD based on data of PCB levels in the plasma of mothers participating in the MARBLES study at UC Davis, a longitudinal study for pregnant women with increased risk for having a child with NDD. This MARBLES mix will be tested for effects on morphometric, biochemical and functional indices of neuronal connectivity in unique mouse models that express an expansion repeat in the FMR1 gene, the single most frequent monogenetic cause of neurodevelopmental impairments, and a human RyR1 gain of function mutation, singly or in combination. These studies will address the critical need to better understand mechanisms by which non-dioxin-like PCBs cause developmental neurotoxicity, and will provide among the first mechanistic data regarding relevant interactions among genes and environment that increase NDD risk. This information will inform rational strategies for minimizing NDD risk by mitigating relevant exposures and will facilitate the development of mechanistically based screening platforms for identifying other gene-environment interactions likely to amplify adverse neurodevelopmental outcomes.

Description (provided by applicant): The Cell and Tissue Analysis Core Facility (formerly Histopathology) was established in 1986 in response to the need of Center investigators for plastic glycol-methacrylate (GMA)-embedded tissue sections for histopathologic evaluations. The plastic tissue sections (1-2 um thick) enable a resolution halfway between standard paraffin-embedded sections and sections observed under low magnification in an electron microscope. In GMA sections, cells and cell components not readily seen in standard paraffin-embedded sections can be easily visualized [e.g., mast cells and basophils (and their granules), capillary endothelial cells and pericytes, and basement membranes]. Since its inception, this Core Facility has expanded the services offered to Center investigators to accommodate the growing need for expert assistance with immunocytochemical analyses of tissue samples and cultured cells as well as the preparation of publication quality photomicrographs. In the advent of the sequencing of the human genome and the rapid advances in genomics, proteomics and metabonomics, it is becoming clear that determining the physiological relevance of these global changes in DNA expression will require imaging of these molecular changes at the level of cells and tissues, using techniques such as in situ hybridization, tissue arraying, FRET, FRAP and imaging of green fluorescent protein (GFP)-labeled proteins in live cells. Thus, the investigators anticipate that the need for the Cell and Tissue Analysis Core Facility will continue to grow in the future. The Core provides: 1) a wide range of histological and cytological services; (2) photographic and digital imaging; and, (3) professional consultation to NIEHS Center members and those in their laboratories. Through the Core facility, Center investigators also have cost-effective access to equipment and services available through the Johns Hopkins University School of Medicine Microscopic Facility. The Core Facility Directors and staff also keep abreast of new developments in histopathology, cell imaging and digital photography and help Center investigators choose and develop the appropriate techniques for their research projects, analyze and interpret their data, and ultimately organize and prepare their results for publication and presentation. The overall objective of the Cell and Tissue Core Facility is to provide histopathology and cell imaging services, so that pathophysiological and toxicological investigations will be backed by morphological data at both the level of cells and tissues.

DESCRIPTION (provided by applicant): Organophosphorus pesticides (OPs) are the most commonly used pesticides in the U.S. and worldwide. Evidence from human and animal studies clearly identifies neurotoxicity as the primary endpoint of concern. However, it has been difficult to predict the risk that repeated low-dose exposure to OPs pose to humans because: 1) a relationship between OP dose and neurobehavioral deficits has yet to be established in humans;2) biomarkers that reliably predict OP-induced neurobehavioral deficits are not available: and 3) the potential for genetic variation to modify exposure sensitivity has not been thoroughly investigated. The proposed studies will test the hypotheses that OP-induced neurobehavioral deficits are dose-related and that measures of oxidative stress and inflammation are better predictors of neurobehavioral deficit than cholinesterase inhibition. These hypotheses will be tested by studying a cohort of pesticide application workers in Egypt's Menoufia Governorate previously reported to exhibit the broadest range of neurobehavioral deficits in humans following OP exposure. This Egyptian cohort is uniquely suited for these studies because, unlike most pesticide exposures, the exposure is simple (a single OP, chlorpyrifos) and consistent within job categories, but with substantial differences between job categories. In aim 1, OP doses will be estimated using PBPK/PD modeling of urinary OP metabolite data collected from 255 Egyptian workers over the application cycle. These workers will also be genotyped for polymorphisms of key enzymes involved in OP metabolism (CYP2B6, CYP2C19 and PON1) to evaluate the potential for genetic variation to modify internal dose. In aim 2, behavioral deficits will be determined in a subset of workers exhibiting a range of OP exposures. Data from aims 1 and 2 will be integrated to determine the relationship between OP dose and neurobehavioral deficits. Rat studies will be conducted in parallel (aim 3) to test candidate biomarkers as predictors of OP-induced neurobehavioral deficits. The specific biomarkers that will be examined include cholinesterase inhibition, urinary isoprostanes as a measure of oxidative stress, and serum levels of C-reactive protein and inflammatory cytokines as measures of inflammation. In aim 4, those biomarkers that predict OP-induced neurobehavioral deficits in rats will be tested to determine if they similarly predict deficits in behavioral performance in Egyptian pesticide workers. The proposed studies will provide critical data needed to develop effective biomarkers of OP exposure, biological response and genetic susceptibility. The availability of such biomarkers would facilitate the identification of at-risk individuals as well as the testing of intervention and treatment strategies, and the need to develop these strategies is underscored by evidence of widespread human exposure to OPs and the credible threat of OPs as chemical agents of terrorism. Project Summary/Abstract - Relevance The goal of the proposed studies is to identify biomarkers of exposure and effect that are predictive of neurobehavioral deficits in humans exposed to organophosphorus pesticides (OPs). In addition, we will examine human genetic variants of the enzymes CYP2B6 and CYP2C19 that influence OP metabolism to not only inform interpretation of OP exposure data, but also provide insights into genetic susceptibilities that modulate neurotoxic responses to OPs. These studies will provide data critically needed to identify at-risk individuals and will provide tools to facilitate the development and evaluation of intervention and treatment strategies for exposure to not only OP pesticides, which are the most commonly used pesticides in the U.S. and worldwide, but also to nerve agents that are considered a credible terrorist threat.

DESCRIPTION (provided by applicant): We have demonstrated that in a guinea pig model, organophosphorus pesticides (OPs) cause airway hyperreactivity that is dose-related and associated with the functional loss of autoinhibitory muscarinic M2 receptors that normally limit acetylcholine release from parasympathetic nerves that innervate airway smooth muscle. We recently reported that sensitization to antigen alters the mechanisms underlying OP-induced airway hyperreactivity to involve IL-5-dependent mechanisms in the sensitized but not the non-sensitized guinea pig. How OPs cause neuronal M2 dysfunction in the non-sensitized animal is not known but our preliminary data indicate that this effect is not mediated by cholinesterase inhibition or direct antagonistic interactions with neuronal M2 receptors. Rather, OPs appear to influence neuronal M2 receptor function indirectly via effects on macrophages since depletion of macrophages using liposome-encapsulated clodronate protects against OP-induced airway hyperreactivity. It is our hypothesis that OPs activate macrophages to upregulate expression and release of inflammatory cytokines previously shown to cause M2 receptor dysfunction in various models of airway hyperreactivity. We propose four Aims to test this hypothesis. In Aim 1, we will use in vivo physiological measurements to confirm that macrophages mediate airway hyperreactivity caused by OPs and determine whether their role changes over time, as has been observed for eosinophils in ozone-induced airway hyperreactivity. Aim 2 will utilize macrophages isolated from bronchoalveolar lavage collected from OP-treated versus untreated guinea pigs guinea pigs to examine the effect of OPs on macrophage expression and release of inflammatory cytokines implicated in airway hyperreactivity. In Aim 3, we will use primary nerve cell cultures to determine whether OP- induced macrophage mediators interact with nerves directly to alter M2 receptor expression or function or the structural plasticity of nerves. Aim 4 will confirm the in vivo physiological relevance of OP-induced macrophage mediators identified in aims 2 and 3. Mechanistic studies are critical to developing preventive and therapeutic approaches for OP-induced airway hyperreactivity, which are likely to differ between sensitized (allergic) and non-sensitized individuals, and for determining the risks to human health posed by OP exposures. The public health implications of these studies are significant in light of the increasing prevalence of asthma, the wide spread exposure of humans and especially children to OPs and the credible threat of terrorist use of OP pesticides and nerve agents. PUBLIC HEALTH RELEVANCE: Recent epidemiological studies suggest a link between exposure to organophosphorus pesticides (OPs) and asthma. Our previous work confirms this link by demonstrating that OPs cause airway hyperreactivity, a major symptom of asthma, in a guinea pig model. The goal of this project is to elucidate the mechanism(s) by which OPs cause airway hyperreactivity, which will be critical to developing effective preventive and therapeutic approaches to OP-induced airway hyperreactivity. Given the documented widespread exposure to OPs not only in the U.S. but worldwide, and the credible threat of terrorist use of OPs, the proposed work is of significant public health relevance.

DESCRIPTION (provided by applicant): The rodenticide tetramethylenedisulfotetramine (TETS) and the organophosphorus (OP) pesticide parathion are considered credible terrorist threat agents. Current medical countermeasures for acute TETS or OP intoxication can prevent mortality but do not sufficiently protect the CNS from persistent seizures and/or permanent injury. Therefore, new and more effective countermeasures must be developed to facilitate better medical treatment of civilians, first responders and military personnel following exposure to acutely toxic levels of TETS, parathion and similar chemical threat agents. The goals of the proposed research are to develop rodent models of TETS- and parathion-induced seizures and then use these models to identify potential therapeutic agents. We seek to identify agents that can protect against the development of seizures or treat seizures once they have begun and/or can prevent irreversible neuronal injury. We plan to evaluate a 2,3- benzodiazepine AMPA receptor antagonist and a soluble epoxide hydrolase inhibitor (sEHi). We have recently shown that AMPA receptor antagonists provide sustained seizure protection and are able to block seizures when administered at later times when there is refractoriness to diazepam, the benzodiazepine typically used to treat acute TETS and OP intoxication. We and others have data indicating that sEHi also exhibit anti- epileptic properties. Perhaps more important in light of recent evidence suggesting that the use of anti- inflammatory compounds in combination with standard antidote significantly decreases neuronal damage in acute OP intoxication, we have demonstrated that sEHi are also potent anti-inflammatory compounds. These observations suggest the potential for AMPA receptor antagonists and sEHi to significantly improve the clinical management of acute TETS and parathion intoxication by extending the therapeutic window and enhancing neuroprotection. To test this hypothesis, we will address two specific aims: (1) Develop rodent models of acute TETS and parathion intoxication;and 2) Determine whether AMPA receptor antagonist and/or sEHi are of therapeutic benefit in acute TETS or parathion intoxication when administered prior to or after exposure to the chemical threat agent. Endpoints that will be measured across all Aims include time to onset, frequency and duration of clonic and tonic seizures, EEG, histological measures of neuropathology and blood levels of TETS, AMPA receptor antagonist and sEHi. AMPA receptor antagonists and sEHi are currently undergoing human clinical trials and have demonstrated an excellent safety record;which will facilitate translation of positive findings in these rodent models to human studies. In summary, this project will develop rodent models of acute TETS and parathion intoxication that will be useful to the field beyond the proposed studies, and it will generate critical information on novel treatment approaches of practical value for two diverse classes of chemical threat agents. PUBLIC HEALTH RELEVANCE: The rodenticide tetramethylenedisulfotetramine (TETS) and the organophosphorus (OP) pesticide parathion are considered credible terrorist threat agents. Current medical countermeasures for acute TETS or OP intoxication can prevent mortality but do not sufficiently protect the CNS from persistent seizures and/or permanent injury. The goal of this research project is to test the hypothesis that AMPA receptor antagonists and/or inhibitors of soluble epoxide hydrolases will significantly improve outcome following exposure to acutely toxic levels of TETS and parathion by extending the therapeutic window for seizure protection and enhancing neuroprotection.

DESCRIPTION (provided by applicant) This pre-doctoral and postdoctoral program will train environmental health scientists through interdisciplinary research and courses in mission areas of the NIEHS. Training faculty consist of 29 active researchers in the Schools of Medicine and Veterinary Medicine and the College of Agricultural and Environmental Science. Areas of emphasis include neuro-, respiratory and reproductive toxicology, intracellular signaling and oxidative stress. Faculty interests overlap in these areas, leading to interaction among laboratories and to interdisciplinary approaches to human biology and disease. A large pool of excellent pre-doctoral applicants is available through the Pharmacology and Toxicology Graduate Group and other graduate groups (covering such disciplines as molecular biology, genetics, cell biology, neuroscience, pathology, epidemiology) to which the training faculty belong, a strength of graduate education at the University of California, Davis. Postdoctoral applicants will be solicited among those attracted to the laboratories of the training faculty and by advertisements in widely read journals. In addition, trainees will be solicited among new graduates of the campus Masters in Public Health Program. Trainees will have access to advanced technologies, including proteomics, genomics and use of genetically modified mice. A second strength of training at the University of California, Davis is the vertical integration of studies of environmentally induced disease. Molecular, cellular, tissue and whole laboratory animal (including knockout mouse) approaches complement use of nonhuman primates and human clinical samples obtained through the funded Clinical and Translational Science Center (CTSC). Integration of research with ongoing activities in the various campus research centers (children's health, air pollution, agrochemical exposures and effects, etc.) and with the developing School of Public Health will provide synergy and promote connections to disease prevention and public health. Trainees will receive enrichment in responsible conduct of research and obtain instruction and practice in scientific writing, including proposals for extramural funding. They will also participate in activities communicating scientific findings (seminars, retreats, national meetings) and in workshops organized by the CTSC. This proposed training builds on an established program with a strong track record of meeting the NIEHS mission to protect public health by connecting scientific advances to environmental exposures and the consequent disease processes. BACKGROUND This is a competing continuation application for a Training Program in environmental health sciences at the University of California, Davis that has been in existence for over 30 years and is administered by the Department of Environmental Toxicology. In the past the award has provided support for approximately 120 trainees. The previous funded cycle supported 10 pre-doctoral trainees. In this competing renewal application support is requested for 6 pre-doctoral and 3 postdoctoral trainees each year. Of those completing the program more than 10 years ago, nearly half are in industry, one quarter function as managers and regulators in federal and state governments, approximately 15% are in academia and 5% have left the environmental health sciences field. There are a total of 29 mentors listed with three of them physician-scientists. Over the past 10 years 75% of the students have come out of the Pharmacology and Toxicology (PTX) Graduate Group and this is expected to continue.

The unifying goal of the UC Davis CounterACT Center of Excellence is to identify improved medical countermeasures for treating acute intoxication with seizure-inducing chemical threat agents. Research will focus on the organophosphorus (OP) cholinesterase inhibitor diisopropylfluorophosphate (DFP) and the GABA-inhibiting agent tramethylenedisulfotetramine (TETS), which arguably encompass the mechanistic spectrum of seizure inducing chemical threats, with the goal of identifying therapeutic approaches with broad-spectrum efficacy. The specific objectives of the Center are to: (1) identify improved treatments for acute seizures and lethality; and (2) identify therapeutic strategies for mitigating seizure-induced brain damage in patients that survive acute intoxication. This will be accomplished by repositioning marketed drugs and drug combinations for treatment of seizures triggered by chemical threat agents, in parallel with evaluation of new antidotes based on strong preliminary efficacy data. The molecules to be tested for anticonvulsant and neuroprotective efficacy range from compounds approaching readiness for IND enabling studies to early stage chemical probes. In all cases, these efforts in translation of anti-seizure agents are supported by innovative work on diagnostics employing emerging in vivo imaging technologies and analytical chemistry for monitoring pathological effects as well as target engagement and therapeutic efficacy. Additional outcomes from Center research that will benefit the CounterACT community include: (1) a high content in vitro/ex vivo platform for rapid screening of compounds to identify anticonvulsant and neuroprotective potential as well as mechanistically relevant novel drug targets; (2) in vivo models of DFP and TETS-induce seizures for studying neuropathic mechanisms and therapeutic rescue of neurologic sequelae triggered by seizurogenic exposures; (3) innovative in vivo imaging modalities for non-invasive longitudinal monitoring of neurologic damage and response to therapeutic candidates; (4) focused metabolomic profiling to identify biomarkers of seizure damage; and (5) innovative immunoassays for the detection of TETS in biological and environmental matrices. A highly integrated, interdisciplinary (pharmacology, medicinal chemistry, neurotoxicology, analytical chemistry, behavioral neuroscience, imaging, cellular/molecular neuroscience) research team with experience in drug discovery and translational research will work cooperatively and synergistically to achieve the Center's goal of identifying improved medical countermeasures that can be readily deployed during a chemical emergency to stop seizures and mitigate the neurological sequelae of seizures triggered by TETS and OPs.

Project Summary ? Administrative Core The overall goal of the UC Davis CounterACT Center of Excellence is to identify and advance improved medical countermeasures for stopping seizures and preventing long-term consequences of acute intoxication with chemical threat agents, specifically organophosphate cholinesterase inhibitors like diisopropylfluorophosphate (DFP), paraoxon and soman, or GABAA receptor blockers like tetramethylenedisulfotetramine (TETS) and picrotoxin. The UC Davis CounterACT Center comprises three projects, three scientific cores, a research education core and multiple committees ? all of which must work closely together to ensure success. The role of the Administrative Core is to oversee and coordinate the scientific and administrative operations of the Center's activities and to foster interactions and synergism among Center research projects and scientific cores, ultimately ensuring the Center meets annual milestones established in collaboration with NIH CounterACT program officer(s). Additionally, the Administrative Core will coordinate interactions with the UC Davis administration and external entities, such as the NIH CounterACT administration, the Center's External Advisory Committee, the larger CounterACT research community, the U.S. Food and Drug Administration (FDA), the Biomedical Advanced Research and Development Authority (BARDA), and commercial partners. The Core's objectives are: (1) Develop a strategic plan for achieving annual Center milestones and ensure its effective and efficient implementation; (2) Provide scientific leadership and logistical support to coordinate and integrate Center activities and promote interactions among Center investigators; (3) Facilitate data and resource sharing among Center investigators and other CounterACT investigators; (4) Provide budgetary oversight and grant management; (5) Ensure the safety and security of personnel, materials, data and facilities; (6) Coordinate the development of intellectual property (IP) strategies and the transitioning of leads for advanced development; (7) Coordinate with FDA, BARDA and other federal government agencies; and (8) Identify, engage and coordinate communications with commercial partners.

The overall goal of Project 2 is to identify effective therapeutic strategies for mitigating the delayed neurological sequelae of acute intoxication with the GABA antagonist tetramethylenedisulfotetramine (TETS) or organophosphorus (OP) cholinesterase inhibitors. In the context of accidental or terrorist release of these nerve poisons, it is likely that emergency medical support will not be available within the first 30-60 min after seizures begin, at which time the neuropathological processes that promote brain damage and ultimately persistent functional deficits have been set in motion. The specific objectives of Project 2 are to: (1) develop in vivo rodent models for assessing persistent neurological damage subsequent to seizures induced by TETS or the OPs diisopropylfluorophosphate (DFP) and parathion, with a focus on developing in vivo imaging modalities that can be used in preclinical and clinical studies to longitudinally monitor progressive neurological effects of intoxication and the efficacy of candidate therapeutics; and (2) identify effective therapeutic strategies for mitigating neurological damage following acute intoxication with these chemical threat agents. Project 2 will test the hypothesis that compounds that are more effective post-exposure anticonvulsants than diazepam will be more effective in mitigating the neurological damage following TETS- and OP-induced seizures than current medical countermeasures and that neuroprotection will be significantly enhanced by combining anticonvulsants with therapeutics that attenuate neuroinflammation and/or stabilize Ca channels. Neurological damage will be assessed up to 28 days post-exposure using: (1) histological and biochemical indices of neuropathology, inflammation/oxidative stress (Core A) and dysregulated calcium signaling (Project 3); (2) behavioral testing of cognitive and emotional behavior and telemetry EEG video monitoring of spontaneous recurrent seizures (conducted in collaboration with Project 1);and (3) in vivo imaging of neuroinflammation by positron emission tomography (PET) and functional connectivity by resting state functional magnetic resonance imaging (fcMRI). Pharmacological tools will include therapeutics already approved for use in humans or currently undergoing clinical trials (referred to as Tier 1 candidates) to facilitate translation of positive findings in rodent models to human studies as well as novel compounds identified in mechanistic screening studies in Core B and Project 3 (referred to as Tier 2 candidates). Anticonvulsants tested in Project 2 will include diazepam and lead compounds identified by Project 1 to terminate TETS- and DFP-induced seizures when administered at times post-exposure when diazepam has lost efficacy. Candidate lead compounds include AMPA receptor (AMPA-R) antagonists. Pharmacological tools for attenuating neuroinflammation and/or stabilizing Ca2+ channels will be the most promising compounds identified in mechanistic screening studies in Core B and Project 3. Lead candidates include the ryanodine receptor (RyR) antagonist dantrolene (Tier 1 therapeutic), inhibitors of soluble epoxide hydrolase (sEH), phosphodiesterase (PDE) and cyclooxygenase (COX) alone or in combination (Tier 2 therapeutics), and the KCa3.1 inhibitor TRAM-34 (Tier 2 therapeutic). Dosing paradigms will be informed by preliminary pharmacokinetic studies in Core B; optimization studies performed in Project 3 to evaluate timing, dose, and drug combinations; and determination by Core A of ADME and brain levels of therapeutic candidates and toxicants. Across all Aims, study design and data analyses will be developed in consultation with Core C. The most effective therapeutic strategies for enhancing neuroprotection in the DFP model will be assessed for neuroprotective efficacy against parathion and live nerve agent (soman). By comparing the efficacy of novel therapeutic approaches across models of acute TETS and OP intoxication, we hope to identify a convergent therapeutic strategy for protecting against diverse classes of chemical threat agents.

PROJECT SUMMARY This is a competing renewal application for a highly successful T32 program that has trained more than 180 predoctoral students in toxicology/environmental health sciences (EHS) over the last 45 years. The objective of this predoctoral program is to train the next generation of environmental health scientists through interdisciplinary research and coursework that address issues of direct relevance to the NIEHS mission. We are requesting 2 years of support for each of 8 predoctoral trainees beginning after their first or second year in a PhD degree program. Trainees are recruited from several UC Davis graduate programs that provide disciplinary training relevant to EHS, including toxicology, cell and molecular biology, exposure assessment, epidemiology, neuroscience, immunology and genetics. Training faculty ? 32 active researchers from 19 departments ? have substantial experience mentoring predoctoral students. Faculty research focuses on mechanisms by which environmental factors contribute to human disease and encompasses diverse areas within EHS, including respiratory toxicology, cancer, neurotoxicology, genotoxicity, epigenetics, and metabolic disorders. Significant interactions between training faculty members promote interdisciplinary approaches to EHS research. Trainees have access to advanced technologies, such as proteomics, epi/genomics and metabolomics, state-of-the-art imaging, genetically modified organisms, and inhalation facilities for rodents and non-human primates. A strength of EHS research at UC Davis is the vertical integration of studies directed toward understanding environmentally induced disease. Molecular, cellular, tissue, and diverse animal models, including nonhuman primate models, complement human clinical samples obtained through the UC Davis Clinical and Translational Science Center (CTSC) and epidemiologic studies. The training program leverages the activities and resources of multiple research centers at UC Davis to provide synergy and promote connections to disease prevention and public health; examples include the NIEHS P30 Environmental Health Sciences Core Center at UC Davis, the MIND Institute, Center for Children's Environmental Health, NCI Comprehensive Cancer Center, Western Center for Agricultural Health and Safety, NIEHS Superfund Program and the NIEHS Center for Nanotechnology Health Implications Research. The training program emphasizes practical instruction in scientific writing and communication of scientific findings to peers and lay audiences through chalk talks, annual retreats, town halls, and national meetings. Trainees are also exposed to emerging concepts, controversies, and technologies in environmental health by participating in a trainee-organized and -managed seminar series that hosts leading environmental health scientists from across the country, as well as through faculty?student interactions during a summer course in which trainees explore a current issue relevant to environmental health under the guidance of training faculty. Trainees will receive training in responsible conduct of research. The training program builds on an excellent track record of training leaders in EHS in academia, government, and the private sector.

Abstract - Career Development Core A major strategic initiative of the UC Davis NIEHS Core Center is to promote the professional development of early stage investigators (ESI) in environmental health science (EHS). To this end, the Career Development Program will provide support and mentoring to ESI with the goal of fostering their development as independent investigators in EHS. To promote the development of new areas of research that align with the Center mission and strategic vision, the Career Development Program will also recruit established investigators new to EHS who bring expertise, innovative approaches or technology that expand EHS research opportunities in the Center. The specific objectives of the Career Development Program are to: (1) Establish an EHS Scholars Program to recruit, support, mentor and promote the professional development of two to three outstanding junior faculty (referred to as ESI EHS scholars) and one to two established investigators new to EHS whose expertise and research interests align with the strategic vision of the Center; and (2) Support activities that foster the career development and research skills of Center investigators in EHS. Services provided by the Program include: (1) a structured mentoring program for ESI EHS Scholars and other Center ESIs who receive funding from the Center's Pilot Project Program; (2) priority access to career development resources available through the UC Davis Clinical and Translational Science Center (CTSC) Mentored Clinical Research Training Program (MCRTP) and the EHS Center's Community Outreach and Engagement Core (COEC); (3) development and implementation of a course on best practices in research program management; and (4) a Center Peer Review Program to provide constructive feedback on grant proposals and manuscripts. The significance of this Program is underscored by an identified need for resources to support and promote the careers of junior faculty within the Center and across UC Davis and a similar need for resources to attract established investigators to EHS research. Promoting the professional development of ESIs interested in EHS-related research and recruiting established investigators new to EHS is critically important for: (1) maintaining and reinvigorating the historic strengths of UC Davis in environmental health science research; (2) expanding current research activities at UC Davis to explore conceptual or technical innovations of relevance to EHS research; and (3) developing new areas of research that align with the Center mission and strategic vision.

? DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is the most prevalent age-related neurodegenerative disease in the United States. More than 90% of cases are idiopathic and there is growing consensus that environmental factors interact with genes of susceptibility to influence the age of onset and progression of this disease. Recent epidemiological studies have reported a positive correlation between exposure to traffic-related air pollution and the occurrence of the hallmark clinical characteristics of AD, including increased expression of pro-inflammatory markers in the brain, diffuse amyloid plaques, neuronal cell loss, and impaired cognition. We hypothesize that traffic- related air pollution emitted from motor vehicles triggers inflammatory responses in the brain that initiate or accelerate the progression of AD. To test this, we will use a unique animal model: the TgF344 rat, which expresses human genes that confer susceptibility to AD. We will expose male and female TgF344 rats and their wildtype littermates to polluted air sampled directly from the Caldecott tunnel in the San Francisco area beginning at postnatal day 28 to up to 12 months of age. Caldecott tunnel air will be delivered to animals housed in a portable vivarium parked adjacent to the tunnel. Control animals will be exposed to clean filtered air. Caldecott tunnel air will be collected every three weeks for gas and particle chemical characterization, and both Caldecott tunnel air and clean filtered air will be monitored continuously for particle size distribution and concentration. Learning and memory as well as neuroinflammation and AD-like pathology will be assessed in animals at 3, 6, 9 and 12 months of age. These studies will provide proof-of-concept data identifying functionally relevant interactions between AD-linked genetic susceptibilities and a ubiquitous environmental risk factor.

Aging is characterized by the development of systemic inflammatory changes, organ dysfunction and frailty. Aging is associated with increased oxidative stress and inflammation, which leads to impaired vascular repair, increased inflammation and increased monocyte adhesion. This is accompanied by the development of senescent (Sen) endothelial cells (EC), which originally were thought to be benign, but are now known to be a source of a sustained inflammatory output known as the senescence associated secretory phenotype (SASP) and other deleterious effects that contribute to the overall decline and frailty seen with aging. The vasculature has been under investigated as a contributor to the development of Alzheimer?s and other dementias, all of which are diseases predominantly associated with aging. We hypothesize that senescent (Sen) endothelial cells (ECs), which accumulate with aging, create a pro-inflammatory environment that adversely affects the blood brain barrier (BBB) and contributes to neuropathology implicated in age-associated dementias such as Alzheimer?s disease (AD). To this end we will use an established, well-characterized in vitro model of human Sen EC to study the effects of the SASP and exosomes derived from Sen EC, on human BBB integrity and neurons in culture. We will also analyze the content, including miRNA, of exosomes produced by Sen vs. early passage (EP) EC derived from the same donor, and determine whether co-culture with Sen ECs, SASP or exosomes increases the susceptibility of different neural cells to the toxicity of A?42 peptides. Lastly, we will investigate whether exogenous administration of exosomes derived from Sen EC compromises BBB integrity and promotes AD-relevant neuropathology in vivo in male and female TgF344-AD rats, which are an early onset Alzheimer's model. Age- and sex-matched congenic wild type rats will be used as controls. BBB integrity, neuroinflammation, neuronal connectivity, neurofibrillary tangles and amyloid plaques will be studied using state of the art imaging modalities as well as biochemical and cellular analysis. This work has the potential to identify new mechanisms contributing to the development of dementias, such as Alzheimer?s, which is approaching epidemic proportions in our aging population. Such findings have the potential to lead to new therapeutic targets.

Placental tissue is normally discarded at birth, but is essentially a molecular time capsule for gene by environmental interactions and dysregulated molecular and cellular pathways that can be revealed at the level of the epigenome. Identifying epigenetic biomarkers at birth that reflect in utero exposures or predict adverse neurodevelopmental outcomes is an important goal that has been limited by prior technologies or lack of relevant tissue availability. Our team of currently collaborating interdisciplinary scientists within the Children?s Center for Environmental Health plans to use existing placental samples from a prospective high-risk cohort study (MARBLES) to identify epigenetic biomarkers at birth for in utero exposure to polychlorinated biphenyls (PCB) and neurodevelopmental outcomes by age three. Using unbiased whole genome bisulfite sequencing (WGBS), we have previously demonstrated that placental tissues retain the distinctive DNA methylation patterns of the preimplantation embryo and so can capture the molecular state in very early development, a feature that is conserved across mammalian species, including mouse. The new hypothesis to be tested in this proposal is that perinatal exposures to PCB adversely impact neurodevelopment and leave a lasting molecular signature over genes relevant to neurodevelopment that can be detected in placenta. The proposed PCB Epigenomic Brain & Behavior Lasting Effects Study (PEBBLES) will combine the analysis of human placental samples from the high-risk MARBLES cohort with the analysis of placenta and brain tissues and sorted cell types derived from a mouse model of perinatal exposure to the same mixture of PCB congeners detected in MARBLES mothers. This study will leverage existing neurological and behavioral analyses and samples to examine the relationship between PCB-induced perturbations of DNA methylation marks with adverse neurotoxic outcomes. Epigenomic analyses of placenta and brain as well as sorted cellular subtypes from each of these tissues will include WGBS for methylome, RNA-seq for transcriptome, and ATAC-seq for chromatin accessibility. Bioinformatic and statistical analyses will integrate the genomic data sets with behavioral and molecular outcome measures and determine whether similar epigenetic marks are observed in placenta that could be used to predict long-lasting adverse brain and behavioral outcomes in humans. !

PROJECT SUMMARY/ABSTRACT Polychlorinated biphenyls (PCBs) remain a significant children?s health concern because they continue to contaminate food and indoor air, especially in schools across the United States. Non-dioxin-like (NDL) PCBs are implicated as environmental risk factors for neurodevelopmental disorders, and the parent grant addresses the critical need to understand the mechanisms by which NDL PCBs interact with genetic susceptibility factors to influence risk for developing these disorders. The long-term goal is to identify and understand factors that influence individual susceptibility to environmentally-mediated childhood disorders and, ultimately, to reduce the burden of these disorders to individuals and society. The objective of the proposed transdisciplinary and translational studies is to expand the scope of the current project to investigate a PCB congener (PCB 11), route of exposure (inhalation) and target organ (developing lung) not addressed in the parent grant. Additionally, this project will employ state-of-the art optogenetic techniques to translate in vitro observations of PCB effects on structural connectivity to functional connectivity in living animals. The consortium enables extensive collaborations among the PIs, including sharing of animal models, unique skills (optogenetics) and expertise (PCB toxicity, inhalation exposure, biodistribution, in vivo imaging). The Specific Aims are to: (1) Test the hypothesis that the disposition and neurotoxicity of PCB 11 differs when maternal exposure occurs via the diet versus inhalation; (2) Use optogenetics to test the hypothesis that developmental exposure to PCB 11 alters sensorimotor learning coincident with changes in neural assemblies and patterns of synaptic connectivity in vivo; and (3) Test the hypothesis that exposure to PCB 11 alters conducting airway epithelial development, airway innervation and airway oxidant stress/responsiveness and that the response is influenced by route of exposure. Aim 1 will fill a data gap on how the route of exposure influences PCB developmental neurotoxicity. Aim 2 will determine whether PCB-induced changes in structural connectivity of primary cultured neurons translate to changes in functional connectivity in the intact living brain that impair behavior. Aim 3 will determine whether PCB 11 interferes with the innervation and function of the developing lung, an understudied target in PCB toxicity. All three aims will generate novel toxicity data for PCB 11, a congener emerging as a prevalent PCB contaminant in complex environmental mixtures, including air and the serum of women at risk for having a child with a NDD. The proposed research is innovative because it uses state-of-the-art methods to: characterize how the route of PCB exposure affects developmental toxicity; confirm that in vitro observations of PCB effects on structural connectivity can be translated to physiological consequences in vivo; and evaluate the developing lung as a target organ for PCBs. These outcomes are significant because they will inform risk assessment for PCB 11, a prevalent environmental contaminant for which there is little toxicity data.

Abstract Alzheimer?s disease (AD) affects an estimated 5.7 million Americans, a number expected to reach 14 million by 2050. Despite several decades of research, the initiation and progression of AD continues to be poorly understood, and we currently lack reliable biomarkers to longitudinally monitor disease progression. Synaptic dysfunction is being evaluated as a potential early biomarker for evaluating AD risk; however, most studies to date have relied on cross-sectional or endpoint, ex vivo analyses. We hypothesize that in vivo imaging measures of synapse density, which will be carefully validated against histologic measures, will be predictive biomarkers of AD pathology that precede detection of amyloid deposition and neurofibrillary tangles by in vivo imaging. A positive outcome from testing this hypothesis would enable the identification of at-risk individuals and the application of therapeutic strategies to arrest disease progression before substantial neuronal loss occurs. Our studies will utilize PET and MR imaging in a novel transgenic rat model that presents key pathologic features of significance in human AD. Our first specific aim will establish the spatial correlation between in vivo imaging measures (synapse density, amyloid deposition and tauopathy via PET and structural measures via MRI) and concurrent histopathology in the Tg344-AD transgenic rat model versus congenic age- matched wildtype animals. Our second specific aim will map the spatiotemporal patterns of synapse density, amyloid deposition, tauopathy and neurodegeneration via in vivo PET and MRI in TgF344-AD rats and age- matched control animals. PET using the radiotracers 18F-UCB-H, 18F-florbetapir and 18F-T807, as proxy measures of synapse density, amyloid-beta deposition and tau accumulation, respectively, and structural MRI, based on T2-weighted scanning, will be performed over the time course of presentation of synaptic and AD- related pathology. Brains from a subset of animals at each time will be analyzed for histopathologic markers of neuronal loss and degeneration to provide ground-truth measures for correlating with the in vivo imaging measures. This study will unleash the potential to: (i) robustly validate in vivo imaging measures of synapse dysfunction as early biomarkers of AD against other imaging measures and histopathology, which is a necessary step towards their evidence-based clinical translation; (ii) provide preliminary data to support future mechanistic hypotheses about the regional and temporal relationships between synapse dysfunction and other AD-associated pathologies, with the ultimate goal of improving our understanding of AD risk; and (iii) understand concordance and discordance between the different in vivo imaging and histopathology measures, which will have implications for therapy design and testing. In summary, this project will provide key translational elements that will inform future human studies assessing the role of synapse loss in AD and for monitoring treatments to preserve synapse density and function.

Inhibition of ?-aminobutyric acid type A (GABAA) receptors (GABAAR) is the presumed mechanism of the seizure-inducing activity of the natural product picrotoxin (PTX), the rodenticide tetramethylenedisulfotetramine (TETS) and the high-energy explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). TETS, which has been banned from production worldwide, is still readily available on the black market, and is associated with thousands of human poisonings per year. It is incredibly stable in the environment. RDX is an environmental contaminant found in both groundwater and soil due to its worldwide military and civilian use, and it is an illicit abuse substance. PTX is of toxicological concern, because like TETS and RDX, it is listed as a credible threat agent by the United States Department of Homeland Security. All three compounds can cause seizures that rapidly progress to status epilepticus and death; however, TETS is by far the most potent with a lethal dose of 7 to 10 mg in humans and an LD50 of 0.1 mg/kg in rodents, which makes it roughly ~40x more potent than PTX and ~1000x more potent than RDX. There currently is no approved medical countermeasure for individuals acutely intoxicated with these convulsant chemicals. While some information is available regarding the molecular site of action and the GABAAR subtype selectivity of PTX, practically nothing is known about the molecular mechanism of action of TETS and RDX other than that they are most probably GABAAR inhibitors. We intend to use molecular modeling, whole-cell patch-clamp electrophysiology and gene knockdown techniques in zebrafish to identify the subunit specificity of TETS and RDX interactions with GABAA receptors and to determine whether it differs from that of picrotoxin. Treatment with subtype selective compounds/drugs should allow us to confirm which GABAA receptor subunit combinations are important for the seizure activity of TETS and RDX, and whether they differ from the receptor subunit profile that mediate PTX action. Detailed understanding of the molecular mechanism(s) of action of TETS and RDX will not only provide novel insight as to the biological reasons for the toxicologic differences between these agents, but will also be important for evaluating the validity of ?read across? risk assessment approaches for GABAA receptor antagonists, and for developing effective medical countermeasures for terminating SE in intoxicated individuals.

Placental tissue, which is normally discarded at birth, is a molecular time capsule that captures gene by environmental interactions and dysregulated molecular and cellular pathways that can be revealed at the level of the epigenome. Identifying epigenetic biomarkers at birth that reflect in utero exposures or predict adverse neurodevelopmental outcomes is an important goal that has been limited by prior technologies or lack of relevant tissue availability. Our team of currently collaborating interdisciplinary scientists within the Children?s Center for Environmental Health plans to use existing placental samples from a prospective high-risk cohort study (MARBLES) to identify epigenetic biomarkers at birth for in utero exposure to polychlorinated biphenyls (PCBs) and neurodevelopmental outcomes at age three years. In the MARBLES cohort, we have shown that taking folic acid in the form of prenatal vitamins specifically in the first month of pregnancy was associated with a 50% reduction in the risk for autism. Our human studies and the work of others also show evidence that folic acid supplementation can counter epigenetic and neurodevelopmental effects of environmental contaminants. Furthermore, using unbiased whole genome bisulfite sequencing (WGBS), we have previously demonstrated that placental tissues retain the distinctive DNA methylation patterns of the preimplantation embryo and so can capture the molecular state in very early development, a feature that is conserved across mammalian species, including mouse. The proposed PCB Epigenomic Brain & Behavior Lasting Effects Study (PEBBLES) combines WGBS analysis of human placental samples from the high-risk MARBLES cohort with a more extensive epigenomic analysis of placenta and brain tissues and sorted cell types derived from a mouse model of perinatal exposure to the same mixture of PCB congeners detected in MARBLES mothers. The hypothesis to be tested in the parent PEBBLES grant is that perinatal PCB exposures adversely impact neurodevelopment and leave a lasting molecular signature over genes relevant to neurodevelopment that can be detected in placenta. The hypothesis to be tested in the nutritional supplement to PEBBLES is that dietary folic acid supplementation will be protective against the adverse effects of PCB exposures at the level of the epigenome in an experimental animal model and human placenta. Bioinformatic and statistical analyses will integrate the genomic data sets with behavioral and molecular outcome measures and determine whether similar epigenetic marks are observed in placenta that could be used to predict long-lasting brain and behavioral outcomes in humans. !

The primary objective of the UC Davis CounterACT Center of Excellence is to identify and advance improved medical countermeasures for rapidly terminating seizures and mitigating the delayed neurologic consequences following acute intoxication with convulsant chemical threat agents. The Center comprises three research projects: Project 1 discovers therapeutic candidates via in vitro mechanistic screens, which are tested for in vivo antiseizure and neuroprotective efficacy by Projects 2 and 3, respectively. The projects rely on three scientific cores to support drug analysis and biomarker detection (Core A), medicinal chemistry and pharmacological testing (Core B), and experimental design and data analysis (Core C). A Research Education Core supports training in countermeasure research, and an Administrative Core oversees and coordinates scientific and administrative operations. The Center focuses on the GABAA receptor antagonist tetramethylenedisulfotetramine (TETS) and the organophosphate cholinesterase inhibitor diisopropylfluorophosphate (DFP), which can trigger convulsions that progress to life threatening status epilepticus (SE). Survivors face significant, long-term morbidity, including mild-to-severe memory loss, affective disorders and recurrent seizures. Current medical countermeasures can reduce mortality in exposed individuals, but they do so with significant side effects and are maximally effective only if administered within minutes of exposure. These limitations underscore the urgent need for improved medical countermeasures. In the first project period, we developed innovative in vitro platforms for mechanism-based screening to identify candidate antiseizure and neuroprotective therapeutics, and novel preclinical models that recapitulate acute seizure activity and neurological deficits observed in humans following acute intoxication with TETS or OPs. Using these models, we discovered: (1) allopregnanolone, a GABAA receptor positive allosteric modulator, was a superior countermeasure for TETS-induced SE, and (2) combining standard-of-care with allopregnanolone and a low dose of perampanel, a potent AMPA receptor antagonist, was more effective than standard-of-care alone in terminating DFP-induced SE. We also discovered that neuropathology was mitigated by post-exposure treatment with dantrolene, a Ca2+ channel stabilizer, or a novel small molecule dual inhibitor of soluble epoxide hydrolase (sEH) and cyclooxygenase-2 (COX-2). Our goals in this second project period are to: (1) advance our antiseizure lead allopregnanolone; (2) continue development of allopregnanolone and perampanel; (3) identify adjunct neuroprotective leads, focusing initially on the dual sEH-COX-2 inhibitor and dantrolene; and (4) conduct mechanistic studies to discover new therapeutic candidates. Our milestones for the second project period are to: (i) produce data and a regulatory strategy to advance allopregnanolone for treatment of GABAAR antagonist-induced seizures; (ii) determine whether combination treatment with allopregnanolone and perampanel warrants development as a lead ?universal antidote?; and (iii) identify lead neuroprotective treatments for improving long-term outcomes.

Project Summary Alzheimer?s disease is the most common age-related neurodegenerative disease, and there is a clear need to develop effective approaches to treat or prevent the cognitive impairment that is the prominent symptom of this disease. Neuroinflammation and mitochondrial impairments have been implicated in the pathogenesis of Alzheimer?s disease, and ketogenic diets have been reported to decrease inflammation and enhance mitochondrial function. This has led to speculation that ketogenic diets may provide an effective approach to treat Alzheimer?s disease. In support of this idea, short-term (?3 months) studies have shown that ketogenic diets improve motor function in mouse models of Alzheimer?s disease. However, memory was not improved with this diet, perhaps because the impact of the ketogenic diet was blunted by the ad libitum feeding approach used in these studies. There is growing appreciation that physiological response to a high fat diet is likely dependent on level of energy intake. Ketogenic diets are inherently high in fat and ad libitum feeding of a high fat diet induces a metabolic stress that may impair cognitive function. To investigate the physiological impact of these diets independent of differences in energy intake, we fed mice a ketogenic diet in isocaloric amounts to a control diet and found that the ketogenic diet increased life span and preserved memory and motor function with advanced age. It remains to be determined whether this feeding approach, which produces sustained ketosis, would also improve memory in rodent models of Alzheimer?s disease. Calorie restriction and intermittent fasting, which produce intermittent periods of ketosis, have also been reported to mitigate cognitive impairments in mouse models of Alzheimer?s disease. This raises the possibility that ketosis does not need to be continuous to have a beneficial impact on memory, which if true, would provide a lifestyle change more likely to be adopted by at-risk individuals. Sustained or intermittent increases in blood ketone levels with consumption of a ketogenic diet may be particularly beneficial if they lessen inflammation. Thus, we hypothesize that isocaloric ketogenic diet feeding approaches that produce either sustained or intermittent ketosis will decrease neuroinflammation and delay the onset or lessen the severity of disease progression in the TgF344-AD rat model of Alzheimer?s disease. We propose two specific aims to test this hypothesis: 1) determine if an isocaloric ketogenic diet feeding approach slows disease progression in the TgF344-AD rat; 2) determine if intermittent feeding of a ketogenic diet is sufficient to delay the onset or lessen the severity of memory and motor deficits in the TgF344-AD rat. These studies will take an important step toward determining if ketogenic diet approaches provide a viable strategy to treat or prevent Alzheimer?s disease.

Abstract Alzheimer?s disease (AD) affects an estimated 5.7 million Americans, a number expected to reach 14 million by 2050. Despite several decades of research, the initiation and progression of AD continues to be poorly understood, indicating the need for novel therapeutic strategies. There is mounting evidence that neuroinflammation plays an important role in the progression of AD. We are proposing to investigate Cmpd 10357, a novel UCD small molecule originally developed as a highly selective anti-cancer agent, for treatment of AD. Cmpd 10357 selectively targets intracellular protein complexes containing the serine protease inhibitor, plasminogen activator inhibitor-1 (PAI-1). PAI-1 functions as the primary inhibitor of tissue plasminogen activator (tPA) and urokinase (uPA). PAI-1 is not expressed in the normal brain, but its expression is markedly increased in cytokine-activated microglia and astrocytes, and increased PAI-1 expression has been demonstrated in AD patients and AD animal models. High levels of PAI-1 expression and secretion by activated microglia and astrocytes inhibits plasmin activation by serine proteases and has been demonstrated to decrease A? protein degradation, thereby promoting plaque formation. We hypothesize that Cmpd 10357 will selectively target and kill activated microglia and astrocytes in the AD brain that express PAI-1, thereby slowing or preventing progression of AD via potentially two mechanisms: (i) reducing neuroinflammation; and/or (ii) improving A? clearance from the brain. To test this hypothesis, we will investigate the drug?s impact on neuroinflammatory status, AD pathology, and cognitive dysfunction in the transgenic TgF344-AD rat model. The objectives are to determine whether weekly intraperitoneal administration of Cmpd 10357 attenuates neuroinflammatory changes and plaque formation in the TgF344-AD rat and assess the impacts of these changes on the progression of AD pathology and cognitive decline. This project leverages an ongoing collaboration between the PIs that has resulted in publications characterizing the anti-inflammatory properties and novel drug mechanisms of action of two new classes of small molecules. Consistent with the goals of the R21 funding mechanism, this project is high risk, but if successful, high payoff in that it will generate proof-of- concept data for a novel therapeutic candidate for AD that potentially works via both A?-independent and dependent mechanisms. Findings from these studies may also validate Cmpd 10357 as a potential therapeutic approach for other neurological diseases with a predominant neuroinflammatory component, as well as a tool compound for addressing the controversy regarding the neuroprotective vs. neurotoxic role of microglia and astrocytes in AD by enabling the selective death of PA1-expressing glia at specific stages in the progression of AD in preclinical models.

PROJECT SUMMARY/ABSTRACT Exposure of the developing brain to polychlorinated biphenyls (PCBs) and disruption of the gut microbiome have independently been implicated in the etiology of neurodevelopmental disorders (NDDs). Both phenomena likely interact by two mechanisms to cause adverse neurodevelopmental outcomes: PCB-mediated changes in the gut microbiome (1) alter the profile of neuroactive microbial metabolites distributed to the developing brain and (2) affect PCB disposition in the developing brain by modifying host and microbial PCB metabolism. A mechanistic understanding of these interactions is required to address the critical need for interventional strategies that effec- tively reduce the impact of PCB-induced NDDs on individuals, families, and society. The long-term goal is to de- termine the role of the gut microbiome?liver?brain axis in modulating susceptibility to PCB effects on the develop- ing brain, with the objective of characterizing how dose-dependent interactions between maternal exposure to an environmentally relevant PCB mixture and the gut microbiome influence neurotoxic outcomes in conventional (CV) vs. germ-free (GF) juvenile mice. We will test the central hypothesis that dysbiosis of the gut microbiome as- sociated with developmental exposure to varying doses of PCBs contributes to adverse neurodevelopmental out- comes later in life. This hypothesis is based on preliminary studies showing that: (1) PCB exposure causes dysbiosis of the gut microbiome; (2) PCBs and their metabolites are present in the rodent brain; (3) the expression of PCB-metabolizing enzymes differs between CV vs. GF mice; (4) PCB disposition differs between CV and GF mice; and (5) PCBs are developmental neurotoxicants in mice. Guided by these preliminary data, the novel hy- pothesis will be tested by (a) characterizing gut microbiome composition and function and neuroactive microbial metabolites; (b) determining how differences in host vs. microbial biotransformation affect disposition of PCBs and their metabolites; and (c) examining neuroinflammation, oxidative stress, synaptic connectivity, and behavior in dams and fetuses/pups derived from (i) CV vs.GF dams exposed to PCBs in the diet and (ii) GF dams who re- ceived a fecal transplant from PCB-exposed or vehicle control CV dams. The proposed research is innovative because it uses a systems biology approach in a state-of-the-art mouse model to assess the role of the gut mi- crobiome?liver?brain axis in modulating neurotoxic outcomes following exposure to an environmental PCB mix- ture. The anticipated outcome of these studies is a new research paradigm demonstrating that developmental exposures to PCBs mediate (1) longitudinal changes in gut microbiome composition and function and (2) alter PCB disposition in the developing brain that influence neurodevelopmental outcomes later in life. This outcome will have a significant impact on public health by informing future studies of cellular mechanisms of the devel- opmental origin of PCB neurotoxicity and, ultimately, provide critical insights regarding the plausibility of micro- biome-based approaches to diagnose and treat NDDs induced by exposure to neurotoxicants.

Project Summary Alzheimer?s disease (AD) is the most prevalent age-related neurodegenerative disease in the United States. More than 90% of cases are idiopathic and there is growing consensus that gene x environment interactions influence the age of onset and progression of this devastating disease. Epidemiological studies have reported a positive correlation between exposure to traffic-related air pollution (TRAP) and the occurrence of the hallmark clinical characteristics of AD. Preclinical studies support a causal relationship between TRAP and increased AD risk, but many of these studies used concentrated ambient particles or diesel exhaust that do not recapitulate the complexity of current real-world TRAP exposures. We have designed a unique exposure model in which TgF344-AD rats expressing human AD susceptibility genes are exposed in real-time to TRAP collected from a major freeway tunnel system, which preserves the gaseous and particulate components of real-world TRAP and captures daily fluctuations in pollutant levels. We will leverage this model to test our central hypothesis that TRAP decreases the time to onset and/or increases severity of AD-like phenotypes via microglial cell activation secondary to lung inflammation by addressing the following specific aims: (1) Determine which vehicle emission component(s) cause neuroinflammation and neurodegeneration in the TgF344-AD rat; and (2) Investigate the role of the lung-brain axis in mediating TRAP effects on AD phenotypes in the genetically susceptible TgF344-AD rat. In Aim 1, male and female TgF344-AD rats will be transported to the tunnel vivarium at 1 month of age and then exposed to gases, particulate matter (PM), or both from light duty only vs. light and heavy-duty vehicle exhaust. Outcomes including blood brain barrier (BBB) integrity, particulate matter in the brain, microglial and astroglial activation, AD-relevant pathology, lung pathology, and spatiotemporal profiles of soluble inflammatory mediators and immune cells in the lung, blood, and brain, will be assessed at 4, 9, 12, and 15 months of age. In Aim 2, we will assess the contributions of pulmonary inflammation and microglial activation to TRAP effects on AD by quantifying AD phenotypes in TRAP-exposed male and female TGF344-AD rats fed chow supplemented with pirfenidone or Senicapoc, which block pulmonary fibrosis via inhibition of TGF-? signaling or inhibition of KCa3.1 ion channels, respectively. Outcomes, which include cognitive behavior plus the endpoints measured in Aim 1, will be assessed at 12 and 15 months of age. Our broad long-term objectives are to inform regulatory and health interventions aimed at reducing AD risk for individuals living, working, or attending school near busy roadways.