Isaac N. Pessah - US grants

The proposed studies will elucidate the structure and function of cardiac, skeletal, and smooth muscle ligand-gated calcium release channels (LGCRC) in isolate membranes and purified receptor preparations. Particular emphasis is placed on defining the mechanisms by which pertinent classes of pharmacological agents and environmental toxicants alter normal channel function in vitro and assesses their toxicological relevance in vivo. Analysis of (3H)ryanodine receptor binding with concomitant spectrophotometric assays of Ca2+ uptake and release from junctional vesicles with the metallochromic Ca2+ indicator antipyrylazo III allows direct inspection of receptor and Ca2+ channel function. Fluorescent and photoaffinity probes will test the major premise that four distinct effector domains modulate the gating behavior of LGCRC. The principal hypotheses tested are: 1. (3H)ryanodine specifically binds to the Ca2+-induced open state of LGCRC, and hence can directly assess modulation of LGCRC by physiologically-relevant ligands or xenobiotics. Photoaffinity labelling of the ryanoid-binding site reveals its position within junctional foot oligomer. 2. The Ca2+ regulatory domain is primarily responsible for gating LGCRC and unmasking the (3H)ryanodine-binding site. Lanthanides compete for the Ca2+-binding sites while thiol-reactive heavy metals and aryldisulfides specifically interact with critical thiols associated with this domain and serve as functional and structural probe. Fluorescent sulfhydryl reagents clarify the role and position of this domain within the native LGCRC oligomer and discriminate major differences among muscle types. 3. The xanthine domain allosterically influences the sensitivity of LGCRC and the (3H)ryanodine binding site to activation by Ca2+- . Antineoplastic anthracyclines specifically bind to this domain causing potent sensitization to activation by Ca2+ which can be antagonized by caffeine. 4. The adenine nucleotide domain enhances (3H)ryanodine receptor occupancy and the intensity of the LGCRC response to Ca2+ and functionally overlaps the xanthine domain. The combined use of physiological, biochemical, and toxicological endpoints will yield important new information about LGCRC structure and function and assess its involvement as a target for pharmacological and toxicological agents and characterize the mechanisms involved.

receptor coupling; calcium channel; muscle contraction; reagent /indicator; pharmacology; ryanodine; protein structure function; chimeric proteins; membrane reconstitution /synthesis; membrane structure; sarcoplasmic reticulum; protein signal sequence; protein transport; tissue /cell culture; bioassay; genetically modified animals; laboratory mouse;

Epidemiological studies indicate that developmental exposure to polychlorinated biphenyls (PCBs) represents a significant risk factor affecting parameters of learning and cognition in humans. Our long term objective is to understand exactly how the major ortho-substituted PCBs found in human tissues and their metabolites alter spatial and temporal aspects of Ca2+ signaling in neurons of hippocampus and how these mechanisms relate to alterations in (1) synaptic plasticity and (2) associative learning and memory. The specific goals of this pilot proposal focus on understanding how PCB170 (2,2',3,3',4,4',5-heptachlorobiphenyl), a major contaminant of human tissues, affects neurodevelopmental toxicity in rats. Preliminary work has revealed that PCB170 is one of the most potent modulators of the ryanodine receptor (RyR2)/immunophilin (FKBP12) Ca2+ channel complex which predominates within hippocampal neurons. Since the RyR2/FKBP12 complex regulates important aspects of neuroplasticity and has been shown to be closely associated with acquisition of spatial learning, the following hypotheses will be tested: HYPOTHESIS I: PCB170 is a potent modulator of the RyR2/FKBP12 complex of primary hippocampal neurons, an activity which alters short-term (functional) and long-term (transcriptional) Ca2+ signaling critical for neuroplasticity. The specific aims are to elucidate: (1) the molecular mechanism(s) by which PCB170 alters the fidelity of neuronal Ca2+ signaling in cultured hippocampal neurons, (2) whether PCB170 alters expression of key proteins involved in Ca2+-dependent signaling, especially the RyR2/FKBP12 complex, whose activity is known to be associated with changes in neuroplasticity of hippocampus, and (3) if perinatal exposure to PCB170 in vivo alters normal developmental expression of the RyR2/FKBP12 complex in hippocampus, and its relationship to induction of long term potentiation. (Year 1) HYPOTHESIS II: Perinatal exposure to PCB170 alters acquisition of hippocampal associative learning, an effect which is closely correlated with perturbations in the function and transcriptional induction of the RyR2/FKBP12 complex in CA1, CA3 and dentate gyrus. The specific aims are to determine if perinatal exposure to PCB170: (1) alters spatial learning in the Morris water maze and (2) alters the normal relationship between acquisition of spatial learning and transcriptional induction of the RyR2/FKBP 12 complex. (Year 2)

The long-term goal of Research Project III is to identify molecular and cellular mechanisms that underlie idiosyncratic responses within autistic children to chemicals to which they are exposed in in utero and during periods of early postnatal brain development. The pressing worldwide concern about the role of vaccine antigens, the mercurial preservative thimerosal, and environmental exposure to mixtures of methylmercury and PCBs, justify detailed analysis of the underlying mechanisms of these factors in autism. We will first focus on three hypotheses relating to synergistic actions of mercurials and PCBs agents, known to be immunotoxic and neurotoxic. The hypotheses to be tested are: Hypothesis I addresses how peripheral blood mononuclear cells (PBMCs) from autistic children exhibit significant differences in their sensitivity and/or pattern of cell activation and cytokine secretion when challenged in vitro with vaccine antigens. How Non-coplanar PCBs of environmental relevance, thimerosal and other environmental agents identified by the Center's units exacerbate these differences will be studied. Hypothesis II determines how organic mercurials (thimerosal and MeHg) and non-coplanar PCBs (PCBs 118, 138, 153, 170, and 180 singly or in combination) act synergistically to influence glia/neural cell signaling pathways leading to altered patterns of dendritic spine growth, dendritic branching and synaptogenesis. Products of antigen- stimulated and control PBMCs (isolated from autistic and non-autistic children) characterized and quantified in Hypothesis I will be used to address their differential effects on neuronal cell growth. Hypothesis III utilizes mice exposed to PCBs and organic mercurials in vivo (PROJECT II) to assess functional and biochemical changes associated with social behavioral deficits. We will identify differences in patterns of evoked potentials and excitability in hippocampus/amygdala slice preparations from mice that have been perinatally or neonatally exposed to PCBs, organic mercurials, singly or in combination in Project II. We will elucidate the underlying biochemical mechanisms of these effects.

We intend to establish a Center for Children's Environmental Health and Disease Prevention Research at U.C. Davis that will investigate environmental risk factors contributing to the incidence and severity of childhood autism. Autism is a neurodevelopmental disorder defined by deficiencies of social reciprocity and communication, and by repetitive behavior, The majority of cases seem likely to arise from a multiplicity of yet unidentified genetic and environmental factors. Surveys in California have indicated an apparent 210% increase in the cases of profound autism in children diagnosed over the last 10 years. Recent estimates indicate the frequency of mild to severe autism may be as high as 1:150. Thus there is growing concern from both parents and health professionals that prenatal and postnatal exposure to xenobiotic (e.g. mercurials, halogenated aromatics, and pesticides) and biotic (e.g. vaccine antigens) factors may act synergistically with unidentified susceptibility genetic factors to produce autistic spectrum disorders. To understand how the interaction of susceptibility genes with exposure to "environmental" chemicals may increase the risk and severity of autism and to identify which combination of chemical exposures confer the greatest threat, we propose to establish an interdisciplinary Center that addresses this complex problem at several levels. Project I proposes the first case-controlled epidemiological study of environmental factors in the etiology of autism. Tissue samples and exhaustive information will be collected from geographically distinct areas of California. Project two proposes to identify for the first time how known neurotoxicants of concern to children's health influence the development of social behavior and mediating brain regions such as the amygdala. Project III integrates elements of Projects I and II in order to examine molecular mechanisms underlying neurodevelopmental disorders associated with human autism and animal models of autism. The three research projects are integrated within a center framework that provides extensive facility core capabilities in xenobiotic/lipid analysis (Core I), cell activation biomarkers (Core II), and molecular biomarkers (Core III). Our ultimate goal is to understand common patterns of dysfunction in autism and elucidate mechanisms by which known neuroimmunotoxicants contribute to abnormal development of social behavior in children so that rational strategies for treatment and prevention can be undertaken.

Description (provided by applicant): The major long-term goal of the Molecular Neurotoxicology Research Core is to understand the mechanisms by which complex environmental mixtures may produce direct and indirect neurotoxicity, and their possible influence on increasing the risk of known neurological disorders. To meet this goal, this research core promotes and facilitates interdisciplinary research. The overall objectives of the Molecular Neurotoxicology Research Core are to: (1) Elucidate the molecular and cellular mechanisms that influence the, normal patterns of neuronal signaling, growth, and death. Particular attention will be given to the interaction between the immune and nervous systems. (2) Define how these parameters are altered in response to environmental neurotoxicants, presented either individually or in combination. In this regard, particular attention will be given to chronic, low-level exposures. (3) Initiate studies to understand how genetic factors known to alter immune or nervous system signaling pathways may enhance or diminish susceptibility to the mechanisms elucidated in (1) and (2). The intention of the investigators is to identify key steps involved in cellular signaling that are influenced by environmental pollutants and elucidate how their altered activity may alter specific aspects of neuronal health.

Description (provided by applicant): The 2nd conference entitled Autism, Genes and the Environment will be held September 13-15, 2003 in Sacramento, California. Two of 12 NIEHS/EPA-sponsored Children's Centers have their primary focus on childhood autism and will co-sponsor the meeting and associated workshops. The U.C. Davis Center for Children's Environmental Health will host the meeting and the Center for Childhood Neurotoxicology and Exposure Assessment administered through UMDNJ will serve as co-sponsor. Additional financial and logistical support for this year's meeting comes from The M.I.N.D. Institute. The intent is to alternate between east and west cost locations to increase the opportunities for participation in one of the few forums that directly address the interplay between genes and environment in the etilolgy of autism. The agenda is designed to bring together environmental epidemiologists, behavioral researchers, and molecular and cellular neurotoxicologists who may not have applied their expertise toward elucidating the influence of environmental exposures to the development of social behavior, with established researchers in the field of autism. In addition to scientists from academia and governmental organizations, the Centers have incorporated a formal program to encourage participation of parents of autistic children, advocates, and caregivers. Focused workshops will be led by M.I.N.D. Institute professionals and affiliates, which are designed to address specific needs of the community. The long-term objective of the forum is to improve communication between toxicologists and researchers in the field of autism, stimulate multidisciplinary collaboration, and better understand the interplay between susceptibility genes and environmental exposure in the etiology of autism. The following topics will be presented at the conference: Critical Periods of Pre- and Post-Natal Development; Environmental Epidemiology of Neurodevelopmental and Autoimmune Disorders; Animal Models of Neuro- and Immuno-toxicity, and Molecular and Cellular Neurotoxicity of Organic Mercury. Three workshops for parents and caregivers will address the following: Dietary Factors Influencing Autistic Behaviors; Pharmacological Interventions, and Behavioral Interventions.

disease /disorder etiology; autism; environmental health; animal colony; Primates; clinical research;

disease /disorder model; phenylketonuria

[unreadable] DESCRIPTION (provided by applicant) [unreadable] The mission of the University of California-Davis Center for Children's Environmental Health (CCEH) is to promote daily interactions among a multidisciplinary team of scientists whose main research interest is to understand the complex web of etiologic factors that contribute to autism. The shared philosophy among Center participants is that a better understanding of the immunological and neurobiological mechanisms associated with this neurodevelopmental disorder can not only lead to a better understanding of the mechanisms that influence it but can also accelerate the discovery of effective intervention strategies. The goals of the CCEH in the next five years are to: (1) better understand the mechanisms by which environmental, immunologic, and molecular factors interact to influence the risk and severity of autism; (2) identify early immunologic, environmental, and genomic markers of susceptibility to autism; (3) develop mouse models of immunologic susceptibility to environmental triggers and define the impact of these triggers on the development of complex behaviors, key brain structures and neurotransmitter receptors relevant to autism; (4) translate the research findings into diagnostic tools that can be used in clinical practice to predict early autism risk; and (5) supply the community with accurate and timely information about autism risk factors. The CCEH has organized three interrelated hypothesis-based Research Projects that are supported by five Facility Cores (Administrative, Community Outreach and Translation, Analytical Chemistry, Molecular Genomics, and Statistics). [unreadable] [unreadable] The projects are: Project 1, Environmental Epidemiology of Autism, will build upon the investigators' discovery of immunologic and molecular biomarkers specific to children with autism found in 2-5 year olds enrolled in the CHARGE (Childhood Autism Risks from Genetics and Environment) study. Working closely with the Community Outreach and Translation Core and Project 2, newborn bloodspots and a second set of blood samples (CHARGE-BACK study) from CHARGE children will examine the stability over time of these biomarkers. CHARGE-BACK blood samples will also provide peripheral immune cells to study how autism alters properties of cell activation, and susceptibility to known immunotoxicants. The investigators will launch a new cohort study called Markers of Autism Risk in Babies-Learning Early Signs (MARBLES) that tracks 200 women at high risk of giving birth to an autistic child, starting from early pregnancy and following the pregnancies and the babies to the age of three years. [unreadable] [unreadable] Project 2, Immunological Susceptibilities in Autism, will work closely with Project 1 to test the overall hypothesis that autistic children have fundamental defects in cellular immunity that ultimately lead to abnormalities in immune dysfunction and heightened susceptibility to environmental triggers. Project 3, Models of Neurodevelopmental Susceptibility, will develop and use mouse models to understand the relationships between immune system dysfunction and perinatal exposure to environmental toxicants in the development of neurobehavioral disorders in sociability and seizure susceptibility. Working closely with Project 2, the investigators will test mouse strains with low (C57BL/6J) or high (SJL mice) susceptibility to autoimmunity to determine how perinatal exposures to methylmercury, noncoplanar PCB, or polybrominated diphenyl ether 47 (BDE 47) influence brain development, complex social behaviors, and immune system function. [unreadable]

The mission of the UC Davis Center for Children's Environmental Health (CCEH) is to promote daily interactions among a multidisciplinary team of scientists whose main research interest is to understand the complex web of ettologic factors that contribute to autism. The shared philosophy among Center participants is that a better understanding of the immunological and neurobiological mechanisms associated with this neurodevelopmental disorder can not only lead to a better understanding of the mechanisms that influence it but can also accelerate the discovery of effective intervention strategies. The goals of the CCEH in the next five years are to: (1) better understand the mechanisms by which environmental, immunologic, and molecular factors interact to influence the risk and severity of autism;(2) identify early immunologic, environmental, and genomic markers of susceptibility to autism;(3) develop mouse models of immunologic susceptibility to environmental triggers and define the impact of these triggers on the development of complex behaviors, key brain structures and neurotransmitter receptors relevant to autism (4) translate our research findings into diagnostic tools that can be used in clinical practice to predict early autism risk;and (5) supply the community with accurate and timely information about autism risk factors. The CCEH has organized three interrelated hypothesis-based research projects that are supported by five facility cores (Administrative, COTC, Analytical Chemistry, Molecular Genomics, and Statistics). The Projects are: Project 1, Environmental Epidemiology of autism, will build upon our discovery of immunologic and molecular biomarkers specific to children with autism found in 2-5 year olds enrolled in the CHARGE (Childhood Autism Risks from Genetics and Environment) study. Working closely with COTC and Project 2, newborn bloodspots and a second set of blood samples (CHARGE-BACK study) from CHARGE children will examine the stability over time of these biomarkers. CHARGE-BACK blood samples will also provide peripheral immune cells to study how autism alters properties of cell activation, and susceptibility to known immunotoxicants. We will launch a new cohort study called Markers of Autism Risk in Babies-Learning Early Signs (MARBLES) that tracks 200 women at high risk of giving birth to an autistic child, starting from early pregnancy and following the pregnancies and the babies to the age of three years. Project 2, Immunological Susceptibilities in Autism, will work dosely with Project 1 to test the overall hypothesis that autistic children have fundamental defects in cellular immunity that ultimately lead to abnormalities in immune dysfunction and heightened susceptibility to environmental triggers. Project 3, Models of Neurodevelopmental Susceptibility, will develop and use mouse models to understand the relationships between immune system dysfunction and perinatal exposure to environmental toxicants in the development of neurobehavioral disorders in sociability and seizure susceptibility. Working closely with Project 2, we will test mouse strains with low (C57BL/6J) or high (SJL mice) susceptibility to autoimmunity to determine how perinatal exposures to methylmercury, noncoplanar PCB, or polybrominated diphenyl ether 47 (BDE 47) influence brain development, complex social behaviors, and immune system function.

The long range goal is to determine if exposure to environmental toxicants early in development contributes[unreadable] to the etiology of neurodevelopmental disorders such as autism. A related goal is to determine whether[unreadable] susceptibility to autoimmune disease increases the neurotoxicity of environmental contaminants and[unreadable] increases the risk for developing disorders such as autism. Understanding how exposure to environmental[unreadable] toxicants may contribute to the etiology of neurodevelopmental disorders is important so that the exposure[unreadable] risks can be identified and minimized. If immune system dysfunction is found to increase the risk of[unreadable] exposure to environmental toxicants, then exposure limits to toxic substances can be lowered, and children[unreadable] with immune system dysfunction who may be at increase risk can be identified and protected. The specific[unreadable] aims are to expose mouse strains with low (C57BL/6J) or high (SJL mice) susceptibility to autoimmunity[unreadable] perinatally to either methylmercury (MeHg), polychlorinated biphenyl 95 (PCB 95) or polybrominated[unreadable] diphenyl ether 47 (BDE 47). We will then compare the effects of toxicant exposure between these mouse[unreadable] strains on brain development, complex social behaviors, and immune system function. The hypothesis is[unreadable] that perinatal exposure to each of these toxic substances will impair brain development and behavior, and[unreadable] that suscepbility to autoimmune disease will increase the neuro- and immunotoxicity of these agents. We[unreadable] will also explore a potentially new model of autism in mice injected prenatally with unique autoantibodies[unreadable] isolated from the serum of mothers who have given birth to two more more autistic children. Brain[unreadable] development will be examined histologically using stereological procedures and immunohistochemistry.[unreadable] Complex social behaviors will be studied using behavioral testing procedures established in our laboratory[unreadable] that measure social recognition, social interaction and social communication in mice. Immune system status[unreadable] will be established by measuring cytokines, chemokines, immunoglobulins, and quantifying immune system[unreadable] response to antigenic stimulation. In addition, seizure susceptibility will be measured in toxicant-exposed[unreadable] mice as well as measures of synaptic excitibility and plasticity in hippocampal brain slices. These studies will[unreadable] provide critical new information on the role of the immune system and its interaction with environmental[unreadable] contaminants in autism and other neurodevelopmental disorders.

[unreadable] DESCRIPTION (provided by applicant) [unreadable] [unreadable] This proposal requests partial support for the 11th International Neurotoxicology Association's biennial meeting (INA-11) to be held at the Asilomar Conference Center in Pacific Grove, California June 10-15, 2007. The broad and long-term goal of the conference is to increase our understanding of fundamental and applied aspects of neurotoxicology, with a strong emphasis on mechanisms of injury and strategies for promoting neuroprotection and recovery from injury. The specific aims of this meeting will be to convene 35 invited speakers from around the globe that represent critical areas of neurotoxicological research with a total of 200 participants for a five day conference in a relatively isolated setting. The program will commence Sunday evening with a keynote lecture by Dr. Fred Gage entitled "Promoting Recovery from Central Nervous System Injury in the Adult Organism". Eight sessions will broadly address current issues in the field. Two sessions on Monday will focus on the neuroprotective role of metallothioneins, neurosteroids, potassium channels, and nitrones. Two sessions on Tuesday will present advances in neurotoxicity screening in mammalian and non-mammalian models, and a third on Thursday morning addresses progress toward using neurobehavioral testing in human risk assessment. The remaining three sessions commencing Thursday afternoon and ending midday Friday address new neurotoxicological concerns including immunological and neurodevelopmental susceptibilities contributing to autism, air pollution, oxidative stress, and neurodegeneration, and how endocrine disruption influences the development of sexually dimorphic nervous systems and associated behaviors. In addition, a symposium organized and presented by students participating in graduate programs in neurotoxicology will round out the oral presentations. Two evening poster sessions will permit all participants to contribute to these topics. A committee will be constituted. The significance of this application is that the INA Conference series has become a major venue that propel research in the international community of researchers and educators involved in all aspects of neurotoxicology. The health relatedness of this application is that the discussions stimulated by the presentations will define the major questions that require experimental resolution in areas that affect human neurological heath, ranging from developmental exposures to degeneration associated with the aging nervous system. [unreadable] [unreadable] [unreadable] [unreadable]

The long-term objective in Project 2 is to gain understanding of the biochemical and biophysical mechanisms by which mutations from each MH/CCD hot spot alter key regulatory functions of the RyR1 Ca2+ channel complex, and how these changes deregulate SR Ca2+ transport in skeletal muscle SR, and dendritic cells that express MH/CCD RyR1. HYPOTHESIS I) MH/CCD mutations alter cytoplasmic and luminal regulation of RyR1 by ligands by changing the activation energy (Ea) needed for conformational transitions of the channel. A1.1. Define differences among 11 MH/CCD and wild type (Wt) genotypes on expression of key triadic proteins, SR loading capacity and their relationship to altered cation interactions at H- and Lsites using equilibrium [3H]ryanodine ([3H]Ry) binding analysis. A1.2. To analyze mechanisms responsible for altered regulation by cytoplasmic Ca2+, Mg2+, and ATP and luminal Ca2+ anc calsequestrin (CSQ) using selected RyR1 channels. A1.3. Compare the differences in activation energy (Ea) for selected MH/CCD mutations. A1.4. Analyze how halothane and dantrolene differentially influence the stability of closed and open states of selected MH/CCD RyR1s. HYPOTHESIS II) MH/CCD mutations disrupt the redox-sensing properties of RyR1. A2.1. To establish if differences in transmembrane redox regulation and GSH/GSSG transport contribute to the pathophysiology of MH/CCD. HYPOTHESIS III) MH/CCD mutations produce a dysfunctional phenotype in dendritic cells. A3.1. Elucidate how MH/CCD mutations alter murine dendritic cell activation, dendritic cell secretion of IL-6 mediated by ATP acting at P2Y receptors, and if and how MH/CCD mutations influence dendritic cell activation of T-cells. A3.2. To extend these studies to DCs cultured from human blood.

DESCRIPTION (provided by applicant): There is considerable public and regulatory concern that developmental exposures to polychlorinated biphenyls (PCBs) cause significant cognitive and behavioral deficits in children, but assessing the risks posed by these compounds has been difficult because the biological mechanisms underlying PCB effects on the developing nervous system have yet to be identified. We have recently demonstrated that developmental exposure of rodents to a commercial PCB mixture impairs dendritic growth and plasticity in vivo coincident with deficits in spatial learning. These effects on neurodevelopment and cognitive function correlate with altered expression and function of ryanodine receptors (RyR) within the central nervous system. RyR regulate calcium-dependent signaling pathways that have been implicated in activity-dependent dendritic growth, which is a critical determinant of neuronal connectivity in the developing brain. The goal of our study is to characterize the mechanisms and structure-activity relationship (SAR) of PCB developmental neurotoxicity by testing the hypothesis that non-coplanar PCBs alter dendritic growth and plasticity by disrupting RyR function. The specific aims are to: 1. Test the relative contributions of RyR perturbation and thyroid hormone deficits in PCB effects on dendritic growth and plasticity in vivo;2. Use primary cultures of hippocampal neurons to identify the molecular mechanisms mediating PCB effects on dendritic growth;3. Determine how non-coplanar PCBs alter the function and expression of proteins that comprise calcium release units in cultured hippocampal neurons;4. Determine whether heritable mutations in ryr1 and ryr2 that increase sensitivity to halogenated compounds in the human population increase susceptibility to PCB developmental neurotoxicity in mice expressing these mutations. These studies address the critical need to better understand mechanisms underlying PCB developmental neurotoxicity. Results will provide a rational basis for characterizing exposure risks and developing biomarkers of exposure and effect. Since RyR genes exhibit a significant number of expressed mutations and polymorphisms in the human population, data supporting RyR as a molecular target of PCBs in the developing nervous system will provide insights into genetic susceptibilities that magnify environmentally induced neurodevelopmental disorders. Polychlorinated biphenyls (PCBs) are persistent, widespread environmental contaminants, and there is compelling evidence that exposure of the developing brain to PCBs can cause learning and memory problems in children. But how PCBs cause these effects is not well understood. The goal of the proposed studies is to link known molecular effects of PCBs (activation of ryanodine receptors) to specific changes in brain development (disruption of dendritic growth). Establishing this link will provide a powerful means for predicting which of the 209 possible PCBs present the greatest risk to the developing brain and may provide novel insights into genetic susceptibilities that magnify environmentally induced neurodevelopmental disorders.

DESCRIPTION (provided by applicant): Polychlorinated biphenyl (PCB) congeners with multiple ortho chlorine substituents are potent sensitizers of the ryanodine receptor (RyR) and this activity is thought to contribute to the developmental neurotoxicity associated with perinatal PCB exposure. Many of these congeners display axial chirality and are present in industrial PCB mixtures as a racemate (a 1:1 ratio of both atropisomers). However, the ratio of the two enantiomers changes in vivo, probably due to enantioselective processes involving cytochrome (CYP) P450 enzymes. Emerging data suggest significant variability in the enantiomeric enrichment of chiral PCBs in the human population. This, when considered in light of our preliminary data demonstrating that PCB atropisomers differentially sensitize the ryanodine receptor (RyR), raises the question of whether enantiomeric enrichment influences the risk for adverse neurodevelopmental outcomes following PCB exposure. We propose three specific aims to test the hypothesis that chiral PCB congeners undergo enantioselective biotransformation catalyzed by P450 enzymes and that these differences in biotransformation influence neurodevelopmental endpoints. In Aim 1, the enantiospecificity of RyR-mediated mechanisms of developmental neurotoxicity will be characterized in vitro. The effects of pure PCB atropisomers on dendritic morphology will be quantified in primary rat hippocampal neuron-glia co-cultures and correlated with cellular PCB levels. The molecular mechanisms responsible for enantiospecific activation of RyR1 and RyR2 channel complexes will be investigated using biochemical, biophysical and cellular analyses. In Aim 2, the species and isoform-dependent enantioselective binding and metabolism of pure PCB atropisomers by P450 enzymes will be investigated using murine and human microsomes and tissue slices, as well as recombinant human P450 enzymes. The P450 isoforms responsible for the enantioselective metabolism of PCBs in microsomes will be identified using P450 inhibitors. Aim 3 will confirm, in vivo, that metabolism by P450 enzymes is responsible for enantiomeric enrichment of PCBs and that PCB 136 atropisomers cause enantioselective RyR-mediated developmental neurotoxicity. First, a pharmacokinetic model will be developed, in mice, to examine the role of metabolism in the enantioselective disposition of PCBs. Subsequent studies will determine whether perinatal exposure to chiral PCBs causes enantiomeric enrichment-dependent effects on hippocampal expression and function of RyR and dendritic arborization. These studies will make a fundamental contribution to understanding the risk associated with human exposure to chiral PCB congeners that are highly toxic to the developing nervous system and will provide an insight into the role of chirality in the disposition and toxicity of many chiral pollutants, such as pesticides and plasticizers. Given the extensive polymorphism in human CYP genes, the proposed studies may also suggest future investigations into gene-environment interactions that modulate susceptibility to PCB developmental neurotoxicity. PUBLIC HEALTH RELEVANCE: Developmental exposures to chiral polychlorinated biphenyls (PCBs) may cause neurodevelopmental toxicity by interfering with dendritic growth and plasticity via mechanisms involving the enantiospecific sensitization of ryanodine receptors. The goal of the proposed research is to investigate how differences in the enantioselective disposition of chiral PCBs in pregnant mice influence neurodevelopmental endpoints in exposed offspring. Because the enantiomer ratio of chiral PCBs is highly variable in human populations, the proposed studies will make a fundamental contribution to understanding the risk associated with human exposure to chiral PCB congeners that are highly toxic to the developing nervous system and will provide an insight into the role of chirality in the disposition and toxicity of a broad range of other organic pollutants, such as many pesticides and plasticizers.

DESCRIPTION (provided by applicant): Despite the fact that little is known about non-genetic causes of autism spectrum disorders (ASD), currently, few rigorous investigations of environmental factors in the etiology of this condition are underway. This proposal extends the epidemiology project initiated under the NIEHS-funded UC Davis Center for Children's Environmental Health (CCEH) known as "MARBLES" (Markers of Autism Risk in Babies - Learning Early Signs). MARBLES has enrolled close to 200 pregnant women who already have a child with ASD and therefore are at high risk for delivering an infant who will also develop ASD. In this R01, we will recruit an additional 250 pregnant mothers and follow their pregnancy and their child. Through longitudinal collection of 1) extensive behavioral, medical, and exposure data, 2) biologic specimens from the mother and child, and 3) detailed psychometric assessments from birth to three years of age, MARBLES has created an infrastructure for addressing etiologic questions and searching for early pathophysiologic markers. The exposures of interest are two classes of compounds common in household products, and having known neuro- or neurodevelopmental toxicity: pyrethroid pesticides and polybrominated diphenyl ethers (flame retardants). First, we will assess associations of self-reported exposure, measurements of internal dose, and toxicologically-derived estimates of biologically effective dose, on the one hand, with risk for ASD or other impairments in neurobehavioral development on the other. Secondly, we will examine whether the exposure or dose estimates are associated with markers of aberrant immune responses or mitochondrial dysfunction and whether these markers predict clinically confirmed child developmental status at three years of age. Thus, this project begins with the macro-level associations typical of black-box epidemiology, refines the estimates of dose, and then explores more deeply into the mechanisms by which environmental exposures might alter neurodevelopment and lead to clinical outcomes. This integrated approach is made possible by an interdisciplinary team that brings together molecular and basic research, epidemiologic population-based approaches, and clinical sciences. If our hypotheses are supported, this will be the first evidence of higher autism risk based on prospective measurements of compounds in commonly used household products, and one of the first to identify modifiable risk factors. The results will thereby open the door to prevention at the individual behavioral and societal level, e.g., potentially through education, product labeling, and/or regulatory action. Results may also lead to targeted interventions to alter children's developmental trajectory. Overall, we expect this study to stimulate a paradigm shift in the field, towards a more multifactorial and mechanistically driven research agenda, with significantly more attention to environmental exposures and the development of inter-disciplinary approaches. PUBLIC HEALTH RELEVANCE: Autism spectrum disorders (ASDs) are of considerable public health importance, with current estimates suggesting that close to 1% of children will develop the condition. Not only is little known about their causes, but also the mechanisms are poorly understood. This study, known as MARBLES (Markers of Autism Risk in Babies - Learning Early Signs), addresses both gaps and may illuminate potential ways to prevent this disorder from occurring. We propose to determine the role for two classes of common household chemical exposures, namely pyrethroid pesticides and brominated flame retardants, using an innovative design that maximizes the efficiency of the study. Additionally, MARBLES will explore the role of maternal immune dysregulation and of mitochondrial dysfunction in the newborn as markers of potential mechanisms leading to aberrant neurodevelopment. By addressing both the need to understand risk factors for ASDs beginning during gestation and the need to elucidate underlying mechanisms, MARBLES has the ability to move the field of autism forward to towards a more multifactorial and mechanistically driven research agenda. From this study, interventions at the individual behavioral or societal level may be suggested.

Project Summary ? Project 1 Phenotypic hallmarks of intoxication with seizure-inducing chemical threat agents include: (1) acute hyperexcitation of neural circuits associated with seizures and status epilepticus (SE); and (2) chronic neuroinflammation and neuropathology that manifest subsequent to seizures. Project 1 is using in vitro models essential for elucidating toxicological and therapeutic mechanisms and for discovering intervention strategies that are more effective and safer than current medical countermeasures (atropine, oxime, high-dose benzodiazepines) for treating acute intoxication and protecting against persistent neuropathology. Our focus is on two classes of threat agents: those that block GABAA receptor (GABAAR) function, e.g., tetramethylene- disulfotetramine (TETS) and picrotoxin (PTX, and organophosphates (OP) that inhibit cholinesterases, e.g., diisopropylfluorophosphate (DFP) and paraoxon (PO). Our hypothesis is that in vitro approaches we developed during the first project period can be used to identify, refine and optimize combinatorial therapies that more effectively target TETS- and OP-triggered seizure mechanisms and control subsequent neuroinflammation. This hypothesis will be tested using in vitro and ex vivo rodent models of both sexes. Aim-1 will determine whether midazolam plus a neurosteroid is more potent than either alone for normalizing TETS and PTX- triggered neuronal network hyperexcitability. In Aim 2 we will test whether subtype selective nicotinic cholinergic receptor (nAChR) inhibitors are superior to our standard midazolam ± neurosteroid combination or synergize with it in mitigating OP-triggered seizure-like activity in adult neuron/glia cocultures. Our reasoning is based on the known cholinergic mechanisms of seizure initiation. Aim 3 will investigate neuroinflammation by evaluating astrogliosis, microglial activation, and neuropathology following exposure to seizure-inducing chemical threat agents and screen for anti-inflammatory compounds that can mitigate these processes. We have developed two cell-based models (astrocyte/microglia and neuron/glia cocultures) from mouse and rat models of both sexes to quantitatively measure differences in neuroinflammation (astrogliosis and microglia activation) and their relationship to neuropathology over acute and prolonged exposures to chemical threat agents. Results obtained from these three aims will be critical for identifying and prioritizing the most promising candidate therapeutics for mitigating seizure-like activity, neuroinflammation and neuropathology to be testedin vivo by Projects 2 and 3.

Project 4 will test the hypothesis that CGG trinucleotide repeats in the FMRI gene, the most prevalent single gene disorder contributing to autism risk, influence susceptibility to non-dioxin-like (NDL) persistent organic pollutants (POPs) identified in Core 3 and pro-inflammatory cytokine profiles identified in Project 3 to predominate in plasma of women participating in the MARBLES study during pregnancy. A major advantage of the neurotypical and susceptible neuronal cell models to be used in our studies is that they originate from the same individual; thus, individual genetic background variation is excluded as a confounding variable. The specific aims are: Aim 1: Produce isoautosomal iPSC-derived neuronal precursor cells (NPCs) possessing a normal FMRI gene and NPCs possessing an active FMRI CGG repeat expansion in the mid-premutation, high-premutafion, and full-mutation (FXS) range. Aim 2: Identify morphological and functional differences between neuronal cultures with a normal FMRI acfive allele and neuronal cultures with an active FMRI CGG repeat expansion in the mid-premutation, high premutation, or full-mutation (FXS) range. Aim 2.1: Identify temporal differences the development of synchronized Ca2+ oscillafions, electrophysiological properties, mitochondrial bioenergefics and oxidative stress among genotypes. Aim 2.2: Determine how funcfional anomalies identified in Aim 2.1 influence Ca2+- dependent signaling pathways required for acfivity dependent dendrific growth, especially the CaMKl->CREB->Wnt and P13K->Akt->TSC1/2->mTOR signaling pathways. Aim 3: Define the spatiotemporal profile of neuropathological sequelae caused by exposures that mimic the gestational environment in mothers participating in the MARBLES study. Aim 3.1: Determine how exposures to individual congeners and complex mixtures that model the most abundant of PBDEs, PCBs, or perfluorinated compounds in maternal plasma alter the morphometric and funcfional outcomes measured in Aim 2. Identify crifical windows of susceptibility among genotypes. Aim 3.2: Determine how exposures to cytokine/chemokine profiles identified in maternal plasma influence the morphometric and functional outcomes measured in Aim 2. Aim 3.3: Determine whether exposures tested in Aim 3.1 and/or Aim 3.2 differentially alter epigeneflc signatures of global and gene specific (FOXP3, MeCP2, Dnmt3a) methylation among genotypes.

? DESCRIPTION (provided by applicant): The UC Davis NIH T35 STAR program focuses on short-term (10 weeks) research training of DVM students. The program emphasizes 5 fundamental research objectives: (1) how to gain knowledge and understanding of one's field of science; (2) how to formulate a scientifically sound and testable hypothesis; (3) how to identify specific objectives, conduct controlled methodical experiments, and develop technical expertise; (4) how to analyze results, derive conclusions, propose additional experiments, and anticipate new directions; and (5) how to convey research findings succinctly and convincingly to others. UC Davis has a rich environment for veterinary (DVM) students to participate in a structured program that introduces basic biomedical, bioengineering, pre-clinical, and transdisciplinary One Health research experiences early in their professional education. The greatest strengths of our short-term training program include the outstanding quality and motivation of our DVM students, the strong highly collaborative multidisciplinary nature of our research programs, and student access to translational research projects that use cutting-edge approaches. This five- year competitive renewal application requests support for a total of 15 DVM students per year for each of five years (a total of 75 students).

It Is the central tenant of Core B and this program project that the most appropriate method to study MH is in murine models that express frequently observed human MH mutations and in genotyped human tissue. Mouse models provide sufficient tissues for molecular, biochemical, cellular, physiological and morphologic analyses by a multidisciplinary team whose collective goal is to understand this myopathy. Genotyped human myotubes will allow direct correlation of the observed phenotypes between humans and mice with the same mutations and fixed and frozen human tissues will allow morphologic and biochemical comparisons. Core B will perform repetitive tasks that are necessary to support all three Projects and Core D. This core will produce and genotype the 4 existing MH knock-in mice, 2 transgenic mice, and breed, genotype and maintain MH mice crossed with dnTrp6 and SERCA1 overexpression transgenic mice. It will also assure that the long-term pharmacological interventions with salicylamine and 4-OH-BDE49 are carried out. It will distribute these mice or frozen muscles for study by all three Projects as needed and provide fixed muscles from these mice at different ages and genders to Core D. Core B will make myoblast cell lines from all heterozygous and homozygous MH knock-in mice, to be used by all 3 projects. If needed homozygous myoblasts will be obtained from El8 embryos from timed matings in cases where there is homozygous lethality as was the case for the R163C and Y522S RyRI mice. Core B will receive, expand and maintain genotyped human MHS myoblasts from Core C and distribute these to the projects. It will also distribute genotyped glutaraldehyde fixed human biopsy samples to Core D and frozen muscle samples to Project 2 received from Core C. Core B has and will receive new MHS mutations from Core C Discovery, prepare mutated cDNA constructs in mammalian expression vectors for project 3, and where possible lentiviral vectors and permanently transduced cells and where not ,HSV vectors to be distributed to projects 1 and 2 for their analyses. The uniform and consistent supply of exactly the same study models to all three Projects and Core D by this Core will allow a truly integrated approach to the study of malignant hyperthermia.

DESCRIPTION (provided by applicant): The long-term goal of this multicenter PPG is to define the mechanisms responsible for the malignant hyperthermia syndrome caused by mutations in RyR1 and Cav1.1 as well as leveraging new discovery of other gene linkage in humans. The scope of investigations will range from extensive phenotyping of MH mice, studies of Ca2+ and Na+ homeostasis in muscle from MH mice and MHS humans and the importance of TRPCs (Project 1), studies of RyRI function, how MHS mutations cause posttranslational modifications, mitochondrial adaptations and metabolic abnormalities (Project 2), and how disruption of the normal interactions between RyR1 and CaVI.1 lead to Ca2+ dysregulation in MH susceptible animals and patients (Project 3), and the influence of MH mutations on the cellular physiology of muscle (Projects 1, 2, & 3). Our research in the previous funding period has led to a unified general hypothesis applicable to any and all MH mutations: MH is caused by primary structural changes in RyRI, or by structural changes in RyRI induced indirectly by a mutation in Cavl.1 or another protein closely associated with RyR1 (as demonstrated by an MH like phenotype in Casq1 null mice). A transformative concept to be investigated by all PPG participants is that a defect in Cav1.1 f-RyR1 bidirectional signaling is a common convergent pathway leading to all MH susceptibility and progressive muscle damage. The focus of this program is tightly linked. All 3 Projects will: 1. Examine the relationship between gender and MH penetrance, 2. Validate the pathology in mouse models in human muscle. 3. Determine if the sequelae of MHS mutations can be reduced or prevented by genetic/pharmacological manipulations that decrease sarcolemmal Ca2+ entry, reduce RyR1 leak, increase SR Ca2+ load or scavenge lipid peroxides resulting from ROS production. 4. Using discovery from Core C establish the mechanisms by which newly discovered mutations not in RyR1 or Cav1.1 disrupt the normal bidirectional signaling between RyR1 and the DHPR leading to a common cascade causing MHS and its associated pathology, each using their unique expertise. In this way we assure that the outcome will be that the whole of this Program is greater than the sum of the individual parts.

ABSTRACT There is urgent public health need to better understand the relative risks and benefits associated with consumption of seafood. The overall mission of this project is to understand the toxicity of marine organohalogen pollutants. We take a powerful approach to understanding and mitigating this risk by asking two questions, central to future efforts to predict and minimize risk. Aim 1 of this project asks how these compounds bioaccumulate, focusing on xenobiotic transporters, which are a key pathway for limiting accumulation of foreign chemicals. We will determine the interactions of the four major human xenobiotic transporters (XTs) with environmentally relevant natural and man-made marine organohalogens. The results will extend and expand the scope of our previous work indicating that several of these compounds can act as potent inhibitors of transporter function. In parallel, we will take advantage of recent progress with heterologous transporter-expression and CRISPR/CAS9 gene editing in sea urchins, to dissect the functional role of XTs in governing bioaccumulation in marine cells. This will be supported by a structure guided approach to determine how evolutionary changes in transporter structure modify interactions with TICs, following up on recent progress towards purification and crystallization of marine XTs in complex with pollutants. Aim 2 of this project will determine the structure activity relationships governing neurotoxicity of marine pollutants. These studies are motivated by preliminary data indicating that naturally produced organohalogens are highly potent inhibitors of ryanodine sensitive Ca2+ channels (RyRs) and Ca2+ ATPase transporters (SERCAs), which are arguably the most direct targets of environmentally relevant organohalogens in the brain. We will use primary cultures of hippocampal neurons cultured from male and female wild type mice to determine how activity at these molecular targets alter neuronal network Ca2+ dynamics and morphology using real-time fluorescence cell imaging and morphometric approaches. In addition, we will determine how hippocampal neurons that express mutation RyR1-R163C known to confer heat stress intolerance, alter sensitivity to organohalogens, and ask whether these effects are gender-specific. These studies will address the critical need to better understand the molecular mechanisms by which naturally occurring and man-made seafood pollutants accumulate in target cells and perturb the Ca2+ dynamics essential for normal neuronal network development.

There is an urgent public health need to better understand the relative risks and benefits associated with consumption of seafood. This project will investigate how natural and man-made organohalogen compounds, some of which are known to be toxic, accumulate in marine food webs on the way to human consumption. Researchers at UC San Diego Scripps Institution of Oceanography and UC Davis will investigate the biological mechanisms behind the uptake and concentration of these compounds, and also the mechanisms that make them toxic. The project will support two postdoctoral researchers, thus directly contributing to the development of early career scienctists. The research is jointly supported by NSF and by the National Institute for Environmental Health Sciences (NIEHS).

The project has two aims. Aim 1 seeks to understand cellular mechanisms that control how toxic marine compounds, specifically organohalogens, bioaccumulate, focusing on xenobiotic transporters as the key pathway. The investigators will determine the interactions of the four major human xenobiotic transporters (XTs) with environmentally relevant natural and man-made marine organohalogens. In parallel, they will take advantage of recent progress with heterologous transporter-expression and CRISPR/CAS9 gene editing in sea urchins, to dissect the functional role of XTs in governing bioaccumulation in marine cells. Aim 2 of the project will determine the structure-activity relationships governing neurotoxicity of marine pollutants. The team will use primary cultures of hippocampal neurons cultured from male and female wild type mice to determine how activity at these molecular targets alter neuronal calcium dynamics and morphology using real-time fluorescence cell imaging and morphometric approaches. In addition, they will determine how hippocampal neurons that express a particular mutation alter sensitivity to organohalogens, and ask whether these effects are gender-specific. These studies will address the critical need to better understand the molecular mechanisms by which naturally occurring and man-made seafood pollutants accumulate in target cells and perturb the calcium dynamics essential for normal neuronal network development.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

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.

Project Summary/Abstract A clear need to train and retain veterinary physician scientists is outlined by several National Research Council reports in 2004, 2005 and 2013 and by the National Institutes of Health Physician-Scientist Workforce Report in 2014. Recruitment and retention of highly qualified biomedical scientists, especially clinician-scientists with the DVM degree continues to be one of the most challenging issues facing not only the broader research community, but specifically academic veterinary programs around the country. In response, UC Davis School of Veterinary Medicine initiated the Veterinary Scientist Training Program (VSTP) in 2000 with the first class of VSTP students matriculated in August 2001, which has become the second continuing program in the nation. To date, 30 graduates have completed the VSTP program with dual DVM/PhD. Currently, the VSTP program has 17 students and admitted 3 students to the class entering in 2019. Fourteen of our graduates have gone on to leadership careers in academia, government agencies, and industry. 12 recent graduates are still at the early stage of their post-DVM residency and/or postdoctoral training. Only four graduates (13.3%) have chosen to become small animal practitioners, but one of them has indicated to return to academia. Thus, the percentage of our VSTP graduates who are using their research training is more than 80%. Our mission is to prepare our students with dual DVM-PhD degrees to become compassionate and exceptional veterinarian- scientists engaged in basic and translational research to advance the health of people, animals, and environment. The goal of our VSTP program is to provide an outstanding environment for both clinical and biomedical research training at the nation's top-ranked School of Veterinary Medicine and College of Agriculture and Environmental Sciences along with the University's internationally-recognized strong programs in Biological Sciences, Biomedical Engineering, and Human Medicine. Most of our training faculty participate in established Centers and Institutes that promote collaborations and employ diverse evidence-based approaches to solving scientific problems through state-of-the-art equipment in individual labs and campus shared facilities. Our VSTP program hosts a number of student-centered activities, some of which are jointly organized with our medical school MD/PhD program, to create a unique learning environment in comparative medicine. Our students are an integral part of this dynamic environment and promote the excellence of the program through their research, outreach and student mentoring. Our objectives and intended outcomes for this T32 training grant are to: 1) prepare all of our trainees to become future leaders in academia, government service and public health, 2) provide greater exposure to career paths outside academia, 3) maintain the average time (8 years) to degree, 4) attract and train a diverse group of dual degree students, and 5) increase the number of dual degree trainees in training at UC Davis from 17 to 24.