2003 Progress Report: Environmental Factors in the Etiology of Autism; Molecular and Cellular Mechanisms of Autism

EPA Grant Number: R829388C006
Subproject: this is subproject number 006 , established and managed by the Center Director under grant R829388
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: CECEHDPR - University of California at Davis Center for the Study of Environmental Factors in the Etiology of Autism
Center Director: Pessah, Isaac N.
Title: Environmental Factors in the Etiology of Autism; Molecular and Cellular Mechanisms of Autism
Investigators: Pessah, Isaac N. , Gershwin, M. Eric , Goth, Samuel R. , Matsumura, Fumio , Van de Water, Judith
Current Investigators: Pessah, Isaac N. , Goth, Samuel R. , Van de Water, Judith
Institution: University of California - Davis
EPA Project Officer: Hahn, Intaek
Project Period: September 30, 2001 through September 29, 2002
Project Period Covered by this Report: September 30, 2002 through September 29, 2003
RFA: Centers for Children's Environmental Health and Disease Prevention Research (2001) RFA Text |  Recipients Lists
Research Category: Health , Children's Health , Health Effects

Objective:

How thimerosal and polybrominated diphenylethers, major environmental concerns to childhood neurodevelopment, influences peripheral immune cells in the B6 murine model has been a primary focus in year 1. Work has begun on assessing the influence of these xenobiotics on the growth and development of glial/hippocampal neurons in culture. The long-term goal is to define common mechanisms in immune and neural cells responsible for developmental toxicity of these environmental agents. Our specific aims are: 1)To define differences in the response of peripheral immune cells (PMBCs) isolated from autistic and non-autistic children in their sensitivity and/or pattern of cell activation and cytokine secretion when challenged in vitro with vaccine antigens, and xenobiotics of environmental relevance (thimerosal, PCBs, and BDEs), singly and in combination.2) Elucidate the mechanisms by which sub-toxic concentrations of (thimerosal, PCBs, and BDEs), singly and in combination, alter growth and activation properties of immune cells from rodents.3) Elucidate the mechanisms by which sub-toxic concentrations of (thimerosal, PCBs, and BDEs), singly and in combination, influence glia/neural cell interactions leading to altered patterns of dendritic spine growth, dendritic branching and synaptogenesis in hippocampal cells from rodents.

Progress Summary:

Aim I. In collaboration with Core II, we have begun to analyze blood samples from the CHARGE cohort. Two types of measurements are being made. First we are measuring the titer of vaccine antibodies such as MMR. Second we are culturing PBMCs to determine how they respond when challenged with vaccine antigens in the presence and absence of thimerosal and/or PCBs as originally described in our proposal. As would be expected, we are in the data acquisition phase of this case control epidemiological study so no interim summary is available at this time. However, in conjunction with Core II, we have made significant progress in identifying significant differences in autoantibody profiles in the serum of autistic children compared to age-matched controls (see Core II report).

Aim II. Immunotoxicity of thimerosal to murine dendritic cells in culture.

We have focused our initial studies on the immunotoxicology of thimerosal toward dendritic cells. We felt this was warranted since dendritic cells (DCs) reside in most peripheral tissues, particularly at sites of interface with the environment (e.g., skin and mucosae). Although DC make up <1% of the total immune cell number circulating in the blood, they contribute a “frontline” towards engaging immune defenses by taking up antigens in peripheral tissues, process them into peptides, and load these peptides onto major histocompatibility complex (MHC) class I and II molecules. DCs then migrate to secondary lymphoid organs and become competent to present antigens to T lymphocytes, thus initiating antigen-specific immune responses, or immunological tolerance. Antigen presentation in DCs is finely regulated: antigen acquisition, peptide/MHC complex expression, expression of costimulatory and adhesion molecules, secretion of cytokines and other small molecules, and migration to lymphocyte-rich lymph nodes in DCs is different compared to other professional antigen-presenting cells. These specializations and others account for these cells’ unique role in the initiation of primary immune responses and for the induction of tolerance, and provide a strong rationale for examining the actions of our test xenobiotics on DC maturation and function. Last year we reported successful isolation and characterization of DCs from murine bone marrow.

Since then we have discovered that DCs, especially immature DCs are extremely sensitive to thimerosal toxicity. Acute (20 hr) exposure to 1 μM thimerosal produces significant cytotoxicity in DCs as measured by decreased cell size and increased propidium iodide (PI) staining. In this respect, the potency of thimerosal is significantly higher than that seen with thiosalicylate, the major metabolic product. An unexpected and significant finding is revealed by the dose-response relationships comparing the percent of PI positive DCs exposed to thimerosal or ethylmercuric chloride which reveals that the former is significantly more acutely toxic (Fig 1). These results also indicate that immature DCs are more susceptible to organic mercury toxicity. Importantly acute exposure to lower concentrations (100nM to 1 μM) revealed induction of significant apoptosis as measured by increases in TUNEL staining, activated caspases, and externalization of annexin V. Thimerosal in the lower dose range appeared to be a slightly more potent pro-apoptotic agent than ethyl mercuric chloride, whereas thiosalicylate was inactive.

In collaboration with Dr. Peter Green, a new Center participant (Core I) with expertise in metal analysis, we have found that immature and mature DCs accumulate significant amounts of mercury during a 20 hr exposure to thimerosal. Specifically immature DCs accumulate 0.67% of the total mercury available in medium containing 100nM thimerosal compared to mature DCs that accumulate 0.47%. These results indicate that an underlying reason for the high susceptibility of DCs to thimerosal is their ability to actively accumulate mercury. We are determining if there are significant differences between thimerosal and ethylmercuric chloride in accumulation of mercury by DCs. These results provide impetus to examine the how thimerosal and ethylmercury alter the ability of DCs to activate T-cells. We have begun to examined how exposure of DCs to these agents alter the response of allogeneic CD4 + T-cells and ovalbumin specific CD8 + T-cells.

One of the major biochemical targets by which thimerosal disrupts cellular Ca2+ signaling is through its interactions with ryanodine-sensitive Ca2+ channel complexes (RyRs), Ca2+ release channels of the microsomal membrane (1). Recent studies have shown mRNA for RyR1 (the skeletal isoform) expressed in immature DCs, although the contribution of RyR1 protein to DC function unknown. We have successfully shown that RyR1 protein is expressed in immature DCs (Fig 3). Moreover we have taken advantage of our RyR1 knockout mice (2) and successfully cultured RyR1-null DCs from fetal liver. We are currently defining how RyR1 null DCs differ in cell surface expression of leukocyte markers and cellular functions compared to wildtype DCs generated from fetal liver. We expect some differences to arise in cellular function as calcium influx accompanies DC mediated IL-12 secretion and apoptotic body phagocytosis. Next we will determine if RyR1-null DCs exhibit altered sensitivity to thimerosal and ethylmercuric chloride. Our goal in year three is to define the mechanisms responsible for the high susceptibility of DCs that lead to apoptosis and their consequences on immune function (e.g. changes in T-cell activation).

Aim III Dynamic regulation of RyRs by Homer

Homer proteins form an adapter system that regulates coupling of group 1 metabotropic glutamate receptors (mGluR) with intracellular inositol trisphosphate receptors (IP 3R), and is modified by neuronal activity. We have shown that Homer proteins also physically associate with ryanodine receptors type 1 (RyR1) and regulate gating responses to Ca2+, depolarization, and caffeine. In contrast to the prevailing notion of Homer function, Homer1c (long form) and Homer1-EVH1 (short form) evoke similar changes in RyR activity. The EVH1 domain mediates these actions of Homer, and is selectively blocked by a peptide that mimics the Homer ligand. 1B5 dyspedic myotubes expressing RyR1 with a point mutation of a putative Homer-binding domain, exhibit significantly reduced (~33%) amplitude in their responses to K + depolarization compared to cells expressing wild-type protein. These results reveal that in addition to its known role as an adapter protein, Homer is a direct modulator of Ca2+ release gain. Homer is the first example of an “adapter” that also modifies signaling properties of its target protein. This study reveals a novel mechanism by which Homer directly modulates the function of its target protein RyR1 and E-C coupling in skeletal myotubes. This form of regulation may be important in other cell types that express Homer and RyR1. Our findings are significant since they are the first to show that both long and short forms of Homer proteins have modulatory activity toward RyR1 and physiological Ca2+ signaling processes that are mediated through their EVH1 domain. Our plans are to elucidate the possible involvement of Homer/RyR complexes in the toxicity of thimerosal, and persistent halogenated organics as outlined in our original proposal.

Development of HSV Helper-free virions for real-time tracking pyramidal neuron growth

We have adapted the use of an HSV virion amplicon containing enhanced FGP cDNA for use with neurons in culture. We have found that we can efficiently transduced pyramidal neurons in culture without toxicity. GFP expression is stable for at least 72 hr post transduction. Figure 4 show a confocal image of a viable pyramidal neuron visualizing expression GFP not only in the soma, but also throughout the terminal dendrites. This new technique will be use to track not only growth parameters in our experimental models, but also provides a non-toxic way of expressing fluorescently tagged synaptic proteins to identify changes in synaptic structure and function produced by the toxics we are studying. Non-coplanar PCBs induce apoptosis by selective activation of ryanodine receptors in cultured pyramidal neurons from rat hippocampus

In collaboration with Dr. Pam Lein (John’s Hopkins) we have found that non-coplanar PCB 47, but not coplanar PCB77, significantly increased DNA fragmentation in hippocampal but not cortical primary neuronal cultures. This effect was blocked by the caspase inhibitors z-VAD-fmk and DEVD-CHO, indicating a Ca2+-dependent mechanism. We further elucidated the underlying molecular target(s) for Ca2+ deregulation. Pharmacological inhibitors known to block a variety plasmalemmal and ER/SR channels, failed to prevent PCB47-induced DNA damage. However treatment of neurons with FLA 365, a selective blocker of ryanodine receptors (RyR) completely prevented DNA damage. These data indicate that non-coplanar PCBs induce apoptosis in hippocampal neurons subsequent to RyR activation and increased reactive oxygen species, and suggest that altered regional profiles of apoptosis may be an important mechanism underlying the developmental neurotoxicity of PCBs.

Noncoplanar PCB 95 Amplifies Ryanodine Receptor Signaling and Ca2+ Entry Mediated by Ionotrophic Glutamate Receptors in Cerebellar Granule NeuronsWe have submitted a manuscript to Molecular Pharmacology (in appendix) that reports results from a study that explored in detail how PCB95 alters excitatory signaling intact cerebellar granule neurons. PCB 95 significantly increased the number of neurons responding to caffeine. PCB 95 sensitization of RyR-mediated responses was further supported by the observations that ryanodine pretreatment blocked response to caffeine and coplanar 2,4,4’,5-tetrachlorobiphenyl (PCB 66), which lacks RyR activity, failed to sensitize neurons. PCB 95 did not significantly alter levels of resting cytosolic Ca2+ nor thapsigargin-sensitive Ca2+ stores, suggesting a more complex mechanism than sensitization from increased cytosolic Ca2+ or an increased endoplasmic reticulum/cytosolic Ca2+ gradient. The immunosuppressant, rapamycin, sensitized neurons to caffeine in an additive and saturable manner with PCB95, suggesting a common mechanism. PCB 95 or rapamycin significantly enhanced Ca2+ responses following N-methyl-D-aspartate (NMDA) and a -amino-3-hydroxy-5-methyl-4-isoxasolepropiate (AMPA) receptor activation. Store depletion or direct blockade of RyR did not affect PCB 95-enhanced responses to NMDA. These results suggest that PCB 95 and rapamycin may enhance NMDA and AMPA Ca2+ signals by modifying a functional association of the FKBP12/RyR complex with a plasma membrane Ca2+ entry channel and identifies a novel mechanism by which these compounds amplify Ca2+ signals in intact neurons.

Future Activities:

We will define the mechanism by which thimerosal, PCBs and BDEs alter the function of dendritic cells and their ability to activate T cells. The neurotoxicity of these agent are being explored in primary neuronal/glial cultures and hippocampal slices. In year two, work will begin defining the responses of PMBCs collected from the CHARGE study.


Journal Articles on this Report : 3 Displayed | Download in RIS Format

Other subproject views: All 23 publications 21 publications in selected types All 21 journal articles
Other center views: All 146 publications 134 publications in selected types All 133 journal articles
Type Citation Sub Project Document Sources
Journal Article Chen L, Esteve E, Sabatier J-M, Ronjat M, De Waard M, Allen PD, Pessah IN. Maurocalcine and peptide A stabilize distinct subconductance states of ryanodine receptor type 1, revealing a proportional gating mechanism. Journal of Biological Chemistry 2003;278(18):16095-16106. R829388 (2006)
R829388 (Final)
R829388C006 (2003)
R829388C006 (2005)
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  • Journal Article Feng W, Tu J, Yang T, Vernon PS, Allen PD, Worley PF, Pessah IN. Homer regulates gain of ryanodine receptor type 1 channel complex. Journal of Biological Chemistry 2002;277(47):44722-44730. R829388 (2006)
    R829388 (Final)
    R829388C006 (2003)
    R829388C006 (2005)
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  • Journal Article Howard AS, Fitzpatrick R, Pessah I, Kostyniak P, Lein PJ. Polychlorinated biphenyls induce caspase-dependent cell death in cultured embryonic rat hippocampal but not cortical neurons via activation of the ryanodine receptor. Toxicology and Applied Pharmacology 2003;190(1):72-86. R829388 (2006)
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    R829388C006 (2003)
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  • Supplemental Keywords:

    Autism, thimerosal, halogenated organics, cell signaling, immunotoxicology, neurotoxicology,, RFA, Health, Scientific Discipline, PHYSICAL ASPECTS, ENVIRONMENTAL MANAGEMENT, Health Risk Assessment, Chemistry, Risk Assessments, Susceptibility/Sensitive Population/Genetic Susceptibility, Disease & Cumulative Effects, Physical Processes, Children's Health, genetic susceptability, Biology, Risk Assessment, chemical exposure, neurotoxic, xenobiotics, biomarkers, neurodevelopment, gene-environment interaction, pesticides, exposure, halogenated aromatics, neurobehavioral, neurodevelopmental, neurotoxicity, children, susceptibility, etiology, neurobehavioral effects, autism, mechanisms, biological markers, exposure assessment, neurological development, biomarker, synergistic interactions

    Progress and Final Reports:

    Original Abstract
  • Final

  • Main Center Abstract and Reports:

    R829388    CECEHDPR - University of California at Davis Center for the Study of Environmental Factors in the Etiology of Autism

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R829388C001 Environmental Factors in the Etiology of Autism; Analytic Biomakers (xenobiotic) Core
    R829388C002 Environmental Factors in the Etiology of Autism; Cell Activation/Signaling Core
    R829388C003 Environmental Factors in the Etiology of Autism; Molecular Biomakers Core
    R829388C004 Environmental Factors in the Etiology of Autism; Childhood Autism Risks from Genetics and the Environment (The CHARGE Study)
    R829388C005 Environmental Factors in the Etiology of Autism; Animal Models of Autism
    R829388C006 Environmental Factors in the Etiology of Autism; Molecular and Cellular Mechanisms of Autism