Scott M. Belcher - US grants

Like many ligand gated ion channels, glutamate receptors (GluRs) are oligomeric glycoprotein complexes composed of multiple membrane spanning subunits. The functional properties of GluRs are largely determined by the subunits that compose the receptor. For GluRs with the appropriate functional properties to form, there must exist mechanisms to regulate subunit assembly. It seems likely therefore, that GluR subunits contain highly specific interaction and recognition domains that function to regulate subunit assembly and possibly subunit stoichiometry of the receptor. The experiments proposed with in this application are aimed at identifying the association and assembly domains of both the NMDA and non-NMDA types of GluR subunits. Subunits of the nicotinic acetylcholine receptor, the inhibitory glycine and gamma-aminobutyric acid receptors contain structural domains that determine subunit stoichiometry and that are necessary for assembly of the receptor. For the subunits composing those channels, the "association domains" are located within the extracellular amino-terminus of the subunit polypeptides. The overall goal of the research proposed in this application is to identify structural domains of NMDA and non-NMDA GluR subunits that interact and that are required for the accurate assembly of functional homomeric and heteromeric receptors. Once assembly domains for the NMDA and AMPA receptor subunits are defined, assembly domain peptides will be tested for their ability to be neuroprotective during periods of EAA activated neuronal death in cultured cerebellar granule cells, a well established model system to study EAA induced neurotoxicity. The neuroprotective properties of both extracellular and intracellularly delivered GluR association domain peptides will be determined. The intracellular delivery of peptides will be facilitated by linking the association domain peptides to a 16 amino acid peptide from the third helix of the antennapedia homeodomain that is rapidly internalized by neurons. The identification of neuroprotective association domain peptides that inhibit excitotoxicity, may be the first steps toward the development of a new class of clinically important neuroprotective agents that target individual subtypes of GluRs.

DESCRIPTION (provided by applicant): The broad long-term overall goal of the proposed research is to understand the physiological role of estrogen (E2) and estrogen receptors (ERs) during neuronal development and to determine to what extent environmental estrogens (EEs) can mimic or inhibit the effects of E2 in these developing neurons. Mechanisms of rapid E2-mediated signal transduction are not well understood. These actions vary greatly from cell type to cell type, and may even vary in the same cell. In addition to reproductive tissues &breast cancers, cells of the nervous system are a clinically very important E2 targets. Environmental estrogens, also known as endocrine disrupting chemicals, are a diverse group of compounds that can mimic or antagonize the normal actions of E2. The extent to which EEs impact the developing nervous system is unclear. Proposed experiments using primary cultures of estrogen-sensitive neonatal rat cerebellar neurons and the developing cerebellum as non-sexually related neuronal models, will address the following Specific Aims - Specific Aim 1 is to determine whether or not structurally diverse EEs modulate ERK1/2 signaling in developing cerebellar neurons. It is hypothesized that in E2-responsive neurons, EEs rapidly modulate ERK1/2 signaling and neuronal physiology in an E2-like fashion, and that differences in the chemical structure of individual EEs determine their ability to activate ERK-signaling &/or antagonize the rapid actions of E2. Specific Aim 2 is to determine the nature of the signaling mechanism underlying E2-mediated rapid activation of ERK-signaling. It is hypothesized that a plasma membrane, localized version of ERp is acting as the mediator of rapid E2/EE-induced ERKsignaling. Membrane E2-binding proteins from primary granule cell and hippocampal neuronal cultures will be affinity purified and identified through LC-tandem mass spectrometry. Immunocytochemical and loss-of-function experiments are proposed to demonstrate the function of identified candidate membrane ERs. The ability of ERa, ERp, and the orphan G-protein coupled receptor GPR30 to act as the membrane ER will be directly addressed.

DESCRIPTION (provided by applicant): Despite increasing recognition that BPA has harmful effects on the reproductive, nervous and immune systems. While higher urine BPA concentrations are associated with cardiovascular disease in humans, the effect(s) of BPA at environmentally relevant concentrations on the heart is unknown. The aim of the proposed studies is to elucidate the in vivo sex-specific impacts that BPA has on cardiac physiology, to understand the underlying modes and mechanisms of action, and ultimately improve heart health through understanding the impact of pathological actions of exposure to BPA as a protypical environmental EDCs. The central hypothesis of the proposed studies is during development BPA negatively impacts cardiac function which may manifest later in life. It is therefore hypothesized that the harmful effects of BPA exposure will impact Ca++ handling, and that developmental exposures may alter the normal sex-specific actions of estrogen in the heart, and negatively impact heart health by increasing arrhythmias, altering hemodynamic function, and increasing cardiac dysfunction in response to physiologic conditions of stress (e.g. catecholamine and pressure overload induced hypertrophy/heart failure). As its primary endpoints, the proposed studies will focus on understanding the cardiac-specific actions of BPA. However, the proposal is far more crosscutting, and not limited to a single organ or system-based endpoint. Along with the cardiac specific end-points, a number of physiological and reproductive endpoints "classically" used to assess estrogenic EDC activity will be characterized. This will allow direct comparison of results obtained in the "estrogen-sensitive" C57BL/6J strain with studies performed in the "insensitive" CD1 (Swiss) mouse. Detailed physiological phenotype data will be collected for all animals and at termination of the study complete necropsy will be performed with tissues from all organs harvested, collected and prepared for histological studies with paired samples preserved for future molecular, epigenetic, and "omics" analysis. As a result of the proposed studies a defining framework and knowledge base necessary to standardize future studies that employee C57BL/6J derived genetic mouse models will be created, and allow systematic investigation of the mechanisms of action of BPA using knockout and transgenic models. PUBLIC HEALTH RELEVANCE: Endocrine disrupting chemicals acting on the estrogen system: estrogenic endocrine-disrupting chemicals (EDCs) are a structurally diverse group of compounds that mimic or antagonize the effects of endogenous estrogens. Bisphenol A (BPA) is an EDC used extensively in the production of consumer products such as polycarbonate plastics, food cans and plastic packaging, dental sealants and water pipes. Analysis of BPA in urine samples showed that BPA is present essentially all of the US population at levels demonstrated to harm negative effects on the reproductive function and have harmful impacts on cancer. Despite increasing recognition that BPA has harmful effects on the reproductive, nervous and immune systems, the effect(s) of BPA at environmentally relevant concentrations on the heart is unknown. Higher urine BPA concentrations are associated with cardiovascular disease in humans. In contrast to the perception that "female hormones" protect women from CV disease, disease of the cardiovascular system is the leading cause of mortality for women in the US. Since 1984, more women than men have died of CV disease every year in the US;fifty-three percent of total CV disease deaths occur in women. Women, especially prior to menopause, have lower rates of coronary heart disease and myocardial infarction (MI). However, women have a much worse prognosis following MI. The overall post-MI mortality rate is higher for women. For peri-menopausal women (<55 years) the post-MI mortality rate is more than double that of same aged men. A significant contributing factor to the worse prognosis following MI for women is arrhythmia. Women have more ventricular fibrillation-complications following MI, and a much higher arrhythmic death rate than men during the first 6 months post-MI Women also have unique sensitivities to arrhythmias. For example, women are at increased risk of arrhythmia during pregnancy coincident with increasing levels of estrogens. To address the role of estrogens in conferring unique benefits or risks associated with CV disease of women, it is critical to understand the differences between the male and female CV system and how endogenous "female hormones" influence cardiac function. Understanding the unique protective or harmful actions of endogenous and environmental estrogens will result in new strategies to decrease the incidence of CV disease and increase survival of both men and women with CV disease. The proposed studies the proposed studies focus on understanding the cardiac-specific actions of BPA and are of very broad and extremely high impact. In fact, the results of these studies could impact understanding related to cardiovascular health of nearly 100% of the US population.

DESCRIPTION (provided by applicant): Bisphenol A (BPA) is an endocrine disruptor and high volume production chemical used extensively in a wide- variety of consumer products and food packaging. Monomeric BPA is a pervasive pollutant to which there is wide-spread human exposure. Numerous studies have demonstrated that BPA may have effects on health and disease. However, there remain numerous data gaps and conflicting results that have fueled controversy and concern about the toxic threat of BPA. Our recently published studies and preliminary data presented in this application, establish that environmentally relevant concentrations of BPA are directly harmful to cardiac function in both mice and rats. There are currently no data available directly assessing impacts on cardiac specific endpoints from large GLP compliant studies of BPA toxicity. As a result, there is a pressing need to consider cardiac specific endpoints for establishing the health risks of BPA exposure. In 2010, a consortium-based collaboration between the NIEHS/National Toxicology Program (NTP) and the FDA was established the Consortium Linking Academic and Regulatory Insights on BPA Toxicity (CLARITY- BPA) to perform a comprehensive GLP-compliant 2 year chronic exposure study of the toxicity of BPA augmented by inclusion of hypothesis driven and disease-specific apical endpoints not typically assessed in standard toxicological assessment. Currently, the CLARITY-BPA study design lacks endpoints specifically related to cardiovascular effects resulting from BPA exposure. This is a major and critical data gap. If awarded, the proposed studies will eliminate the critical absence of cardiac specific endpoints from this comprehensive GLP-compliant assessment of BPA toxicity. In so doing, the results from the proposed analysis will: 1) fill a critical data gap that would otherwise remain unaddressed; and 2) contribute critical information that would otherwise not be available for the risk assessment and regulatory process. The Specific AIM of the studies proposed in this application is to add cardiac specific end points assessing the impact of BPA on cardiac histopathology to the comprehensive GLP-compliant 2-year CLARITY-BPA chronic exposure study investigating the toxicity of BPA. That Specific Aim will be addressed by ensuring proper isolation and utilization of cardiac tissue from the GLP compliant CLARITY-BPA 2-year chronic study of oral BPA toxicity for histological and cellular morphometric approaches and to assess hypertrophy, remodeling and fibrosis of hearts from each study group. From hearts of each sex, LV wall thickness; myocyte size and volume, and degree of fibrosis will be assessed and compared to controls. The addition of cardiac specific endpoints to the on-going GLP-compliant 2-year CLARITY-BPA toxicity study will add critical information that would otherwise not be available for the risk assessment and regulatory process. Including analysis of the proposed cardiac endpoints in the CLARITY-BPA study will afford a more informed regulatory decision-making process that will result in improved global human health.

Obesity rates in the US have reached epidemic proportions. Although lifestyle factors are clearly primary contributors, fetal metabolic reprogramming by environmental chemicals collectively called ?obesogens? has been hypothesized to exacerbate obesity risk. Data collaboratively generated by the three co-PIs have identified the newly introduced fire retardant mixture Firemaster® 550 (FM 550) as an emerging contaminant in US homes and that (in rats) perinatal exposure to FM 550 results in obesity, and hallmarks of metabolic syndrome including altered exploratory behaviors, disrupted glucose sensitivity and cardiac hypertrophy. There is pressing need to assess the toxicity of FM 550 because it is at least the second most common fire retardant used in residential furniture and baby products with ubiquitous exposure in the US, yet its potential toxicological effects are not well characterized. Working as an interdisciplinary, multi- PI team, comprising an environmental chemist, a neuroendocrinologist and an endocrine pharmacologist, we submitted an R01 application to test the hypothesis that FM 550 is an obesogen, and perinatal exposure induces hallmarks of metabolic syndrome (e.g. hypertension, type-2 diabetes and cardiovascular disease) via metabolic reprograming. To strengthen the R01 application and generate additional data required to address reviewer concerns, work in this R56 application will (1) characterize the toxicokinetics of FM 550 in female rats (pregnant and non- pregnant) to specifically assess the potential for fetal transfer; and (2) characterize the hallmarks of metabolic disrupting and behavioral effects of FM550 (one dose) in exposed offspring of both sexes. Importantly, whether or not the increase in body weight (identified as sexually dimorphic in the parent R01) observed in our pilot study is accompanied by increased food intake and/or changes in overall activity will be determined. These are key markers of metabolic reprogramming and will thus help address the central hypothesis of the parent R01 that FM550 is an ?obesogen? and can predispose exposed offspring to metabolic disease. Examining phenotypic sex differences is a fundamental feature of this application, and the data will ultimately be used to inform future work (and a specific aim of the R01 application) exploring the sex specific mechanisms underlying adipogenesis and the metabolic disrupting activity of FM 550, its components, and primary metabolites. Understanding the contributions of each FM 550 component to an obesogenic phenotype is critical because some have large volume applications as plasticizers in a wide variety of consumer products (e.g. polyvinyl chloride (PVC), circuit boards, hydraulic fluids, adhesives, nail polish). Collectively the proposed studies will contribute to our long term efforts to secure R01 funding for this work but also provide new knowledge required for evaluating potential human health effects of developmental FM 550 exposures including: fate and transport in tissues; obesogenic potential of the mixture and its individual components across a wide dose range; sex-specific mechanism of action; and the possible long term metabolic health consequences of early life exposure in both sexes.