Grantee Research Project Results

2014 Progress Report: Identification and Scientific Validation of AOPS Involving Genomic and Nongenomic Intracellular Thyroid Hormone Signaling in Neurodevelopment

EPA Grant Number: R835550
Title: Identification and Scientific Validation of AOPS Involving Genomic and Nongenomic Intracellular Thyroid Hormone Signaling in Neurodevelopment
Investigators: Lein, Pamela J , Fritsche, Ellen
Institution: University of California - Davis
Current Institution: University of California - Davis , Leibniz Research Institute for Environmental Medicine
EPA Project Officer: Aja, Hayley
Project Period: October 1, 2013 through September 30, 2016
Project Period Covered by this Report: December 9, 2013 through October 31,2014
Project Amount: $800,000
RFA: Development and Use of Adverse Outcome Pathways that Predict Adverse Developmental Neurotoxicity (2012) RFA Text | Recipients Lists
Research Category: Human Health , Chemical Safety for Sustainability

Objective:

The goal of this project is to identify adverse outcome pathways (AOPs) for chemicals that cause developmental neurotoxicity (DNT) by interfering with thyroid hormone (TH) signaling. Disrupted thyroid hormone (TH) signaling is widely postulated as a mechanism of DNT, but understanding how chemicals that disrupt TH signaling cause adverse neurodevelopmental outcomes has been hindered by significant gaps in our knowledge of which neurodevelopmental processes are sensitive to modulation by TH and the intracellular signaling pathways that mediate TH effects on the developing brain. The objectives of this project are to: (1) identify specific neurodevelopmental processes regulated by TH and determine whether these differ significantly between species; (2) elucidate genomic and/or non-genomic intracellular signaling pathways that mediate TH effects on neurodevelopment; (3) identify 'pathways of toxicity' by which chemicals interfere with TH-mediated neurodevelopment; and (4) link pathways of toxicity with adverse neurodevelopmental outcomes in an in vivo model (zebrafish).

Progress Summary:

Progress on the project is proceeding on schedule and as intended based on the original grant submission and research plans. Aims of the project have not changed from the original application. Preliminary data generated for individual Aims include:

Aim 1: Identify specific neurodevelopmental processes regulated by thyroid hormone (TH) and determine whether these differ significantly between species. Quantitative PCR analyses have been completed to determine basal expression levels of TH signaling components, including TH receptors, deiodinases, TH transporters, co-repressors and co-activators and TH-responsive genes as a function of developmental age in human and rat neurospheres, primary rat neuronal cell cultures and zebrafish. These data indicate developmentally regulated expression patterns of these signaling molecules that differ between species. Analyses of the effect of varying thyroid hormone levels on the expression of these signaling molecules has been initiated. We also have made significant progress in assessing the effects of T4 and T3 on early neurodevelopmental events in neurospheres (cell proliferation, migration, differentiation into neurons versus oligodendrocytes) and on later neurodevelopmental events in primary neuronal cortical and hippocampal cell cultures (axon outgrowth, dendrite outgrowth, glial cell proliferation and synaptogenesis).

Aim 2: Elucidate genomic and/or non-genomic intracellular signaling pathways that mediate TH effects on neurodevelopment. A major output of year 1 was the successful synthesis of the TH receptor antagonist, NH3, which will be necessary for interrogating signaling pathways that mediate TH effects on neurodevelopment. We also have begun to assemble reagents needed to study genomic and non-genomic signaling pathways.

Aim 3: Identify 'pathways of toxicity' by which chemicals interfere with TH-mediated neurodevelopment. We have initiated studies of the effects of the potentially TH disrupting compound brominated flame retardant BDE-99 on TH-regulated gene expression and neurodevelopmental processes in neurospheres and in primary rat cultures.

Aim 4: Link pathways of toxicity with in vivo neurodevelopment and behavior. We have established several transgenic lines of zebrafish (Danio rerio) that have mutations in thyroid hormone production, which will allow us to study the effects of hyper- and hypo-thyroidism on specific neurodevelopmental endpoints as well as express reporter genes for studying oligodendroglial cell proliferation and maturation. We are developing and validating neurobehavioral assays and high content imaging protocols for screening for effects of chemicals on apoptosis.

Future Activities:

In project year 2, we anticipate identifying the neurodevelopmental processes most sensitive to TH signaling and beginning to elucidate the genomic and non-genomic pathways that mediate TH effects on these endpoints. For assessing TH disruption on early neurodevelopment, we will establish a human neurosphere-based screening assay. We also will develop an assay for measuring TH levels in zebrafish, and complete in situ hybridization studies in zebrafish to localize TH signaling molecules, in particular the deiodinases and transport proteins. This will set us up for identifying pathways of toxicity in project year 3 and determining whether effects observed in vitro are replicated in vivo in the zebrafish.

Journal Articles:

No journal articles submitted with this report: View all 22 publications for this project

Supplemental Keywords:

developmental neurotoxicity, neurospheres, primary neuronal cell cultures, thyroid hormone, zebrafish

Progress and Final Reports:

Original Abstract

2015 Progress Report

Final Report

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The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.