Final 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 , Leibniz Research Institute for Environmental Medicine
EPA Project Officer: Lasat, Mitch
Project Period: October 1, 2013 through September 30, 2016
Project Amount: $800,000
RFA: Development and Use of Adverse Outcome Pathways that Predict Adverse Developmental Neurotoxicity (2012) RFA Text |  Recipients Lists
Research Category: Health , Human Health , Safer Chemicals

Objective:

The goal of this project was to identify adverse outcome pathways (AOPs) for chemicals that cause developmental neurotoxicity (DNT) by interfering with thyroid hormone (TH) signaling. Disrupted 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 specific neurodevelopmental processes that are sensitive to modulation by TH and the intracellular signaling pathways that mediate TH effects on the developing brain. The objectives of this project were to: (1) identify specific neurodevelopmental processes regulated by thyroid hormone (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 in vivo neurodevelopment and behavior.

Summary/Accomplishments (Outputs/Outcomes):

(1) Using quantitative PCR analysis, we demonstrated that human and rat neurospheres, primary rat neuronal cell cultures and zebrafish all express key TH signaling components, including TH receptors, deiodinases, TH transporters, co-repressors and co-activators and TH-responsive genes, and that expression levels varied as a function of developmental age. Analyses of the effect of exogenous T4 and T3 on the expression of these signaling components identified hairless, deiodinase 3, myelin basic protein (in human), and myelin-associated oligodendrocyte basic protein (in rat) as TH responsive genes in human and rat neurospheres but not in primary rat cortical cultures. In zebrafish, expression of deiodinase 3, thyroid hormone receptor β, and the TH transporter Mct8 were induced by increasing T4 and T3 levels, whereas expression of the TH transporters Lat1 and Lat2 as well as the thyroid stimulating hormone β subunit were downregulated following treatment with T4 and T3. Collectively, our findings suggest that TH signaling components are robustly expressed in the developing nervous system of multiple species at multiple stages of neurodevelopment; however, there are key species- and developmental stage-specific differences in both basal expression of TH signaling molecules, and the effects of altered levels of TH on gene expression profiles. Thus, model selection will be critical in setting up screens for TH disrupting chemicals.

(2) TH directly influences a restricted subset of specific neurodevelopmental processes. We analyzed the effects of varying concentrations of T4 and T3, and conversely, the removal of TH and pharmacological blockade of TR signaling, on early neurodevelopmental events in neurospheres (neural progenitor cell (NPC) proliferation, migration, differentiation into neurons and oligodendrocytes), and on later neurodevelopmental events in primary rat cortical and hippocampal cell cultures (axon outgrowth, dendrite outgrowth, glial cell proliferation and synaptogenesis). Of these various neurodevelopmental endpoints expressed in these different model systems, TH signaling influenced only NPC proliferation and oligodendrocyte differentiation/maturation in the neurosphere models. The later stages of neurodevelopment modeled in primary rat cortical and hippocampal neurons were not altered by TH. The thyroid hormone receptor (TR) antagonist NH-3 was observed to antagonize TH-induced gene expression of the TRa-regulated genes hairless and deiodinase3 in differentiated human and rat NPC demonstrating functionality of genomic TH signaling in primary NPC derived from these species. In analogy, NPC prepared from TRa knockout mice lacks most of the T3-induced hairless and deiodinase3 gene expression changes. NH-3 also interferes with T3-induced oligodendrocyte maturation in human NPCs, suggesting the involvement of canonical TR-dependent genomic signaling pathways in human oligodendrocyte maturation. In rat NPC, NH-3 antagonizes the T3-dependent oligodendrocyte differentiation suggesting TR involvement in this process. In mice, TH does not only stimulate oligodendrocyte maturation, but also induces their formation. Using NPCs prepared from TR knockout mice, it was shown that in mice TRα, but not TRβ, mediates this T3-dependent oligodendrocyte formation and maturation. To elucidate the involvement of non-genomic pathways in human and rat oligodendrogenesis, we tested the effects of the PI3K inhibitor LY294002 and the MAP kinase inhibitor U0126 in NPC differentiated for 5 days. None of these inhibitors altered TH-dependent oligodendrogenesis in either species. Yet, the PI3K and the MAPK pathways are involved in TH-independent human, but not rat, oligodendrogenesis. In addition to glia cell differentiation, we studied the effects of NH-3 (canonical TH signaling), PI3K and MAPK (non-genomic TH signaling) inhibition on T4-reduced proliferation of human NPCs. The TR antagonist NH-3 by itself did not affect hNPC proliferation. Co-treatment of cultures with T3 or T4 and NH-3 did not change the TH-induced reduction of hNPC proliferation showing that the canonical TH signaling pathway does not mediate TH-reduced hNPC proliferation. Inhibition of the PI3K or MAPK pathway reduced hNPC proliferation without effects on cell viability. The co-exposure of T3 or T4 with LY294002 or U0126 did not show an additive effect on proliferation inhibition. These data suggest that non-genomic signaling via PI3K and MAPK mediate TH-dependent hNPC proliferation inhibition.

(3) We developed an "Oligodendrocyte Maturation Assay" to identify human- and rodent-specific neural TH signaling disruptors. With this assay, we study oligodendrocyte maturation by forming the ratio of hMBP, or rodent mog or mobp gene expression/% differentiated O4+ cells. This procedure is necessary because NPC differentiate into different cell types simultaneously and when the number of differentiated oligodendrocytes change, oligodendrocyte-specific gene expression will also change automatically due to change in cell number. This ratio is called QM for "maturation quotient." We found that the flame retardant BDE-99 interfered with mouse oligodendrocyte formation independent of TH signaling and TH-induced oligodendrocyte maturation (reduction of QM) possibly due to TH signaling disruption. BDE-99 inhibited human oligodendrocyte formation independent of TH interaction by an unknown mode-of-action, while it did not affect T3-dependent human oligodendrocyte maturation (unaltered QM). BDE-99 did not reduce TH-induced hairless and deiodinase3 gene expression in either species also pointing to a TR-independent mode-of-action. This is supported by the finding that BDE-99 affected oligodendrocyte differentiation in all mouse NPC genotypes: in the wildtype as well as in the TRa and TRb knockout NPC. The putative TH disruptors perfluorooctanoic acid (PFOA) and tetrabromobisphenol A (TBBPA) were also studied for their abilities to interfere with TR-dependent gene expression and with TH-dependent oligodendrocyte maturation in the "Oligodendrocyte Maturation Assay." PFOA did not inhibit T3-induced human or rat Hairless or Deiodinase3 gene expression and showed no effects on oligodendrocyte differentiation or QM. Thus, we did not identify PFOA as a TH signaling disruptor. In contrast, TBBPA reduces T3- induced QM concentration-dependently and significantly (LOAEC 0.5 µM, 100% reduction of T3-induced QM at 2 µM). At the same time, it significantly reduces T3-induced Hairless and Deiodinase3 gene expression suggesting interference with TRa-dependent T3 signaling. In rat NPC, TBBPA does not reduce T3-induced hairless or deiodinase3 gene expression pointing to species-specific interference with TH signaling.

(4) To link pathways of toxicity with in vivo neurodevelopment, we characterized a phenotype for developmental hyperthyroidism in larval zebrafish. Our initial assessment focused on the first 5 days of development in zebrafish, which encompasses the range of neurodevelopmental processes investigated our in vitro models. Zebrafish treated with exogenous T4 or T3 were evaluated for teratology, mortality, changes in expression of TH signaling genes, and influence on photomotor behavior. Both T4 and T3 treatments caused behavioral changes, measured as a difference in swimming distance in response to alternating light and dark conditions at concentrations and time points that did not cause significant mortality or gross malformations. These TH effects were blocked by treatment with the TR antagonist NH-3, indicating that T4 and T3 effects on photomotor behavior are mediated by nuclear thyroid hormone receptors. To establish a developmental hypothyroid phenotype, we obtained a conditional thyroid ablation zebrafish transgenic line Tg(tg:nVenus-2a-nfnB)wp.rt8 (provided by Parichy lab, University of Washington), which allowed us to generate TH-deficient zebrafish embryos. We developed an analytical method to quantify T3 and T4 in zebrafish to confirm thyroid insufficiency in these transgenic zebrafish. Using these thyroid ablation fish, we showed that developmental hypothyroidism also interferes with photomotor behavior independent of teratological effects.

(5) To follow up on results obtained in NPCs showing that oligodendrocyte formation and maturation are influenced by altered TH levels and by chemical TH disruptors, we developed methods to assess oligodendrocyte development in zebrafish. Transgenic zebrafish expressing fluorescent markers for oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes, Tg(Mbp:GFP;Sox10;TagRFP), were obtained from the Appel Lab (University of Colorado Denver) to investigate the influence of developmental hyperthyroidism and hypothyroidism on oligodendrocyte development in vivo. Our findings demonstrate that developmental hyperthyroidism increases the number of mature oligodendrocytes in the spinal cord and increases the number of oligodendrocyte precursor cells and mature oligodendrocytes in the brain at 5 days post fertilization. These changes in oligodendrocyte number and maturation coincide with TH-dependent changes in photomotor behavior. We are currently assessing the effects of hypothyroidism on oligodendrocyte maturation in zebrafish and setting up a screen of known thyroid hormone disrupting chemicals to determine whether these chemicals interfere with oligodendrocyte maturation in larval zebrafish at concentrations that do not cause teratological effects. Data from the latter study will be compared to concentrations at which these chemicals interfere with behavioral readouts in zebrafish.

Conclusions:

Collectively, the findings from this project suggest that, contrary to expectation based on well-documented effects of hypothyroidism on neurodevelopment in humans and experimental animal models, when tested in in vitro models of neurodevelopment, thyroid hormone influences a very limited number of neurodevelopmental processes, specifically NPC proliferation, and oligodendrocyte maturation. Importantly, the TH-sensitive endpoints identified in human and rodent neurospheres, were confirmed in an in vivo model, the larval zebrafish. Data from the latter suggest that TH-dependent changes in oligodendrocyte proliferation and maturation are linked to changes in swimming behavior. Data from both the neurosphere models and the zebrafish model further suggest that TH effects on oligodendrocyte proliferation and maturation are mediated, at least in part, by canonical thyroid hormone-mediated genomic mechanisms. These data have significant implications regarding adverse outcome pathways for TH-dependent DNT, and provide critical insights regarding appropriate model systems to adapt as screening platforms for TH disrupting chemicals with DNT potential.


Journal Articles on this Report : 5 Displayed | Download in RIS Format

Other project views: All 19 publications 5 publications in selected types All 5 journal articles
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Journal Article Chen H, Streifel KM, Singh V, Yang D, Mangini L, Wulff H, Lein PJ. From the cover:BDE-47 and BDE-49 inhibit axonal growth in primary rat hippocampal neuron-glia co-cultures via ryanodine receptor-dependent mechanisms. Toxicological Sciences 2017;156(2):375-386. R835550 (Final)
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  • Journal Article Dach K, Bendt F, Huebenthal U, Giersiefer S, Lein PJ, Heuer H, Fritschea E. BDE-99 impairs differentiation of human and mouse NPCs into the oligodendroglial lineage by species-specific modes of action. Scientific Reports 2017;7:44861 (11 pp.). R835550 (Final)
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  • Journal Article Harrill JA, Chen H, Streifel KM, Yang D, Mundy WR, Lein PJ. Ontogeny of biochemical, morphological and functional parameters of synaptogenesis in primary cultures of rat hippocampal and cortical neurons. Molecular Brain 2015;8:10 (15 pp.). R835550 (Final)
    R835432 (2014)
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  • Journal Article Mengelinga BJ, Wei Y, Dobrawa LN, Streekstra M, Louisse J, Singh V, Singh L, Lein PJ, Wulff H, Murk AJ, Furlow JD. A multi-tiered, in vivo, quantitative assay suite for environmental disruptors of thyroid hormone signaling. Aquatic Toxicology 2017;190:1-10. R835550 (Final)
    R835164 (Final)
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  • Journal Article Singh L, Pressly B, Mengeling BJ, Fettinger JC, Furlow JD, Lein PJ, Wulff H, Singh V. Chasing the elusive benzofuran impurity of the THR antagonist NH-3: synthesis, isotope labeling, and biological activity. Journal of Organic Chemistry 2016;81(5):1870-1876. R835550 (Final)
    R835164 (Final)
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  • Supplemental Keywords:

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

    Progress and Final Reports:

    Original Abstract
  • 2014 Progress Report
  • 2015 Progress Report