Grantee Research Project Results

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. We have completed quantitative PCR analyses determining 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 using product-specific copy number standards. These analyses were performed with additional reference genes to facilitate cross-species comparison for the planned publication, which made it necessary to prepare new samples. These data elucidated the developmental expression patterns and indicated that the ontogenetic expression profiles of key signaling components vary between the models investigated. Analyses of the effect of exogenous T4 and T3 on the expression of these signaling components has been completed in the human and rat neurospheres, primary rat neuronal cell cultures, and zebrafish. In human and rat neurospheres, we identified hairless, deiodinase 3, myelin basic protein (in human), and myelin-associated oligodendrocyte basic protein (Mobp) as TH responsive genes. In primary rat cortical cultures, varying T4 and T3 did not greatly alter expression of the genes analyzed. In zebrafish, expression of deiodinase 3, thyroid hormone receptor β, and the TH transporter MCT8 were induced by elevated T4 and T3 exposure, 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.

 

We completed analysis of 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). These data indicate that TH concentration may affect cell survival and glial cell proliferation and differentiation in vitro. Primary cerebellar neuron cultures were used as an additional in vitro model that may be more sensitive to changes in thyroid hormone level. These data indicate differences in dendrite arbor growth of Purkinje cells with varying TH concentration.

 

Aim 2: Elucidate genomic and/or non-genomic intracellular signaling pathways that mediate TH effects on neurodevelopment.The thyroid hormone receptor (TR) antagonist NH-3 was observed to interfere with T3-induced oligodendrocyte maturation in human NPCs, suggesting the involvement of TRs in human oligodendrocyte maturation. Using NPCs prepared from TR knockout mice, it was shown that TRα, but not TRβ, mediates the T3 induction of oligodendrocyte formation and maturation from murine NPCs. Furthermore, NH-3 inhibited T3-induced TRα-mediated transcription of hairless in NPCs in a concentration-dependent manner. We also have begun to perform experiments with TR antagonist NH-3 on proliferation of NPC cells. To elucidate the involvement of non-genomic pathways, we started to test PI3K inhibitor LY294002 and MAP kinase inhibitor U0126 on proliferation of human and rat NPCs. Analogous experiments with NH-3, LP294002 and U0126 will be performed for migration and differentiation in human and rat neurospheres. We also have begun assessing the effects of NH-3 and TH on neurodevelopmental events in primary rat cortical cell cultures (axon outgrowth and dendrite outgrowth).

 

Aim 3: Identify ‘pathways of toxicity’ by which chemicals interfere with TH-mediated neurodevelopment. We have developed the oligodendrocyte maturation assay to identify human and murine specific endocrine disruptors. This assay will be implemented for rat NPCs as well. We found that the flame retardant BDE-99 interfered with murine oligodendrocyte formation and maturation. BDE-99 inhibited human oligodendrocyte formation independent of TH interaction, while it did not affect human oligodendrocyte maturation. NH-3, however, inhibited human oligodendrocyte maturation. Analogous studies will be performed with rat NPCs. We also started to evaluate the effects of the putative TH disruptors tetrabromobisphenol A (TBBPA) and perfluorooctanoic acid (PFOA) on proliferation, migration and differentiation into neurons and oligodendrocytes in human and rat NPCs.

 

Aim 4: Link pathways of toxicity with in vivo neurodevelopment and behavior. In order to link pathways of toxicity with in vivo neurodevelopment, a phenotype for developmental hyperthyroidism in zebrafish has been characterized. Our initial assessment focused on the first 5 days of development in zebrafish, which encompasses the range of neurodevelopmental processes investigated in vitro. Zebrafish treated with exogenous T4 or T3 have been 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 do not cause gross malformation. To determine whether the observed effects are mediated by nuclear thyroid hormone receptors, these treatments will be repeated in the presence of the TR antagonist NH-3. In order to establish a developmental hypothyroid phenotype, we have obtained a conditional thyroid ablation zebrafish transgenic line Tg(tg:nVenus-2a-nfnB)^wp.rt8 (provided by Parichy lab, University of Washington), which will allow us to generate TH-deficient zebrafish embryos and assess the neurodevelopmental endpoints mentioned above.

 

To follow up on the results obtained in NPCs showing that oligodendrocyte formation and maturation was influenced by altered TH levels and TH disruptors, the Lein lab has 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. This zebrafish line has been raised in the lab and currently is being used to optimize methods to quantify oligodendrocyte formation and maturation in the zebrafish central nervous system. This will facilitate the investigation of TH disrupting compounds and their influence on oligodendrocyte development and subsequent myelination in vivo.

In project year 3, we will elucidate the genomic and non-genomic pathways that mediate TH effects on neurodevelopmental endpoints (proliferation, migration and differentiation). The oligodendrocyte maturation assay will be implemented for rat NPCs and in zebrafish. Furthermore, we will identify TH disrupting chemicals in human and rat NPCs, and in rat primary cerebellar cultures, and study their toxicity pathways in regard to disruption of genomic and non-genomic TH signaling. We also will assess the influence of potential TH disrupting chemicals on these neurodevelopmental endpoints. In zebrafish, the impact of developmental hypothyroidism will be characterized using teratological evaluation, behavioral testing, and gene expression analyses. The influence of THs, T4 and T3, as well as the TR antagonist NH-3 on oligodendrocyte development will be determined. This endpoint then will be used to determine whether suspected TH-disruptors also alter this endpoint and whether disruption correlates to teratological and behavioral evaluations. Methods to investigate additional endpoints shown to be sensitive to TH in vitro will be established in zebrafish.