The long-term goal of this research is to understand how developmental exposure to the arylhydrocarbon receptor agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), interferes with estradiol (E2)-dependent sexual differentiation of neural functions. We will use the AhR- and ER-rich anterorventral periventricular nucleus (AVPV) as a model to study such interactions. In rodents, sexual differentiation of the AVPV occurs during perinatal life when E2 synthesized in the male AVPV triggers apoptosis, thereby decreasing the size of the adult male AVPV and abolishing the potential for luteinizing hormone (LH) surge release. Developmental exposure to TCDD blocks apoptosis in the male AVPV and prevents defeminization of the LH surge mechanism. We recently identified CUG triplet repeat binding protein 2 (Cugbp2) as an AVPV gene that is expressed at higher levels in males and oppositely regulated by E2 and TCDD. Consistent with evidence that Cubp2 promotes apoptosis, E2 upregulates and TCDD downregulates its expression. Importantly, Cugbp2 is an RNA-binding protein that regulates translation, thereby altering protein levels. Until now, most research on sexual differentiation of the brain has focused on genomic, non-genomic and epigenetic regulation of gene expression. Our recent findings suggest that E2 and TCDD also act through Cugbp2 to affect post- transcriptional control of protein levels. In the proposed studies, we will use LC MS/MS TMT proteomics and bioinformatics strategies to identify protein targets, and CLIP-Seq to identify Cugbp2 binding sites in the transcriptome of the postnatal day 2 AVPV. We will then focus on those targets that, like Cugbp2, differ between the sexes, are oppositely regulated by TCDD and E2, and are unaffected in the sexually dimorphic nucleus of the preoptic area. Using bioinformatics tools, we will identify functional clusters and pathways likely regulated by these proteins. Finally, we will use siRNA to deplete Cugbp2 from hypothalamic cell lines that model AVPV neurons and use those cells to verify that effects of E2 and TCDD on identified proteins require Cugbp2. Our findings will provide new avenues for studying sexual differentiation of the brain and determining how exposure to TCDD disrupts differentiation. Moreover, Cugbp2 has recently been implicated in late-onset Alzheimer?s disease and also shown to play a role in neuronal apoptosis after global ischemia and reperfusion. Thus, our findings may provide new targets for therapeutics to alter the risk of neurodegenerative diseases and to prevent permanent damage related to stroke. Finally, results of our studies will provide information important for mechanism-based risk assessment of AhR ligands.
We recently identified Cugbp2, an RNA-binding protein, as a sex-specific gene target of dioxins and estrogens in the developing brain region that controls ovulation. The proposed research will use proteomics technologies to identify proteins regulated by dioxin and estradiol. Our findings will be important for the mechanism-based risk assessment as well as mitigation of neuroendocrine disruption that can lead to male infertility, as well as polycystic ovary syndrome, premature ovarian failure and hypothalamic infertility.
