Reproductive Endocrine Related Mood Disorders-Differential Sensitivity

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This report includes work arising from the following clinical protocols: NCT00005011, NCT00056901, NCT00059228, NCT00082043, NCT00100360, NCT00001177, NCT00001259, and NCT00001481. Our studies have documented that PMDD symptoms are eliminated by ovarian suppression and stimulated by administration of ovarian steroids, yet appear in the context of levels of ovarian steroids indistinguishable from those in women without PMDD. Additionally, we demonstrated that the change in levels of combined estradiol and progesterone from low to high, and not the steady state levels, triggered the onset of PMDD symptoms in women with this condition whose PMDD was in remission after GnRH agonist-induced ovarian suppression. Our findings provide a new target on which interventions could be focused (namely increasing neurosteroid levels from the follicular to the luteal phase). We will conduct a placebo-controlled treatment trial investigating the effects of stabilizing neurosteroid levels with dutasteride (a 5alpha reductase enzyme inhibitor which prevents the production of the neurosteroid, allopregnanolone from progesterone that is implicated in the alterations of neurotransmitter function that could lead to PMDD) in women with PMDD. We also employed our ovarian steroid manipulation studies (GnRH agonist studies) to explore possible neural substrates of risk in women with PMDD. We have identified the subgenual anterior cingulate cortex (SGCC) to be differentially regulated by ovarian steroids in women with PMDD compared with control women (i.e., regional cerebral blood flow rCBF during a resting state exam is decreased during estradiol or progesterone exposure when PMDD symptoms recur). The degree of altered rCBF also correlated with gene expression in the ESC/E(Z) pathway of genes a family of genes which we previously identified to be differentially expressed (increased) in cell lines from women with PMDD compared to controls, and members of which are differentially regulated by estradiol and progesterone in cell culture (see below). Additionally, the results of a whole transcriptome sequencing study of the SGCC shows evidence that sex steroid receptors are present in this tissue and, therefore, it is plausible that SGCC activity could be regulated by ovarian steroids. These data suggest both the relevance of differences in the response of the SGCC to ovarian steroids with consequent differential activation of brain networks that mediate the affective responsivity of women with PMDD as well as providing a biologically plausible target for neuromodulatory interventions in PMDD. These behavioral and neuroimaging data also set the stage for the performance of in vitro cellular studies of ovarian steroid responsivity in women with PMDD. In collaboration with Dr. David Goldman at NIAAA, we developed lymphoblastoid cell lines (LCLs) and human induced-pluripotent cell lines (h-IPSCs) from women with and without PMDD who had participated in our GnRH agonist-induced ovarian suppression studies. The behavioral outcomes observed during these protocols serve to refine the hormone-sensitive phenotype beyond simply the established clinical diagnoses. Our in vitro experimental strategies attempt to recapitulate the endocrine events that trigger mood symptoms in women with PMDD. In our first study, pathway analyses of the LCL transcriptome revealed, among others, over-expression of ESC/E(Z) complex genes (an ovarian steroid-regulated gene silencing complex) in untreated LCLs from women with PMDD, with more than half of these genes over-expressed as compared to controls. In contrast, protein expression of ESC/E(Z) genes was decreased in untreated PMDD LCLs. Finally, mRNA expression of several ESC/E(Z) complex genes were increased by P in controls only and decreased by E in PMDD LCLs. These findings provided the first evidence of a plausible biological substrate for the differential behavioral response to E/P in women with PMDD. Indeed, these data suggest that women with PMDD have an intrinsic abnormality in their epigenetic capacity that could manifest in an alteration in their ability to translate environmental events into long-term changes in gene expression. We have continued to pursue these findings in PMDD. During the past year we have pursued both the mechanism underlying the altered expression of the ESC/E(Z) pathway and the effects of ovarian steroid exposures on gene expression in PMDD versus controls. First, preliminary evidence from a whole small RNA sequencing (targeting micro-RNA expression) shows a significantly increased expression of several micro-RNAs in LCLs from women with PMDD compared to those in controls. Additionally, several of these differentially expressed micro-RNAs target the ESC/E(Z) pathway, and, therefore, could contribute to the observed dissociation between increased expression of the ESC/E(Z) pathway genes and decreased protein levels of this same pathway in PMDD. Additionally, several of these micro-RNAs target the other genes relevant for both affective dysregulation and tissue-specific steroid signaling including vascular endothelial growth factor (VEGF). Second, we performed transcriptomic analyses of LCLs derived from women with PMDD and asymptomatic controls cultured under untreated (steroid-free), estradiol-treated, and progesterone-treated conditions. Weighted gene correlation network analysis (WGCNA) of transcriptomes identified four gene modules with significant differences in the response patterns in women with PMDD (versus control LCLs) that also differed across hormone exposures, including one enriched for neuronal functions. Exploratory enrichment analysis of hub genes underlying neuronal enrichment signals revealed multiple pathways governing intracellular Ca2+ dynamics. Next, in a gene-level analysis comparing transcriptional response to hormone across diagnoses, a statistical model (i.e., generalized linear model) identified 1522 genes differentially responsive to estradiol (E2-DRGs). Among the top 10 E2-DRGs was an interacting gene network (NUCB1, DST, GCC2, GOLGB1) involved in endoplasmic reticulum (ER)-Golgi function. qRT-PCR validation reproduced a diagnosis by hormone interaction (reflecting differential expression patterns in LCLs from women with PMDD compared with those from controls during estradiol exposure), for NUCB1, a regulator of cellular Ca2+ and ER stress. Finally, we used a thapsigargin (Tg) challenge to test whether estradiol induces differences in Ca2+ homeostasis and ER stress response in PMDD. Untreated PMDD LCLs had a 1.36-fold decrease in Tg-induced XBP1 splicing response (a measure of intracellular calcium signaling) compared to controls, and a 1.62-fold decreased response in the E2-exposed condition. These data suggest that estradiol-dependent aberrations in cellular Ca2+ dynamics and ER stress may contribute to the pathophysiology of PMDD and could guide treatment development to focus on medications regulating intracellular calcium signaling (known to be involved with affective state regulation) in PMDD. In a parallel series of functional genomic studies employing LCLs and h-IPSCs developed from women with PPD and in a separate group of women with first-onset postpartum psychosis (PPP) we have completed whole transcriptome sequencing. Whole transcriptome RNA-sequencing is completed in LCLs from controls and women with PPD under conditions of untreated baseline, supraphysiologic (high) combined estradiol and progesterone (replicating pregnancy), and hormone withdrawal (replicating hormonal events of parturition). Preliminary findings revealed multiple differentially expressed genes in all conditions meeting FDR corrections, particularly in the high combined estradiol and progesterone conditions between cases and controls.