Reproductive Endocrine Related Mood Disorders-Differential Sensitivity

National Institute Of Mental Health

Investigators

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Abstract

This report includes work arising from the following clinical protocols: NCT00005011, NCT00056901, NCT00059228, NCT00082043, NCT00100360, NCT00001177, NCT00001259, and NCT00001481. The studies conducted within this project are relevant to our understanding of womens behavioral health, sex differences in psychiatric illness and the course of affective disorders in general. The innovation in our work is both conceptual as well as applied. In the past, RMDs were considered to be hormone excess or deficiency states, prompting treatments to correct these inferred hormonal imbalances. We address critical mechanistic questions about the role of reproductive hormones on brain function and behavior in RMDs. We employ steroid receptor modulators to isolate and characterize the effects of specific steroids on behavior in both women with RMDs and well-characterized asymptomatic controls. These clinical studies have demonstrated that a change in steroid hormone secretion induces affective symptoms in most women clinically diagnosed with RMDs, but the identical hormonal intervention has no effect in asymptomatic controls. The alteration in affective state in RMDs represents an abnormal behavioral response to normal physiology. Thus, our work has reframed the understanding of these conditions from hormonal excess or deficiency states to that of ovarian steroid-sensitive behavioral states (i.e., atypical endocrinopathies). We also employ both multimodal neuroimaging studies and cellular models to investigate the physiologic basis of the hormone sensitive phenotype in RMDs. We have characterized neuronal hubs that are differentially regulated by ovarian steroids in women with RMDs, developed a steroid metabolomics platform to examine the steroid metabolome in PMDD, demonstrated an impact of genotypic variation in steroid-regulated genes on the central nervous system (CNS) response to steroids (in a brain region-specific manner), and identified underlying cellular mechanisms for the alterations in steroid signaling in both PMDD and PMD (the mechanisms underlying several of which are being actively pursued). These findings are unique and serve to inform our understanding of the pathophysiology of these conditions and their potential comorbidities, their clinical management and use of hormone therapies. Finally, having developed and tested these methods in women with RMDs, we will employ these techniques in our new studies of postpartum psychosis a condition potentially lethal to both mother and child. Findings to date include: 1) the differential affective and behavioral response to normal physiologic events in women with PMDD compared with control women a behavioral phenotype in which we have explored the underlying biologic risks. 2) 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). 3) 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.