Age-dependent Effects of 17ß-estradiol on the dynamics of estrogen receptor ß (ERß) protein-protein interactions in the ventral hippocampus.

Recent clinical evidence suggests that the neuroprotective and beneficial effects of hormone therapy may be limited by factors related to age and reproductive status. The patient's age and length of time without circulating ovarian hormones are likely to be key factors in the specific neurological outcomes of hormone therapy. However, the mechanisms underlying age-related changes in hormone efficacy have not been determined. We hypothesized that there are intrinsic changes in estrogen receptor ß (ERß) function that determine its ability to mediate the actions of 17ß-estradiol (E2) in brain regions such as the ventral hippocampus. In this study, we identified and quantified a subset of ERß protein interactions in the ventral hippocampus that were significantly altered by E2 replacement in young and aged animals, using two-dimensional differential gel electrophoresis coupled with liquid chromatography-electrospray ionization-tandem mass spectrometry. This study demonstrates quantitative changes in ERß protein-protein interactions with E2 replacement that are dependent upon age in the ventral hippocampus and how these changes could alter processes such as transcriptional regulation. Thus, our data provide evidence that changes in ERß protein interactions are a potential mechanism for age-related changes in E2 responsiveness in the brain after menopause.

An emerging role for microRNAs in sexually dimorphic neurobiological systems.

Over the past 20 years, our understanding of the basic mechanisms of gene regulation has vastly expanded due to the unexpected roles of small regulatory RNAs, in particular microRNAs (miRNAs). miRNAs add another layer of complexity to the regulation of effector molecules for nearly every physiological process, making them excellent candidate molecules as therapeutic targets, biomarkers, and disease predictors. Hormonal contributions to mature miRNA expression, biosynthetic processing, and downstream functions have only just begun to be investigated. Elucidating the physiological consequences of miRNA sexual dimorphism, and their associated regulatory processes, may be key toward understanding both normal and pathological processes in the brain. This short review provides a basic overview of miRNA biosynthesis, their role in normal brain development, and potential links to neurological diseases. We conclude with a brief discussion of the current knowledge of sex-specific miRNA processes in both the brain and the heart to conceptually integrate the relevance of miRNAs with the overarching theme ("sex differences in health and disease: brain and heart connections") of this special topics issue.

Estrogen signaling and the aging brain: context-dependent considerations for postmenopausal hormone therapy.

Recent clinical studies have spurred rigorous debate about the benefits of hormone therapy (HT) for postmenopausal women. Controversy first emerged based on a sharp increase in the risk of cardiovascular disease in participants of the Women's Health Initiative (WHI) studies, suggesting that decades of empirical research in animal models was not necessarily applicable to humans. However, a reexamination of the data from the WHI studies suggests that the timing of HT might be a critical factor and that advanced age and/or length of estrogen deprivation might alter the body's ability to respond to estrogens. Dichotomous estrogenic effects are mediated primarily by the actions of two high-affinity estrogen receptors alpha and beta (ER α & ER ß ). The expression of the ERs can be overlapping or distinct, dependent upon brain region, sex, age, and exposure to hormone, and, during the time of menopause, there may be changes in receptor expression profiles, post-translational modifications, and protein:protein interactions that could lead to a completely different environment for E2 to exert its effects. In this review, factors affecting estrogen-signaling processes will be discussed with particular attention paid to the expression and transcriptional actions of ER ß in brain regions that regulate cognition and affect.

MicroRNAs in the aging female brain: a putative mechanism for age-specific estrogen effects.

Menopause is characterized by the rapid age-related decline of circulating 17ß-estradiol (E(2)) levels in women, which can sometimes result in cognitive disorders such as impaired memory and increased anxiety. Hormone therapy (HT) is a widely used treatment for the adverse effects associated with menopause; however, evidence suggests that HT administered to postmenopausal women age 65 years and over can lead to increased risks for cognitive disorders. We hypothesized that these age-related changes in E(2) action are due to posttranscriptional gene regulation by microRNAs (miRNAs). miRNAs are a class of small noncoding RNAs that regulate gene expression by binding to the 3'-untranslated region of target mRNAs and subsequently target these transcripts for degradation. In the present study, 3- and 18-month-old female rats were oophorectomized (OVX) and treated 1 week after surgery with 2.5 µg E(2) once per day for 3 days. Total RNA was isolated from the ventral and dorsal hippocampus, central amygdala, and paraventricular nucleus. Our results showed that E(2) differentially altered miRNA levels in an age- and brain region-dependent manner. Multiple miRNA target prediction algorithms revealed putative target genes that are important for memory and stress regulation, such as BDNF, glucocorticoid receptor, and SIRT-1. Indeed, quantitative RT-PCR analyses of some of the predicted targets, such as SIRT1, showed that the mRNA expression levels were the inverse of the targeting miRNA, thereby confirming the prediction algorithms. Taken together, these data show that E(2) regulates miRNA expression in an age- and E(2)-dependent manner, which we hypothesize results in differential gene expression and consequently altered neuronal function.

Effects of kisspeptin on parameters of the HPA axis.

The hypothalamo-pituitary-adrenal (HPA) and hypothalamo-pituitary-gonadal (HPG) axes have an intricate cross talk that results in the inhibition of reproductive functions during periods of chronic physiological or psychological stress. Recent studies have shown that kisspeptin neurons have projections to many non-reproductive areas of the brain including the paraventricular nucleus (PVN) of the hypothalamus, thereby providing evidence of an anatomical framework for kisspeptin to regulate the HPA axis. In this study, we tested as to whether kisspeptin modulates the HPA axis at three potential levels of regulation: (1) transcription of stress-related genes CRH, AVP, and oxytocin (OXY); (2) release of neuropeptides from PVN-derived neuronal cells via mobilization of intracellular calcium stores; and (3) in vivo regulation of the HPA axis under basal and stress-induced conditions in adult male rats. Overall, our data showed that kisspeptin did not alter basal, or stress-induced HPA axis activity (plasma corticosterone (CORT) and adrenocorticotropin hormone (ACTH)) in adult male rats and had modest, yet significant effects on CRH, AVP, and OXY gene expressions.

Alcohol dysregulates corticotropin-releasing-hormone (CRH) promoter activity by interfering with the negative glucocorticoid response element (nGRE).

EtOH exposure in male rats increases corticotropin-releasing hormone (CRH) mRNA in the paraventricular nucleus of the hypothalamus (PVN), a brain region responsible for coordinating stress and anxiety responses. In this study we identified the molecular mechanisms involved in mediating these effects by examining the direct effects of EtOH on CRH promoter activity in a neuronal cell line derived from the PVN (IVB). In addition, we investigated the potential interactions of EtOH and glucocorticoids on the CRH promoter by concomitantly treating cells with EtOH and the glucocorticoid receptor (GR) antagonist RU486, and by sequentially deleting GR binding sites within glucocorticoid response element (GRE) on the CRH promoter. Cells were transiently transfected with a firefly luciferase reporter construct containing 2.5 kb of the rat wild type (WT) or mutated CRH promoter. Our results showed that EtOH treatment induced a biphasic response in CRH promoter activity. EtOH exposure for 0.5 h significantly decreased promoter activity compared to vehicle treated controls, whereas promoter activity was significantly increased after 2.0 h of EtOH exposure. Treatment with RU486, or deletion of the GR binding sites 1 and 2 within the GRE, abolished the EtOH-induced increase in the promoter activity, however did not affect EtOH-induced decrease in CRH promoter activity at an earlier time point. Overall, our data suggest that alcohol exposure directly regulates CRH promoter activity by interfering with the normal feedback mechanisms of glucocorticoids mediated by GR signaling at the GRE site of the CRH promoter.

Binge-pattern alcohol exposure during puberty induces long-term changes in HPA axis reactivity.

Adolescence is a dynamic and important period of brain development however, little is known about the long-term neurobiological consequences of alcohol consumption during puberty. Our previous studies showed that binge-pattern ethanol (EtOH) treatment during pubertal development negatively dysregulated the responsiveness of the hypothalamo-pituitary-adrenal (HPA) axis, as manifested by alterations in corticotrophin-releasing hormone (CRH), arginine vasopressin (AVP), and corticosterone (CORT) during this time period. Thus, the primary goal of this study was to determine whether these observed changes in important central regulators of the stress response were permanent or transient. In this study, juvenile male Wistar rats were treated with a binge-pattern EtOH treatment paradigm or saline alone for 8 days. The animals were left undisturbed until adulthood when they received a second round of treatments consisting of saline alone, a single dose of EtOH, or a second binge-pattern treatment paradigm. The results showed that pubertal binge-pattern EtOH exposure induced striking long-lasting alterations of many HPA axis parameters. Overall, our data provide strong evidence that binge-pattern EtOH exposure during pubertal maturation has long-term detrimental effects for the healthy development of the HPA axis.

Differential fibroblast growth factor 8 (FGF8)-mediated autoregulation of its cognate receptors, Fgfr1 and Fgfr3, in neuronal cell lines.

Fibroblast growth factors (FGFs) mediate a vast range of CNS developmental processes including neural induction, proliferation, migration, and cell survival. Despite the critical role of FGF signaling for normal CNS development, few reports describe the mechanisms that regulate FGF receptor gene expression in the brain. We tested whether FGF8 could autoregulate two of its cognate receptors, Fgfr1 and Fgfr3, in three murine cell lines with different lineages: fibroblast-derived cells (3T3 cells), neuronal cells derived from hippocampus (HT-22 cells), and neuroendocrine cells derived from hypothalamic gonadotropin-releasing hormone (GnRH) neurons (GT1-7 cells). GnRH is produced by neurons in the hypothalamus and is absolutely required for reproductive competence in vertebrate animals. Several lines of evidence strongly suggest that Fgf8 is critical for normal development of the GnRH system, therefore, the GT1-7 cells provided us with an additional endpoint, Gnrh gene expression and promoter activity, to assess potential downstream consequences of FGF8-induced modulation of FGF receptor levels. Results from this study suggest that the autoregulation of its cognate receptor represents a common downstream effect of FGF8. Further, we show that Fgfr1 and Fgfr3 are differentially regulated within the same cell type, implicating these two receptors in different biological roles. Moreover, Fgfr1 and Fgfr3 are differentially regulated among different cell types, suggesting such autoregulation occurs in a cell type-specific fashion. Lastly, we demonstrate that FGF8b decreases Gnrh promoter activity and gene expression, possibly reflecting a downstream consequence of altered FGF receptor populations. Together, our data bring forth the possibility that, in addition to the FGF synexpression group, autoregulation of FGFR expression by FGF8 represents a mechanism by which FGF8 could fine-tune its regulatory actions.