Permanent middle cerebral artery occlusion (MCAO) causes neuronal cell death in the striatum and cortex. In rodents, estradiol treatment protects the cortex from cell death in an estrogen receptor alpha (ERα) dependent manner. ERα is only transiently expressed in the cortex during neonatal development and is very low in uninjured adult cortex. Following MCAO, ERα mRNA expression is upregulated in the cortex of female rats, but the mechanism of this increase is still unknown. It is also unknown whether a similar increase in ERα expression in seen in males. In the following studies, male and vehicle or estradiol-treated ovariectomized (OVX) female rats underwent MCAO to investigate the regulation of ERα expression after ischemia. 24 hours after surgery, mRNA or genomic DNA was collected from 1mm micropunches taken from 300µm brain sections for quantitative RT-PCR or methylation-specific (MSP) PCR, respectively. Additionally, adjacent 20µm sections were processed for ERα immunohistochemistry. In OVX females, ERα mRNA and protein were increased in the ischemic cortex, but unchanged in males. We hypothesized that this increase in ERα in females is due to a reversal of gene silencing by DNA methylation. Using MSP targeting of CpG islands within the 5' UTR of the rat ERα gene, we found that ischemia decreased methylation in the ischemic cortex of both groups of females, but there was no change in methylation in males. Using chromatin immunoprecipitation, we found that MeCP2 associates with ERα 5'UTR corresponding with the methylation status of the promoter. These data are the first to demonstrate a difference in the regulation of ERα expression in response to MCAO between males and females and that methylation of the ERα gene corresponds with mRNA levels in the brain.

Estrogen plays a critical role in brain development and is responsible for generating sex differences in cognition and emotion. Studies in rodent models have shown high levels of estrogen binding in non-reproductive areas of the brain during development, including the cortex and hippocampus, yet binding is diminished in the same areas of the adult brain. These binding studies demonstrated that estrogen receptors decline in the cortex during development but did not identify which of the two estrogen receptors was present. In the current study, we examined the expression of estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) in the mouse cortex during the first month of life. Messenger RNA was isolated from cortical tissue taken from C57BL/6 mice on postnatal day (PND) 1, 4, 10, 18 and 25 and expression levels were determined by real-time PCR. ERα mRNA expression in the mouse cortex at PND 25 was significantly reduced as compared to PND 1 (p<0.01). ERβ mRNA expression at PND25 was significantly increased as compared to PND 1 (p<0.05). Although the increase in ERβ mRNA was statistically significant, the ERβ levels were extremely low in the isocortex compared to ERα mRNA levels, suggesting that ERα may play a more critical role in the developmental decrease of estradiol binding than ERβ. Additionally, we measured ERα mRNA expression in organotypic explant cultures of cortex taken from PND 3 mice. Explants were maintained in vitro for 3 weeks. mRNA was isolated at several time points and ERα and ERβ mRNA was measured by real-time RT-PCR. ERα and ERβ mRNA levels reflected a similar pattern in vitro and in vivo, suggesting that signals outside the cortex are not needed for this developmental change. This study lays the groundwork for an understanding of the mechanisms of the developmental regulation of ERα mRNA.

Estrogen has long been known to play an important role in coordinating the neuroendocrine events that control sexual development, sexual behavior and reproduction. Estrogen actions in other, nonreproductive areas of the brain have also been described. It is now known that estrogen can also influence learning, memory, and emotion and has neurotrophic and neuroprotective properties. The actions of estrogen are largely mediated through at least two intracellular estrogen receptors. Both estrogen receptor-alpha and estrogen receptor-beta are expressed in a wide variety of brain regions. Estrogen receptor-alpha (ERα), however, undergoes developmental and brain region-specific changes in expression. The precise molecular mechanisms that regulate its expression at the level of gene transcription are not well understood. Adding to the complexity of its regulation, the estrogen receptor gene contains multiple promoters that drive its expression. In the cortex in particular, the ERα mRNA expression is dynamically regulated during postnatal development and again following neuronal injury. Epigenetic modification of chromatin is increasingly being understood as a mechanism of neuronal gene regulation. This review examines the potential regulation of the ERα gene by such epigenetic mechanisms.