Melinda E. Wilson

The Epigenetics of Sex Differences in the Brain

Epigenetic changes in the nervous system are emerging as a critical component of enduring effects induced by early life experience, hormonal exposure, trauma and injury, or learning and memory. Sex differences in the brain are largely determined by steroid hormone exposure during a perinatal sensitive period that alters subsequent hormonal and nonhormonal responses throughout the lifespan. Steroid receptors are members of a nuclear receptor transcription factor superfamily and recruit multiple proteins that possess enzymatic activity relevant to epigenetic changes such as acetylation and methylation. Thus steroid hormones are uniquely poised to exert epigenetic effects on the developing nervous system to dictate adult sex differences in brain and behavior. Sex differences in the methylation pattern in the promoter of estrogen and progesterone receptor genes are evident in newborns and persist in adults but with a different pattern. Changes in response to injury and in methyl-binding proteins and steroid receptor coregulatory proteins are also reported. Many steroid-induced epigenetic changes are opportunistic and restricted to a single lifespan, but new evidence suggests endocrine-disrupting compounds can exert multigenerational effects. Similarly, maternal diet also induces transgenerational effects, but the impact is sex specific. The study of epigenetics of sex differences is in its earliest stages, with needed advances in understanding of the hormonal regulation of enzymes controlling acetylation and methylation, coregulatory proteins, transient versus stable DNA methylation patterns, and sex differences across the epigenome to fully understand sex differences in brain and behavior.

Estrogens: Trophic and protective factors in the adult brain

Our appreciation that estrogens are important neurotrophic and neuroprotective factors has grown rapidly. Although a thorough understanding of the molecular and cellular mechanisms that underlie this effect requires further investigation, significant progress has been made due to the availability of animal models in which we can test potential candidates. It appears that estradiol can act via mechanisms that require classical intracellular receptors (estrogen receptor alpha or beta) that affect transcription, via mechanisms that include cross-talk between estrogen receptors and second messenger pathways, and/or via mechanisms that may involve membrane receptors or channels. This area of research demands attention since estradiol may be an important therapeutic agent in the maintenance of normal neural function during aging and after injury.

Minireview: Neuroprotective Effects of Estrogen—New Insights into Mechanisms of Action

An accumulating body of evidence clearly establishes that estradiol is a potent neuroprotective and neurotrophic factor in the adult: it influences memory and cognition, decreases the risk and delays the onset of neurological diseases such as Alzheimer’s disease, and attenuates the extent of cell death that results from brain injuries such as cerebrovascular stroke and neurotrauma. Thus, estradiol appears to act at two levels: 1) it decreases the risk of disease or injury; and/or 2) it decreases the extent of injury incurred by suppressing the neurotoxic stimulus itself or increasing the resilience of the brain to a given injury. During the past century, the average life span of women has increased dramatically, whereas the time of the menopause has remained essentially constant. Thus, more women will live a larger fraction of their lives in a postmenopausal, hypoestrogenic state than ever before. Clearly, it is critical for us understand the circumstances under which estradiol exerts protective actions a...

Estradiol is a protective factor in the adult and aging brain: understanding of mechanisms derived from in vivo and in vitro studies

We have shown that 17beta-estradiol exerts profound protective effects against stroke-like ischemic injury in female rats. These effects are evident using physiological levels of estradiol replacement in ovariectomized rats and require hormone treatment prior to the time of injury. The protective actions of estradiol appear to be most prominent in the cerebral cortex, where cell death is not apparent until at least 4 h after the initiation of ischemic injury and where cell death is thought to be apoptotic in nature. Middle-aged rats remain equally responsive to the protective actions of estradiol. The maintenance of responsiveness of the cerebral cortex to the neuroprotective actions of estradiol was unexpected since responsiveness of the hypothalamus to estradiol decreases dramatically by the time animals are middle-aged. We believe that the protective actions of estradiol require the estrogen receptor-alpha, since estradiol does not protect in estrogen receptor-alpha knockout mice. We have also implemented a method of culturing cerebral cortical explants to assess the protective effects of estradiol in vitro. This model exhibits remarkable parallelisms with our in vivo model of brain injury. We have found that 17beta-estradiol decreases the extent of cell death and that this protective effect requires hormone pretreatment. Finally, 17alpha-estradiol, which does not interact effectively with the estrogen receptor, does not protect; and addition of ICI 182,780, an estrogen receptor antagonist, blocks the protective actions of estradiol. We have begun to explore the molecular and cellular mechanisms of estradiol-mediated protection. In summary, our findings demonstrate that estradiol exerts powerful protective effects both in vivo and in vitro and suggest that these actions are mediated by estrogen receptors.

HIV protease inhibitors promote atherosclerotic lesion formation independent of dyslipidemia by increasing CD36-dependent cholesteryl ester accumulation in macrophages

Protease inhibitors decrease the viral load in HIV patients, however the patients develop hypertriglyceridemia, hypercholesterolemia, and atherosclerosis. It has been assumed that protease inhibitor-dependent increases in atherosclerosis are secondary to the dyslipidemia. Incubation of THP-1 cells or human PBMCs with protease inhibitors caused upregulation of CD36 and the accumulation of cholesteryl esters. The use of CD36-blocking antibodies, a CD36 morpholino, and monocytes isolated from CD36 null mice demonstrated that protease inhibitor-induced increases in cholesteryl esters were dependent on CD36 upregulation. These data led to the hypothesis that protease inhibitors induce foam cell formation and consequently atherosclerosis by upregulating CD36 and cholesteryl ester accumulation independent of dyslipidemia. Studies with LDL receptor null mice demonstrated that low doses of protease inhibitors induce an increase in the level of CD36 and cholesteryl ester in peritoneal macrophages and the development of atherosclerosis without altering plasma lipids. Furthermore, the lack of CD36 protected the animals from protease inhibitor-induced atherosclerosis. Finally, ritonavir increased PPAR-gamma and CD36 mRNA levels in a PKC- and PPAR-gamma-dependent manner. We conclude that protease inhibitors contribute to the formation of atherosclerosis by promoting the upregulation of CD36 and the subsequent accumulation of sterol in macrophages.

HDL-associated estradiol stimulates endothelial NO synthase and vasodilation in an SR-BI–dependent manner

Cardiovascular diseases remain the leading cause of death in the United States. Two factors associated with a decreased risk of developing cardiovascular disease are elevated HDL levels and sex - specifically, a decreased risk is found in premenopausal women. HDL and estrogen stimulate eNOS and the production of nitric oxide, which has numerous protective effects in the vascular system including vasodilation, antiadhesion, and anti-inflammatory effects. We tested the hypothesis that HDL binds to its receptor, scavenger receptor class B type I (SR-BI), and delivers estrogen to eNOS, thereby stimulating the enzyme. HDL isolated from women stimulated eNOS, whereas HDL isolated from men had minimal activity. Studies with ovariectomized and ovariectomized/estrogen replacement mouse models demonstrated that HDL-associated estradiol stimulation of eNOS is SR-BI dependent. Furthermore, female HDL, but not male HDL, promoted the relaxation of muscle strips isolated from C57BL/6 mice but not SR-BI null mice. Finally, HDL isolated from premenopausal women or postmenopausal women receiving estradiol replacement therapy stimulated eNOS, whereas HDL isolated from postmenopausal women did not stimulate eNOS. We conclude that HDL-associated estrodial is capable of the stimulating eNOS. These studies establish a new paradigm for examining the cardiovascular effects of HDL and estrogen.

Estradiol protects against injury-induced cell death in cortical explant cultures: a role for estrogen receptors.

Estradiol has been shown to exert trophic and protective actions in the brain. Our laboratory has shown that in vivo, low physiological levels of estradiol protect the female rat brain against ischemic injury. In the present study, we used organotypic cortical explant cultures to begin to decipher the mechanisms of estradiols actions. Injury was induced by exposure to kainic acid or potassium cyanide/2-deoxyglucose (KCN/2-DG) for varying lengths of time, and cell death was monitored by LDH release at 2, 6, 12, 24, 48, 72 and 96 h after injury. We found that exposure to 1 mM KCN/2 mM 2-DG for 2 h produced consistent delayed cell death that was detectable by 24 h. The presence of 17beta-estradiol (E2) during the 7 days prior to injury significantly reduced the extent of cell death; whereas, administration of E2 at the time of injury did not protect. The protective effects of estradiol were dose dependent. Low doses of E2 (1, 10, and 30 nM) significantly reduced cell death; however, higher concentrations of E2 (>60 nM) had no protective effect. The observations that low levels of E2 protect against cell death, and that pretreatment is required suggest that the protective actions of estradiol may involve estrogen receptors. Therefore, we examined the ability of 17alpha-estradiol, which does not efficiently activate the estrogen receptor, and the addition of the estrogen receptor antagonist, ICI 182,780, to influence the extent of cell death induced by KCN/2-DG. 17alpha-Estradiol failed to protect, and ICI 182,780 prevented E2 from protecting against cell death. Furthermore, E2 pretreatment is required for more than 24 h to be neuroprotective. Our results clearly show that in cortical explant cultures, estradiol protects cells against ischemic injury, and suggest that these protective actions involve estrogen receptors.

Age differentially influences estrogen receptor α (ERα) and estrogen receptor β (ERβ) gene expression in specific regions of the rat brain

Abstract Estradiols ability to influence neurochemical events that are critical to female reproductive cyclicity and behavior decreases with age. We tested the hypothesis that decreases in estrogen receptor-α (ERα) and/or ERβ mRNA explain the brains declining responsiveness to estradiol. We assessed ERα and ERβ mRNA levels in intact and ovariectomized estradiol-treated rats. ERβ mRNA was detected in several brain regions and decreased by middle-age in the cerebral cortex and supraoptic nucleus of estradiol-treated rats. ERβ mRNA levels exhibited a diurnal rhythm in the suprachiasmatic nucleus of young and middle-aged rats and this rhythm was blunted in old rats. We examined ERα mRNA in the periventricular preoptic, medial preoptic, ventromedial and arcuate nuclei, and it was decreased only in the periventricular preoptic nucleus of the old rats. In summary, the expression of ERα and ERβ mRNAs is differentially modulated in the aging brain and changes are region specific.

Estradiol is a neuroprotective factor in in vivo and in vitro models of brain injury

Many clinical studies suggest that estrogen enhances memory and cognition and protects against neurodegenerative diseases and injury associated with stroke or stress. These results are strongly supported by experiments performed in animal models using both in vivo and in vitro methods. We present here data from our lab that establishes that physiological levels of estradiol exert profound protective actions against ischemic injury. Further we will present evidence that these effects may be mediated through estrogen receptors that may influence the bcl-2 family of genes.

Estrogen receptor-alpha gene expression in the cortex: Sex differences during development and in adulthood

17β-estradiol is a hormone with far-reaching organizational, activational and protective actions in both male and female brains. The organizational effects of early estrogen exposure are essential for long-lasting behavioral and cognitive functions. Estradiol mediates many of its effects through the intracellular receptors, estrogen receptor-alpha (ERα) and estrogen receptor-beta (ERβ). In the rodent cerebral cortex, estrogen receptor expression is high early in postnatal life and declines dramatically as the animal approaches puberty. This decline is accompanied by decreased expression of ERα mRNA. This change in expression is the same in both males and females in the developing isocortex and hippocampus. An understanding of the molecular mechanisms involved in the regulation of estrogen receptor alpha (ERα) gene expression is critical for understanding the developmental, as well as changes in postpubertal expression of the estrogen receptor. One mechanism of suppressing gene expression is by the epigenetic modification of the promoter regions by DNA methylation that results in gene silencing. The decrease in ERα mRNA expression during development is accompanied by an increase in promoter methylation. Another example of regulation of ERα gene expression in the adult cortex is the changes that occur following neuronal injury. Many animal studies have demonstrated that the endogenous estrogen, 17β-estradiol, is neuroprotective. Specifically, low levels of estradiol protect the cortex from neuronal death following middle cerebral artery occlusion (MCAO). In females, this protection is mediated through an ERα-dependent mechanism. ERα expression is rapidly increased following MCAO in females, but not in males. This increase is accompanied by a decrease in methylation of the promoter suggesting a return to the developmental program of gene expression within neurons. Taken together, during development and in adulthood, regulation of ERα gene expression in the cortex can occur by DNA methylation and in a sex-dependent fashion in the adult brain.