Temporal and region-specific tau hyperphosphorylation in the medulla and forebrain coincides with development of functional changes in male obese Zucker rats.

Metabolic syndrome (MetS) is associated with development of tauopathies that contribute to cognitive decline. Without functional leptin receptors, male obese Zucker rats (OZRs) develop MetS, and they have increased phosphorylated tau (ptau) with impaired cognitive function. In addition to regulating energy balance, leptin enhances activation of the hippocampus, which is essential for spatial learning and memory. Whether spatial learning and memory are always impaired in OZRs or develop with MetS is unknown. We hypothesized that male OZRs develop MetS traits that promote regional increases in ptau and functional deficits associated with those brain regions. In the medulla and cortex, tau-pSer199,202 and tau-pSer396 were comparable in juvenile (7-8 wk old) lean Zucker rats (LZRs) and OZRs but increased in 18- to 19-wk-old OZRs. Elevated tau-pSer396 was concentrated in the dorsal vagal complex of the medulla, and by this age OZRs had hypertension with increased arterial pressure variability. In the hippocampus, tau-pSer199,202 and tau-pSer396 were still comparable in 18- to 19-wk-old OZRs and LZRs but elevated in 28- to 29-wk-old OZRs, with emergence of deficits in Morris water maze performance. Comparable escape latencies observed during acquisition in 18- to 19-wk-old OZRs and LZRs were increased in 28- to 29-wk-old OZRs, with greater use of nonspatial search strategies. Increased ptau developed with changes in the insulin/phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway in the hippocampus and cortex but not medulla, suggesting different underlying mechanisms. These data demonstrate that leptin is not required for spatial learning and memory in male OZRs. Furthermore, early development of MetS-associated autonomic dysfunction by the medulla may be predictive of later hippocampal dysfunction and cognitive impairment.NEW & NOTEWORTHY Male obese Zucker rats (OZRs) lack functional leptin receptors and develop metabolic syndrome (MetS). At 16-19 wk, OZRs are insulin resistant, with increased ptau in dorsal medulla and impaired autonomic regulation of AP. At 28-29 wk OZRs develop increased ptau in hippocampus with deficits in spatial learning and memory. Juvenile OZRs lack elevated ptau and these deficits, demonstrating that leptin is not essential for normal function. Elevated ptau and deficits emerge before the onset of diabetes in insulin-resistant OZRs.

Long-term hypogonadism diminishes the neuroprotective effects of dietary genistein in young adult ovariectomized rats after transient focal ischemia.

Increasing age disproportionately increases the risk of stroke among women compared to men of similar age, especially after menopause. One of the reasons for this observation is a sharp drop in circulating estrogens. However, the timing of initiation of estrogen replacement after menopause is associated with mixed beneficial and detrimental effects, hence contributing to widespread mistrust of estrogen use. Agents including soy isoflavones are being assessed as viable alternatives to estrogen therapy. In this study, we hypothesized that the neuroprotective effects of genistein, a soy isoflavone are less sensitive to the length of hypogonadism in young adult ovariectomized rats following cerebral ischemia. We expected that long-term hypogonadism will worsen motor and cognitive function, increase post-stroke inflammation with no effect on the neuroprotection of genistein. We compared the effect of treatment with dietary genistein (GEN) on short-term (2 weeks) and long-term hypogonadism (12 weeks) in young adult ovariectomized Sprague-Dawley rats on sensorimotor function, cognition and inflammation after focal ischemia. Dorsal Silastic implant of 17ß-estradiol (E2) was used as a control for hormone therapy. Long-term hypogonadism stroked rats performed worse than the short-term hypogonadism stroked rats on the motor and cognitive function tests. GEN did not improve neurological assessment and motor learning after either short-term or long-term hypogonadism. GEN improved cognitive flexibility after short-term hypogonadism but not after the long-term. Both GEN and E2 reduced tissue loss after short-term hypogonadism and reduced GFAP expression at the contralateral side of ischemia after long-term hypogonadism. The length of hypogonadism may differentially influence the neuroprotective effects of both GEN and E2 on the motor and cognitive functions in young adult rats.

Dietary genistein and 17ß-estradiol implants differentially influence locomotor and cognitive functions following transient focal ischemia in middle-aged ovariectomized rats at different lengths of estrogen deprivation.

Genistein possesses estrogenic activity and has been considered a potential replacement for estrogen replacement therapy after menopause. In the current study, we investigated the neuroprotective effects of dietary genistein at varied lengths of estrogen deprivation in middle-aged ovariectomized Sprague-Dawley rats under ischemic conditions. Two weeks of treatment with dietary genistein at 42 mg/kg but not 17ß-estradiol implants improved cognitive flexibility (Morris water maze test) after short-term estrogen deprivation (2 weeks) but not long-term estrogen deprivation (12 weeks). 17ß-estradiol implants but not dietary genistein improved locomotor asymmetry (cylinder test) after long-term but not short-term estrogen deprivation. Dietary genistein but not 17ß-estradiol implant improved early phase motor learning (rotarod test) after long-term estrogen deprivation. Neither 17ß-estradiol implant nor dietary genistein reduced infarct size after either short-term or long-term estrogen deprivation. Genistein, however, reduced ionized calcium-binding adaptor molecule-1 (Iba1) expression, a marker of brain inflammation, at the ipsilateral side of stroke injury after short-term but not long-term estrogen deprivation. This study suggests that the neuroprotective effects of dietary genistein on motor and cognitive functions are distinctly influenced by the length of estrogen deprivation following focal ischemia. SIGNIFICANCE: There is an increasing postmenopausal population opting for homeopathic medicines for the management of menopausal symptoms due to the perceived distrust in estrogen use as hormone replacement. Basic and clinical studies support the notion that early, but not delayed, hormone replacement after menopause is beneficial. Furthermore, evidence suggests that delaying hormone replacement augments the detrimental, rather than the beneficial effects of estrogens. Because of the active consideration of soy isoflavones including genistein as alternatives to estrogen replacement, it is necessary to understand the ramifications of soy isoflavones use when their administration is begun at various times after menopause.

Chronic Testosterone Deprivation Sensitizes the Middle-Aged Rat Brain to Damaging Effects of Testosterone Replacement.

INTRODUCTION: An increasing number of middle-aged men are being screened for low testosterone levels and the number of prescriptions for various forms of testosterone replacement therapy (TRT) has increased dramatically over the last 10 years. However, the safety of TRT has come into question with some studies suggesting increased morbidity and mortality. OBJECTIVE: Because the benefits of estrogen replacement in postmenopausal women and ovariectomized rodents are lost if there is an extended delay between estrogen loss and replacement, we hypothesized that TRT may also be sensitive to delayed replacement. METHODS: We compared the effects of testosterone replacement after short-term (2 weeks) and long-term testosterone deprivation (LTTD; 10 weeks) in middle-aged male rats on cerebral ischemia, oxidative stress, and cognitive function. We hypothesized that LTTD would increase oxidative stress levels and abrogate the beneficial effects of TRT. RESULTS: Hypogonadism itself and TRT after short-term castration did not affect stroke outcome compared to intact rats. However, after long-term hypogonadism in middle-aged male Fischer 344 rats, TRT exacerbated the detrimental behavioral effects of experimental focal cerebral ischemia, whereas this detrimental effect was prevented by administration of the free-radical scavenger tempol, suggesting that TRT exacerbates oxidative stress. In contrast, TRT improved cognitive performance in non-stroked rats regardless of the length of hypogonadism. In the Morris water maze, peripheral oxidative stress was highly associated with decreased cognitive ability. CONCLUSIONS: Taken together, these data suggest that TRT after long-term hypogonadism can exacerbate functional recovery after focal cerebral ischemia, but in the absence of injury can enhance cognition. Both of these effects are modulated by oxidative stress levels.

Genistein: mechanisms of action for a pleiotropic neuroprotective agent in stroke.

Genistein is a plant estrogen promoted as an alternative to post-menopausal hormone therapy because of a good safety profile and its promotion as a natural product. Several preclinical studies of cerebral ischemia and other models of brain injury support a beneficial role for genistein in protecting the brain from injury whether administered chronically or acutely. Like estrogen, genistein is a pleiotropic molecule that engages several different mechanisms to enhance brain health, including reduction of oxidative stress, promotion of growth factor signaling, and immune suppression. These actions occur in endothelial, glial, and neuronal cells to provide a coordinated beneficial action to ischemic challenge. Though many of these protective actions are associated with estrogen-like actions of genistein, additional activities on other receptors and intracellular targets suggest that genistein is more than a mere estrogen-mimic. Importantly, genistein lacks some of the detrimental effects associated with post-menopausal estrogen treatment and may provide an alternative to hormone therapy in those patients at risk for ischemic events.

Delayed puberty and estrogen resistance in a woman with estrogen receptor α variant.

Although androgen resistance has been characterized in men with a normal chromosome complement and mutations in the androgen-receptor gene, a mutation in the gene encoding estrogen receptor α (ESR1) was previously described only in one man and not, to our knowledge, in a woman. We now describe an 18-year-old woman without breast development and with markedly elevated serum levels of estrogens and bilateral multicystic ovaries. She was found to have a homozygous loss-of-function ESR1 mutation in a completely conserved residue that interferes with estrogen signaling. Her clinical presentation was similar to that in the mouse orthologue knockout. This case shows that disruption of ESR1 causes profound estrogen resistance in women. (Funded by the National Institutes of Health.).

Treatment of Stroke at a Delayed Timepoint with a Repurposed Drug Targeting Sigma 1 Receptors.

Sigma 1 receptors are intracellular chaperone proteins that have been explored as a subacute treatment to enhance post-stroke recovery. We recently identified the antitussive oxeladin as a selective sigma 1 receptor agonist with the ability to stimulate the release of brain-derived neurotrophic factor from neurons in vitro. In this study, we hypothesized that oral oxeladin citrate would stimulate BDNF secretion and improve stroke outcomes when administered to male rats starting 48 h after transient middle cerebral artery occlusion. Oxeladin did not alter blood clotting and crossed the blood brain barrier within 30 min of oral administration. Rats underwent 90 min of transient middle cerebral artery occlusion. Forty-eight hours later rats began receiving daily oxeladin (135 mg/kg) for 11 days. Oxeladin significantly improved neurological function on days 3, 7, and 14 following MCAO. Infarct size was not altered by a single dose, but the final extent of infarct after 14 days was decreased. However, there was no significant reduction in astrogliosis or microgliosis compared to vehicle-treated control rats. In agreement with in vitro studies, oxeladin increased the amount of mature BDNF in the cerebral cortex 2, 6, and 24 h after single oral dose. However, the increase in BDNF did not result in increases in cellular proliferation in the subventricular zone or dentate gyrus when compared to vehicle-treated controls. These results suggest that oxeladin may reduce the extent of infarct expansion in the subacute phase of stroke, although this action does not appear to involve a reduction in inflammation or increased cell proliferation.

Brain-derived neurotrophic factor for high-throughput evaluation of selective Sigma-1 receptor ligands.

The Sigma-1 receptor (S1R) is an endoplasmic reticulum (ER) chaperone protein that has been implicated in attenuating inflammatory stress-mediated brain injuries. Selective S1R agonists represent a new class of therapeutic agent for treating neuropsychiatric and neurodegenerative disorders, however, to date, no S1R ligand has been approved for therapeutic purposes. We used three potential methods on known and potential S1R ligands to develop an unambiguous high-throughput cell screen for S1R activity. We screened known and potential S1R ligands using radioligand binding and previously reported markers of S1R activity including BDNF release, modulation of IP3 mediated calcium release, and modulation of NGF-induced neurite sprouting. Here, we present results several prototypical S1R compounds and some compounds with the potential for drug repurposing. Using an in-situ ELISA approach we demonstrated that these compounds could stimulate S1R-mediated BDNF release, which is a valuable therapeutic property since BDNF plays a critical role in neuronal support. These compounds were classified as S1R agonists because the BDNF response was comparable to the prototypical agonist 4-PPBP and because it could be reversed by a S1R selective concentration of the antagonist BD1063. When modulation of IP3 mediated calcium response and NGF-induced neurite sprouting were used as a measure of S1R activation, we were unable to reproduce the published results and determined that they are not reliable measures for evaluating functional properties of S1R ligands.

Soluble epoxide inhibition is protective against cerebral ischemia via vascular and neural protection.

Inhibition of soluble epoxide hydrolase (SEH), the enzyme responsible for degradation of vasoactive epoxides, protects against cerebral ischemia in rats. However, the molecular and biological mechanisms that confer protection in normotension and hypertension remain unclear. Here we show that 6 weeks of SEH inhibition via 2 mg/day of 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA) in spontaneously hypertensive stroke-prone (SHRSP) rats protects against cerebral ischemia induced by middle cerebral artery occlusion, reducing percent hemispheric infarct and neurodeficit score without decreasing blood pressure. This level of cerebral protection was similar to that of the angiotensin-converting enzyme inhibitor, enalapril, which significantly lowered blood pressure. SEH inhibition is also protective in normotensive Wistar-Kyoto (WKY) rats, reducing both hemispheric infarct and neurodeficit score. In SHRSP rats, SEH inhibition reduced wall-to-lumen ratio and collagen deposition and increased cerebral microvessel density, although AUDA did not alter middle cerebral artery structure or microvessel density in WKY rats. An apoptosis mRNA expression microarray of brain tissues from AUDA-treated rats revealed that AUDA modulates gene expression of mediators involved in the regulation of apoptosis in neural tissues of both WKY and SHRSP rats. Hence, we conclude that chronic SEH inhibition protects against cerebral ischemia via vascular protection in SHRSP rats and neural protection in both the SHRSP and WKY rats, indicating that SEH inhibition has broad pharmacological potential for treating ischemic stroke.

Dietary genistein and equol (4', 7 isoflavandiol) reduce oxidative stress and protect rats against focal cerebral ischemia.

High soy diets reduce injury in rat models of focal cerebral ischemia and are proposed as alternatives to hormone replacement therapy for postmenopausal women. The present study tests the hypothesis that the major soy isoflavone genistein and the daidzein metabolite equol are neuroprotective in transient focal cerebral ischemia in male and ovariectomized (OVX) female rats by inhibiting oxidative stress. Genistein is the primary circulating soy isoflavone in humans, whereas equol is the primary circulating isoflavone in rats. Male and OVX female Sprague-Dawley rats were fed an isoflavone-reduced diet alone or supplemented with genistein (500 ppm) or equol (250 ppm) for 2 wk prior to 90-min transient middle cerebral artery occlusion followed by reperfusion under isoflurane anesthesia. Indices of oxidative stress were determined 24 h after reperfusion, and cerebral injury was evaluated 3 days after reperfusion. Genistein and equol significantly reduced infarct size in both sexes. Further studies in OVX female rats revealed that this neuroprotection was accompanied by a decrease in NAD(P)H oxidase activity and superoxide levels in the brain. In addition, equol reduced plasma thiobarbituric acid reactive substances, and neurological deficits up to 7 days after injury. There were no significant differences in cerebral blood flow among treatment groups. In conclusion, dietary soy isoflavones are neuroprotective in transient focal cerebral ischemia in male and OVX female rats. These isoflavones may protect the brain via increases in endogenous antioxidant mechanisms and reduced oxidative stress.

The role of aldosterone in mediating the dependence of angiotensin hypertension on IL-6.

Knockout (KO) of IL-6 has been shown to attenuate ANG II hypertension, and mineralocorticoid receptors (MR) have been reported to contribute to the increase in IL-6 during acute ANG II infusion. This study determined whether that MR action is sustained with chronic ANG II infusion and whether it plays a role in mediating ANG II hypertension. ANG II infusion (90 ng/min) increased plasma IL-6 from 1.6 +/- 0.6 to 22.7 +/- 2.2 and 19.9 +/- 3.2 pg/ml on days 7 and 14, respectively, and chronic MR blockade with spironolactone attenuated that only at day 7 (7.2 +/- 2.2 pg/ml). ANG II increased MAP (19 h/day with telemetry) approximately 40 mmHg, but in ANG II+spironolactone mice (25 or 50 mg*kg(-1)*day(-1)), mean arterial pressure (MAP) was not significantly different despite a tendency for lower pressure the first 6 days. To isolate further the mineralocorticoid link to IL-6 and blood pressure, DOCA-salt hypertension was induced in IL-6 KO and wild-type (WT) mice. Plasma IL-6 increased from 4.1 +/- 1.7 to 34.5 +/- 7.0 pg/ml by day 7 of DOCA treatment in the WT mice but was back to control levels by day 14. An IL-6 bioassay using the murine B9, B-cell hybridoma cell line demonstrated that plasma IL-6 measurements reflected actual IL-6 bioactivity. The hypertension was not different and virtually superimposable in WT vs. IL-6 KO mice, averaging 145 +/- 2 and 144 +/- 3 mmHg, respectively. Both experiments confirm chronic stimulation of IL-6 by mineralocorticoids but show that it is transient. In addition, IL-6 was not required for mineralocorticoid hypertension. This suggests that aldosterone contributes to the increase in plasma IL-6 in the early stage of ANG II hypertension but that the blood pressure actions of IL-6 in that model are linked most likely to ANG II rather than aldosterone.

Protective effect of estrogen in endothelin-induced middle cerebral artery occlusion in female rats.

Estrogen is a powerful endogenous and exogenous neuroprotective agent in animal models of brain injury, including focal cerebral ischemia. Although this protection has been demonstrated in several different treatment and injury paradigms, it has not been demonstrated in focal cerebral ischemia induced by intraparenchymal endothelin-1 injection, a model with many advantages over other models of experimental focal ischemia. Reproductively mature female Sprague-Dawley rats were ovariectomized and divided into placebo and estradiol-treated groups. Two weeks later, halothane-anesthetized rats underwent middle cerebral artery (MCA) occlusion by interparenchymal stereotactic injection of the potent vasoconstrictor endothelin 1 (180pmoles/2microl) near the middle cerebral artery. Laser-Doppler flowmetry (LDF) revealed similar reductions in cerebral blood flow in both groups. Animals were behaviorally evaluated before, and 2 days after, stroke induction, and infarct size was evaluated. In agreement with other models, estrogen treatment significantly reduced infarct size evaluated by both TTC and Fluoro-Jade staining and behavioral deficits associated with stroke. Stroke size was significantly correlated with LDF in both groups, suggesting that cranial perfusion measures can enhance success in this model.

N-terminal truncations in sex steroid receptors and rapid steroid actions.

Sex steroid receptors act as ligand activated nuclear transcription factors throughout the body, including the brain. However, post-translational modification of these receptors can direct them to extranuclear sites, including the plasma membrane, where they are able to initiate rapid signaling. Because of the conserved domain structure of these receptors, alternative exon splicing can result in proteins with altered nuclear and extranuclear actions. Although much attention has focused on internal and C-terminal splice variants, both estrogen and androgen receptors undergo N-terminal truncations, as well. These truncated proteins not only influence the transcriptional activity of the full-length receptors, but also associate with caveolin and initiate signaling at the plasma membrane. Such actions may have important physiological consequences in neuronal, endothelial, and cancer signaling and cell survival.

Chronic intermittent hypoxia induces hormonal and male sexual behavioral changes: Hypoxia as an advancer of aging.

Sleep apnea is a common sleep disorder characterized by intermittent periods of low blood oxygen levels. The risk for sleep apnea increases with age and is more prevalent in men than women. A common comorbidity of sleep apnea includes male sexual dysfunction, but it is not clear if a causal relationship exists between sleep apnea and sexual dysfunction. Possible mechanisms that link these two disorders include oxidative stress and testosterone. Oxidative stress is elevated in clinical patients with sleep apnea and in rodents exposed to chronic intermittent hypoxia (CIH), an animal model for apnea-induced hypopnea. Further, oxidative stress levels increase with age. Therefore, age may play a role in sleep apnea-induced sexual dysfunction and oxidative stress generation. To investigate this relationship, we exposed gonadally intact 3 (young) and 12 (middle-aged) month old male F344/BN F1 hybrid male rats to 8 days of CIH, and then examined male sexual function. Plasma was used to assess circulating oxidative stress and hormone levels. Middle-aged male rats had lower testosterone levels with increased sexual dysfunction and oxidative stress, independent of CIH. However, CIH decreased testosterone levels and increased sexual dysfunction and oxidative stress only in young gonadally intact male rats, but not in gonadectomized young rats with physiological testosterone replacement. In sum, CIH had a greater impact on younger gonadally intact animals, with respect to sexual behaviors, testosterone, and oxidative stress. Our data indicate CIH mimics the effects of aging on male sexual behavior in young gonadally intact male rats.

A high soy diet enhances neurotropin receptor and Bcl-XL gene expression in the brains of ovariectomized female rats.

Estrogen is a powerful neuroprotective agent with the ability to induce trophic and antiapoptotic genes. However, concerns about negative overall health consequences of estrogen replacement after menopause have led to the adoption of other strategies to obtain estrogen's benefits in the brain, including the use of selective estrogen receptor modulators, high soy diets, or isoflavone supplements. This study sought to determine the ability of a high soy diet to induce neuroprotective gene expression in the female rat brain and compare the actions of soy with estrogen. Adult ovariectomized female rats were treated with 3 days of high dose estrogen or 2 weeks of a soy-free diet, a high soy diet, or chronic low dose estrogen. Different brain regions were microdissected and subjected to real time RT-PCR for neuroprotective genes previously shown to be estrogen-regulated. The principle findings are that a high soy diet led to the widespread increase in the mRNA for neurotropin receptors TrkA and p75-NTR, and the antiapoptotic Bcl-2 family member Bcl-X(L). Immunohistochemistry confirmed increases in both TrkA and Bcl-X(L). Chronic low dose estrogen mimicked some of these effects, but acute high dose estrogen did not. The effects of a high soy diet were particularly evident in the parietal cortex and hippocampus, two regions protected by estrogen in animal models of neurological disease and injury. These results suggest that a high soy diet may provide beneficial effects to the brain similar to low dose chronic estrogen treatment such as that used for postmenopausal hormone replacement.

Presence of Androgen Receptor Variant in Neuronal Lipid Rafts.

Fast, nongenomic androgen actions have been described in various cell types, including neurons. However, the receptor mediating this cell membrane-initiated rapid signaling remains unknown. This study found a putative androgen receptor splice variant in a dopaminergic N27 cell line and in several brain regions (substantia nigra pars compacta, entorhinal cortex, and hippocampus) from gonadally intact and gonadectomized (young and middle-aged) male rats. This putative splice variant protein has a molecular weight of 45 kDa and lacks an N-terminal domain, indicating it is homologous to the human AR45 splice variant. Interestingly, AR45 was highly expressed in all brain regions examined. In dopaminergic neurons, AR45 is localized to plasma membrane lipid rafts, a microdomain involved in cellular signaling. Further, AR45 protein interacts with membrane-associated G proteins Gαq and Gαo. Neither age nor hormone levels altered AR45 expression in dopaminergic neurons. These results provide the first evidence of AR45 protein expression in the brain, specifically plasma membrane lipid rafts. AR45 presence in lipid rafts indicates that it may function as a membrane androgen receptor to mediate fast, nongenomic androgen actions.

Transcriptional regulation by phytoestrogens in neuronal cell lines.

Widespread epidemiological data support the notion that high isoflavone intake is safe and may provide health benefits similar to estrogen. Evidence from rodents shows that certain phytoestrogens can act as estrogen receptor (ER) ligands in the brain. This study sought to determine the estrogenic profile of food-borne phytoestrogens in neuronal cell lines using physiologically attainable concentrations. At sub-micromolar concentrations genistein, daidzein, and zearalenone stimulated ERalpha and ERbeta-dependent transcription in Neuro2A cells co-transfected with ERs and simple and complex estrogen-response-element (ERE) containing promoters, although compounds were more active in the presence of ERbeta. In SN56, neuronblastoma cells expressing endogenous ERs, only genistein mimicked estrogen regulation of progesterone receptor steady state mRNA levels. Unlike pharmaceutical SERMs, phytoestrogens did not stimulate an AP-1-dependent promoter. Micromolar concentrations of phytoestrogens did not antagonize physiological estrogen concentrations or antagonist activation of an AP-1-dependent promoter. These results demonstrate that food-borne phytoestrogens, particularly those found in soy, act as ERE-, but not AP-1-dependent transcriptional activators in neurons in the absence of estrogen, and dietary levels of these compounds do not act as antagonists to physiological estrogen concentrations.

Estrogen receptor-alpha (ERalpha), but not ERbeta, modulates estrogen stimulation of the ERalpha-truncated variant, TERP-1.

Estrogens regulate pituitary gene expression through two nuclear receptors (ERs), ERalpha and ERbeta. Rodent pituitary also expresses high levels of the pituitary-specific ERalpha isoform, truncated ER product-1 (TERP-1), which modulates the response of both ER forms to 17beta-estradiol (E2). Under physiological conditions, E2 stimulates TERP-1 expression from an ERalpha intronic promoter containing several potential binding sites for ERs. To evaluate the role of intact ER proteins on TERP-1 expression, we measured basal expression and steroid stimulation of TERP-1 in wild-type (WT) mice and mice in which either the ERalpha (ERalphaKO) or the ERbeta (ERbetaKO) gene was disrupted. TERP-1 mRNA expression was assessed by semiquantitative RT-PCR, and protein expression was evaluated by immunoblots. Both TERP-1 mRNA and protein were expressed in pituitaries from castrate WT, ERalphaKO, and ERbetaKO male and female mice. E2 stimulated TERP-1 mRNA expression in WT and ERbetaKO mice of both sexes, but had no effect on TERP-1 mRNA in either male or female ERalphaKO mice. Testosterone treatment also stimulated TERP-1 in WT, ERalphaKO, and ERbetaKO male mice. We conclude that ERalpha is critical for E2 stimulation, but not basal expression, of the TERP promoter, and that testosterone may act through the androgen receptor to stimulate the TERP-1 promoter in males.

Central inflammatory response to experimental stroke is inhibited by a neuroprotective dose of dietary soy.

Dietary soy and soy isoflavones are neuroprotective in experimental cerebral ischemia. Because the isoflavones in soy that are responsible for this neuroprotective effect act as phytoestrogens, we hypothesized that they would mimic the beneficial effects of estrogens on the innate inflammatory response to cerebral ischemia. Ovariectomized Sprague-Dawley rats were fed a soy free diet or a diet containing high dietary levels of soy for 5 weeks, after which they were subjected to transient middle cerebral artery occlusion (tMCAO) for 90min. Dietary soy was associated with a reduced inflammatory response in the cerebral cortex during the acute innate period 4 and 24h after tMCAO, including significant (>2-fold) reductions in interleukins 1 beta, 2, and 13, and the chemokine CXCL1. However, there was no effect of soy on tumor necrosis factor-alpha or interferon-gamma. Dietary soy was also associated with a 40 percent reduction in the nuclear translocation of p65 nuclear factor kappa B despite an increase in the expression of p65 RELA mRNA. In support of an early effect on the innate immune response to stroke, soy-fed rats had 44 percent fewer activated microglia in the infarct core than soy free rats. Interestingly, despite increased expression following injury, the steady state mRNA levels of inflammatory factors were not altered in soy-fed rats even though inflammatory proteins were. These data suggest that dietary soy isoflavones, like estrogens, inhibit of the innate immune response to injury. However, post-transcriptional mechanisms may play an important role in the mechanism of this action. Coupled with previously published data, these results support an early and rapid effect of dietary soy on the evolution of brain injury following stroke.