Derek A. Schreihofer

Estrogen receptors and ischemic neuroprotection: who, what, where, and when?

Estrogens, particularly 17β-estradiol (E2), are powerful neuroprotective agents in animal models of cerebral ischemia. Loss of endogenous E2 in women at menopause or after surgical oopherectomy leads to an increase risk of stroke, neurodegenerative disease, and cognitive decline. However, several clinical trials found detrimental effects of E2 therapy after menopause, including increased stroke risk and dementia. Recent animal and human studies now support the critical period hypothesis for E2 neuroprotection whereby E2 therapy must begin soon after the loss of endogenous E2 production to have a beneficial effect. Although a wide array of mechanisms has been proposed for estradiol (E2)-dependent neuroprotection in cerebral ischemia and neurodegenerative disease, most of these mechanisms involve interactions of E2 with one of its cognate receptors, estrogen receptor alpha (ERα), estrogen receptor beta (ERβ), or the G protein-coupled estrogen receptor (GPER). However, these receptors are not uniformly distributed throughout the brain, across different cell types, and within cellular compartments. Such differences likely play a role in the ability of E2 and ER selective ligands to protect the brain from ischemia. This review examines the changes in ER expression and location that may underlie the loss of E2 neuroprotection seen with aging and long-term estrogen deprivation (LTED). Recent results suggest that the loss of ERα that accompanies aging and LTED plays an important role in the loss of E2-dependent neuroprotection. This article is part of a Special Issue entitled Hormone Therapy.

Cell Models for the Study of Sex Steroid Hormone Neurobiology.

To date many aspects of neurons and glia biology remain elusive, due in part to the cellular and molecular complexity of the brain. In recent decades, cell models from different brain areas have been established and proven invaluable toward understanding this complexity. In the field of steroid hormone neurobiology, an important question is: what is the profile of steroid hormone receptor expression in these specific cell lines? Currently, a clear summary of such receptor profiling is lacking. For this reason, we summarized in this review the expression of estrogen, progesterone, and androgen receptors in several widely used cell lines (glial and neuronal) derived from the forebrain and midbrain, based on our own data and that from the literature. Such information will aid in the selection of specific cell lines used to test hypotheses related to the biology of estrogens, progestins, and/or androgens.

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.

Presence of Androgen Receptor Variant in Neuronal Lipid Rafts

Abstract 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.

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 8u202fdays 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.

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

HighlightsSex steroid receptors undergo numerous alternative splicing events.N‐terminal deletions result in receptors that can inhibit transcription from full‐length receptors.N‐terminal deleted receptors also localize to the plasma membrane and initiate rapid signaling.N‐terminal deleted receptors may play a role in cell survival. &NA; 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.

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. GRAPHICAL ABSTRACT

Duration of isoflurane-based surgical anesthesia determines severity of brain injury and neurological deficits after a transient focal ischemia in young adult rats

Tremendous efforts and funds invested in discovery of novel drug treatments for ischemic stroke have so far failed to deliver clinically efficacious therapies. The reasons for these failures are not fully understood. An indiscriminate use of isoflurane-based surgical anesthesia with or without nitrous oxide may act as an unconstrained, untraceable source of data variability, potentially causing false-positive or false-negative results. To test this hypothesis, a common transient suture middle cerebral artery occlusion (tMCAO) model of ischemic stroke in young adult male rats was used to determine the impact of a typical range of anesthesia durations required for this model on data variability (i.e., infarct volume and neurological deficits). The animals were maintained on spontaneous ventilation. The study results indicated that: (1) Variable duration of isoflurane anesthesia prior, during and after tMCAO is a significant source of data variability as evidenced by measurements of infarct volume and neurological deficits; and (2) Severity of brain injury and neurological deficits after tMCAO is inversely related to the duration of isoflurane anesthesia: e.g., in our study, a 90min isoflurane anesthesia nearly completely protected brain tissues from tMCAO-induced injury and thus, would be expected to obscure the effects of stroke treatments in pre-clinical trials. To elevate transparency, rigor and reproducibility of stroke research and minimize undesirable effects of isoflurane on the outcome of novel drug testing, we propose to monitor, minimize and standardize isoflurane anesthesia in experimental surgeries and make anesthesia duration a required reportable parameter in pre-clinical studies. Specifically, we propose to adopt 20-30min as an optimal anesthesia duration that both minimizes neuroprotective effects of isoflurane and permits a successful completion of surgical procedures in a suture tMCAO model of ischemic stroke in rodents. As the mechanisms and neuroprotective, metabolic and immune effects of general anesthesia are not fully understood, the results of this study cannot be blindly generalized to other anesthetics, animal species and experimental models.

Neuroprotection by Dietary Isoflavones and Their Role in Cerebral Ischemia

Abstract Dietary isoflavones derived from soybeans and other legumes have long been part of the Asian diet and are considered a safe, healthy protein source. More recently, the estrogenic nature of isoflavones has been explored for postmenopausal symptoms in women with equivocal results. However, the beneficial effects of dietary isoflavones in the brain have received new attention with the recognition that these compounds can be neuroprotective in experimental cerebral ischemia and other models of neurodegenerative diseases, such as Alzheimer’s disease. Several overlapping mechanisms likely underlie these protective mechanisms, including transcriptional activation by estrogen receptors and multiple antioxidant effects on the mitochondria. These generalized mechanisms support all members of the neurovascular unit and may provide both prophylactic and acute neuroprotection. Human studies support a role for soy isoflavones in cardiovascular health, but the neuroprotective properties of isoflavones in humans are yet to be elucidated.