Original Contribution17β-Estradiol prevents cell death and mitochondrial dysfunction by an estrogen receptor-dependent mechanism in astrocytes after oxygen–glucose deprivation/reperfusion
Highlights
► Primary cultured brain astrocytes were deprived of oxygen and glucose in vitro. ► Estrogen protects from cell death via estrogen receptor α. ► Estrogen inhibits reactive oxygen species production. ► Estrogen preserves mitochondrial function and cell ATP. ► The protective actions of estrogen are mediated by estrogen receptors.
Introduction
The female sex hormone estrogen exerts profound neuroprotective effects against ischemic brain injury, but the targets and underlying mechanisms of estrogen-mediated protection remain obscure [1], [2]. In the past few decades, most efforts have been devoted to understanding how estrogen affects neurons in both in vitro and in vivo models, with less attention paid to astrocytes, the most abundant cell type in the brain [3]. Astrocytes function as the principal housekeeping cells of the central nervous system. They are dynamically involved in many important activities in brain, such as synaptic transmission, metabolic and ionic homeostasis, inflammatory response, antioxidant defense, structural and nutritive support of neurons, and formation and maintenance of the blood–brain barrier [4]. Because their end feet surround capillaries, astrocytes are the first cells to suffer ischemic insult among all types of brain cells [5], [6]. Astrocytes interact with neurons by cross talk, both physiologically and pathologically [4], [7]. Proper astrocyte function is particularly important for neuronal survival under ischemic conditions. Dysfunction of astrocytes resulting from ischemic insult significantly influences the responses of other brain cells to injury [7], [8]. It is believed that astrocytes are critical determinants in stroke pathophysiology [9]. Thus, it is of great importance and significance to understand the role of astrocytes in estrogen-mediated protection against ischemic injury.
Astrocytes have been shown to be the targets of estrogen and are postulated to play a key role in estrogen-mediated protection of the brain [10], [11]. 17β-Estradiol (E2), the predominant form of estrogen, exerts multiple regulatory actions on astrocytes including, but not limited to, regulating astrocytic morphology and function and modulating the release of neurotrophic factors and inflammatory molecules [12]. Moreover, astrocytes have the ability to synthesize estrogen. It has been shown that expression of the E2 synthetic enzyme aromatase and 17β-estradiol production are both increased in astrocytes under pathological conditions [13]. E2 also has been found to regulate the synthesis of other steroids in astrocytes, such as progesterone [14], [15]. Many studies indicate that it is astrocytes that mediate the protective actions of E2 by releasing tumor growth factor β-1 [16], [17]. E2 has been shown to regulate the expression of aquaporin-4 in parenchymal reactive astrocytes and perivascular glial processes, and this may specifically relate to regulation of brain injury from ischemic stroke [18]. The protective effects of E2 against ischemic damage induced by middle cerebral artery occlusion are most prominent in the cortex, implicating astrocytes as major targets for E2 protection against brain ischemic injury [19], [20]. Protective effects of E2 against ischemic insult in vitro also have been shown in astrocytes and cortical explant culture [21], [22]. However, the effects of E2 on astrocytes during ischemia are not completely understood.
One of the most important functions of astrocytes is energy support, largely depending on mitochondria. Mitochondria are unique organelles involved in energy production and cell life–death regulation. During mitochondrial energy production, an inevitable product, reactive oxygen species (ROS), is generated in the mitochondrial matrix [23]. It has been shown that mitochondrial energy production is decreased and ROS production is increased in brain cells under pathological conditions, which may account for the etiology and development of age-related diseases in central nervous system, including stroke [24]. Excessive ROS can affect mitochondrial enzymes, lipids, and DNA, causing deleterious effects leading to mitochondrial dysfunction. It has been suggested that the degree of mitochondrial impairment in cerebral ischemia may be a critical determinant of the final extent of neuronal injury [25].
Increasing evidence suggests that E2 regulates mitochondrial function, which may play a central role in the protective action of E2 [26], [27], [28]. We previously found that E2 decreases mitochondrial oxidative stress in cerebral blood vessels as well as in brain tissue under physiological conditions [29], [30], [31]. More recently, we found E2 to protect mitochondrial function in cerebral endothelial cells after ischemic insult in vitro [32]. In astrocytes, recent studies have shown that E2 influences mitochondrial gene expression and respiratory chain activity and regulates mitochondrial function [33], [34]. However, little is known about the effects of E2 on mitochondria in astrocytes during ischemia. To this end, this study was designed to investigate the impact of physiological levels of E2 on cell viability and mitochondrial function under ischemic-like conditions of oxygen–glucose deprivation (OGD)/reperfusion in primary cultured astrocytes. In addition, by using a specific estrogen receptor (ER) antagonist and agonists, we evaluated the specific role of ER in E2-mediated effects.
Section snippets
Primary astrocyte cultures
Primary cultures of mouse cerebral cortical astrocytes were prepared as described previously with few modifications [35]. Briefly, meninges-free cortices were collected from 1- to 3-day-old Swiss Webster mice. Cells were dispersed by mechanical and enzymatic dissociation using a solution containing 0.05% trypsin (Invitrogen, Carlsbad, CA, USA). The cells were then suspended in plating medium consisting of minimal essential medium (MEM; Gibco, Grand Island, NY, USA) containing 10% fetal bovine
E2 attenuates OGD/reperfusion-induced cell death in primary cultures of astrocytes
Astrocyte cell death is one of the common ultimate consequences under ischemic conditions where energy depletion and metabolic disruption are severe [7]. In this study, the model of OGD/reperfusion was applied in primary cultures of cortical astrocytes to simulate ischemic insult. Six hours of OGD with 24 h of reperfusion treatment resulted in obvious cell death in astrocytes; cell viability of vehicle-treated cells exposed to OGD/reperfusion was decreased by 36.5% compared to normoxic control
Discussion
Estrogen has proven protective properties against ischemic injury in animal models of stroke. However, the targets and the mechanisms of estrogen-mediated protective action still remain poorly understood. In this study, the effects of E2 on primary cultures of astrocytes after ischemic insult were examined in an ischemia-like in vitro model of oxygen and glucose deprivation. It was essential to select the time points at which the impact of OGD was clear, but not so great that any protective
Acknowledgments
This study was supported by the U.S. National Heart, Lung, and Blood Institute (R01 HL-50775) and the National Natural Science Foundation of China (81102424, 81020108031, 31079630, and 30973558). Jiabin Guo is the recipient of a doctoral scholarship from the China Scholarship Council (20073020). We thank Dr. Hongzheng Yin and Allan Jay Acab for technical help in cell culture and live imaging experiments. We also thank Dr. Douglas Wallace and the U.C. Irvine Center for Molecular and
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2019, Journal of Steroid Biochemistry and Molecular BiologyCitation Excerpt :Generally, the neuroprotective action of estradiol is due to genomic (with changes in gene expression) and non-genomic (activation of cellular signaling such as kinases) pathways via estrogen receptors. Various neuroprotective mechanisms of estradiol have been demonstrated: the upregulation of antiapoptotic proteins such as Bcl-2 and Bcl-w, the downregulation of proapoptotic proteins such as Bad and Bim [15,16], the elevation of the levels of brain-derived neurotrophic factor [17], the activation of the kinase cascade for survival (e.g., ERK and phosphoinositide-3-kinase/Akt) [18] and the protection of mitochondria via the attenuation of reactive oxygen species (ROS) [19]. Thus, estrogen in the brain is considered to be a strong endogenous neuroprotectant that can suppress diverse neuronal damages.
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2019, Free Radical Biology and MedicineCitation Excerpt :Our data also revealed that the ERα-dependent signaling cascade is an important control switch for redox modulation because E2 protects cells against ROS. Previous research revealed that E2 protects mitochondria against ROS but also decreases mitochondrial membrane potential and induces ATP depletion under ischemic conditions in which oxygen and glucose transfer were insufficient [38]. However, under high glucose conditions, our data identified that E2 through ERα translocation upregulates antioxidant enzymes that directly target high glucose-mediated ROS generation, finally decreasing autophagic cell death.