DOI 10.1515/revneuro-2013-0055      Rev. Neurosci. 2014; 25(2): 255–268

Cuifen Wang, Chao Jie and Xiaoniu Dai*

Possible roles of astrocytes in estrogen neuroprotection during cerebral ischemia Abstract: 17β-Estradiol (E2), one of female sex hormones, has well-documented neuroprotective effects in a variety of clinical and experimental disorders of the central cerebral ischemia, including stroke and neurodegenerative diseases. The cellular mechanisms that underlie these protective effects of E2 are uncertain because a number of different cell types express estrogen receptors in the central nervous system. Astrocytes are the most abundant cells in the central nervous system and provide structural and nutritive support of neurons. They interact with neurons by cross-talk, both physiologically and pathologically. Proper astrocyte function is particularly important for neuronal survival under ischemic conditions. Dysfunction of astrocytes resulting from ischemia significantly influences the responses of other brain cells to injury. Recent studies demonstrate that estrogen receptors are expressed in astrocytes, indicating that E2 may exert multiple regulatory actions on astrocytes. Cerebral ischemia induced changes in the expression of estrogen receptors in astrocytes. In the present review, we summarize the data in support of possible roles for astrocytes in the mediation of neuroprotection by E2 against cerebral ischemia. Keywords: astrocytes; cerebral ischemia; estrogen receptors; neuroprotection; 17β-estradiol. *Corresponding author: Xiaoniu Dai, Department of Physiology, Medical School of Southeast University, Nanjing, Jiangsu Province, China, e-mail: [email protected] Cuifen Wang: Department of Physiology, Medical School of Southeast University, Nanjing, Jiangsu Province, China; and Center for Diagnostic Nanosystems, Marshall University, Huntington, WV 25755, USA Chao Jie: Department of Physiology, Medical School of Southeast University, Nanjing, Jiangsu Province, China

Introduction Stroke affects 15 million people worldwide each year and is the leading cause of disability in the US. A number of studies have documented that women are ‘protected’ against stroke relative to men, at least until the years of menopause, when estrogen levels decline due

to follicular depletion and stroke incidence increases in women (Roquer et  al., 2003; Murphy et  al., 2004). Ischemic stroke occurs with greater frequency in men vs. women regardless of country of origin and ethnic culture (Lang and McCullough, 2008). The underlying mechanisms involved in these sex differences remain unclear, but exposure to gonadal hormones, particularly estrogen, has been thought to play a major role (McCullough et al., 2005; Lang and McCullough, 2008). The gonadal steroid 17β-estradiol (E2), one kind of estrogen, is known to be a female sex hormone (Dai et al., 2007). The biological effects of E2 on the reproductive axis have been established for a long time. Over the past 30 years, biomedical science has begun to appreciate that E2 and their receptors display important roles beyond the reproductive system and acts on the central nervous system (CNS), cardiovascular health, the immune system, the adipose/metabolic system, bone, and mineral metabolism (Turgeon et al., 2006; Brown et al., 2009). Numerous studies have shown that E2 exerts powerful neuroprotective actions on multiple brain regions, including the cerebral cortex, hippocampus, striatum, basal forebrain, and cerebellum (Brown et al., 2009). E2 has also demonstrated a variety of actions on the brain by regulating spine density (Gould et al., 1990), synaptic number, and synthesis of neurotrophic factors (Sohrabji et al., 1995; Smith and McMahon, 2006; Morissette et al., 2008). In addition to its well-documented neuroregulatory effects, clinical investigations have provided the evidences that postmenopausal women are more vulnerable than young women to neurodegenerative diseases such as Alzheimer’s (Boada et al., 2012; Mateos et al., 2012) and Parkinson’s (Al Sweidi et al., 2012; Bourque et  al., 2012) diseases, cerebral ischemic injury such as stroke, and memory or cognitive dysfunctions. Studies on sex differences in stroke in humans are few, focusing primarily on incidence, age of first stroke, and outcome. Astrocytes in the mammalian brain are not only important supporters of neurons but also actively involved and required for effective function of the CNS (Somjen, 1988). They are critical to synaptic transmission, regulation of neural immune responses, structural and nutritive support of neurons, and neuronal survival (Pellerin and Magistretti, 2004; Takano et  al., 2009; Tasker et  al.,

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256      C. Wang et al.: Estrogen, astrocytes and neuroprotection 2012). Recent evidences show that astrocytes are targets for estrogen action (Arevalo et al., 2010; Kuo et al., 2010), as reflected by the fact that astrocytes express both estrogen receptor α (ERα) and estrogen receptor β (ERβ) on their plasma membranes (mERs) and in cytosol (Chaban et al., 2004; Garcia-Ovejero et al., 2005). Activation of the mER initiates a rapid, free cytoplasmic calcium concentration ([Ca2+]) flux via the phospholipase C/inositol trisphosphate pathway (Chaban et  al., 2004). Recently, the transmembrane ER, G protein-coupled ER (GPR) 30, was reported to mediate nongenomic and rapid signaling in astrocytes (Kuo et al., 2010), contributing to the neuroprotective effects of E2. In the past few decades, most efforts have been devoted to understand how E2 affects neurons in both in vitro and in vivo models, with attentions paid to astrocytes, the most abundant cell type in the brain. This review provides a brief overview of the neuroprotective effect of E2 on cerebral ischemia, with a specific focus on the emerging role of E2 action in astrocytes.

Neuroprotective action of estrogen during cerebral ischemia Several reports have investigated the prevalence of stroke and documented that stoke increases with age and is higher in men than in women (Falkeborn et  al., 1993; Prencipe et  al., 1997; Boix et  al., 2006). That E2 may be neuroprotective first arose from studies in intact animals on sex differences in brain injuries. Studies on cerebral ischemia-induced brain damage following carotid artery occlusion revealed that female gerbils had a lower incidence and less severe brain damage than male ones did (Hall et  al., 1991). Subsequent studies found that young adult female rats and mice have smaller infarct volume as compared with young adult males following middle cerebral artery occlusion (MCAO) (Alkayed et al., 1998). Compared with male rats, female rats have also been reported to have greater survival rates following diffuse traumatic brain injury (Roof and Hall, 2000). Studies of animals undergoing focal and global cerebral ischemia provided evidences for a limited time window for E2 neuroprotection. One-week pretreatments that maintain low physiological E2 levels (10–25 pg/ml) (Suzuki et al., 2007; Zhang et al., 2009) and 1 week treatment with proestrous levels of E2 (64 pg/ml) (Zhang et  al., 2011) protected the brain from cerebral ischemia immediately after ovariectomy in young mice or rats. However, high physiological hormone levels of E2 (60– 80 pg/ml) exacerbate focal ischemia-induced infarcts

in reproductively senescent, middle-aged females (Selvamani and Sohrabji, 2010a,b). Other evidences under some circumstances showed that E2 can retain its neuroprotective action after long-term loss of ovarian function (Alkayed et al., 2000; Toung et al., 2004; De Butte-Smith et  al., 2009; Lebesgue et  al., 2010; Wappler et  al., 2010; Inagaki et al., 2012). These findings suggest that the dose, timing, and type of E2 administration may be critical factors. Functional and behavioral studies elucidated that E2 enhances hippocampal synaptic transmission and synaptic plasticity (Dai et al., 2007; Inagaki et al., 2012) and improves visual and spatial working memory deficits after global ischemia in the rats (Gulinello et al., 2006). An elegant study in an estrogen-deplete animal model (e.g., mice in which the estrogen biosynthetic gene, aromatase, has been knocked out) revealed that intact and ovariectomized aromatase knockout mice have increased MCAO-induced infarct size and similar results also caused by administration of aromatase inhibitors (McCullough et al., 2003). Aromatase protein was increased after MCAO, indicating the potential for aromatase to promote the survival of cells after experimental stroke by local synthesis of estrogens (Carswell et  al., 2005). In addition, serum E2 levels are inversely correlated with ischemic stroke damage in intact animals, and treatment of intact female mice with an anti-ER compound, ICI182,780, significantly enhanced stroke infarct size (Sawada et al., 2000). These results emphasize that both gonadal-derived circulating and brain-derived estrogen have an important protective effect for the brain against ischemic insult. Several groups have explored the potential roles of ERs in mediation of the neuroprotective effects of E2. Administration of the potent ER antagonist ICI182,780 dramatically increased infarct size in intact female rats following MCAO (Sawada et  al., 2000) and also blocked neuroprotection by estrogen in global ischemia (Wilson et al., 2000; Miller et al., 2005), supporting a role for ERs in the mediation of the neuroprotective effects of E2. With respect to which receptor mediates E2 neuroprotection against cerebral ischemia, however, the results have been somewhat contradictory. The majority of the literatures suggest that ERα has the primary and critical mediator role for E2-induced neuroprotection. Evidence supporting a neuroprotective role for ERα has come from studies demonstrating that estrogen-mediated neuroprotection was lost in ER-α knockout (ERαKO) mouse with gonadectomy procedures (Dubal et al., 2001, 2006; Merchenthaler et al., 2003; Elzer et  al., 2010). In addition, antisense knockdown studies confirmed a critical role for ERα, but not ERβ, in mediating E2 neuroprotection in the hippocampal CA1 region in rats following global cerebral ischemia

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(Zhang et  al., 2009). Furthermore, our laboratory and other groups demonstrated that a selective ERα agonist, 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT), but not ERβ agonist, 2,3-bis(4-hyroxyphenyl) propionitrile (DPN), exerts neuroprotection and rescue the ischemiainduced deficit in LTP in the hippocampal CA1 region following global cerebral ischemia (Miller et  al., 2005; Dai et  al., 2007). However, these findings are in contrast to the results from reports that used ERαKO mouse (Sampei et  al., 2000), ER-β knockout mouse (Dubal et  al., 2001; Merchenthaler et  al., 2003), and selective ERβ agonist WAY 200070-3 or DPN (Carswell et al., 2004; Miller et al., 2005), suggesting that ERβ may have a neuroprotective role in certain situations. In addition, a naturally occurring plant-derived phytoestrogen, genistein, has also been shown to exert neuroprotection in the hippocampus against global cerebral ischemia, and this effect was blocked by treatment with an ERβ-specific antagonist, 4-[2-phenyl-5,7-bis(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-3-yl]phenol (Donzelli et al., 2010). Recently, several studies have supported the role of GPR30 in neuroprotection of E2 using a selective agonist for GPR30, G-1 (Bologa et al., 2006; Gingerich et al., 2010). G-1 pretreatment significantly ameliorated glutamateinduced neuronal cell death in hippocampal cell cultures (Gingerich et al., 2010). G-1 has also been shown to exert neuroprotection against focal cerebral ischemia in female mouse (Zhang et  al., 2010) and to play an important role in the ability of E2 to protect the cerebrovasculature against ischemia/reperfusion injury (Murata et al., 2013). Although these studies are intriguing, they rely on exogenous agonist studies and do not demonstrate a conclusive role for GPR30 in mediating endogenous E2 neuroprotective actions. Definitive conclusions on the role of GPR30 in mediating E2 neuroprotection must await the results from studies using GPR30 KO mouse, as well as selective GPR30 antagonist and knockdown approaches.

Estrogen receptors ERs are critical to our understanding of the mechanisms of estrogen action. Currently, there are two main documented ER subtypes. Although ERα and ERβ are quite similar in structure, their distributions throughout the body and the brain are unique (Dubal and Wise, 2002). In the CNS, these ERs have similar patterns of expression in the preoptic area, the cortical amygdaloid nuclei, and the bed nucleus of the stria terminalis (Lebesgue et al., 2009). ERα expression is found exclusively in the ventromedial

hypothalamic nucleus and the subfornical organ, and it is predominant in mouse hippocampus, whereas ERβ is predominant in hippocampus and the cerebral cortex (Mitra et al., 2003). Other localization studies have demonstrated that ERα is expressed most densely in the hypothalamus, hippocampus, and preoptic area, with moderate to light density in the cerebral cortex (Shughrue and Merchenthaler, 2000; McEwen et  al., 2001), whereas ERβ is documented predominantly in the cortex, throughout the hippocampus, in the olfactory bulb, septum, preoptic area, nucleus of striata terminalis, amygdala, paraventricular hypothalamus, thalamus, ventral tegmental area, substantia nigra, and cerebellum (Shughrue et  al., 1997; Zhang et  al., 2002a; Perez et  al., 2003). These unique regional distributions of ERα and ERβ suggest that these receptors play very different roles in the brain. ERα and ERβ are ligand-activated receptors that work with the help of two DNA consensus elements to regulate gene transcription and function as part of a vast family of proteins called ligand-activated transcription factors. Their actions have been documented to include either genomic or nongenomic mechanisms (Brann et al., 2007, 2012). The genomic mechanism could take two different routes: either direct or indirect. When not bound to a ligand, ERs are found as monomers that associate with heat shock protein (Hsp-90) and immunophilins, forming a multiprotein complex (Xu et  al., 1999). Once E2 binds a complementary receptor, the phosphorylation of its many different serine/threonine residues is induced, which causes them to lose the Hsp-90 and change their conformation to promote their homodimerization or heterodimerization and translocation into the nucleus. A hydrophobic clef is also revealed to bind transcriptional coactivators that help by initiating a chromatin structural change in target promoters (McKenna and O’Malley, 2002; Wu et al., 2005). Once inside, ERs interact with estrogen response elements (EREs) on the regulatory sequences of target genes to either suppress or activate their transcription at the same time as being limited to both promoter and cell specificities (Xu et al., 1999; Christian et al., 2008; Suuronen et  al., 2008). However, they can also function without EREs with the help of ER tethering and coactivation of transcription factors bound to the target DNA, such as the transcriptional factor cAMP-response element binding (CREB) protein (Coleman et  al., 2003). ERs can also interact with fos/jun transcription factors so they can regulate transcription through activator protein-1 (McKenna and O’Malley, 2002; Kalaitzidis and Gilmore, 2005). The indirect route is initiated by disinhibiting mitogen-activated protein kinases (MAPKs) (Singer et al., 1999; Nilsson et al., 2001; Zhao and Brinton, 2007; Ji et al., 2011)

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258      C. Wang et al.: Estrogen, astrocytes and neuroprotection and protein kinase B (PKB/Akt) signaling cascades (Singh et  al., 2000; Jover-Mengual et  al., 2007), thus leading to the activation of CREB protein (McEwen, 2000; Carlstrom et al., 2001; Cheong et al., 2012; Kwakowsky et al., 2013). Distinct from the classic ERα and ERβ, a novel and putative third ER, GPR (GPR30, also known as GPER1), has been described. This seven-transmembrane receptor is localized in the plasma membrane and endoplasmic reticulum of neurons (Funakoshi et  al., 2006; Matsuda et al., 2008) and expressed in various regions of the brain, including the hippocampus, cortex, and striatum, and may thus have a role in mediating E2 actions (Matsuda et al., 2008). GPR30 has a high affinity for E2 but low affinity for 17α-estradiol (Revankar et al., 2005; Matsuda et al., 2008). Upon binding to GPR30, E2 transactivates epidermal growth factor receptor followed by extracellular signal-regulated kinases 1 and 2 activation (Filardo et al., 2000). GPR30 up-regulates nerve growth factor and Bcl-2 (Kanda and Watanabe, 2003a,b) and promotes neuronal calcium release (Brailoiu et  al., 2007). The functions of GPR30 have been largely studied with its selective ligand, G1 (Bologa et al., 2006), establishing the role of GPR30 in transcriptional regulation of genes involved in cell proliferation and tumorigenesis (Albanito et al., 2007).

Astrocyte function in the CNS Historically, astrocytes have been regarded as nonexcitable cells that primarily serve a support and structural role in CNS function. However, investigations over the last 10–15  years have been changing this perception. Astrocytes function as the principal housekeeping cells of the CNS (Guo et  al., 2012) and are dynamically involved in many important activities in the 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 (BBB) (Tasker et  al., 2012). Astrocytes also interact by cross-talk with neurons both physiologically and pathophysiologically and play a very important role during extracellular clearance of neurotransmitters (Tan et  al., 2012). Key mediators of astrocyte-neuron signaling are glutamate and ATP/adenosine (Nedergaard et al., 2003). Astrocytes actively propagate Ca2+ signals to neighboring neurons, whose level of synaptic activity can be actively modulated (Parpura et al., 1994). Astrocytes have also been implicated in the local control of blood flow, but it is not established how ischemia affects the ability of astrocytes to modulate

vascular tone (Iadecola and Nedergaard, 2007). Proper astrocyte function is particularly important for neuronal survival under ischemic conditions, and dysfunction of astrocytes resulting from ischemic insult significantly influences the responses of other brain cells to injury (Takano et al., 2009; Guo et al., 2012). It is believed that astrocytes are critical determinants in stroke pathophysiology (Wu et al., 2000) and contribute novel therapeutic approaches for cerebral ischemic impairment.

Estrogen action on the astrocytes Several studies have revealed the existence of bidirectional communication between neurons and astrocytes. Neuronal activity regulates the astrocyte Ca2+ signal, which in turn is involved in astrocyte-to-astrocyte signaling. In addition, astrocytes integrate and process synaptic information and release several neuroactive molecules that influence synaptic function and plasticity (Panatier et al., 2006; Perea et al., 2009). Within the brain, astrocytes are the major cellular target of E2, as reflected by the fact that astrocytes express both ERα (Garcia-Ovejero et  al., 2002; Bondar et al., 2009) and ERβ (Azcoitia et al., 1999; Huppmann et al., 2008) receptors either on their plasma membranes or in cytosol (Bondar et  al., 2009; FuenteMartin et  al., 2013). E2 can regulate astrocyte-to-astrocyte and astrocyte-to-neuron communication (Micevych et al., 2010). Although E2 may act directly on astrocytes, the final response of astrocytes to the hormone in vivo is probably influenced by interactions with neurons (Azcoitia et  al., 2010). Neuronal signals are known to regulate the effects of E2 on astrocytes. Cell adhesion molecules, such as polysialylated neural cell adhesion molecule and neurotransmitters, such as GABA (Mong et al., 2002; Azcoitia et al., 2010), have been identified to be involved in E2-induced neuron-to-astrocyte signaling. In addition, the action of E2 on astrocytes is probably influenced by the hormonal action on other glial cells, such as microglia (Vegeto et  al., 2006; Tapia-Gonzalez et  al., 2008), or on the vascular endothelium (Stirone et  al., 2003). E2 regulates the morphology of astrocytes and the expression of different molecules that are relevant for the physiological and pathological responses of these cells (Arevalo et al., 2010). Furthermore, soluble factors released by astrocytes from target brain areas influence the neuritogenic effect of E2 on cultured hypothalamic neurons (Cambiasso et  al., 2000). E2 exerts rapid signaling events in astrocytes via the regulation of the activation of kinase signaling pathways,

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such as the MAPK (Ivanova et  al., 2001; Zhang et  al., 2002b) or the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway (Dhandapani et al., 2005). E2 regulates the expression of neuroprotective astrocyte proteins, particularly Hsp (Lu et  al., 2002), and the glutamate transporters (GLT) GLT-1 and glutamate aspartate transporter (GLAST) (Cimarosti et  al., 2005; Pawlak et  al., 2005; Lee et al., 2009), as well as a variety of growth factors, including TGF-α (Galbiati et al., 2002), TGF-β1 (Melcangi et al., 2001), basic fibroblast growth factor (FGF) (Melcangi et  al., 2001), and insulin-like growth factor I (GarciaSegura and McCarthy, 2004; Acs et al., 2009). Evidence of an estrous-related astrocytic response in the hippocampal formation demonstrated that the number of tyrosine kinase A receptor (TrkA)-immunoreactive astrocytes fluctuates across the estrous cycle, reaching greatest numbers at estrus after the peak plasma E2 concentration of proestrous (McCarthy et  al., 2002). E2 replacement in OVX animals also significantly increased the number of TrkA immunoreactive astrocytes in the hippocampal formation (McCarthy et  al., 2002). E2 also affects the expression of cytoskeletal proteins in astrocytes, and this action may be relevant for normal astrocyte physiology and the regulation of synaptic plasticity. For instance, the effects of E2 on the morphology and expression of glial fibrillary acidic protein (GFAP) in hypothalamic astrocytes are linked to the regulation of synaptic connectivity (Garcia-Segura et  al., 1994). E2 modulates neuronal plasticity through direct effects on GFAP transcription that, in turn, modify GFAP-containing intermediate filaments and reorganize astrocytic laminin (Rozovsky et al., 2002). A recent study showed that age increases ERα in cortical astrocytes and impairs neurotrophic support action of astrocytes in male and female rats (Arimoto et al., 2013). However, treatment with the ERβ agonist LY3201 increased activated astrocytes but did not alter microglia or oligodendrocytes (Tan et  al., 2012). Activation of the astrocytes mER by E2 initiates a rapid, free cytoplasmic calcium release (Chaban et al., 2004), which was significantly attenuated in ERαKO mouse (Kuo et al., 2010).

Several literatures substantiate the neuroprotective effects of E2 on astrocytes against cerebral ischemia; the main mechanisms of astrocytes mediating E2 and selective ER modulator (SERM) neuroprotective action against cerebral ischemia are presented in Figure 1. Brain aromatase levels were shown to increase in astrocytes in the peri-infarct area at 24 h and 8 days after MCAO, suggesting that enhanced production of brain-derived estrogen may facilitate survival of the neurons following brain ischemia insult (Carswell et  al., 2005). Martinez and de Lacalle (2007) demonstrated that a direct influence of gonadal hormones on the morphology and functional response of astrocytes and E2 administration may contribute to structural recovery of damaged cholinergic neurons by blocking GFAP expression in the basal forebrain, and this may be crucial to prevent Alzheimer’s disease. E2 also protects cultured astrocytes from OGD/reperfusion-induced injury by ER-dependent mechanisms and particularly highlighted an important role of ERα in protecting astrocyte viability (Guo et al., 2012). Oxygen deprivation increased the expression of hypoxia inducible factor-1 (Al-Bader et  al., 2011), which interacts with ERα, and this interaction down-regulates the transcriptional activity of ERα (Cho et al., 2005) but not of ERβ (Yi et al., 2009). Results from primary astrocyte culture demonstrated that 1  h of hypoxia and glucose deprivation significantly decreased the level of full-length ERα (Al-Bader et al., 2011). Since it is believed that E2 signaling can be protective during cerebral ischemia/hypoxia, this indicates a possibility that E2 can exert limited protective effects on astrocytes during the short recovery phase. Down-regulation in ERα during hypoxia and glucose deprivation also is one of the possible reasons for an observed reduced ability of astrocytes to take up glutamate via a Na+-dependent mechanism during hypoxia (Dallas et al., 2007). Recently, the transmembrane ER, GPR30, was also reported to mediate nongenomic and rapid E2 signaling in astrocytes (Lee et al., 2012). G1, a selective agonist of GPR30, increased astrocytic GLT-1, which would prevent excitotoxic-induced neuronal death, expressed via PI3K-NF-κB and cAMP-PKA-CREB pathways

Role of astrocytes in estrogen neuroprotection during cerebral ischemia ER expression in astrocytes was detected in the CNS, suggesting that astrocytes may be physiological targets of E2 actions (Azcoitia et  al., 1999; Dhandapani et  al., 2005).

Figure 1 Possible roles of astrocytes in mediation of estrogen neuroprotection action during cerebral ischemia.

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260      C. Wang et al.: Estrogen, astrocytes and neuroprotection in astrocytes, suggesting that GPR30 in astrocytes is a potential target to be explored for developing therapeutics of neuronal injury (Lee et al., 2012). During the initial phase of cerebral ischemia, ranging from minutes to hours, local astrocytes become activated, secrete a number of inflammatory mediators, and thereby take over the regime to control early repair and protection mechanisms (Arevalo et al., 2010; Johann and Beyer, 2013). Several literatures demonstrated that the antiinflammatory action of E2 on astrocytes may contribute to the neuroprotective effects after cerebral ischemia. E2 and SERMs, such as raloxifene and tamoxifen, have a potential therapeutic value in controlling astrogliosis, a key component of the inflammatory cellular response to CNS injury, including ischemia and traumatic brain injury, and these may remain beneficial, even in aged animals that have experienced a prolonged depletion of ovarian hormones (Barreto et al., 2009). In contrast, chronic deprivation of gonadal hormones was associated with a strong increase in astrocyte response to ischemia (McAsey et al., 2006; Cordeau et  al., 2008). Santos-Galindo et  al. (2011) demonstrated that cortical primary astrocytes derived from male and female mice pups have a different expression of inflammatory markers in response to lipopolysaccharide (LPS). Studies using E2, DPN (ERβ agonist), and 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (ERα agonist/ERβ antagonist) suppressed the LPS-induced increase in interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and matrix metallopeptidase (MMP)-9 activities, which were used as surrogate markers for inflammation in astrocytes, whereas PPT (ERα agonist) reduced only IL-1β, but not TNFα and MMP-9. These results indicate that ligands for either ER attenuated some aspects of the inflammatory response in astrocytes (Lewis et al., 2008). E2 also decreases the activation of nuclear factor (NF)-κB, potent immediate-early transcriptional regulator of numerous proinflammatory genes, induced amyloid β (Aβ) peptide, and LPS in cultured astrocytes (Dodel et al., 1999). E2 inhibits autoimmune neuroinflammation and NF-κB-dependent monocyte chemoattractant protein-1/ CCL2, a proinflammatory chemokine that drives the local recruitment of inflammatory myeloid cells, expressed in astrocytes, indicating that E2 is capable of counteracting NF-κB activation in astrocytes after stroke, traumatic brain injury, and inflammatory challenges (Giraud et al., 2010; Spence et al., 2011). E2 also causes a decrease in the expression of nitric oxide synthase and the inflammatory markers TNF-α, IL-6, and interferon-γ-inducible protein10 (IP-10) in cultured astrocytes incubated with LPS (Kipp et al., 2007; Tenenbaum et al., 2007; Cerciat et al., 2010). The down-regulation of the production of cytokines and

chemokines, such as TNF-α, IL-6, and IP-10, by reactive astrocytes may be involved in the neuroprotective mechanisms of E2, at least under chronic neurodegenerative conditions. These effects, in turn, may lead to a delay or even prevention of tissue damage and neuronal cell loss during the early phase of traumatic challenge and allows the self-repairing mechanisms of the organism to grapple with the life-threatening situation. Astrocytes modulate the rapidly increasing oxidative stress in neurons after deprivation of blood supply with the subsequent deficiency of glucose and oxygen as well as the decrease in ATP content after cerebral ischemia (Sims and Muyderman, 2010; Johann and Beyer, 2013). With respect to the role of mitochondrial fusion and fission in energy metabolism, apoptosis, and proliferation, astrocyte mitochondria resemble a perfect intracellular target for steroids to modulate these processes, thereby promoting cell vitality after damage. E2 affects mitochondrial fusion and fission gene transcription in cortical astrocytes in a gender-specific way, thereby influencing mitochondrial function differently in both genders (Arnold et al., 2008). In astrocytes, studies have shown that E2 influences mitochondrial gene expression and respiratory chain activity and regulates mitochondrial function (Araujo et  al., 2008; Johann et  al., 2010), suggesting the potency of E2 to counteract pathological damage by stabilizing mitochondrial performance. The important features of brain damage after cerebral ischemia are as follows: elevation of mitochondrial permeability transition, dissipation of ionic gradients across the plasma membrane, enhanced formation and reduced clearance of radical oxygen species, loss of mitochondrial antioxidant glutathione, and release of apoptogenic proteins (for further information, see Sims and Muyderman, 2010). The degree of mitochondrial impairment in cerebral ischemia may be a critical determinant of the final extent of neuronal injury (Sims and Muyderman, 2010). It is not clear yet whether the mitochondria contain ERs. If so, E2 may directly act on mitochondria. Several literatures have shown evidences that E2 directly affects mitochondrial properties related to oxidative stress as a result of cerebral ischemic damage (Brinton, 2008; Zhang et  al., 2009; Eichner and Giguere, 2011). Mitochondrial protection in astrocytes is fundamental for maintaining the energetic balance of the brain and antioxidant production that contributes to neuronal protection (Dugan and Kim-Han, 2004). Paraoxonase 2 (PON2), a member of a gene family, is located primarily in mitochondria and exerts a potent antioxidant effect, protecting mouse CNS cells against oxidative stress (Devarajan et al., 2011; Giordano et al., 2011). PON2 levels are higher in astrocytes

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than in neurons and in cortical microglia (Giordano et al., 2011). Astrocytes and neurons from male mice were significantly more sensitive (by threefold to fourfold) to oxidative stress-induced toxicity than the same cells from female mice (Giordano et al., 2011). These results suggest that PON2 is a novel major intracellular factor that protects CNS cells against oxidative stress and confers gender-dependent susceptibility to such stress. The lower expression of PON2 in males may have broad ramifications for susceptibility to diseases involving oxidative stress and neurodegenerative diseases (Giordano et  al., 2011, 2013). E2 preserves mitochondrial function in the early phase (up to 60 min) of ischemic injury in astrocytes, indicating mitochondria as potential specific subcellular targets for estrogen-mediated action in the CNS during ischemia (Guo et  al., 2012). Other studies have reported the sex differences of astrocytes and in their vulnerability to oxidative damage (Guevara et al., 2011; Santos-Galindo et al., 2011; Sundar Boyalla et al., 2011; Loram et al., 2012; Schwarz and Bilbo, 2012). Female astrocytes are more resistant than male astrocytes to oxygen and glucose deprivation and to cell death induced by oxidants (Liu et al., 2007), while male astrocytes are more sensitive to the toxic effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (Sundar Boyalla et al., 2011). These studies suggest that mitochondrial protection would be an important common mechanism for E2-mediated protection against ischemic brain injury, and strategies aimed to protect astrocytic mitochondria might be useful in developing therapies to attenuate ischemic brain injury. There are additional effects of E2 on astrocytes that inhibit cerebral edema and narrow down the spread of the cortical damage after stroke and acute brain trauma. This estrogenic action may have particular relevance in stroke. It is well known that the brain is separated from the systemic circulation by the BBB, which impedes the entry of various substances that are not lipid soluble. The BBB is a complex entity that starts with tight junctions between vascular endothelial cells that interface with astrocytes (Yang et  al., 2005; Ayus et  al., 2008); therefore, astrocytes are ideally situated to function as relay cells in neurovascular communication (Attwell et al., 2010). Astrocytes surround synapses and can thus be stimulated by neuronal activity, whereas their endfoot processes envelop blood vessels and can signal to the smooth muscle cells that control vessel diameter (Attwell et  al., 2010). This structure performs many functions, and much of them are accomplished through a concentration of aquaporine (AQP) water channels, particularly AQP4 water channels (Ayus et  al., 2008). E2 possesses an antiedema action that is characterized by reduced

astrocyte swelling and a reduction of AQP4 abundance (Rutkowsky et al., 2011). BBB disruption coincides with a strong induction of AQP4 in perivascular astrocytes after physiopathological stress to the brain (Johann and Beyer, 2013). E2 enhanced AQP4 expression in parenchymal reactive astrocytes and perivascular astrocytes processes, preventing brain edema and BBB disruption (Tomas-Camardiel et  al., 2005). Regulation of AQP4 expression in astrocytes by E2 might also be the reason for sex-specific volume regulation in hyponatremia in the CNS and adaptation to brain damage after cerebral ischemia (Ayus et al., 2008). These findings suggest that E2 readily penetrates into the brain and can therefore target astrocytes as well as BBB endothelial cells and deserves further attention as a therapeutic approach to reduce cerebral edema in the ischemic penumbra (Rutkowsky et al., 2011). Finally, astrocytes sense and respond to nerve cell activity by rising intracellular calcium levels. Estrogen curbs intracellular calcium responses in astroglia, which might have a direct functional consequence on astrocyteto-neuron communication as well as control extracellular ion concentrations and neural network activity under pathological conditions (Kuppers et  al., 2001; Rao and Sikdar, 2006; Lebesgue et  al., 2009). E2 also enhances the expression of the astrocyte GLTs, GLAST (glutamate/ aspartate transporter), and GLT-1, which could function as a key neuroprotective effect in the brain by reducing damaging extracellular glutamate levels (Pawlak et al., 2005). The effects of E2 on postischemic astrocyte responses have also been studied using both traditional immunohistochemistry and biophotonic/bioluminescent imaging, resulting in marked reductions in GFAP-positive cells in animals treated with E2 (Barreto et al., 2007, 2009; Cordeau et al., 2008). After brain injury, astrocytes acquire a reactive phenotype characterized by a series of morphological and molecular modifications, including the expression of the cytoskeletal protein vimentin (Barreto et al., 2009).

Conclusions E2 has well-documented neuroprotective effects in a variety of clinical and experimental disorders of the central cerebral ischemia, including stroke and neurodegenerative diseases, but the underlying mechanisms of estrogen-mediated protection remain obscure. Astrocytes are complex cells that are increasingly implicated as playing essential roles in normal CNS function and the response to injury by providing trophic support to

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262      C. Wang et al.: Estrogen, astrocytes and neuroprotection neurons in the presence and absence of injury in part through regulation by sex hormones. Astrocytes express ERs and they are physiological and pathophysiological targets for E2. Accumulating evidences demonstrated that astrocytes are unique and critical effecter cells of estrogen-mediated neuroprotective action in diverse CNS disorders. Studies on E2 action on astrocytes have potential to uncover new therapeutic approach to reduce cerebral ischemic damage.

Acknowledgments: This work was supported in part by grants from the National Natural Science Foundation of China (30770573), the 973 program from the Minister of Science and Technology in China (2007CB512304), and the SRF for ROCS, SEM (2008-101).

Received November 18, 2013; accepted January 29, 2014; previously published online February 22, 2014

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268      C. Wang et al.: Estrogen, astrocytes and neuroprotection

Cuifen Wang is taking her post-doc research at School of Pharmacy, Marshall University, West Virginia, USA. She is skilled in neural degenerative diseases with immunohistochemistry and fluorescence microscopy in brain tissues and employs a variety of biochemical and molecular biological techniques to develop new chemicals against cerebral diseases.

Xiaoniu Dai is a lecture at the Department of Physiology at the Medical School of Southeast University, Nanjing, China. He got his PhD from the Medical School of Nagoya University, Japan. His research interest is primarily in learning-memory mechanisms with focus on the effects of neurosteroids on synaptic transmission and synaptic plasticity by using electrophysiological techniques, including whole cell patch clamping and field excitatory postsynaptic potentials (fEPSPs) recording from brain slice.

Jie Chao is a professor at Department of Physiology at the Medical School of Southeast University, Nanjing, China. He got his PhD from University of Kansas Medical Center. He has ample experience with the technique of intravital microscopy, as well as molecular biologic techniques in neuroscience and respiration physiology. He is firstauthor or correspond-author of more than 20 peer-reviewed papers of his field. The primary objective of Jie’s laboratory is to explore, in vivo and in vitro, molecular and cellular mechanisms underlying the inflammation of silicosis.

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