Kun Don Yi

Estrogen attenuates nuclear factor-kappa B activation induced by transient cerebral ischemia

The protective effects of estrogens have been widely reported in a number of animal and cell culture models, but the molecular mechanisms of this potent neuroprotective activity are not well understood. Both in vitro and in vivo studies indicate that in the central nervous system and peripheral tissues, estrogen treatment reduces cytokine production and inflammatory responses. Nuclear factor-kappa B (NFkappaB) plays an essential role in the regulation of post-ischemic inflammation, which is detrimental to recovery from an ischemic stroke. We investigated the role of NFkappaB in neuronal survival in rats that received transient middle cerebral artery (MCA) occlusion, and observed that this transient cerebral ischemia induced substantial apoptosis and inflammatory responses, including IkappaB phosphorylation, NF-kappaB activation and iNOS over-expression. 17 beta-estradiol (E2) treatment produced strong protective effects by reducing infarct volume, neuronal apoptosis, and inflammatory responses. These findings provide evidence for a novel molecular and cellular interaction between the sex hormone and the immunoresponsive system. These studies also provide evidence that suppression of post-ischemic inflammation may play a critical role in estrogen-mediated neuroprotection.

Mitochondrial mechanisms of estrogen neuroprotection.

Mitochondria have become a primary focus in our search not only for the mechanism(s) of neuronal death but also for neuroprotective drugs and therapies that can delay or prevent Alzheimers disease and other chronic neurodegenerative conditions. This is because mitochrondria play a central role in regulating viability and death of neurons, and mitochondrial dysfunction has been shown to contribute to neuronal death seen in neurodegenerative diseases. In this article, we review the evidence for the role of mitochondria in cell death and neurodegeneration and provide evidence that estrogens have multiple effects on mitochondria that enhance or preserve mitochondrial function during pathologic circumstances such as excitotoxicity, oxidative stress, and others. As such, estrogens and novel non-hormonal analogs have come to figure prominently in our efforts to protect neurons against both acute brain injury and chronic neurodegeneration.

Estrogens directly potentiate neuronal L-type Ca2+ channels

L-type voltage-gated Ca2+channels (VGCC) play an important role in dendritic development, neuronal survival, and synaptic plasticity. Recent studies have demonstrated that the gonadal steroid estrogen rapidly induces Ca2+ influx in hippocampal neurons, which is required for neuroprotection and potentiation of LTP. The mechanism by which estrogen rapidly induces this Ca2+ influx is not clearly understood. We show by electrophysiological studies that extremely low concentrations of estrogens acutely potentiate VGCC in hippocampal neurons, hippocampal slices, and HEK-293 cells transfected with neuronal L-type VGCC, in a manner that was estrogen receptor (ER)-independent. Equilibrium, competitive, and whole-cell binding assays indicate that estrogen directly interacts with the VGCC. Furthermore, a L-type VGCC antagonist to the dihydropyridine site displaced estrogen binding to neuronal membranes, and the effects of estrogen were markedly attenuated in a mutant, dihydropyridine-insensitive L-type VGCC, demonstrating a direct interaction of estrogens with L-type VGCC. Thus, estrogen-induced potentiation of calcium influx via L-type VGCC may link electrical events with rapid intracellular signaling seen with estrogen exposure leading to modulation of synaptic plasticity, neuroprotection, and memory formation.

Neuroprotective Effects of a Novel Non–Receptor-Binding Estrogen Analogue In Vitro and In Vivo Analysis

Background and Purpose— Although estrogens are neuroprotective, hormonal effects limit their clinical application. Estrogen analogues with neuroprotective function but lacking hormonal properties would be more attractive. The present study was undertaken to determine the neuroprotective effects of a novel 2-adamantyl estrogen analogue, ZYC3. Methods— Cytotoxicity was induced in HT-22 cells by 10 mmol/L glutamate. 17&bgr;-Estradiol (E2) or ZYC3 was added immediately before the exposure to glutamate. Cell viability was determined by calcein assay. The binding of E2 and ZYC3 to human &agr; (ER&agr;) and &bgr; (ER&bgr;) estrogen receptors was determined by ligand competition binding assay. Ischemia/reperfusion injury was induced by temporary middle cerebral artery occlusion (MCAO). E2 or ZYC3 (100 &mgr;g/kg) was administered 2 hours or immediately before MCAO, respectively. Infarct volume was determined by 2,3,5-triphenyltetrazolium chloride staining. Cerebral blood flow was recorded during and within 30 minutes after MCAO by a hydrogen clearance method. Results— ZYC3 significantly decreased toxicity of glutamate with a potency 10-fold that of E2. ZYC3 did not bind to either ER&agr; or ER&bgr;. Infarct volume was significantly reduced to 122.4±17.6 and 83.1±19.3 mm3 in E2 and ZYC3 groups, respectively, compared with 252.6±15.6 mm3 in the ovariectomized group. During MCAO, both E2 and ZYC3 significantly increased cerebral blood flow in the nonischemic side, while no significant differences were found in the ischemic side. However, E2 and ZYC3 significantly increased cerebral blood flow in both sides within 30 minutes after reperfusion. Conclusions— Our study shows that ZYC3, a non–receptor-binding estrogen analogue, possesses both neuroprotective and vasoactive effects, which offers the possibility of clinical application for stroke without the side effects of estrogens. It also suggests that both the neuroprotective and vasoactive effects of estrogen are receptor independent.

Role of protein phosphatases and mitochondria in the neuroprotective effects of estrogens

In the present treatise, we provide evidence that the neuroprotective and mito-protective effects of estrogens are inexorably linked and involve the ability of estrogens to maintain mitochondrial function during neurotoxic stress. This is achieved by the induction of nuclear and mitochondrial gene expression, the maintenance of protein phosphatases levels in a manner that likely involves modulation of the phosphorylation state of signaling kinases and mitochondrial pro- and anti-apoptotic proteins, and the potent redox/antioxidant activity of estrogens. These estrogen actions are mediated through a combination of estrogens receptor (ER)-mediated effects on nuclear and mitochondrial transcription of protein vital to mitochondrial function, ER-mediated, non-genomic signaling and non-ER-mediated effects of estrogens on signaling and oxidative stress. Collectively, these multifaceted, coordinated action of estrogens leads to their potency in protecting neurons from a wide variety of acute insults as well as chronic neurodegenerative processes.

Estrogen Receptor-Independent Neuroprotection via Protein Phosphatase Preservation and Attenuation of Persistent Extracellular Signal-Regulated Kinase 1/2 Activation

The mechanism of estrogen-mediated neuroprotection is not yet clear. Estrogens have a variety of modes of action, including transducing signaling events such as activation and/or suppression of the mitogen-activated protein kinase (MAPK) pathway. We have previously shown protein phosphatases to be involved in 17β-estradiol-mediated neuroprotection. In the present study, we assessed the role of estrogen receptors (ERs) in estrogen-mediated neuroprotection from oxidative/excitotoxic stress and the consequential effects on MAPK signaling. Okadaic acid and calyculin A, nonspecific serine/threonine phosphatase inhibitors, were exposed to cells at various concentrations in the presence or absence of 17α-estradiol, the enantiomer of 17β-estradiol, 2-(1-adamantyl)-3-hydroxyestra-1,3,5(10)-trien-17-one (ZYC3; non-ER-binding estrogen analog), and/or glutamate. All three compounds, which we have shown to have little or no binding to ERα and ERβ, were protective against glutamate toxicity but not against okadaic acid and calyculin A toxicity. In addition, in the presence of effective concentrations of these inhibitors, the protective effects of these estrogen analogs were lost. Glutamate treatment caused a 50% decrease in levels of protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A), and protein phosphatase 2B (calcineurin) (PP2B). Coadministration of ZYC3 with glutamate prevented the decreases in PP1, PP2A, and PP2B levels. Furthermore, glutamate treatment caused a persistent increase in phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 that corresponds with the decrease protein levels of serine/threonine phosphatases. ZYC3 blocked this persistent increase in ERK phosphorylation. These results suggest that estrogens protect cells against glutamate-induced oxidative stress through an ER-independent mediated mechanism that serves to preserve phosphatase activity in the face of oxidative insults, resulting in attenuation of the persistent phosphorylation of ERK associated with neuronal death.

Role of Protein Phosphatases in Estrogen-Mediated Neuroprotection

The signaling pathways that mediate neurodegeneration are complex and involve a balance between phosphorylation and dephosphorylation of signaling and structural proteins. We have shown previously that 17β-estradiol and its analogs are potent neuroprotectants. The purpose of this study was to delineate the role of protein phosphatases (PPs) in estrogen neuroprotection against oxidative stress and excitotoxicity. HT-22 cells, C6-glioma cells, and primary rat cortical neurons were exposed to the nonspecific serine/threonine protein phosphatase inhibitors okadaic acid and calyculin A at various concentrations in the presence or absence of 17β-estradiol and/or glutamate. Okadaic acid and calyculin A caused a dose-dependent decrease in cell viability in HT-22, C6-glioma, and primary rat cortical neurons. 17β-Estradiol did not show protection against neurotoxic concentrations of either okadaic acid or calyculin A in these cells. In the absence of these serine/threonine protein phosphatase inhibitors, 17β-estradiol attenuated glutamate toxicity. However, in the presence of effective concentrations of these protein phosphatase inhibitors, 17β-estradiol protection against glutamate toxicity was lost. Furthermore, glutamate treatment in HT-22 cells and primary rat cortical neurons caused a 50% decrease in levels of PP1, PP2A, and PP2B protein, whereas coadministration of 17β-estradiol with glutamate prevented the decrease in PP1, PP2A, and PP2B levels. These results suggest that 17β-estradiol may protect cells against glutamate-induced oxidative stress and excitotoxicity by activating a combination of protein phosphatases.

Protein phosphatase 1, protein phosphatase 2A, and calcineurin play a role in estrogen-mediated neuroprotection.

It is becoming increasingly clear that protein phosphatases are important modulators of cellular function and that disruption of these proteins are involved in neurodegenerative disease processes. Serine/threonine protein phosphatases (PP) such as protein phosphatase PP1, PP2A, and calcineurin are involved in hyperphosphorylation of tau- as well as beta-amyloid-induced cell death. We have previously shown serine/threonine protein phosphatases to be involved in estrogen-mediated neuroprotection. The purpose of this study was to delineate the role of PP1, PP2A, and calcineurin in the mechanism of estrogen mediated neuroprotection against oxidative stress and excitotoxicity. Treatment with protein phosphatases inhibitor II, endothall, or cyclosporin A, which are specific inhibitors of PP1, PP2A, and calcineurin, respectively, did not have an effect on cell viability. However, in combination, these inhibitors adversely affected cell survival, which suggests the importance of serine/threonine protein phosphatases in maintenance of cellular function. Inhibitors of PP1, PP2A, and calcineurin attenuated the protective effects of estrogen against glutamate-induced -neurotoxicity but did not completely abrogate the estrogen-mediated protection. The attenuation of estrogen-induced neuroprotection was achieved through decrease in the activity of theses serine/threonine phosphatases without the concomitant decrease in protein expression. In an animal model, transient middle cerebral artery occlusion caused a 50% decrease in levels of PP1, PP2A, and PP2B ipsilateral to the lesion in a manner that was prevented by estradiol pretreatment. Therefore, we conclude that in the face of cytotoxic challenges in vitro and in vivo, estrogens maintain the function of PP1, PP2A, and calcineurin.

Aromatase Is Increased in Astrocytes in the Presence of Elevated Pressure

After traumatic brain injury (TBI), a progressive injury and death of neurons and glia leads to decreased brain function. Endogenous and exogenous estrogens may protect these vulnerable cells. In this study, we hypothesized that increased pressure leads to an increase in aromatase expression and estrogen production in astrocytes. In this study, we subjected rat glioma (C6) cells and primary cortical astrocytes to increased pressure (25 mm Hg) for 1, 3, 6, 12, 24, 48, and 72 h. Total aromatase protein and RNA levels were measured using Western analysis and RT-PCR, respectively. In addition, we measured aromatase activity by assaying estrone levels after administration of its precursor, androstenedione. We found that increased pressure applied to the C6 cells and primary cortical astrocytes resulted in a significant increase in both aromatase RNA and protein. To extend these findings, we also analyzed aromatase activity in the primary astrocytes during increased pressure. We found that increased pressure resulted in a significant (P < 0.01) increase in the conversion of androstenedione to estrone. In conclusion, we propose that after TBI, astrocytes sense increased pressure, leading to an increase in aromatase production and activity in the brain. These results may suggest mechanisms of brain estrogen production after increases in pressure as seen in TBI patients.

Mechanism of Okadaic Acid Induced Neuronal Death and the Effect of Estrogens

Serine/threonine protein phosphatases are important mediators of general cellular function as well as neurodegenerative processes. We have previously shown inhibition of protein phosphatases to be as neurotoxic as glutamate‐induced neuronal death but resistant to neuroprotection by estrogens. In this study, the mechanism by which phosphatase inhibition via okadaic acid (OA) induced neurotoxicity is explored. Neurons were exposed to OA or glutamate in the presence or absence of various protein kinases inhibitors, and/or one of four estrogens. Both OA and glutamate induced cell death via increased reactive oxygen species, protein carbonylation, lipid peroxidation, caspase‐3 activity, and mitochondrial dysfunction. All estrogens attenuated glutamate‐mediated responses, but not OA‐induced responses. In addition, inhibition of protein kinase C and mitogen‐activated protein kinase pathway was neuroprotective against glutamate but not OA toxicity. Interestingly, inhibition of mitogen‐activated protein kinase pathway with PD98096 or U0126 caused a decrease in reactive oxygen species production suggesting that activation of ERK1/2 could further exacerbate the oxidative stress caused by glutamate‐induced toxicity; however, these inhibitors had no effect on OA‐induced toxicity. Collectively, these results indicate that both glutamate and OA neurotoxicities are mediated by persistent activation of ERK1/2 and/or protein kinase C and a resulting oxidative stress, and that protein phosphatase activity is an important and necessary aspect of estrogen‐mediated neuroprotection.