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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 Alzheimer's 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. Keywordsestrogens; estradiol; non-feminizing estrogens mitochondria; neuroprotection; estrogen receptors; Alzheimer's disease Mitochondrial and cell death mechanismsMitochondrial oxidative phosphorylation is essential for neurons to meet their high ATP demand, and neuronal viability is imperiled when this ATP production is even transiently diminished. In addition to a bioenergetic crisis, mitochondrial impairment also produces a concomitant increase in production of reactive oxygen species [1]. Mitochondrial failure is the key event in the pathogenic cascade leading to ischemia-induced cell death from both necrosis and apoptosis [1,2]. Under conditions of oxidative stress and excessive cytoplasmic Ca 2+ loading, mitochondria undergo a loss of the impermeability of the inner mitochondrial membrane that completely collapses the mitochondrial membrane potential (ΔΨm), a process called permeability transition. Such irreversible collapse of ΔΨm is accompanied by mitochondrial swelling and release of cytochrome c into the cytoplasm, where it activates certain caspases and induces apoptotic cell death [2,3].Normally, antioxidant defense systems reduce radical-induced damage by scavenging free radicals. However, accelerated mitochondrial radical production can overwhelm these Send correspondence to: James W. Simpkins, Ph.D., Department of Pharmacology & Neuroscience, Room RES-334J, University of North Texas Health Science Center, 3500 Camp Bowie Bvld., Fort Worth, TX 76107, Phone 817-735-0498, jsimpkin@hsc.unt.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptBiochim Biophys Acta. Author manuscript; availa...

L-type voltage-gated Ca 2؉ 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 Ca 2؉ influx in hippocampal neurons, which is required for neuroprotection and potentiation of LTP. The mechanism by which estrogen rapidly induces this Ca 2؉ 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, dihydropyridineinsensitive 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.estrogen receptors ͉ signaling ͉ estradiol ͉ memory A large body of evidence shows that estrogens exert multiple rapid effects on the structure and function of neurons in a variety of brain regions, including the hippocampus (1). For example, estrogens rapidly potentiate kainite-induced currents in hippocampal neurons from wild-type (2) as well as from estrogenreceptor (ER)-␣ knockout (3) mice and induce rapid spine synapse formation in the CA1 hippocampus of ovariectomized (OVX) rats (4). Furthermore, acute application of estrogens to hippocampal slices increases NMDA and AMPA receptor transmission (5), induces long-term potentiation (LTP) and long-term depression (LTD) (6), and rapidly modulates neuronal excitability in rat medial amygdala (7) and hippocampus(8).It is well known that estrogens interact with cell membrane components and initiate signaling events leading to a rise in intracellular Ca 2ϩ , and activation of Src kinase, G protein-coupled receptor (GPCR), MAPK, PI3K/AKT, PKA, and adenylyl cyclase (9). The mechanism(s) by which estrogens induce these rapid and diverse effects remains largely unknown. Ca 2ϩ is a second messenger that can trigger the modification of synaptic efficacy. A plasticity-induction protocol like repetitive low-frequency synaptic stimulation (10) induces the elevation of postsynaptic intracellular Ca 2ϩ . The level of intracellular Ca 2ϩ concentration can activate numerous kinases like CAMK, PKA, PKC, MAPK, PI3K, or phosphatases (11-15), which, respectively, phosphorylate or dephosphorylate ion channels, transcription factors, and other proteins that are involved in synaptic plasticity and memory formation. Because voltage-gated Ca 2ϩ channels (VGCC)-mediated extracellular Ca ...

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 17␤-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 17␤-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. 17␤-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, 17␤-estradiol attenuated glutamate toxicity. However, in the presence of effective concentrations of these protein phosphatase inhibitors, 17␤-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 17␤-estradiol with glutamate prevented the decrease in PP1, PP2A, and PP2B levels. These results suggest that 17␤-estradiol may protect cells against glutamate-induced oxidative stress and excitotoxicity by activating a combination of protein phosphatases.

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␤-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 ␣ (ER␣) and ␤ (ER␤) 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 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␣ or ER␤. Infarct volume was significantly reduced to 122.4Ϯ17.6 and 83.1Ϯ19.3 mm 3 in E2 and ZYC3 groups, respectively, compared with 252.6Ϯ15.6 mm 3 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. (Stroke. 2002; 33:2485-2491.)