Shao-Hua Yang

Mitochondrial localization of estrogen receptor β

Estrogen receptors (ERs) are believed to be ligand-activated transcription factors belonging to the nuclear receptor superfamily, which on ligand binding translocate into the nucleus and activate gene transcription. To date, two ERs have been identified: ERα and ERβ. ERα plays major role in the estrogen-mediated genomic actions in both reproductive and nonreproductive tissue, whereas the function of ERβ is still unclear. In this study, we used immunocytochemistry, immunoblotting, and proteomics to demonstrate that ERβ localizes to the mitochondria. In immunocytochemistry studies, ERβ was detected with two ERβ antibodies and found to colocalize almost exclusively with a mitochondrial marker in rat primary neuron, primary cardiomyocyte, and a murine hippocampal cell line. The colocalization of ERβ and mitochondrial markers was identified by both fluorescence and confocal microscopy. No translocation of ERβ into the nucleus on 17β-estradiol treatment was seen by using immunocytochemistry. Immunoblotting of purified human heart mitochondria showed an intense signal of ERβ, whereas no signals for nuclear and other organelle markers were found. Finally, purified human heart mitochondrial proteins were separated by SDS/PAGE. The 50,000–65,000 Mr band was digested with trypsin and subjected to matrix-assisted laser desorption/ionization mass spectrometric analysis, which revealed seven tryptic fragments that matched with those of ERβ. In summary, this study demonstrated that ERβ is localized to mitochondria, suggesting a role for mitochondrial ERβ in estrogen effects on this important organelle.

Depletion of GGA3 stabilizes BACE and enhances β-secretase activity

Summary β-site APP-cleaving enzyme (BACE) is required for production of the Alzheimers disease (AD)-associated Aβ protein. BACE levels are elevated in AD brain, and increasing evidence reveals BACE as a stress-related protease that is upregulated following cerebral ischemia. However, the molecular mechanism responsible is unknown. We show that increases in BACE and β-secretase activity are due to posttranslational stabilization following caspase activation. We also found that during cerebral ischemia, levels of GGA3, an adaptor protein involved in BACE trafficking, are reduced, while BACE levels are increased. RNAi silencing of GGA3 also elevated levels of BACE and Aβ. Finally, in AD brain samples, GGA3 protein levels were significantly decreased and inversely correlated with increased levels of BACE. In summary, we have elucidated a GGA3-dependent mechanism regulating BACE levels and β-secretase activity. This mechanism may explain increased cerebral levels of BACE and Aβ following cerebral ischemia and existing in AD.

Increased β-secretase activity and expression in rats following transient cerebral ischemia

Abstract The aberrant proteolytic processing of the amyloid precursor protein (APP) by β- and γ-secretases is key to amyloid plaque formation in Alzheimers disease (AD). Identification of an aspartyl protease as the β-secretase (β-site APP cleaving enzyme, BACE) involved in APP processing provides a pharmaceutical target for potential AD treatment. In the present studies, we demonstrate that transient cerebral ischemia in female rats caused a 30% increase in β-secretase activity. α-Secretase activity did not increase significantly. We examined protein levels of BACE1, and its analogue BACE2, in ischemic brain extracts. BACE1 protein levels increased 67%, while BACE2 protein level did not change after such a transient ischemia. Immunohistochemical studies demonstrated that BACE1 protein was increased in the ischemic neocortex, when compared with its contralateral cortex. Further, colocalization assessment indicated that BACE1 strongly associated with staining for the apoptotic marker, TUNEL. These results may partially explain epidemiological study, which demonstrate a higher incidence of dementia after stroke. Further, our results support the hypothesis that apoptosis and aberrant APP processing are correlated events in AD brain, and suggest that inhibition of BACE may have a therapeutic effect in the prevention of dementia after stroke recovery.

Alternative Mitochondrial Electron Transfer as a Novel Strategy for Neuroprotection

Neuroprotective strategies, including free radical scavengers, ion channel modulators, and anti-inflammatory agents, have been extensively explored in the last 2 decades for the treatment of neurological diseases. Unfortunately, none of the neuroprotectants has been proved effective in clinical trails. In the current study, we demonstrated that methylene blue (MB) functions as an alternative electron carrier, which accepts electrons from NADH and transfers them to cytochrome c and bypasses complex I/III blockage. A de novo synthesized MB derivative, with the redox center disabled by N-acetylation, had no effect on mitochondrial complex activities. MB increases cellular oxygen consumption rates and reduces anaerobic glycolysis in cultured neuronal cells. MB is protective against various insults in vitro at low nanomolar concentrations. Our data indicate that MB has a unique mechanism and is fundamentally different from traditional antioxidants. We examined the effects of MB in two animal models of neurological diseases. MB dramatically attenuates behavioral, neurochemical, and neuropathological impairment in a Parkinson disease model. Rotenone caused severe dopamine depletion in the striatum, which was almost completely rescued by MB. MB rescued the effects of rotenone on mitochondrial complex I-III inhibition and free radical overproduction. Rotenone induced a severe loss of nigral dopaminergic neurons, which was dramatically attenuated by MB. In addition, MB significantly reduced cerebral ischemia reperfusion damage in a transient focal cerebral ischemia model. The present study indicates that rerouting mitochondrial electron transfer by MB or similar molecules provides a novel strategy for neuroprotection against both chronic and acute neurological diseases involving mitochondrial dysfunction.

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.

Hypoperfusion induces overexpression of β-amyloid precursor protein mRNA in a focal ischemic rodent model

Silent stroke is one of the risk factors of dementia. In the present study, we used a novel focal ischemic animal model to investigate the effects of comparatively small changes of cerebral blood flow (CBF) on the expression of beta-amyloid precursor protein (APP) mRNA. Focal ischemia was achieved by introducing a 4-0 monofilament to the bifurcation of anterior and middle cerebral arteries. Brain samples were harvested from ischemic core and penumbra of cortices at 1, 4 and 7 days following ischemia. The expression of APP mRNA was assessed by RT-PCR. The CBF was decreased to 50% for 1 day after stroke and recovered to 90% at the fourth day after stroke. The changes of CBF were accompanied by an increase in the expression of APP mRNA. APP mRNA increased to 208% and 152% in the penumbra and core ischemic regions, respectively, on the fourth day after MCAO and remained high through the seventh day of ischemia. This study suggests brain hypoperfusion enhances APP mRNA expression and may contribute to the progression of cognitive impairment after silent stroke.

Pyruvate protects mitochondria from oxidative stress in human neuroblastoma SK-N-SH cells.

Oxidative stress is implicated in neurodegenerative diseases including stroke, Alzheimers disease and Parkinsons disease, and has been extensively studied as a potential target for therapeutic intervention. Pyruvate, a natural metabolic intermediate and energy substrate, exerts antioxidant effects in brain and other tissues susceptible to oxidative stress. We tested the protective effects of pyruvate on hydrogen peroxide (H(2)O(2)) toxicity in human neuroblastoma SK-N-SH cells and the mechanisms underlying its protection. Hydrogen peroxide insult resulted in 85% cell death, but co-treatment with pyruvate dose-dependently attenuated cell death. At concentrations of >or=1 mM, pyruvate totally blocked the cytotoxic effects of H(2)O(2). Pyruvate exerted its protective effects even when its administration was delayed up to 2 h after H(2)O(2) insult. As a scavenger of reactive oxygen species (ROS), pyruvate dose-dependently attenuated H(2)O(2)-induced ROS formation, assessed from 2,7-dichlorofluorescein diacetate fluorescence. Furthermore, pyruvate suppressed superoxide production by submitochondrial particles, and attenuated oxidative stress-induced collapse of the mitochondrial membrane potential. Collectively, these results suggest that pyruvate protects neuronal cells through its antioxidant actions on mitochondria.

Transient Cerebral Ischemia Induces Aberrant Neuronal Cell Cycle Re-entry and Alzheimer's Disease-like Tauopathy in Female Rats

Aberrant mitosis occurs in many tauopathy-related neurodegenerative diseases and is believed to precede the formation of neurofibrillary tangles. In this study, we report for the first time that transient cerebral ischemia induces aberrant mitotic proteins and hyperphosphorylation of tau protein with neurofibrillary tangle-like conformational epitopes in adult female rat cortex. Following transient cerebral ischemia in rats, initiation of apoptosis precedes and is potentially integrated with subsequent aberrant mitosis and tau hyperphosphorylation. Furthermore, inhibition of mitosis-related cyclin-dependent kinases (Cdks) by roscovitine significantly reduced the hyperphosphorylation of tau. Administration of the female sex steroid and potent neuroprotective agent, 17β-estradiol, reduced ischemia-reperfusion-induced cerebral damage and the subsequent aberrant mitosis and tauopathies. These results provide a neuropathological basis for the higher prevalence of dementia in stroke patients and support the hypothesis that apoptosis and aberrant mitosis are integrated pathological events in neurons that may play a critical role in the development of Alzheimers disease and other tauopathy-related neuropathology.

Estrogen Actions on Mitochondria-Physiological and Pathological Implications

Estrogens are potent neuroprotective hormones and mitochondria are the site of cellular life-death decisions. As such, it is not surprising that we and others have shown that estrogens have remarkable effects on mitochondrial function. Herein we provide evidence for a primary effect of estrogens on mitochondrial function, achieved in part by the import of estrogen receptor beta (ERbeta) into the mitochondria where it mediates a number of estrogen actions on this vital organelle. ERbeta is imported into the mitochondria, through tethering to cytosolic chaperone protein and/or through direct interaction with mitochondrial import proteins. In the mitochondria, ERbeta can affect transcription of critical mitochondrial genes through the interaction with estrogen response elements (ERE) or through protein-protein interactions with mitochondrially imported transcription factors. The potent effects of estrogens on mitochondrial function, particularly during mitochondrial stress, argues for a role of estrogens in the treatment of mitochondrial defects in chronic neurodegenerative diseases like Alzheimers disease (AD) and Parkinsons disease (PD) and more acute conditions of mitochondrial compromise, like cerebral ischemia and traumatic brain injury.