Mitochondrial permeability transition (MPT) is an inner membrane permeabilization event, which can result in irreversible de-energization and swelling of mitochondria, leading to release of pro-death factors. Mitochondrial Ca2+ overload is the best-characterized trigger of MPT and has been implicated in the pathogenesis of diverse paradigms of neuronal death, such as ischemia-reperfusion injury, where a large influx of cytosolic Ca2+ triggers mitochondrial Ca2+ overload. While uncontrolled MPT can result in mitochondrial disruption, under certain conditions, MPT could provide mitochondria with a Ca2+ release outlet, allowing Ca2+ recycling and protecting mitochondria from Ca2+ overload. Estrogen receptors (ER) have been implicated in various paradigms of neuronal injury, and MPT modulation could be one of the mechanisms whereby they exert their role. Our studies revealed an unprecedented role of the ERβ in modulating MPT. In mouse brain mitochondria, estrogen decreases mitochondrial Ca2+ capacity in an ERβ and cyclophilin-D (CyPD, an MPT activator) dependent manner. Mitochondria from ERβ knock out (ERβKO) mice have reduced sensitivity to cyclosporine A, a potent CyPD inhibitor and CyPD genetic ablation in ERβKO does not further increase Ca2+ capacity. These results point to ERβ as a novel regulator of Ca2+-dependent MPT that functionally interacts with CyPD. In this application, we will test the hypothesis that ERβ localized in mitochondria (mERβ) regulates MPT, independently of transcriptional effects. The goals are to investigate the mechanisms of MPT modulation by mERβ and to test the effects of MPT modulation by mERβ in models of neuronal injury that involve mitochondrial Ca2+ toxicity, such as oxygen glucose deprivation (OGD) and glutamatergic toxicity. To this end we propose 1) to study the mechanisms of regulation of Ca2+-mediated MPT by ERβ. This regulation will be investigated using a multipronged approach, involving biochemical and molecular studies. 2) To assess the role of ERβ MPT regulation in neuronal Ca2+-mediated injury. Evidence suggests that Ca2+ dependent MPT and its regulator CyPD are involved in ischemic neuronal injury. We will use neuronal OGD and exposure to glutamatergic agents, both well-known paradigms of neuronal toxicity involving mitochondrial Ca2+ overload, to test the effects of genetic and pharmacological modulation of ERβ. The impact of the project will be two-fold: first, it will elucidate novel mechanisms of MPT regulation; second, it will assess if MPT modulation by mERβ could be protective in neuronal injury.