This is an application for renewal of the project entitled the pathogenic role of mutant SOD1 in mitochondria. Mitochondrial Ca2+ overload leads to deleterious consequences, including mitochondrial de-energization, structural changes, and bioenergetic failure, which can result in cell death. The overarching hypothesis of this application is that mitochondrial Ca2+ overload plays a fundamental role in the pathogenesis of familial ALS (FALS) associated with SOD1 mutations. Mitochondrial dysfunction, one of the cardinal features of ALS, causes increased susceptibility to Ca2+ overload. Our preliminary results show impaired mitochondrial Ca2+ handling and susceptibility to overload in the CNS of mutant SOD1 transgenic mice. They also show that female mutant SOD1 mice are partially protected against mitochondrial Ca2+ overload by a pathway of Ca2+ release involving estrogen, mitochondrial estrogen receptor (ER), and cyclophilin D (CyPD), a modulator of mitochondrial Ca2+ induced damage. In this application, we provide a mechanistic hypothesis for the involvement of mitochondria and for the gender differences in FALS. The broad goals are to define the mechanisms of mitochondrial Ca2+ handling regulation and to develop approaches to prevent Ca2+-overload in FALS mitochondria. Our specific aims are to: 1) Define the role of the estrogen receptor in Ca2+ handling in FALS mitochondria by investigating, in vivo and ex vivo, ER2 KO mice crossed with G93A mutant SOD1 mice. Then, with the G85R mutant SOD1 mouse model, to determine if the mechanisms of estrogen-ER2 and CyPD dependent neuroprotection are common to different SOD1 mutants. 2) Define the biochemical and molecular basis of estrogen-ER regulation of mitochondrial Ca2+ handling and the effects of mutant SOD1. In the mouse crosses established in aim 1, we will determine: i) how estrogen-ER2 modulates mitochondrial bioenergetics and the CyPD- dependent Ca2+ release pathway, ii) the mitochondrial localization of the ER2 and the ER- CyPD interactions with SOD1, by mitochondrial fractionation, immuno-electron microscopy, and immuno-precipitation. 3) Test different complementary approaches to prevent Ca2+ overload in FALS mitochondria and improve disease in mutant SOD1 mice by: i) chronic administration of estrogen to male SOD1 mutant mice, ii) boosting mitochondrial bioenergetics with expression of a mitochondrial soluble adenylyl cyclase that enhances oxidative phosphorylation, iii) mild mitochondrial uncoupling achieved with overexpression of uncoupling protein 2 (UCP2).