Mitochondrial calcium homeostasis in SOD1-familial ALS

描述（由申请人提供）：这是一份题为“突变体SOD1在线粒体中的致病作用”的项目更新申请。线粒体Ca2+超载会导致有害的后果，包括线粒体失能、结构变化和生物能量衰竭，这可能导致细胞死亡。该应用的总体假设是线粒体Ca2+超载在与SOD1突变相关的家族性ALS （FALS）发病机制中起着重要作用。线粒体功能障碍，ALS的主要特征之一，导致Ca2+超载易感性增加。我们的初步结果显示，突变SOD1转基因小鼠的CNS中线粒体Ca2+处理受损和易感性超载。他们还表明，雌性突变型SOD1小鼠通过涉及雌激素、线粒体雌激素受体（ER）和亲环蛋白D （CyPD）的Ca2+释放途径部分保护线粒体Ca2+过载，CyPD是线粒体Ca2+诱导损伤的调节剂。在这个应用中，我们为线粒体的参与和FALS的性别差异提供了一个机制假设。广泛的目标是确定线粒体Ca2+处理调节的机制，并制定防止FALS线粒体Ca2+过载的方法。我们的具体目标是：1)通过体内和体外研究ER2 KO小鼠与G93A突变体SOD1小鼠杂交，确定雌激素受体在FALS线粒体Ca2+处理中的作用。然后，利用G85R突变SOD1小鼠模型，确定雌激素- er2和CyPD依赖性神经保护机制是否在不同SOD1突变体中具有共性。2)明确雌激素-内质网调控线粒体Ca2+处理的生化和分子基础以及突变体SOD1的影响。在目的1中建立的小鼠杂交中，我们将确定：i)雌激素-ER2如何调节线粒体生物能量学和CyPD依赖的Ca2+释放途径，ii) ER2的线粒体定位以及ER- CyPD与SOD1的相互作用，通过线粒体分离，免疫电镜和免疫沉淀。3)测试不同的互补方法，以防止Ca2+超载FALS线粒体和改善SOD1突变小鼠的疾病：1)对雄性SOD1突变小鼠长期给予雌激素，ii)通过表达线粒体可溶性腺苷酸环化酶增强氧化磷酸化来提高线粒体生物能量，iii)通过过表达解偶联蛋白2 （UCP2）实现轻度线粒体解偶联。

DESCRIPTION (provided by applicant): 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). PUBLIC HEALTH RELEVANCE: Mitochondria play a fundamental role in regulating neuronal life and death. One of the main functions of mitochondria is to take care of intracellular calcium (Ca2+). Mitochondrial dysfunction is one of the cardinal features of ALS and causes increased susceptibility to Ca2+ overload. Mitochondrial Ca2+ overload leads to deleterious consequences, including mitochondrial failure, which can result in cell death. The leading hypothesis of this study is that mitochondrial Ca2+ overload plays a fundamental role in the pathogenesis of familial ALS (FALS) associated with SOD1 mutations. Therefore the broad goals of this project are to define the mechanisms of mitochondrial Ca2+ handling regulation and to develop approaches to prevent Ca2+-overload in FALS mitochondria to ameliorate the disease.