Mitochondrial calcium homeostasis in SOD1-familial ALS

描述(申请人提供)：这是一个项目的续展申请，题为“突变的SOD1在线粒体中的致病作用”。线粒体钙超载会导致有害后果，包括线粒体失能、结构改变和生物能衰竭，从而导致细胞死亡。这一应用的首要假设是线粒体钙超载在SOD1突变相关的家族性肌萎缩侧索硬化症(FAL)的发病机制中起着基础性作用。线粒体功能障碍是肌萎缩侧索硬化症的主要特征之一，导致对钙超载的易感性增加。我们的初步结果显示，在突变的SOD1转基因小鼠的中枢神经系统中，线粒体钙处理能力和对超载的敏感性受到了损害。他们还表明，雌性突变SOD1小鼠通过涉及雌激素、线粒体雌激素受体(ER)和亲环素D(CyPD)的钙释放途径，部分地保护线粒体免受线粒体钙超载的影响。在这一应用中，我们为线粒体的参与和FALS的性别差异提供了一个机制假说。其主要目标是明确线粒体钙处理调节的机制，并开发防止FALS线粒体钙超载的方法。我们的具体目的是：1)通过体内外研究ER2KO小鼠与G93A突变的SOD1小鼠杂交，明确雌激素受体在FALS线粒体钙处理中的作用。然后，用G85R突变型SOD1小鼠模型，确定雌激素-ER2和CyPD依赖的神经保护机制是否在不同的SOD1突变型中是共同的。2)明确雌激素调节线粒体Ca~(2+)处理的生化和分子基础以及突变体SOD的作用。在目标1中建立的小鼠杂交中，我们将通过线粒体分离、免疫电子显微镜和免疫沉淀来确定：i)雌激素-ER2如何调节线粒体生物能量学和CyPD依赖的钙释放途径；ii)线粒体ER2的定位以及ER-CyPD与SOD1的相互作用。3)测试不同的互补方法，以防止FALS线粒体中的钙超载，并改善突变的SOD1小鼠的疾病：i)对SOD1突变的雄性小鼠长期服用雌激素，ii)通过表达线粒体可溶性腺酰环化酶来增强线粒体的生物能量学，以增强氧化磷酸化，iii)通过过度表达解偶联蛋白2(UCP2)实现轻微的线粒体解偶联。 与公共健康相关：线粒体在调节神经元的生死中起着基础性作用。线粒体的主要功能之一是照顾细胞内的钙离子(Ca2+)。线粒体功能障碍是肌萎缩侧索硬化症的主要特征之一，导致对钙超载的易感性增加。线粒体钙超载会导致有害后果，包括线粒体衰竭，这可能导致细胞死亡。本研究的主要假设是线粒体钙超载在SOD1突变相关的家族性肌萎缩侧索硬化症(FAL)的发病机制中起重要作用。因此，本项目的主要目标是明确线粒体钙处理调节的机制，并开发防止FALS线粒体钙超载的方法，以改善疾病。

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.