Using mitochondrial Ca2+ uptake as a therapeutic target for ALS

肌萎缩侧索硬化症(ALS)是一种致命的成人起病的神经退行性疾病，其特征是 进行性运动神经元(MN)丧失，肌肉失神经，最终瘫痪。目前，没有有效的 目前已有治疗方法可用于阻止或逆转ALS疾病的进展及其确切的分子机制 肌萎缩侧索硬化症的发病机制仍不清楚。先前的研究表明线粒体呼吸链减少 MN和骨骼肌(SM)的活性、线粒体超微结构改变和线粒体功能障碍 在ALS患者和小鼠模型中。肌萎缩侧索硬化症的第一个病理征象出现在神经肌肉接头(NMJ)， 突触前MN轴突与突触后SM终板相连。到目前为止，信号是否会导致 最初的NMJ损伤来自MN或SM，目前尚不清楚。在这个项目中，我们的目标是确定组织特异性 SM和MN线粒体钙摄取在疾病发生发展中的作用及治疗 降低线粒体钙摄取对ALS小鼠NMJ和SM功能的影响我们假设 SM和MN中线粒体Ca~(2+)的错误处理积极参与ALS的发病机制 抑制线粒体钙摄取可减轻线粒体损伤，保护NMJ/肌肉功能。至 验证这一假设，我们将使用转基因小鼠进行诱导，SM或MN特异表达一种显性 线粒体钙单一转运蛋白的负性形式特异性和选择性地降低线粒体钙摄取 在SM和MN中，在hSOD1G93A小鼠和C9-500(C9orf72)小鼠中，两种小鼠模型与大多数 肌萎缩侧索硬化症的普遍遗传原因。核心假设将在两个具体目标上得到检验。目标一号将决定 SM或MN线粒体钙摄取在生存、运动功能、NMJ功能和活体肌肉中的作用 在hSOD1G93A和C9-500小鼠中的表现。目标2将评估组织特异性抑制的影响 NMJ上SM或MN线粒体钙摄取与肌肉结构、MN存活率、肌肉内收缩力 HSOD1G93A和C9-500小鼠SM的特性、线粒体结构和线粒体生物能量学这 项目将：1)从细胞水平提供SM和NMJ功能的系统、纵向表征 HSOD1G93A和C9-500小鼠疾病进展不同阶段的整体动物水平；2)确定 SM或MN线粒体钙摄取缺陷对NMJ结构/功能改变的影响程度， HSOD1G93A和C9-500小鼠的疾病发生和发展；3)提供关于 使用相同的遗传模型和遗传模型，在ALS表型中SM和MN中线粒体钙摄取的相对作用 确定导致NMJ破坏的信号来源(来自SM或MN或两者)；4)提供机械性 线粒体钙处理不当是否是ALS小鼠疾病进展的触发或靶点的证据， 无论引起突变(线粒体相关还是非线粒体相关)；最重要的是， 5)测试潜在的新治疗靶点的有效性(线粒体钙摄取，或线粒体钙 治疗肌萎缩侧索硬化症。

Amyotrophic lateral sclerosis (ALS) is a fatal, adult-onset neurodegenerative disease characterized by progressive motor neuron (MN) loss, muscle denervation, and eventually, paralysis. Currently, no effective treatments are available to stop or reverse ALS disease progression and the precise molecular mechanisms underlie ALS pathogenesis remain elusive. Prior studies revealed decreased mitochondrial respiratory chain activity, altered mitochondrial ultrastructure, and mitochondrial dysfunction in both MN and skeletal muscle (SM) in ALS patients and mouse models. The first sign of ALS pathology occurs at the neuromuscular junction (NMJ), where presynaptic MN axons connect with postsynaptic SM end plates. To date, whether signals resulting in the initial NMJ damage are from MN or SM remain unclear. In this project, we aim to determine the tissue-specific causative role of mitochondrial Ca2+ uptake in SM and MN in disease onset and progression, and the therapeutic efficacy of reducing mitochondrial Ca2+ uptake on NMJ and SM function in ALS mice. We hypothesize that mitochondrial Ca2+ mishandling in both SM and MN actively contribute to ALS disease pathogenesis and that attenuating mitochondrial Ca2+ uptake mitigates mitochondrial damage and preserves NMJ/muscle function. To test this hypothesis, we will use transgenic mice with inducible, SM or MN-specific expression of a dominant negative form of the mitochondrial Ca2+ uniporter to specifically and selectively reduce mitochondrial Ca2+ uptake in SM and MN in hSOD1G93A mice and C9-500 (C9orf72) mice, two mouse models associated with the most prevalent genetic causes for ALS. The central hypothesis will be tested in two Specific Aims. Aim 1 will determine the role of mitochondrial Ca2+ uptake in SM or MN in survival, motor function, NMJ function and in vivo muscle performance in hSOD1G93A and C9-500 mice. Aim 2 will assess the impact of tissue-specific inhibition of mitochondrial Ca2+ uptake in SM or MN on NMJ and muscle structure, MN survival, muscle intrinsic contractile properties, mitochondrial structure and mitochondrial bioenergetics in SM of hSOD1G93A and C9-500 mice. This project will: 1) provide a systematic, longitudinal characterization of SM and NMJ function from a cellular level to whole animal level at different stages of disease progression in hSOD1G93A and C9-500 mice; 2) determine the degrees to which defects in mitochondrial Ca2+ uptake in SM or MN contribute to altered NMJ structure/function, disease onset and progression in hSOD1G93A and C9-500 mice; 3) provide the first detailed dissection on the relative role of mitochondrial Ca2+ uptake in SM and MN in ALS phenotype using the same genetic models and determine the origin of the signals that result in NMJ destruction (from SM or MN or both); 4) provide mechanistic evidence for whether mitochondrial Ca2+ mishandling is a trigger or a target for disease progression in ALS mice, regardless of the causing mutations (mitochondrial related or non-mitochondrial related); and most importantly, 5) test the validity of a potential new therapeutic target (mitochondrial Ca2+ uptake, or the mitochondrial Ca2+ uniporter) for the treatment of ALS.