Tissue-specific role of aberrant mitochondrial Ca2+ uptake in respiratory and limb muscle dysfunction in ALS

肌萎缩侧索硬化症（ALS）是一种致命的，成人发病的神经退行性疾病，其特征在于 进行性运动神经元（MN）丧失、肌肉去神经支配，并最终瘫痪。目前，没有有效的 治疗可用于停止或逆转ALS疾病进展和精确的分子机制 ALS发病机制的基础仍然是难以捉摸的。先前的研究表明线粒体功能障碍是早期的全球性疾病， 在ALS患者和小鼠模型的MN和骨骼肌中的病理学。ALS病理学的第一个迹象 发生在神经肌肉接头（NMJ），在那里突触前MN轴突与突触后骨骼肌连接。 肌肉终板迄今为止，导致最初NMJ损伤的信号是来自MN还是骨骼肌， 仍然不清楚。呼吸衰竭是ALS患者死亡的主要原因。然而，有限的研究 目前对呼吸肌MN丢失机制的研究较少，对呼吸肌MN丢失机制的研究较少。 弱点在这个项目中，我们的目的是确定线粒体Ca 2+摄取在组织特异性致病作用， 四肢和呼吸肌的MN丢失和肌肉功能障碍以及减少MN丢失和肌肉功能障碍的治疗效果 线粒体Ca 2+摄取对ALS小鼠疾病进展和呼吸功能的影响。我们假设 骨骼肌和MN中异常的线粒体Ca 2+摄取协同地有助于肢体和 呼吸肌无力和线粒体Ca 2+摄取的组织特异性衰减将减轻Ca 2 +- 诱导线粒体功能障碍，促进MN存活并保护肢体和膈肌功能。到 为了验证这一假设，我们将使用具有可诱导的骨骼肌或MN特异性表达的转基因小鼠， 线粒体Ca 2+单向转运蛋白的显性负性形式，以特异性和选择性地减少线粒体Ca 2+转运蛋白的表达。 hSOD 1G 93 A小鼠骨骼肌和MN中的Ca 2+摄取。中心假设将在两个特定的 目标。目的1将检验骨骼肌线粒体Ca 2+摄取的组织特异性衰减的假设， 肌肉或MN-小鼠存活，改善运动和呼吸功能，保留NMJ传输 和整体肌肉表现的影响。目的2将检验组织特异性抑制 骨骼肌或MN中的线粒体Ca 2+摄取促进MN存活，保护NMJ结构，肌肉 肌萎缩侧索硬化症患者肢体和呼吸肌的收缩和Ca 2+信号传导特性以及线粒体功能 小鼠该项目将：1）提供肢体和呼吸肌的系统性纵向表征， 在hSOD 1G 93 A中疾病进展的不同阶段，从细胞水平到整个动物水平的NMJ功能 小鼠; 2）提供了第一个关于骨骼肌线粒体Ca 2+摄取的相对作用的详细解剖 和MN在ALS表型中使用相同的遗传模型，并确定导致 NMJ破坏（来自肌肉或MN或两者）; 3）为线粒体Ca 2+是否 处理不当是ALS小鼠疾病进展的触发因素或靶点;以及4）测试潜在新疗法的有效性。 本发明涉及用于治疗ALS的治疗靶点（线粒体Ca 2+摄取，或线粒体Ca 2+单向转运体）。

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 mitochondrial dysfunction as an early global pathology in both MN and skeletal muscle 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 skeletal muscle end plates. To date, whether signals resulting in the initial NMJ damage are from MN or skeletal muscle remain unclear. Respiratory failure is the leading cause of death in ALS patients. However, limited research has been focused on mechanisms of respiratory MN loss, and even less on mechanisms of respiratory muscle weakness. In this project, we aim to determine the tissue-specific causative role of mitochondrial Ca2+ uptake in MN loss and muscle dysfunction in both limb and respiratory muscles and the therapeutic efficacy of reducing mitochondrial Ca2+ uptake on disease progression and respiratory function in ALS mice. We hypothesize that aberrant mitochondrial Ca2+ uptake in both skeletal muscle and MN synergistically contribute to limb and respiratory muscle weakness and that tissue-specific attenuation of mitochondrial Ca2+ uptake will mitigate Ca2+- induced mitochondrial dysfunction, promote MN survival and preserve limb and diaphragm muscle function. To test this hypothesis, we will use transgenic mice with inducible, skeletal muscle or MN-specific expression of a dominant negative form of the mitochondrial Ca2+ uniporter to specifically and selectively reduce mitochondrial Ca2+ uptake in skeletal muscle and MN in hSOD1G93A mice. The central hypothesis will be tested in two Specific Aims. Aim 1 will test the hypothesis that tissue-specific attenuation of mitochondrial Ca2+ uptake in skeletal muscle or MN prolongs mouse survival, improves motor and breathing function, preserves NMJ transmission and overall muscle performance in ALS mice. Aim 2 will test the hypothesis that tissue-specific inhibition of mitochondrial Ca2+ uptake in skeletal muscle or MN promotes MN survival, preserves NMJ structure, muscle contractile and Ca2+ signaling properties and mitochondrial function in both limb and respiratory muscles in ALS mice. This project will: 1) provide a systematic, longitudinal characterization of limb and respiratory muscle and NMJ function from a cellular level to whole animal level at different stages of disease progression in hSOD1G93A mice; 2) provide the first detailed dissection on the relative role of mitochondrial Ca2+ uptake in skeletal muscle and MN in ALS phenotype using the same genetic model and determine the origin of the signals that result in NMJ destruction (from muscle or MN or both); 3) provide mechanistic evidence for whether mitochondrial Ca2+ mishandling is a trigger or a target for disease progression in ALS mice; and 4) test the validity of a potential new therapeutic target (mitochondrial Ca2+ uptake, or the mitochondrial Ca2+ uniporter) for the treatment of ALS.