Defects of mitochondrial dynamics in ALS

神经元依靠一种微调的运输机械来保持其胞体和广泛的 进程已连接。越来越多的证据表明，细胞器运输在一些疾病中受到损害。 运动神经元(MN)，在那里细胞组件必须沿着轴突长距离移动，而且 转运缺陷可能是MN在肌萎缩侧索硬化症中受到特异性影响的原因之一 (ALS)。这一建议的中心假设是线粒体动力学受损(即，运输， 融合，裂变)是ALS MN的主要损害：当运输受损时，线粒体不能运输 通常往返于能量利用的关键部位，如突触终末，导致 线粒体定位错误和功能障碍，进而导致能量耗竭，钙离子受损 动态平衡，最终导致细胞退化。 在这个方案中，我们将研究转基因MN的线粒体动力学缺陷。 表达突变SOD1的动物模型，导致家族性肌萎缩侧索硬化症。我们将用一本小说， 针对线粒体、(mito-Dendra)和活的共聚焦细胞的可光激活的荧光蛋白 成像。我们将研究线粒体动力学缺陷、线粒体 结构异常和生物能量障碍。我们的初步数据强烈表明 SOD1突变型MN线粒体动力学异常，这种异常与 受损的生物能量学。首先，我们将描述突变型SOD1如何影响线粒体运输 并确定线粒体转运缺陷是否是MN特有的，或者是否影响其他神经 单元类型。此外，由于ALS涉及MN以外的其他细胞类型，我们将确定 与ALS发病机制直接相关的星形胶质细胞和小胶质细胞在ALS的发病机制中起着重要作用 MN中线粒体的动力学和功能。第二，我们将确定线粒体缺陷如何 突变型SOD1MN的动力学影响神经肌肉连接处与肌细胞的相互作用 (NMJ)，在分区神经支配的MN-肌肉共培养中。第三，验证线粒体 动力学损伤是MN退变的主要缺陷，我们将确定线粒体的作用 在正常、野生型MN中维持MN和NMJ的转运，其中线粒体顺行 运输受到了遗传途径的损害，与突变的SOD1无关。

Neurons depend on a finely tuned transport machinery to keep their cell bodies and extensive processes connected. Increasing evidence suggests that organelle transport is impaired in diseases of motor neurons (MN), where cellular components have to move long distances along axons, and that transport defects may contribute to why MN are specifically affected in amyotrophic lateral sclerosis (ALS). The central hypothesis of this proposal is that impaired mitochondrial dynamics (i.e., transport, fusion, fission) is a primary lesion in ALS MN: when transport is impaired, mitochondria cannot traffic normally to and from crucial sites of energy utilization, such as synaptic terminals, resulting in mitochondrial mislocalization and dysfunction, which in turn causes energy depletion, impaired calcium homeostasis, and ultimately cell degeneration. In this proposal, we will investigate mitochondrial dynamics defects in primary MN from transgenic animal models expressing mutant SOD1, which causes a familial form of ALS. We will use a novel, photo-activatable, fluorescent protein targeted to mitochondria, (mito-Dendra), and live confocal cell imaging. We will investigate the correlations between mitochondrial dynamics defects, mitochondrial structural abnormalities and bioenergetic dysfunction. Our preliminary data strongly suggest that mitochondrial dynamics is abnormal in SOD1 mutant MN and that this abnormality correlates with impaired bioenergetics. First, we will characterize how mutant SOD1 affects mitochondrial transport and determine whether mitochondrial transport defects are specific to MN or if they affect other neural cell types. Furthermore, since ALS involves other cell types besides MN, we will determine whether astrocytes and microglia, which are directly implicated in ALS pathogenesis, play a role in impairing mitochondrial dynamics and function in MN. Second, we will determine how defective mitochondrial dynamics in mutant SOD1 MN affects the interactions with muscle cells at the nuromuscular junction (NMJ), in compartmentalized innervated MN-muscle co-cultures. Third, to verify that mitochondrial dynamics impairment is a primary defect in MN degeneration we will establish the role of mitochondrial transport in maintaining MN and NMJs in normal, wild type, MN, where anterograde mitochondrial transport has been impaired by a genetic approach, independent of mutant SOD1.