Defects of mitochondrial dynamics in ALS

神经元依赖于一个微调的运输机制，以保持他们的细胞体和广泛的 进程连接。越来越多的证据表明，细胞器运输受损的疾病， 运动神经元（MN），其中细胞成分必须沿沿着长距离移动， 运输缺陷可能是肌萎缩侧索硬化症中MN特别受影响的原因 （ALS）。该提议的中心假设是受损的线粒体动力学（即，运输， 核融合、核分裂）是ALS MN的一种原发性损害：当转运受损时，线粒体不能 正常地往返于能量利用的关键部位，如突触末梢，导致 线粒体定位错误和功能障碍，这反过来又会导致能量消耗， 体内平衡，最终导致细胞退化。 在这个建议中，我们将研究转基因小鼠原发性MN中的线粒体动力学缺陷， 表达突变SOD 1的动物模型，其导致家族性ALS。我们会用一本小说， 光活化荧光蛋白靶向线粒体（mito-Dendra）和活的共聚焦细胞 显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功能障碍。我们的初步数据有力地表明， 线粒体动力学在SOD 1突变MN中异常，这种异常与 生物能量受损首先，我们将描述突变SOD 1如何影响线粒体转运 并确定线粒体转运缺陷是否是MN特有的，或者它们是否影响其他神经系统， 细胞类型。此外，由于ALS涉及除了MN之外的其他细胞类型，我们将确定是否 直接参与ALS发病机制的星形胶质细胞和小胶质细胞在损害ALS的神经功能中发挥作用。 MN中线粒体动力学和功能。其次，我们将确定缺陷线粒体 突变体SOD 1 MN的动力学影响与神经肌肉接头处肌细胞的相互作用 (NMJ)在区室化神经支配的MN-肌肉共培养物中。第三，为了验证线粒体 动力学损伤是MN变性的主要缺陷，我们将建立线粒体的作用， 在正常野生型MN中维持MN和NMJ的转运，其中顺行线粒体 运输已被遗传方法，独立于突变体SOD 1受损。

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.