Defects of mitochondrial dynamics in ALS

描述（由申请人提供）：神经元依赖于精细调节的运输机制来保持其细胞体和广泛的过程连接。越来越多的证据表明，运动神经元（MN）疾病中的细胞器运输受损，其中细胞成分必须沿着轴突长距离移动，并且运输缺陷可能有助于为什么MN在肌萎缩性侧索硬化症（ALS）中特别受到影响。该提议的中心假设是，线粒体动力学受损（即运输、融合、裂变）是ALS MN的主要病变：当运输受损时，线粒体不能正常往返于能量利用的关键部位，如突触末端，导致线粒体定位错误和功能障碍，进而导致能量消耗、钙稳态受损，最终导致细胞变性。在这项提议中，我们将从表达突变体SOD1的转基因动物模型中研究原发性MN的线粒体动力学缺陷，这导致了家族性ALS。我们将使用一种新的，光激活的，荧光蛋白靶向线粒体，（mitto - dendra）和活共聚焦细胞成像。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功能障碍之间的相关性。我们的初步数据强烈表明，线粒体动力学在SOD1突变体MN中是异常的，这种异常与生物能量学受损有关。首先，我们将描述突变SOD1如何影响线粒体运输，并确定线粒体运输缺陷是MN特异性的还是影响其他神经细胞类型。此外，由于ALS除MN外还涉及其他细胞类型，我们将确定直接参与ALS发病机制的星形胶质细胞和小胶质细胞是否在MN中损害线粒体动力学和功能中发挥作用。其次，我们将确定在区隔化神经支配的MN-肌肉共培养中，突变SOD1 MN中线粒体动力学缺陷如何影响神经肌肉接点（NMJ）与肌肉细胞的相互作用。第三，为了验证线粒体动力学损伤是MN变性的主要缺陷，我们将在正常野生型MN中建立线粒体运输在维持MN和NMJs中的作用，在正常野生型MN中，顺行线粒体运输通过遗传方法受损，独立于突变的SOD1。与公共卫生相关：线粒体是细胞内致力于能量代谢的细胞器。线粒体必须沿着神经元运输，并放置在需要能量的地方。线粒体运输缺陷导致疾病。本提案探索了临床研究的新领域，结合了新颖的实验方法，利用最近开发的荧光显微镜技术。更好地了解ALS中线粒体动力学和功能的变化将有助于确定治疗途径。此外，我们正在开发的用于研究线粒体运输缺陷的系统和模型不仅适用于ALS，也适用于许多其他神经退行性疾病。

DESCRIPTION (provided by applicant): 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 neuromuscular 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. PUBLIC HEALTH RELEVANCE: Mitochondria are intracellular organelles dedicated to energy metabolism. Mitochondria must be transported along neurons and positioned where energy is needed. Defective mitochondrial transport results in disease. This proposal explores the new field of clinical research using a combination of novel experimental approaches, taking advantage of recently developed fluorescent microscopy techniques. A better understanding of the changes in the dynamics and function of mitochondria in ALS will contribute to identifying avenues of treatment. Furthermore, the system and models that we are developing to study mitochondrial transport defects will be applicable not only to ALS, but also to many other neurodegenerative disorders.