Defects of mitochondrial dynamics in ALS

描述(申请人提供)：神经元依靠微调的运输机械来保持它们的细胞体和广泛的过程连接。越来越多的证据表明，在运动神经元疾病(MN)中，细胞器运输受到损害，细胞成分必须沿着轴突长距离移动，运输缺陷可能是肌萎缩侧索硬化症(ALS)中MN受到特殊影响的原因之一。这一设想的核心假设是，线粒体动力学受损(即运输、融合、分裂)是ALS MN的主要损害：当运输受损时，线粒体无法正常往返于突触终末等能量利用的关键部位，导致线粒体错误定位和功能障碍，进而导致能量耗竭、钙稳态受损，最终导致细胞退化。在这项建议中，我们将研究原代MN中表达突变SOD1的转基因动物模型中的线粒体动力学缺陷，该突变导致家族性ALS。我们将使用一种新颖的、可光激活的、针对线粒体的荧光蛋白(mito-Dendra)，并进行实时共聚焦细胞成像。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，在SOD1突变的MN中，线粒体动力学是异常的，这种异常与生物能量学受损有关。首先，我们将确定突变的SOD1是如何影响线粒体运输的，并确定线粒体运输缺陷是MN特有的还是影响其他类型的神经细胞。此外，由于ALS涉及MN以外的其他细胞类型，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的线粒体动力学和功能受损中发挥作用。其次，我们将确定在分区神经支配的MN-肌肉共培养中，突变的SOD1MN中的线粒体动力学缺陷如何影响神经肌肉接头(NMJ)处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退变的主要缺陷，我们将在正常、野生型MN中建立线粒体运输在维持MN和NMJ中的作用，其中线粒体顺行运输已被一种遗传方法损害，独立于突变的SOD1。公共卫生相关性：线粒体是专用于能量新陈代谢的细胞内细胞器。线粒体必须沿着神经元运输，并定位在需要能量的地方。线粒体运输缺陷会导致疾病。这一建议探索了临床研究的新领域，使用了新的实验方法的组合，利用了最近发展的荧光显微镜技术。更好地了解肌萎缩侧索硬化症中线粒体的动态和功能变化将有助于确定治疗途径。此外，我们正在开发的研究线粒体运输缺陷的系统和模型不仅适用于ALS，而且还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.