Defects of mitochondrial dynamics in ALS

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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.