Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of motor neurons, ultimately leading to paralysis and death in three to five years after onset. Despite almost two decades of research, no clear mechanism has emerged to explain both the tissue specificity and the pathogenesis of this disease. Recent work by our lab revealed a tissue-specific defect in mitochondrial protein import in a rodent model of superoxide dismutase 1 (SOD1)-mediated familial ALS. This defect in import is accompanied by increased levels of the translocase of the outer mitochondrial membrane (TOM) proteins TOM20, TOM22, and TOM40, which are three core components of the machinery responsible for the import of nearly all nuclear-encoded mitochondrial proteins. The fact that mutant SOD1 uniquely inhibits protein import in mitochondria isolated from spinal cord presents an attractive explanation for both the tissue specificity and mechanism of disease. However, nothing is known about how mutant SOD1 inhibits protein import in spinal cord mitochondria or how this inhibition affects TOM component levels. By applying established techniques to novel questions about mutant SOD1, ALS, and mitochondria, the experiments in this proposal will answer fundamental mechanistic questions about the role mutant SOD1 plays in protein import and ALS, ultimately shifting the knowledge from observation to mechanism. Such a shift would springboard mitochondrial protein import to the forefront of ALS disease research, with the ultimate goal of focusing therapy development for this incurable disease. ! ! In Aim I, we will determine how mutant SOD1 inhibits mitochondrial protein import by testing the hypothesis that mutant SOD1 physically blocks precursor protein translocation through the mitochondrial outer membrane. First, we will utilize a protease-sensitive SOD1 mutant to assess the degree of protein import inhibition in mitochondria. We will then ask whether mutant SOD1 occupies the TOM pore or inhibits mitochondria- targeted precursor proteins from binding to the TOM machinery. ! In Aim II, we will elucidate the relationship between the reduced import capacity and increased levels of TOM proteins observed in ALS. First, to determine if increased TOM proteins represent a compensatory mechanism to a global import deficit in mitochondria, import will be inhibited in cell culture via pharmacologic and protein- based approaches followed by quantification of TOM mRNA and protein levels. Second, to determine if increased levels of TOM proteins correspond to increased import capacity, mitochondrial protein import will be assessed in immortalized cell lines overexpressing these TOM components. Finally, we will apply this model applies to additional forms of ALS by examining TOM protein levels in sporadic and familial TDP-43 human spinal cords.
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the selective loss of motor neurons, ultimately leading to paralysis and death in three to five years after onset. Despite almost two decades of research, no clear mechanism has emerged to explain both the tissue specificity and the pathogenesis of this disease. Building off previous work from our group implementing mitochondrial protein import as a major factor in ALS pathogenesis, this proposal aims to elucidate the mechanism responsible for defective mitochondrial protein import seen in a rodent model of ALS and seeks to apply those findings to human sporadic ALS. Success in these endeavors would springboard mitochondria to the forefront of ALS research and would provide a mechanistic foundation for future therapeutic development.
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