Amyotrophic lateral sclerosis (ALS) is a fatal, adult-onset neurodegenerative disease characterized by progressive motor neuron (MN) loss, muscle denervation, and eventually, paralysis. Patients typically die from respiratory failure 3-5 years from the onset of symptoms. Currently, no effective treatments are available to stop or reverse ALS disease progression. The Cu,Zn, superoxide dismutase gene (SOD1) was the first gene shown to cause ALS. However, the precise molecular mechanisms by which SOD disease mutations underlie ALS pathogenesis remain frustratingly elusive. Prior studies revealed decreased mitochondrial respiratory chain activity, altered mitochondrial ultrastructure, and mitochondrial dysfunction in both MN and skeletal muscle (SM) in ALS patients and hSOD1G93A transgenic mice, the most commonly used ALS mouse model. Given that depolarization of mitochondria in SM adjacent to the neuromuscular junction (NMJ) is one of the earliest phenotypes observed in hSOD1G93A mice and SM-specific expression of hSOD1G93A leads to MN degeneration, an early/active role of SM in MN damage has been proposed to underlie ALS pathogenesis. In this project, we aim to determine the involvement of a SM-specific pathway, specifically the pathophysiological role of aberrant mitochondrial Ca2+ uptake in SM, in disease onset and progression in hSOD1G93A mice. We will also assess the therapeutic efficacy of reducing mitochondrial Ca2+ uptake in SM on NMJ competence and SM function in hSOD1G93A mice. We hypothesize that mitochondrial dysfunction in SM actively contributes to ALS pathogenesis and that attenuating mitochondrial Ca2+ uptake in SM will prevent mitochondria damage and preserve NMJ/muscle function. To test this hypothesis, we generated a new mouse model that permits inducible, SM-specific expression of a dominant negative form of the mitochondrial Ca2+ uniporter (dnMCU) to reduce mitochondrial Ca2+ uptake in SM of hSOD1G93A mice. The central hypothesis will be tested in two Specific Aims.
Aim 1 will determine the role of mitochondrial Ca2+ uptake in SM in the survival and single muscle fiber phenotypes of hSOD1G93A mice, including intracellular Ca2+ release properties, mitochondrial function and NMJ structure.
Aim 2 will evaluate the rescue of NMJ function, contractile strength, and overall muscle performance by inhibiting mitochondrial Ca2+ uptake in SM of hSOD1G93A mice. This project will: 1) provide the first systematic, longitudinal characterization of SM and NMJ function from a cellular to whole animal level at different stages of disease progression in hSOD1G93A mice, 2) determine the degree to which defects in mitochondrial Ca2+ uptake in SM contribute to altered NMJ structure/function and disease onset and progression in hSOD1G93A mice, 3) provide evidence for whether mitochondrial dysfunction in SM is a trigger or epiphenomenon of disease progression in hSOD1G93A mice, and most importantly, 4) test the efficacy of a potential new therapeutic target (inhibition of MCU in SM) for the treatment of ALS.
The global objective of this proposal is to assess the role of mitochondrial Ca2+ uptake in skeletal muscle on Amyotrophic Lateral Sclerosis (ALS) pathogenesis. Specifically, we will determine the impact of aberrant mitochondrial Ca2+ uptake in skeletal muscle on the survival, exercise performance, and intracellular Ca2+ release properties, mitochondrial function in skeletal muscle fibers, muscle force generation, and neuromuscular junction structure/function at different stage of disease progression in an established mouse model of ALS. Results from this project will provide pre-clinical evidence needed to determine if reducing excessive mitochondrial Ca2+ uptake in skeletal muscle is a viable therapeutic target for ALS treatment.