Amyotrophic lateral sclerosis (ALS) is a fatal, adult-onset neurodegenerative disease characterized by progressive motor neuron (MN) loss, muscle denervation, and eventually, paralysis. Currently, no effective treatments are available to stop or reverse ALS disease progression and the precise molecular mechanisms underlie ALS pathogenesis remain elusive. Prior studies revealed mitochondrial dysfunction as an early global pathology in both MN and skeletal muscle in ALS patients and mouse models. The first sign of ALS pathology occurs at the neuromuscular junction (NMJ), where presynaptic MN axons connect with postsynaptic skeletal muscle end plates. To date, whether signals resulting in the initial NMJ damage are from MN or skeletal muscle remain unclear. Respiratory failure is the leading cause of death in ALS patients. However, limited research has been focused on mechanisms of respiratory MN loss, and even less on mechanisms of respiratory muscle weakness. In this project, we aim to determine the tissue-specific causative role of mitochondrial Ca2+ uptake in MN loss and muscle dysfunction in both limb and respiratory muscles and the therapeutic efficacy of reducing mitochondrial Ca2+ uptake on disease progression and respiratory function in ALS mice. We hypothesize that aberrant mitochondrial Ca2+ uptake in both skeletal muscle and MN synergistically contribute to limb and respiratory muscle weakness and that tissue-specific attenuation of mitochondrial Ca2+ uptake will mitigate Ca2+- induced mitochondrial dysfunction, promote MN survival and preserve limb and diaphragm muscle function. To test this hypothesis, we will use transgenic mice with inducible, skeletal muscle or MN-specific expression of a dominant negative form of the mitochondrial Ca2+ uniporter to specifically and selectively reduce mitochondrial Ca2+ uptake in skeletal muscle and MN in hSOD1G93A mice. The central hypothesis will be tested in two Specific Aims. Aim 1 will test the hypothesis that tissue-specific attenuation of mitochondrial Ca2+ uptake in skeletal muscle or MN prolongs mouse survival, improves motor and breathing function, preserves NMJ transmission and overall muscle performance in ALS mice. Aim 2 will test the hypothesis that tissue-specific inhibition of mitochondrial Ca2+ uptake in skeletal muscle or MN promotes MN survival, preserves NMJ structure, muscle contractile and Ca2+ signaling properties and mitochondrial function in both limb and respiratory muscles in ALS mice. This project will: 1) provide a systematic, longitudinal characterization of limb and respiratory muscle and NMJ function from a cellular level to whole animal level at different stages of disease progression in hSOD1G93A mice; 2) provide the first detailed dissection on the relative role of mitochondrial Ca2+ uptake in skeletal muscle and MN in ALS phenotype using the same genetic model and determine the origin of the signals that result in NMJ destruction (from muscle or MN or both); 3) pr...