ROS-related mitochondrial dysfunction in skeletal muscle of an ALS mouse model during the disease progression

In amyotrophic lateral sclerosis (ALS), mitochondrial dysfunction and oxidative stress form a vicious cycle that promotes neurodegeneration and muscle wasting. To quantify the disease-stage-dependent changes of mitochondrial function and their relationship to the generation of reactive oxygen species (ROS), we generated double transgenic mice (G93A/cpYFP) that carry human ALS mutation SOD1(G93A) and mt-cpYFP transgenes, in which mt-cpYFP detects dynamic changes of ROS-related mitoflash events at individual mitochondria level. Compared with wild type mice, mitoflash activity in the SOD1(G93A) (G93A) mouse muscle showed an increased flashing frequency prior to the onset of ALS symptom (at the age of 2 months), whereas the onset of ALS symptoms (at the age of 4 months) is associated with drastic changes in the kinetics property of mitoflash signal with prolonged full duration at half maximum (FDHM). Elevated levels of cytosolic ROS in skeletal muscle derived from the SOD1(G93A) mice were confirmed with fluorescent probes, MitoSOX (TM) Red and ROS Brite (TM) 570. Immunoblotting analysis of subcellular mitochondrial fractionation of G93A muscle revealed an increased expression level of cyclophilin D (CypD), a regulatory component of the mitochondrial permeability transition pore (mPTP), at the age of 4 months but not at the age of 2 months. Transient overexpressing of SOD1(G93A) in skeletal muscle of wild type mice directly promoted mitochondria] ROS production with an enhanced mitoflash activity in the absence of motor neuron axonal withdrawal. Remarkably, the SOD1(G93A)-induced mitoflash activity was attenuated by the application of cyclosporine A (CsA), an inhibitor of CypD. Similar to the observation with the SOD1(G93A) transgenic mice, an increased expression level of CypD was also detected in skeletal muscle following transient overexpression of SOD1(G93A) Overall, this study reveals a disease-stage-dependent change in mitochondria] function that is associated with CypD-dependent mPTP opening; and the ALS mutation SOD1(G93A) directly contributes to mitochondrial dysfunction in the absence of motor neuron axonal withdrawal.