LAUSR

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease. Oxidative stress is implicated in ALS pathophysiology. Using ROS Brite™ 570, a cytosolic ROS detector, we found a significant increase in ROS production of muscle fibers derived from the ALS G93A mice. Mitochondria are the major source of ROS production. In order to detect early changes in ROS-related mitochondrial metabolic function, we generated double transgenic mice (G93A/cpYFP) that carry human ALS mutation SOD1G93A and mt-cpYFP transgenes, in which mt-cpYFP monitors dynamic changes of ROS-related mitoflash events at the single mitochondrion level. Remarkably, G93A muscle cells show early and disease stage-dependent changes in mitoflash events. The mitoflash activity at the age of 2 months (before ALS symptom onset) is marked by an increased flashing area in G93A muscle fibers, while their kinetics properties remain unchanged. After ALS onset (3-month old), there are drastic changes in the kinetics of mitoflash signal with prolonged FDHM (full duration at half maximum) of the mitoflash signal. Thus, mitoflash provides a sensitive and quantitative biomarker for evaluating ROS-related mitochondrial dysfunction. It is known that uncontrolled opening of mitochondrial permeability transition pore (mPTP) is a key step to promote mitochondrial ROS production. While the molecular composition of mPTP is still incompletely understood, studies have shown that cyclophilin D (CypD) promotes the opening of mPTP and phosphorylated form of GSK-3β participates in maintaining mPTP in the closed state. We used subcellular fractionation to isolate mitochondria from skeletal muscle of G93A mice, and found an increased CypD expression level in muscle mitochondria, while the phosphorylated form of GSK-3β was significantly reduced in mitochondria of G93A muscle. Thus, both may act as triggering factors for mPTP opening in G93A muscle.