Therefore it can be concluded that imaging or staining methods did not bias the results

of these plasmids was transfected into the FDB muscle of normal mice. Seven days after transfection, FDB muscle fibers expressing mt-SOD1-Dendra or mtSOD1G93A-Dendra were isolated and incubated with TMRE. doi: 10.1371/journal.pone.0082112.g003 fluorescent protein inside mitochondria. In contrast, not a single fiber expressing mt-SOD1-Dendra had aggregated fluorescent protein. In addition, 63% of fibers expressing mtSOD1G93A-Dendra showed fragmented mitochondria compared to only 11% of fibers expressing mt-SOD1-Dendra. The time-dependent migration of Dendra fluorescent proteins was monitored using the method described earlier. Representative images are shown in 5 Mitochondrial Dynamics in ALS Skeletal Muscle doi: 10.1371/journal.pone.0082112.g004 significant movement of the fluorescent protein during the 20 min recording after the photoactivation, suggesting mitochondrial dynamics in those regions are more severely impaired. Overall, our data show that muscle fibers in normal mice expressing mutant mt-SOD1G93A-Dendra have significantly reduced mitochondrial dynamics compared to control fibers expressing mt-SOD1-Dendra. doi: 10.1371/journal.pone.0082112.g005 Abnormal mitochondrial dynamics is associated with mitochondrial inner membrane depolarization depolarization alone altered mitochondrial dynamics. We found that incubation of normal muscle fibers with 200 nM FCCP partially depolarized the inner membrane potential evoking a reduction of TMRE fluorescence intensity of about 19%. To evaluate mitochondrial dynamics, the same FCCP challenge was applied to fibers expressing wild type SOD1 fusion protein. After 20-min FCCP incubation, a small fiber region was photoactivated and the migration of red fluorescent protein from this region was purchase 2883-98-9 measured. 6 Mitochondrial Dynamics in ALS Skeletal Muscle Mutant SOD1G93A promotes mitochondrial fission events by enhancing the function of mitochondrial fission protein Drp1 Normal mitochondrial dynamics rely on the balance between fusion and fission processes. Reduced migration rate of SOD1G93A-Dendra fusion protein implies a net decrease in fusion events or a net increase in fission events. Mitochondrial fusion and fission events are regulated by key proteins such as Mitofusin and dynamin-related protein 1 respectively. We examined whether inhibition of mitochondrial fission protein Drp1 could restore normal mitochondrial dynamics in fibers expressing mutant SOD1G93A. Two groups of normal mice were transfected with mt-SOD1G93A-Dendra at the site of FDB muscle. In the next 7 days, one group received intraperitoneal injection of Mdivi-1, a specific inhibitor of Drp1 as described in Method section. The control group received the intraperitoneal injection of same amount of DMSO. After the 7-day period, the FDB muscles were removed and mitochondrial dynamics were determined. Representative images are shown in doi: 10.1371/journal.pone.0082112.g006 Discussion In this study, we discovered that skeletal muscle of an ALS mouse model G93A has abnormal mitochondrial dynamics at the age of two months when there is no significant axonal withdrawal 11336787 and disease symptoms. We further demonstrated that similar abnormality of mitochondrial dynamics can be directly induced by overexpressing mutant SOD1G93A 15102954 protein in skeletal muscle of normal mice. Our data suggest that SOD1G93A mutation drives ALS-associated muscle pathology in the absence of motor neuron degeneration. Mitochondrial dynamics involves mitochondrial fissi