SOD1G93A Mutant forms Aggregates Inside Mitochondria and Alters Mitochondrial Structure and Dynamics in Skeletal Muscle

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular degenerative disease. Mutations in SOD1 (superoxide dismutase 1) is a major cause for most familial ALS. The transgenic mouse model (G93A) overexpressing mutant SOD1G93A recapitulates many features of the human disease. Immunohistochemical analysis of motor neurons in G93A mice showed accumulation of SOD1 inside swollen mitochondria (Deng 2006). Our earlier study also found similar morphological changes in skeletal muscle of G93A mice (Zhou 2010). SOD1 is a cytosolic protein with a small portion inside mitochondria under normal conditions. To elucidate why mutant SOD1 causes mitochondrial dysfunction, we targeted SOD1 directly into mitochondria by making constructs (mt-SOD1-Dendra and mt-SOD1G93A-Dendra) with addition of a mitochondrial targeting peptide at the N-terminus of SOD1/SOD1G93A and a photoswitchable fluorescent protein (Dendra2, Addgene) at the C-terminus. Those constructs were acutely expressed in skeletal muscle of normal mice. 7 days after transfection, muscle fibers expressing SOD1G93A showed aggregated fluorescent proteins inside mitochondria (SOD1G93A: 91%, n= 24; SOD1: 0%, n=16) and 3-fold more fibers with fragmented mitochondria (SOD1G93A: 67%; SOD1: 19%). Mitochondrial dynamics was also evaluated. After Dendra was photo-converted from green to red in a fiber area (∼ 10 μm x 10 μm), the migration of Dendra out of the original area was evaluated in both longitudinal and transversal directions. Migration over one sarcomere distance (∼2.2 μm) was defined as one migration step (ms). A significant reduction of mitochondrial dynamics was found in fibers expressing SOD1G93A (1.4 ± 0.3 ms, n=7) compared with SOD1 (5.1 ± 1.2 ms, n=8) in 20 min. The result is consistent with the finding in G93A mice. While normal fibers showed 18.7 ± 2.3 ms of mitochondrial migration (n=13), G93A fibers only showed 2.0 ± 0.4 ms (n=12).