Amyotrophic lateral sclerosis (ALS) is usually a fatal, progressive paralysis arising from the premature death of motor neurons. strength provides no benefit in slowing disease onset or progression. can be beneficial remains untested, however, because in this report the effect on limb muscles was short lived, yielding neither a survival benefit nor preservation of muscle mass throughout disease progression. In contrast, muscle hypertrophy induced by brokers such as IGF-1 (8C10) or growth hormone (8C10) led to significant life extensions in ALS transgenic mice. For the IGF-1 studies, not only was there muscle hypertrophy, but also there was concomitant stimulation of muscle satellite cell proliferation and an increase of centrally nucleated muscle fibers, indicating regeneration (8). Added to these findings, a number of studies have shown that exercise is beneficial in ALS transgenic animals (11C13), with exercise and IGF-1 exhibiting a synergistic effect resulting in an increase Dasatinib small molecule kinase inhibitor in median life span by 83 days (11C13). These prior efforts raised the likelihood that potential therapies that limited the synthesis of the disease-causing mutant SOD1 gene or that increased growth factor production might be acting in whole or in part on the Dasatinib small molecule kinase inhibitor muscle. Determining whether an initiating toxic insult or a determinant of disease progression after onset is usually developed within muscle is important, because if so, muscle is an attractive target for therapeutic development in ALS, especially by virus-mediated Dasatinib small molecule kinase inhibitor gene therapy approaches. Here we use three genetic approaches to test the role of muscle in PLA2G3 mutant SOD1 disease onset and progression. Results Reducing Mutant SOD1 Within Muscle Does Not Affect Disease. After intramuscular injection and its retrograde transport to spinal motor neurons (14), adeno-associated computer virus (AAV) encoding an siRNA against SOD1 (Fig. 1= 10 each) (Fig. 1and = 10, average SE). Hindlimb grip strength was recorded after injection of either viral construct. As previously exhibited (15), animals injected with AAV-siRNA maintained grip strength between 75 and 95 days compared with untreated SOD1G93A mice that lost grip strength (Fig. 1and and = 4; Cre?, = 3) in quadriceps femoris ( 0.05; unpaired test. For = 0.11. (and testing was performed to confirm the biological activity of follistatin to inhibit myostatin. (= 4 common SE). To directly assess whether increased muscle proliferation affected disease course in a mouse model of inherited ALS caused by mutation in SOD1, AAVCfollistatin, or AAVCGFP (1 1011 viral genomes per injection) were injected bilaterally via intramuscular delivery into the hindlimb quadriceps and tibialis anterior muscles of 16 animals (age, 40 days; Dasatinib small molecule kinase inhibitor equal distribution of male and female). Both sets of mice reached end stage disease at 126 days. Despite this lack of difference in survival, follistatin-treated muscles showed gross changes, including widespread increased muscle mass, compared with the GFP-treated animals (Fig. 4= 10C15 animals each), including the tibialis anterior, gastrocnemius, medial quadriceps, and triceps muscles, were significantly increased ( 0.05) after injection of AAVCfollistatin (Fig. 4and = 15 per group). (= 10C15 per group, common SE). (= 8 common SE). To determine whether the muscle weight increase was due to hyperplasia, hypertrophy, and/or muscle sparing in the ALS animals, myofiber numbers within the gastrocnemius muscle were counted in serial sections of AAVCfollistatin- or AAVCGFP-treated groups (= 8 animals). This analysis revealed that at end stage (126C127 days), approximately twice as many myofibers were present in the gastrocnemius of follistatin-treated animals compared with GFP-treated animals (= 8 animals) ( 0.01) (Fig. 5and = 8 per group, average SE). (and = 8 per group, common SE). Increased muscle mass, increased myofiber number, and hypertrophy of muscle fibers translated, as expected, into increased strength in the follistatin-treated animals (Fig. 6and = 0.06; 2 = 3.504) (Fig. 6and = 0.06 (= 15; litter-matched; the number of female mice equaled the number of male mice). Discussion An understanding of the role of muscle in ALS has practical implications for treating disease. Reduction in mutant SOD1 accumulation in muscle did not affect disease onset or progression. In contrast to its effects in motor neurons and microglia (4),.