Secreted miRNAs from primary myocytes

Amyotrophic lateral sclerosis (ALS) is the most common adult-onset paralytic disorder, characterized primarily by a progressive loss of motor neurons in whose degeneration skeletal muscle involvement has been demonstrated. Skeletal muscle is a plastic tissue that responds to insults through proliferation and differentiation of satellite cells. The process of skeletal muscle degeneration and regeneration are finely regulated by signals that regulate satellite cell proliferation and differentiation. It is known that satellite cell differentiation is impaired in ALS, but little is known about the involvement of miRNAs and their role in intercellular communication in skeletal muscle in ALS. Here we demonstrated impaired differentiation of satellite cells derived from ALS mice related to the impairment of myogenic p38MAPK and PKA/pCREB signaling pathways that can be regulated by miR-882 and -134-5p. These miRNAs participate in autocrine signaling in association with miR-26a-5p, that, secreted from WT and captured by ALS myoblasts, enhances ALS-related myoblast differentiation by repressing Smad4-related signals. These findings underscore the urgency of better understanding the role of intercellular communication and the molecular mechanisms underlying ALS pathogenesis and progression. They also suggest that miRNAs could be targeted as potential therapeutic agents to enhance myofiber regeneration. Primary cultures of myocytes were performed collecting leg skeletal muscle from from different mice. Each sample described in this research represent a biological replicate. Experiments were performed using three different co-cultures to evaluate miRNAs secreted in the culture medium. Performed co-cultures were hSOD1(WT)/hSOD1(WT), hSOD1(WT)/hSOD1(G93A) and hSOD1(G93A)/hSOD1(G93A). Tg mice overexpressing WT hSOD1 (strain B6SJL(Tg-SOD1)2Gur/J, abbreviated as hSOD1(WT) or the G93A mutant of the protein (B6SJL(Tg-SOD1*G93A)1Gur/J mice (model for Amyotrophyc Lateral Sclerosis), here abbreviated as hSOD1(G93A), were from Jackson Laboratories (cat. n. 002297 and 002726, respectively). Colonies were preserved by breeding Tg hemizygote males with wild-type B6SJLF1/J hybrid females. Newborns were genotyped as previously described 80 using the MyTaq Extract-PCR kit (Bioline). Primary myoblasts from newborn (1-3 days) hSOD1(WT) or hSOD1(G93A) mice were prepared as previously described 81. Briefly, the posterior limb muscles were removed, and cells isolated by three successive treatments with 0.1% trypsin in phosphate-buffered saline (PBS; 140 mM NaCl, 2 mM KCl, 1.5 mM KH2PO4, 8 mM Na2HPO4, pH 7.4) for 30 min at 37 °C, seeded at a density of 2.5 × 105 cells onto 13 mm-coverslips coated with collagen (0.1% w/v in PBS) for immunocytochemistry, or at a density of 1 × 106 cells onto 35 mm-collagen-coated plates for biochemical assays, and cultured in proliferation medium (Ham's F12 with 10% foetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin) for 2 days (2 Days In Vitro: 2 DIV). At this time point, proliferation medium was replaced by differentiation medium (Dulbecco’s Modified Eagle Medium containing 2% horse serum, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin) for myoblast differentiation into myotubes. All reagents for cell culture were from Euroclone.