A conserved annexin A6-mediated membrane repair mechanism in muscle, heart, and nerve.

Membrane instability and disruption underlie myriad acute and chronic disorders. Anxa6 encodes the membrane-associated protein annexin A6 and was identified as a genetic modifier of muscle repair and muscular dystrophy. To evaluate annexin A6's role in membrane repair in vivo, we inserted sequences encoding green fluorescent protein (GFP) into the last coding exon of Anxa6. Heterozygous Anxa6gfp mice expressed a normal pattern of annexin A6 with reduced annexin A6GFP mRNA and protein. High-resolution imaging of wounded muscle fibers showed annexin A6GFP rapidly formed a repair cap at the site of injury. Injured cardiomyocytes and neurons also displayed repair caps after wounding, highlighting annexin A6-mediated repair caps as a feature in multiple cell types. Using surface plasmon resonance, we showed recombinant annexin A6 bound phosphatidylserine-containing lipids in a Ca2+- and dose-dependent fashion with appreciable binding at approximately 50 µM Ca2+. Exogenously added recombinant annexin A6 localized to repair caps and improved muscle membrane repair capacity in a dose-dependent fashion without disrupting endogenous annexin A6 localization, indicating annexin A6 promotes repair from both intracellular and extracellular compartments. Thus, annexin A6 orchestrates repair in multiple cell types, and recombinant annexin A6 may be useful in additional chronic disorders beyond skeletal muscle myopathies.

Reduction of Autophagic Accumulation in Pompe Disease Mouse Model Following Gene Therapy.

BACKGROUND: Pompe disease is a fatal neuromuscular disorder caused by a deficiency in acid α-glucosidase, an enzyme responsible for glycogen degradation in the lysosome. Currently, the only approved treatment for Pompe disease is enzyme replacement therapy (ERT), which increases patient survival, but does not fully correct the skeletal muscle pathology. Skeletal muscle pathology is not corrected with ERT because low cation-independent mannose-6-phosphate receptor abundance and autophagic accumulation inhibits the enzyme from reaching the lysosome. Thus, a therapy that more efficiently targets skeletal muscle pathology, such as adeno-associated virus (AAV), is needed for Pompe disease. OBJECTIVE: The goal of this project was to deliver a rAAV9-coGAA vector driven by a tissue restrictive promoter will efficiently transduce skeletal muscle and correct autophagic accumulation. METHODS: Thus, rAAV9-coGAA was intravenously delivered at three doses to 12-week old Gaa-/- mice. 1 month after injection, skeletal muscles were biochemically and histologically analyzed for autophagy-related markers. RESULTS: At the highest dose, GAA enzyme activity and vacuolization scores achieved therapeutic levels. In addition, resolution of autophagosome (AP) accumulation was seen by immunofluorescence and western blot analysis of autophagy-related proteins. Finally, mice treated at birth demonstrated persistence of GAA expression and resolution of lysosomes and APs compared to those treated at 3 months. CONCLUSION: In conclusion, a single systemic injection of rAAV9-coGAA ameliorates vacuolar accumulation and prevents autophagic dysregulation.