The cytosolic sialidase Neu2 is degraded by autophagy during myoblast atrophy

Background: The sialiclase Neu2 is a cytosolic enzyme which is fully expressed in mature muscle myofibers. Methods: To investigate Neu2 expression during muscle atrophy, we employed an in vitro model consisting of terminally differentiated C2C12 myotubes exposed to different pro-atrophic stimuli that triggered catabolic pathways involved in proteasome activation or autophagy. Results: Neu2 expression was unchanged in myotubes treated with TNF-alpha, a cytokine known to activate the proteasome. However, Neu2 transcript levels and enzymatic activity were downregulated in starved or dexamethasone-treated myotubes that showed proteosomal activation and several hallmarks of macro-autophagy, such as formation of autophagosomes, the accumulation of LC3 dots and bulk degradation of long-lived proteins. Neu2 activity and protein levels were rescued upon cotreatment with the lysosomotropic agent NH4Cl, the autophagy inhibitor 3-methyladenine or cathepsin inhibitors, as well as by insulin administration, but were unaffected upon pharmacological inhibition of the proteasome. Moreover, HA- or GST-Neu2 recombinant fusion proteins were rapidly degraded in vitro by purified cathepsin L and B. Overall. we may conclude that Neu2 is degraded by lysosomal enzymes in myotubes undergoing autophagy-mediated atrophy. General significance: This study demonstrates that Neu2 enzyme degradation occurs in atrophic myotubes via macroautophagy and independently of proteasome activation. (C) 2009 Elsevier B.V. All rights reserved.