The cytosolic sialidase Neu2 is degraded by autophagy during myoblast atrophy.
BACKGROUND
The sialidase 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 macroautophagy, 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.


CONCLUSIONS
This study demonstrates that Neu2 enzyme degradation occurs in atrophic myotubes via macroautophagy and independently of proteasome activation.
Department of Biomedical Sciences and Biotechnology, University of Brescia, Italy.
Stefania
S
Rossi
Elena
E
Stoppani
Wim
W
Martinet
Andrea
A
Bonetto
Paola
P
Costelli
Roberta
R
Giuliani
Francesca
F
Colombo
Augusto
A
Preti
Sergio
S
Marchesini
Alessandro
A
Fanzani
D000468:Alkalies / Q000378:metabolism; D000818:Animals; D001284:Atrophy; D001343:Autophagy / Q000187:drug effects; D002401:Cathepsin B / Q000378:metabolism; D056668:Cathepsin L; D002403:Cathepsins / Q000378:metabolism; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology*; D003907:Dexamethasone / Q000494:pharmacology; D015536:Down-Regulation / Q000187:drug effects; D007328:Insulin / Q000494:pharmacology; D008247:Lysosomes / Q000187:drug effects / Q000201:enzymology / Q000648:ultrastructure; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology* / Q000648:ultrastructure; D009439:Neuraminidase / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D051381:Rats; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D000468:Alkalies; D007328:Insulin; D014409:Tumor Necrosis Factor-alpha; D003907:Dexamethasone; C528920:Neu2 protein, rat; D009439:Neuraminidase; D002403:Cathepsins; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; D056668:Cathepsin L; C534278:Ctsl protein, mouse; C534280:Ctsl protein, rat