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Mitophagy is a key process regulating mitochondrial quality control. Several mechanisms have been proposed to regulate mitophagy, but these have mostly been studied using stably expressed non-native proteins in immortalized cell lines. In skeletal muscle, mitophagy and its molecular mechanisms require more thorough investigation. To measure mitophagy directly, we generated a stable skeletal muscle C2C12 cell line, expressing a mitophagy reporter construct (mCherry-green fluorescence protein-mtFIS1101-152 ). Here, we report that both carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treatment and adenosine monophosphate activated protein kinase (AMPK) activation by 991 promote mitochondrial fission via phosphorylation of MFF and induce mitophagy by ~20%. Upon CCCP treatment, but not 991, ubiquitin phosphorylation, a read-out of PTEN-induced kinase 1 (PINK1) activity, and Parkin E3 ligase activity toward CDGSH iron sulfur domain 1 (CISD1) were increased. Although the PINK1-Parkin signaling pathway is active in response to CCCP treatment, we observed no change in markers of mitochondrial protein content. Interestingly, our data shows that TANK-binding kinase 1 (TBK1) phosphorylation is increased after both CCCP and 991 treatments, suggesting TBK1 activation to be independent of both PINK1 and Parkin. Finally, we confirmed in non-muscle cell lines that TBK1 phosphorylation occurs in the absence of PINK1 and is regulated by AMPK-dependent signaling. Thus, AMPK activation promotes mitophagy by enhancing mitochondrial fission (via MFF phosphorylation) and autophagosomal engulfment (via TBK1 activation) in a PINK1-Parkin independent manner.

Original publication

DOI

10.1096/fj.201903051R

Type

Journal article

Journal

FASEB J

Publication Date

2020

Volume

34

Pages

6284 - 6301

Keywords

AMP-Activated Protein Kinases/genetics/*metabolism Animals Carbonyl Cyanide m-Chlorophenyl Hydrazone/pharmacology Enzyme Activation HeLa Cells Humans Mice *Mitochondrial Dynamics *Mitophagy Muscle, Skeletal/drug effects/metabolism/*pathology Protein Kinases/genetics/*metabolism Protein Serine-Threonine Kinases/genetics/*metabolism Proton Ionophores/pharmacology Signal Transduction Ubiquitin/metabolism Ubiquitin-Protein Ligases/genetics/*metabolism Ubiquitination *endogenous *skeletal muscle *tandem ubiquitin-binding entity (TUBE) *ubiquitin