AMPK activation induces mitophagy and promotes mitochondrial fission while activating TBK1 in a PINK1-Parkin independent manner

Alex P. Seabright, Nicholas H. F. Fine, Jonathan P. Barlow, Samuel O. Lord, Ibrahim Musa, Alexander Gray, Jack A. Bryant, Manuel Banzhaf, Gareth G. Lavery, D. Grahame Hardie, David J. Hodson, Andrew Philp, Yu-Chiang Lai (Lead / Corresponding author)

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Abstract

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 languageEnglish
Number of pages18
JournalFASEB journal : official publication of the Federation of American Societies for Experimental Biology
Early online date22 Mar 2020
DOIs
Publication statusE-pub ahead of print - 22 Mar 2020

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Keywords

  • endogenous
  • mitophagy
  • skeletal muscle
  • tandem ubiquitin-binding entity (TUBE)
  • ubiquitin

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