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

TY - JOUR

T1 - AMPK activation induces mitophagy and promotes mitochondrial fission while activating TBK1 in a PINK1-Parkin independent manner

AU - Seabright, Alex P.

AU - Fine, Nicholas H. F.

AU - Barlow, Jonathan P.

AU - Lord, Samuel O.

AU - Musa, Ibrahim

AU - Gray, Alexander

AU - Bryant, Jack A.

AU - Banzhaf, Manuel

AU - Lavery, Gareth G.

AU - Hardie, D. Grahame

AU - Hodson, David J.

AU - Philp, Andrew

AU - Lai, Yu-Chiang

N1 - This study was supported by the BBSRC funded Midlands Integrative Biosciences Doctoral Training Partnership (MIBTP) and MRC Versus Arthritis Centre for Musculoskeletal Ageing Research. GGL was supported by a Wellcome Trust Senior Fellowship (104612/Z/14/Z). DGH was supported by an Investigator Award from the Wellcome Trust (204766/Z/16/Z). DJH was supported by MRC (MR/N00275X/1 & MR/S025618/1) as well as Diabetes UK (17/0005681) project grants. This project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (Starting Grant 715884 to DJH) © 2020 The Authors. The FASEB Journal published by Wiley Periodicals, Inc. on behalf of Federation of American Societies for Experimental Biology.

PY - 2020/5/4

Y1 - 2020/5/4

N2 - 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.

AB - 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.

KW - endogenous

KW - mitophagy

KW - skeletal muscle

KW - tandem ubiquitin-binding entity (TUBE)

KW - ubiquitin

UR - https://www.scopus.com/pages/publications/85082034250

U2 - 10.1096/fj.201903051R

DO - 10.1096/fj.201903051R

M3 - Article

C2 - 32201986

SN - 0892-6638

VL - 34

SP - 6284

EP - 6301

JO - FASEB journal : official publication of the Federation of American Societies for Experimental Biology

JF - FASEB journal : official publication of the Federation of American Societies for Experimental Biology

IS - 5

ER -