Mitophagy and its signalling mechanisms in skeletal muscle

Seabright, Alex Peter ORCID: 0000-0002-2071-6722 (2021). Mitophagy and its signalling mechanisms in skeletal muscle. University of Birmingham. Ph.D.

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Abstract

The reasons why skeletal muscle health undergoes unfavourable decline with increasing age are not well understood. One hypothesis that may explain the demise of skeletal muscle during ageing is linked to a reduced ability to selectively remove damaged and dysfunctional mitochondria in a process called mitophagy. At present, much of our understanding of the molecular mechanisms that regulate mitophagy is derived from work conducted in immortalised, cancer cell lines made to overexpress non-native proteins. Conversely, far less is known about the endogenous mechanisms that coordinate mitophagy in skeletal muscle. Thus, the aim of this thesis is to gain a deeper understanding of the endogenous signalling mechanisms that govern mitophagy in skeletal muscle. Herein, after making a stable C2C12 mito-QC skeletal muscle cell line, we discover that 5'-AMP-activated protein kinase (AMPK) activation by a small molecule, 991, promotes mitochondrial fission and induces mitophagy. From a mechanistic perspective, we demonstrate that AMPK mediates the phosphorylation and activation of mitochondrial fission factor (MFF), unc-51-like autophagy activating kinase 1 (ULK1) and TANK-binding kinase 1 (TBK1). Activation of MFF facilitates mitochondrial fission while activation of ULK1 and TBK1 helps to coordinate autophagosomal engulfment. Both of these steps are vital for efficient mitophagy. Meanwhile, having developed a ubiquitin pull-down assay to examine the most widely studied signalling nodes known to regulate mitophagy in immortalised cell lines, PTENinduced kinase 1 (PINK1) and Parkin, we reveal some interesting findings. While it is possible to stimulate endogenous PINK1 kinase and Parkin E3 ligase activity in C2C12 skeletal muscle cells, we demonstrate that such activation requires prolonged, nonphysiological treatment with the chemical protonophore carbonyl cyanide m chlorophenyl hydrazone (CCCP). Lastly, we integrate the findings contained within this thesis into the current literature while evaluating how mitophagy is regulated in muscle through the steps of mitochondrial fission, ubiquitylation and autophagosomal engulfment. In doing so, we identify the most critical questions within the field that are yet to be answered. Altogether, this thesis improves understanding of the molecular signalling mechanisms that regulate mitophagy in skeletal muscle.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):