Mammalian mitophagy - from in vitro molecules to in vivo models

Abstract

The autophagic turnover of mitochondria, termed mitophagy, is thought to play an essential role in not only maintaining the health of the mitochondrial network but also that of the cell and organism as a whole. We have come a long way in identifying the molecular components required for mitophagy through extensive in vitro work and cell line characterisation, yet the physiological significance and context of these pathways remain largely unexplored. This is highlighted by the recent development of new mouse models that have revealed a striking level of variation in mitophagy, even under normal conditions. Here, we focus on programmed mitophagy and summarise our current understanding of why, how and where this takes place in mammals.

Keywords: NIX; Parkin; autophagy; development; disease; metabolism; mito-QC; mitochondria; mitophagy; mouse models.

Figure 1

The requirements for mitophagy. Cartoon depicting the molecular events occurring during mitophagy. (1) Fission of the mitochondrial network into individual mitochondrion/mitochondrial fragments; (2) Marking of these to prime them for recognition by the autophagy machinery; (3) Formation of the autophagic phagophore to engulf the primed mitochondrion. Once engulfed, the mitochondrion‐containing autophagosome (mitophagosome) fuses with a lysosome to form the mitolysosomes where degradation and recycling occurs.

Figure 2

Mitophagy reporters. Schematic detailing the different reporters used to monitor mitophagy. In mito‐QC, a tandem mCherry‐GFP tag is targeted to the OMM. Under normal cytosolic conditions the mitochondria display both mCherry and GFP fluorescence. However, upon mitophagy, the low pH of the mitochondrion‐containing lysosome (mitolysosome) is sufficient to quench the GFP, but not mCherry, signal. In mt‐Keima, monomeric Keima protein is targeted to the mitochondrial matrix. Under normal cytosolic conditions, mt‐Keima is excited by light peaking at 400 nm, whereas this increases to 568 nm under lysosomal conditions, i.e. upon mitophagy. mt‐Keima's emission spectra remains the same, at 620 nm, regardless of this pH shift. For MitoTimer, the fluorophore DsRed1‐E5 was targeted to the mitochondrial matrix. Though strictly not designed to monitor mitophagy, it provides a powerful way in which to monitor the age of mitochondria as fluorescence shifts from green to red over a period of 48 h.

Figure 3

Mitophagy in the cortex of the adult kidney. Upper panels show a confocal micrograph (tile scan) of a section of adult kidney cortex from the mito‐QC reporter mouse under basal conditions. Note the distinct and polarised degree of mitophagy, as visualised by the ring‐like mCherry (red) signals, in a subset of kidney tubules (PCTs). Scale bar, 200 μm. Lower panels show a magnified mito‐QC PCT cell detailing the GFP and mCherry mitochondrial signals. Arrows highlight mCherry‐only signal co‐localising with LAMP1, a marker for lysosomes, confirming mitophagy. Scale bar, 5 μm. For details see McWilliams et al. 121.