ROS-induced mitochondrial depolarization initiates PARK2/PARKIN-dependent mitochondrial degradation by autophagy

Abstract

Reactive oxygen species (ROS) have been implicated as a signal for general autophagy. Both mitochondrial-produced and exogenous ROS induce autophagosome formation. However, it is unclear whether ROS are required for the selective autophagic degradation of mitochondria, a process called mitophagy. Recent work using carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial-uncoupling reagent, has been shown to induce mitophagy. However, CCCP treatment may not be biologically relevant since it causes the depolarization of the entire mitochondrial network. Since mitochondria are the main ROS production sites in mammalian cells, we propose that short bursts of ROS produced within mitochondria may be involved in the signaling for mitophagy. To test this hypothesis, we induced an acute burst of ROS within mitochondria using a mitochondrial-targeted photosensitizer, mitochondrial KillerRed (mtKR). Using mtKR, we increased ROS levels in the mitochondrial matrix, which resulted in the loss of membrane potential and the subsequent activation of PARK2-dependent mitophagy. Importantly, we showed that overexpression of the mitochondrial antioxidant protein, superoxide dismutase-2, can squelch mtKR-induced mitophagy, demonstrating that mitochondrial ROS are responsible for mitophagy activation. Using this assay, we examined the impact of mitochondrial morphology on mitophagy. It was shown recently that elongated mitochondria are more resistant to mitophagy through unknown mechanisms. Here, we show that elongated mitochondria are more resistant to ROS-induced damage and mitophagy compared with fragmented mitochondria, suggesting that mitochondrial morphology has an important role in regulating ROS and mitophagy. Together, our results suggest that ROS-induced mitochondrial damage may be an important upstream activator of mitophagy.

Tools and techniques to measure mitophagy using fluorescence microscopy

“…A recent report showed that ROS generated from a defined set of mitochondria are sufficient to cause depolarization, and that these mitochondria are subsequently degraded by mitophagy. 23 This report further highlights the close link between mitochondrial ROS generation, mitochondrial membrane potential, and mitophagy. Further work will help to clarify the exact role of mitochondrial membrane potential as a marker and/or signal for the induction of mitophagy.…”

“…The second family are the GABA A receptor-associated proteins (GABARAP) which has four members: the first is GABARAP, the second member is GABARAPL1 (GABA(A) receptor-associated protein like 1)/ATG8L/GEC1, the third member is GABARAPL2 (GABA(A) receptor-associated protein like 2)/GATE-16/GEF2, with the final member GABARAPL3 (GABA(A) receptor-associated protein like 3). 28 Examples where mitochondria have been labeled along with MAP1LC3 as either a fluorescent protein (FP) chimera 19,21,23,29 or by using antibodies raised against MAP1LC3 30 are noted. MAP1LC3 reporters can also be combined with many other mitochondrial/mitophagy fluorescent markers, including TMRM, 21 mitochondrial-targeted RFP, 19 PARK2 as an FP chimera, 30,31 PINK1 (Flag epitope-tagged PINK1), 30 and MitoTracker ® dyes.…”

“…In this example, cells were treated with 20 μM CCCP for 6 h prior to confocal imaging. Studies of this nature often use a line profile analysis 23,38 as demonstrated in Figure 3B. This approach allows for a correlation of intensity peaks that mirrors colocalization of fluorescent molecules.…”

“…As outlined above, many studies in the field of mitophagy involve a determination of colocalization between mitochondrial labels and markers for autophagy (for example MAP1LC3 19,21,23,29,30 ) or other salient proteins such as PARK2 30,31 or PINK1 30 The recruitment of mitochondria has also been analyzed through colocalization of other autophagy markers such as Atg8 or GABARAPL1 with Atg32 26,27 or BNIP3L, 15 respectively. Quantification of the extent to which markers colocalize provides a numerical index that can be subjected to statistical analysis.…”

“…However, the use of CCCP causes a global loss of mitochondrial membrane potential and therefore has potential off-target effects. 23 For this reason, the targeted depolarization or damage of a limited number of mitochondria presents a more physiologically relevant mechanism by which to stimulate mitophagy. 23 The discovery and cloning of light-activated ion channels, known as channel rhodopsins, has led to advances in the study of electrically excitable cells given the ability to precisely and non-invasively depolarize a subset of cells in a field.…”