Mitophagy of damaged mitochondria occurs locally in distal neuronal axons and requires PINK1 and Parkin.
Bottom Line: In PINK1(-/-) axons, damaged mitochondria did not accumulate Parkin and failed to be engulfed in autophagosomes.Similarly, initiation of mitophagy was blocked in Parkin(-/-) axons.Local mitophagy likely provides rapid neuroprotection against oxidative stress without a requirement for retrograde transport to the soma.
Affiliation: Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138 F.M. Kirby Neurobiology Center, Children's Hospital Boston, Boston, MA 02115.Show MeSH
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Mentions: To study mitophagy in neurons, we set out to induce mitochondrial damage in a spatiotemporally controlled manner, while preserving the overall integrity of the mitochondrial network. To this end, mt-KR, a genetically encoded photosensitizer targeted to mitochondria, was used to selectively damage a subset of axonal mitochondria. Light-induced activation of mt-KR causes local ROS-mediated damage to mitochondria (Bulina et al., 2006; Ertürk et al., 2014) and has been previously used to induce mitophagy in HeLa cells (Yang and Yang, 2011; Wang et al., 2012). We expressed mt-KR in rat hippocampal neurons and, with a 555-nm laser, irradiated a 10-µm section of an axon, which typically included three to four mitochondria, to activate and thereby photobleach mt-KR in those mitochondria, while sparing those in adjacent segments (Fig. 1 A). Mitochondrial damage results in stereotypical changes in mitochondrial morphology, such as swelling and fragmentation (Legros et al., 2002; Kaasik et al., 2007). To observe mitochondrial morphology after mt-KR photobleaching, we coexpressed mitochondrially targeted GFP (mt-GFP). Within 10 min of mt-KR activation, a fragmented or rounded morphology arose in 22% of the laser-illuminated mitochondria compared with 4% of the mitochondria outside the illuminated region (P < 0.0001; Fig. 1, B and C). In contrast, irradiation of mitochondria expressing mitochondrially targeted DsRed (mt-DsRed) did not cause fragmentation or rounding (Fig. S1 A). Activation of mt-KR also increased the fluorescence intensity of mt-GFP within those mitochondria (Fig. 1 B), possibly because of dequenching of the fluorophores in the swollen matrix of damaged mitochondria. Consistent with oxidative damage as the mechanism of mt-KR, we rarely detected changes in mitochondrial morphology when mt-KR was activated in Hibernate E medium, which contains antioxidants.
Affiliation: Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138 F.M. Kirby Neurobiology Center, Children's Hospital Boston, Boston, MA 02115.