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 determine whether these lysosomes would be recruited to autophagosomes containing mitochondria, hippocampal neurons were plated in microfluidic devices and sparsely transfected with mt-BFP, RFP-LC3, and LAMP1-YFP. RFP-LC3 is more stable than GFP-LC3 and labels both autophagosomes and autolysosomes (Bains and Heidenreich, 2009), accounting for frequent double labeling of lysosomes with both LAMP1-YFP and RFP-LC3. 50 min after application of Antimycin A to the perfusion chamber, the fraction of axonal mitochondria colocalizing with RFP-LC3–positive vesicles increased from 5% before treatment to 20% after (P < 0.05; Fig. 4, A–C). Similarly, mitochondrial colocalization increased significantly with vesicles containing either LAMP1-YFP alone (from 9 to 28%, P < 0.01) or containing both RFP-LC3 and LAMP1-YFP (from 5 to 19%, P < 0.05). In neurons preincubated with lysosomal inhibitors (5 µM pepstatin A and 10 µM E64D) to prevent the loss of mitochondrial markers after autophagolysosome formation, 60% of mitochondria became LAMP1-YFP–positive after Antimycin A treatment (P < 0.05; Fig. 4 C). Thus, local lysosomal degradation of mitochondria was rapid and extensive. In one example of live imaging (Fig. 4, D and E), an RFP-LC3–positive autophagosome first formed on a fragmented mitochondrion 25 min after the addition of Antimycin A. Then, a moving RFP-LC3– and LAMP1-YFP–positive lysosome stopped at the site of the autophagosome-associated mitochondrion. 17 min later, the mitochondrial BFP signal was significantly diminished, which could indicate acidification of the autolysosome or degradation of its mitochondrial content, but was not cause by photobleaching of mt-BFP because other mitochondria in the field retained their BFP fluorescence (Fig. 4, F and G). Thus, mobile axonal lysosomes are recruited to autophagosomes to mediate local degradation of their damaged mitochondrial content.
Affiliation: Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138 F.M. Kirby Neurobiology Center, Children's Hospital Boston, Boston, MA 02115.