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Mitophagy of damaged mitochondria occurs locally in distal neuronal axons and requires PINK1 and Parkin.

Ashrafi G, Schlehe JS, LaVoie MJ, Schwarz TL - J. Cell Biol. (2014)

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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138 F.M. Kirby Neurobiology Center, Children's Hospital Boston, Boston, MA 02115.

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Axonal lysosomes are recruited to autophagosomes containing damaged mitochondria. (A) RFP-LC3 and LAMP1-YFP–positive autolysosomes colocalize with mitochondria depolarized with 40 µM Antimycin A (Ant A). Arrowheads (cyan in the top axon and white in the bottom axon) denote LC3- and LAMP1-positive mitochondria. (B) Line scan analysis of the upper axon in A. Peaks marked by arrowheads correspond to those with cyan arrowheads in A. (C) Frequency of mitochondrial colocalization with autophagosome and lysosome markers before and 50 min after Antimycin A addition to neurons with or without preincubation with lysosomal inhibitors Pepstatin A and E64d. n = 98–171 mitochondria from five microfluidic devices. *, P < 0.05; **, P < 0.001. (D and E) Time-lapse images starting 25 min after application of Antimycin A depict an RFP-LC3–positive autophagosome (red arrowheads) forming on a fragmented mitochondrion (cyan arrowheads). A moving LAMP1-YFP–positive lysosome (green arrowhead) stopped at the mitochondrion, and the mitochondrial signal subsequently diminished. (F and G) Neighboring mitochondria imaged in the same field as D retained their BFP signal. Orange and brown arrowheads denote corresponding points in images and line scans. Error bars represent means ± SEM. AU, arbitrary unit. Bars, 5 µm.
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fig4: Axonal lysosomes are recruited to autophagosomes containing damaged mitochondria. (A) RFP-LC3 and LAMP1-YFP–positive autolysosomes colocalize with mitochondria depolarized with 40 µM Antimycin A (Ant A). Arrowheads (cyan in the top axon and white in the bottom axon) denote LC3- and LAMP1-positive mitochondria. (B) Line scan analysis of the upper axon in A. Peaks marked by arrowheads correspond to those with cyan arrowheads in A. (C) Frequency of mitochondrial colocalization with autophagosome and lysosome markers before and 50 min after Antimycin A addition to neurons with or without preincubation with lysosomal inhibitors Pepstatin A and E64d. n = 98–171 mitochondria from five microfluidic devices. *, P < 0.05; **, P < 0.001. (D and E) Time-lapse images starting 25 min after application of Antimycin A depict an RFP-LC3–positive autophagosome (red arrowheads) forming on a fragmented mitochondrion (cyan arrowheads). A moving LAMP1-YFP–positive lysosome (green arrowhead) stopped at the mitochondrion, and the mitochondrial signal subsequently diminished. (F and G) Neighboring mitochondria imaged in the same field as D retained their BFP signal. Orange and brown arrowheads denote corresponding points in images and line scans. Error bars represent means ± SEM. AU, arbitrary unit. Bars, 5 µm.

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.


Mitophagy of damaged mitochondria occurs locally in distal neuronal axons and requires PINK1 and Parkin.

Ashrafi G, Schlehe JS, LaVoie MJ, Schwarz TL - J. Cell Biol. (2014)

Axonal lysosomes are recruited to autophagosomes containing damaged mitochondria. (A) RFP-LC3 and LAMP1-YFP–positive autolysosomes colocalize with mitochondria depolarized with 40 µM Antimycin A (Ant A). Arrowheads (cyan in the top axon and white in the bottom axon) denote LC3- and LAMP1-positive mitochondria. (B) Line scan analysis of the upper axon in A. Peaks marked by arrowheads correspond to those with cyan arrowheads in A. (C) Frequency of mitochondrial colocalization with autophagosome and lysosome markers before and 50 min after Antimycin A addition to neurons with or without preincubation with lysosomal inhibitors Pepstatin A and E64d. n = 98–171 mitochondria from five microfluidic devices. *, P < 0.05; **, P < 0.001. (D and E) Time-lapse images starting 25 min after application of Antimycin A depict an RFP-LC3–positive autophagosome (red arrowheads) forming on a fragmented mitochondrion (cyan arrowheads). A moving LAMP1-YFP–positive lysosome (green arrowhead) stopped at the mitochondrion, and the mitochondrial signal subsequently diminished. (F and G) Neighboring mitochondria imaged in the same field as D retained their BFP signal. Orange and brown arrowheads denote corresponding points in images and line scans. Error bars represent means ± SEM. AU, arbitrary unit. Bars, 5 µm.
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fig4: Axonal lysosomes are recruited to autophagosomes containing damaged mitochondria. (A) RFP-LC3 and LAMP1-YFP–positive autolysosomes colocalize with mitochondria depolarized with 40 µM Antimycin A (Ant A). Arrowheads (cyan in the top axon and white in the bottom axon) denote LC3- and LAMP1-positive mitochondria. (B) Line scan analysis of the upper axon in A. Peaks marked by arrowheads correspond to those with cyan arrowheads in A. (C) Frequency of mitochondrial colocalization with autophagosome and lysosome markers before and 50 min after Antimycin A addition to neurons with or without preincubation with lysosomal inhibitors Pepstatin A and E64d. n = 98–171 mitochondria from five microfluidic devices. *, P < 0.05; **, P < 0.001. (D and E) Time-lapse images starting 25 min after application of Antimycin A depict an RFP-LC3–positive autophagosome (red arrowheads) forming on a fragmented mitochondrion (cyan arrowheads). A moving LAMP1-YFP–positive lysosome (green arrowhead) stopped at the mitochondrion, and the mitochondrial signal subsequently diminished. (F and G) Neighboring mitochondria imaged in the same field as D retained their BFP signal. Orange and brown arrowheads denote corresponding points in images and line scans. Error bars represent means ± SEM. AU, arbitrary unit. Bars, 5 µm.
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.

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.

View Article: PubMed Central - HTML - PubMed

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
Related in: MedlinePlus