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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.

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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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Mitophagy of damaged axonal mitochondria is impaired in the absence of PINK1. (A–D) GFP-LC3–positive autophagosomes form on mitochondria of PINK1+/+ (A and B) but not PINK1−/− (C and D) rat hippocampal axons depolarized for 35 min with 40 µM Antimycin A (Ant A). (B and D) Cyan arrowheads denote GFP-LC3–positive mitochondria. (E) Frequency of autophagosome formation before and after Antimycin A treatment. Orange and brown arrowheads denote corresponding points in images and line scans. (F) Quantification of mitochondrial size before and after 20 min of Antimycin A treatment indicates that mitochondrial remodeling upon damage is PINK1 independent. Untreated mitochondria in PINK1−/− axons are smaller than those in PINK1+/+ or PINK1-FLAG–expressing PINK1−/− axons. n = 108–342 mitochondria from five to nine microfluidic devices per genotype. *, P < 0.05; **, P < 0.001; ****, P < 0.0001. Error bars represent means ± SEM. AU, arbitrary unit. Bars, 5 µm.
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fig9: Mitophagy of damaged axonal mitochondria is impaired in the absence of PINK1. (A–D) GFP-LC3–positive autophagosomes form on mitochondria of PINK1+/+ (A and B) but not PINK1−/− (C and D) rat hippocampal axons depolarized for 35 min with 40 µM Antimycin A (Ant A). (B and D) Cyan arrowheads denote GFP-LC3–positive mitochondria. (E) Frequency of autophagosome formation before and after Antimycin A treatment. Orange and brown arrowheads denote corresponding points in images and line scans. (F) Quantification of mitochondrial size before and after 20 min of Antimycin A treatment indicates that mitochondrial remodeling upon damage is PINK1 independent. Untreated mitochondria in PINK1−/− axons are smaller than those in PINK1+/+ or PINK1-FLAG–expressing PINK1−/− axons. n = 108–342 mitochondria from five to nine microfluidic devices per genotype. *, P < 0.05; **, P < 0.001; ****, P < 0.0001. Error bars represent means ± SEM. AU, arbitrary unit. Bars, 5 µm.

Mentions: We then asked whether PINK1 is similarly required for autophagosome formation on damaged mitochondria. In PINK1+/+ axons, 40 µM Antimycin A increased the fraction of mitochondria colocalizing with GFP-LC3 autophagosomes from 3 to 18% (P < 0.01; Fig. 9, A, B, and E). In PINK1−/− axons, however, there was a smaller Antimycin A–induced change in the fractions of mitochondria colocalizing with autophagosomes (from 3 to 8%), and that change did not reach statistical significance (P = 0.09; Fig. 9, C–E). This represented significantly less recruitment of GFP-LC3 autophagosomes in PINK1−/− axons compared with PINK1+/+ axons (P < 0.05). Expression of exogenous PINK1-FLAG rescued autophagosome formation on mitochondria in PINK1−/− axons. The fraction of GFP-LC3–positive mitochondria increased from 6 to 16% after Antimycin A treatment (P < 0.05; Fig. 9 E).


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)

Mitophagy of damaged axonal mitochondria is impaired in the absence of PINK1. (A–D) GFP-LC3–positive autophagosomes form on mitochondria of PINK1+/+ (A and B) but not PINK1−/− (C and D) rat hippocampal axons depolarized for 35 min with 40 µM Antimycin A (Ant A). (B and D) Cyan arrowheads denote GFP-LC3–positive mitochondria. (E) Frequency of autophagosome formation before and after Antimycin A treatment. Orange and brown arrowheads denote corresponding points in images and line scans. (F) Quantification of mitochondrial size before and after 20 min of Antimycin A treatment indicates that mitochondrial remodeling upon damage is PINK1 independent. Untreated mitochondria in PINK1−/− axons are smaller than those in PINK1+/+ or PINK1-FLAG–expressing PINK1−/− axons. n = 108–342 mitochondria from five to nine microfluidic devices per genotype. *, P < 0.05; **, P < 0.001; ****, P < 0.0001. Error bars represent means ± SEM. AU, arbitrary unit. Bars, 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig9: Mitophagy of damaged axonal mitochondria is impaired in the absence of PINK1. (A–D) GFP-LC3–positive autophagosomes form on mitochondria of PINK1+/+ (A and B) but not PINK1−/− (C and D) rat hippocampal axons depolarized for 35 min with 40 µM Antimycin A (Ant A). (B and D) Cyan arrowheads denote GFP-LC3–positive mitochondria. (E) Frequency of autophagosome formation before and after Antimycin A treatment. Orange and brown arrowheads denote corresponding points in images and line scans. (F) Quantification of mitochondrial size before and after 20 min of Antimycin A treatment indicates that mitochondrial remodeling upon damage is PINK1 independent. Untreated mitochondria in PINK1−/− axons are smaller than those in PINK1+/+ or PINK1-FLAG–expressing PINK1−/− axons. n = 108–342 mitochondria from five to nine microfluidic devices per genotype. *, P < 0.05; **, P < 0.001; ****, P < 0.0001. Error bars represent means ± SEM. AU, arbitrary unit. Bars, 5 µm.
Mentions: We then asked whether PINK1 is similarly required for autophagosome formation on damaged mitochondria. In PINK1+/+ axons, 40 µM Antimycin A increased the fraction of mitochondria colocalizing with GFP-LC3 autophagosomes from 3 to 18% (P < 0.01; Fig. 9, A, B, and E). In PINK1−/− axons, however, there was a smaller Antimycin A–induced change in the fractions of mitochondria colocalizing with autophagosomes (from 3 to 8%), and that change did not reach statistical significance (P = 0.09; Fig. 9, C–E). This represented significantly less recruitment of GFP-LC3 autophagosomes in PINK1−/− axons compared with PINK1+/+ axons (P < 0.05). Expression of exogenous PINK1-FLAG rescued autophagosome formation on mitochondria in PINK1−/− axons. The fraction of GFP-LC3–positive mitochondria increased from 6 to 16% after Antimycin A treatment (P < 0.05; Fig. 9 E).

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