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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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Initiation of axonal mitophagy requires Parkin. (A and B) GFP-LC3–positive autophagosomes (white and cyan arrowheads) form on mitochondria of wild-type (Parkin+/+) axon segments treated with 20 µM Antimycin A (Ant A). Cyan arrowheads point to mitochondria in the axon analyzed in B. (C and D) In Parkin−/− axons, mitochondria did not acquire autophagosomes. Orange and brown arrowheads denote corresponding points in images and line scans. (E) Frequency of autophagosome formation axons before and after Antimycin A. n = 104–106 mitochondria from four to five microfluidic devices per genotype. (F) Quantification of mitochondrial size before and after 20 min of 20 µM Antimycin A treatment indicates that damage-induced mitochondrial remodeling is Parkin independent. The sizes of the fragmented mitochondria may be overestimates because of the limited resolution of the microscope. n = 76–99 mitochondria from four to five microfluidic devices per genotype. *, P < 0.05; **, P < 0.001. Error bars represent means ± SEM. AU, arbitrary unit. Bars, 5 µm.
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fig7: Initiation of axonal mitophagy requires Parkin. (A and B) GFP-LC3–positive autophagosomes (white and cyan arrowheads) form on mitochondria of wild-type (Parkin+/+) axon segments treated with 20 µM Antimycin A (Ant A). Cyan arrowheads point to mitochondria in the axon analyzed in B. (C and D) In Parkin−/− axons, mitochondria did not acquire autophagosomes. Orange and brown arrowheads denote corresponding points in images and line scans. (E) Frequency of autophagosome formation axons before and after Antimycin A. n = 104–106 mitochondria from four to five microfluidic devices per genotype. (F) Quantification of mitochondrial size before and after 20 min of 20 µM Antimycin A treatment indicates that damage-induced mitochondrial remodeling is Parkin independent. The sizes of the fragmented mitochondria may be overestimates because of the limited resolution of the microscope. n = 76–99 mitochondria from four to five microfluidic devices per genotype. *, P < 0.05; **, P < 0.001. Error bars represent means ± SEM. AU, arbitrary unit. Bars, 5 µm.

Mentions: The recruitment of expressed Parkin to axonal mitochondria raised the question of whether endogenous Parkin was required for damage-induced mitophagy. We therefore compared mitophagy in hippocampal axons of Parkin+/+ and Parkin−/− mice (Goldberg et al., 2003). In microfluidic chambers, GFP-LC3 was coexpressed with mt-DsRed to monitor autophagosome formation. In Parkin+/+ axons, the fraction of mitochondria colocalizing with GFP-LC3–positive autophagosomes increased from 4% before to 33% after 35 min of Antimycin A treatment in the perfusion chamber (P < 0.05; Fig. 7, A, B, and E). In contrast, in Parkin−/− axons, Antimycin A did not increase mitochondrial colocalization with autophagosomes (P = 1; Fig. 7, C–E). Damage-induced mitophagy could be rescued in Parkin−/− axons by expressing mCherry-Parkin (Fig. S4 A). Thus, depolarization-induced initiation of mitophagy in neuronal axons both triggers Parkin recruitment and requires Parkin. We also examined damage-induced mitochondrial fragmentation and found that treatment with Antimycin A for 20 min caused a significant decrease in mitochondrial size in both Parkin+/+ and Parkin−/− axons (P < 0.01; Fig. 7 F). We conclude that Parkin is not required for the remodeling of depolarized mitochondria, only for the subsequent recruitment of the autophagosome.


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)

Initiation of axonal mitophagy requires Parkin. (A and B) GFP-LC3–positive autophagosomes (white and cyan arrowheads) form on mitochondria of wild-type (Parkin+/+) axon segments treated with 20 µM Antimycin A (Ant A). Cyan arrowheads point to mitochondria in the axon analyzed in B. (C and D) In Parkin−/− axons, mitochondria did not acquire autophagosomes. Orange and brown arrowheads denote corresponding points in images and line scans. (E) Frequency of autophagosome formation axons before and after Antimycin A. n = 104–106 mitochondria from four to five microfluidic devices per genotype. (F) Quantification of mitochondrial size before and after 20 min of 20 µM Antimycin A treatment indicates that damage-induced mitochondrial remodeling is Parkin independent. The sizes of the fragmented mitochondria may be overestimates because of the limited resolution of the microscope. n = 76–99 mitochondria from four to five microfluidic devices per genotype. *, P < 0.05; **, P < 0.001. Error bars represent means ± SEM. AU, arbitrary unit. Bars, 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4151150&req=5

fig7: Initiation of axonal mitophagy requires Parkin. (A and B) GFP-LC3–positive autophagosomes (white and cyan arrowheads) form on mitochondria of wild-type (Parkin+/+) axon segments treated with 20 µM Antimycin A (Ant A). Cyan arrowheads point to mitochondria in the axon analyzed in B. (C and D) In Parkin−/− axons, mitochondria did not acquire autophagosomes. Orange and brown arrowheads denote corresponding points in images and line scans. (E) Frequency of autophagosome formation axons before and after Antimycin A. n = 104–106 mitochondria from four to five microfluidic devices per genotype. (F) Quantification of mitochondrial size before and after 20 min of 20 µM Antimycin A treatment indicates that damage-induced mitochondrial remodeling is Parkin independent. The sizes of the fragmented mitochondria may be overestimates because of the limited resolution of the microscope. n = 76–99 mitochondria from four to five microfluidic devices per genotype. *, P < 0.05; **, P < 0.001. Error bars represent means ± SEM. AU, arbitrary unit. Bars, 5 µm.
Mentions: The recruitment of expressed Parkin to axonal mitochondria raised the question of whether endogenous Parkin was required for damage-induced mitophagy. We therefore compared mitophagy in hippocampal axons of Parkin+/+ and Parkin−/− mice (Goldberg et al., 2003). In microfluidic chambers, GFP-LC3 was coexpressed with mt-DsRed to monitor autophagosome formation. In Parkin+/+ axons, the fraction of mitochondria colocalizing with GFP-LC3–positive autophagosomes increased from 4% before to 33% after 35 min of Antimycin A treatment in the perfusion chamber (P < 0.05; Fig. 7, A, B, and E). In contrast, in Parkin−/− axons, Antimycin A did not increase mitochondrial colocalization with autophagosomes (P = 1; Fig. 7, C–E). Damage-induced mitophagy could be rescued in Parkin−/− axons by expressing mCherry-Parkin (Fig. S4 A). Thus, depolarization-induced initiation of mitophagy in neuronal axons both triggers Parkin recruitment and requires Parkin. We also examined damage-induced mitochondrial fragmentation and found that treatment with Antimycin A for 20 min caused a significant decrease in mitochondrial size in both Parkin+/+ and Parkin−/− axons (P < 0.01; Fig. 7 F). We conclude that Parkin is not required for the remodeling of depolarized mitochondria, only for the subsequent recruitment of the autophagosome.

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