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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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Damaged axonal mitochondria colocalize with autophagosomes. (A–D) Cyan and white arrowheads denote GFP-LC3–positive autophagosomes colocalizing with mitochondria. (A) Within the outlined laser-illuminated region of activated mt-KR, one mt-BFP–labeled mitochondrion colocalized with a GFP-LC3–positive autophagosome; the BFP signal from the engulfed mitochondrion was present 20 min after illumination but disappeared by 40 min, even though the autophagosome was present. In the insets are the merged images for the engulfed mitochondrion. (B) Line scan analysis of A. (C) GFP-LC3–positive autophagosomes formed on mitochondria depolarized with 40 µM Antimycin A (Ant A) in the perfusion chamber of a microfluidic device. (D) Line scan analysis of C. Here and subsequently, orange and brown arrowheads denote corresponding points in images and line scans, and mitochondria marked with cyan arrowheads correspond to similarly marked peaks in line scans. (E) Frequency of autophagosome formation before and after Antimycin A or mock treatments. n = 182–244 mitochondria from four microfluidic devices per condition. Here and in subsequent graphs, each point represents the average percentage of positive mitochondria for a single microfluidic chamber. *, P < 0.05. Error bars represent means ± SEM. AU, arbitrary unit. Bars, 5 µm.
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fig3: Damaged axonal mitochondria colocalize with autophagosomes. (A–D) Cyan and white arrowheads denote GFP-LC3–positive autophagosomes colocalizing with mitochondria. (A) Within the outlined laser-illuminated region of activated mt-KR, one mt-BFP–labeled mitochondrion colocalized with a GFP-LC3–positive autophagosome; the BFP signal from the engulfed mitochondrion was present 20 min after illumination but disappeared by 40 min, even though the autophagosome was present. In the insets are the merged images for the engulfed mitochondrion. (B) Line scan analysis of A. (C) GFP-LC3–positive autophagosomes formed on mitochondria depolarized with 40 µM Antimycin A (Ant A) in the perfusion chamber of a microfluidic device. (D) Line scan analysis of C. Here and subsequently, orange and brown arrowheads denote corresponding points in images and line scans, and mitochondria marked with cyan arrowheads correspond to similarly marked peaks in line scans. (E) Frequency of autophagosome formation before and after Antimycin A or mock treatments. n = 182–244 mitochondria from four microfluidic devices per condition. Here and in subsequent graphs, each point represents the average percentage of positive mitochondria for a single microfluidic chamber. *, P < 0.05. Error bars represent means ± SEM. AU, arbitrary unit. Bars, 5 µm.

Mentions: It has been reported that damaged mitochondria move retrograde toward the soma, presumably, to undergo mitophagy there (Miller and Sheetz, 2004; Cai et al., 2012). However, our observations of mitochondrial motility arrest (Wang et al., 2011) and the accumulation of aged mitochondrial proteins in distal processes (Ferree et al., 2013) argue against this model. In addition, a recent study has shown autophagosome formation in distal regions of undamaged neurons in culture (Maday et al., 2012). To determine whether damaged mitochondria in distal axons could locally recruit autophagosomes, we expressed the autophagosome marker GFP-LC3 along with mt-KR in hippocampal neurons. GFP-LC3 is a cytosolic protein, but upon induction of autophagy, it gets lipidated and incorporated into autophagosomes forming distinct puncta (Mizushima and Komatsu, 2011). Mitochondrially targeted BFP (mt-BFP) was also expressed to mark mitochondria. After activation of mt-KR, we observed GFP-LC3 accumulation on mitochondria, indicating the formation of mitochondrion-containing autophagosomes (Fig. 3, A and B). In some instances, the autophagosomal mitochondrion eventually disappeared, presumably, as a result of the formation of autolysosomes. Our findings establish that the initial stages of mitophagy, specifically colocalization with LC3-positive autophagosomes, can occur locally in axons.


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)

Damaged axonal mitochondria colocalize with autophagosomes. (A–D) Cyan and white arrowheads denote GFP-LC3–positive autophagosomes colocalizing with mitochondria. (A) Within the outlined laser-illuminated region of activated mt-KR, one mt-BFP–labeled mitochondrion colocalized with a GFP-LC3–positive autophagosome; the BFP signal from the engulfed mitochondrion was present 20 min after illumination but disappeared by 40 min, even though the autophagosome was present. In the insets are the merged images for the engulfed mitochondrion. (B) Line scan analysis of A. (C) GFP-LC3–positive autophagosomes formed on mitochondria depolarized with 40 µM Antimycin A (Ant A) in the perfusion chamber of a microfluidic device. (D) Line scan analysis of C. Here and subsequently, orange and brown arrowheads denote corresponding points in images and line scans, and mitochondria marked with cyan arrowheads correspond to similarly marked peaks in line scans. (E) Frequency of autophagosome formation before and after Antimycin A or mock treatments. n = 182–244 mitochondria from four microfluidic devices per condition. Here and in subsequent graphs, each point represents the average percentage of positive mitochondria for a single microfluidic chamber. *, P < 0.05. Error bars represent means ± SEM. AU, arbitrary unit. Bars, 5 µm.
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fig3: Damaged axonal mitochondria colocalize with autophagosomes. (A–D) Cyan and white arrowheads denote GFP-LC3–positive autophagosomes colocalizing with mitochondria. (A) Within the outlined laser-illuminated region of activated mt-KR, one mt-BFP–labeled mitochondrion colocalized with a GFP-LC3–positive autophagosome; the BFP signal from the engulfed mitochondrion was present 20 min after illumination but disappeared by 40 min, even though the autophagosome was present. In the insets are the merged images for the engulfed mitochondrion. (B) Line scan analysis of A. (C) GFP-LC3–positive autophagosomes formed on mitochondria depolarized with 40 µM Antimycin A (Ant A) in the perfusion chamber of a microfluidic device. (D) Line scan analysis of C. Here and subsequently, orange and brown arrowheads denote corresponding points in images and line scans, and mitochondria marked with cyan arrowheads correspond to similarly marked peaks in line scans. (E) Frequency of autophagosome formation before and after Antimycin A or mock treatments. n = 182–244 mitochondria from four microfluidic devices per condition. Here and in subsequent graphs, each point represents the average percentage of positive mitochondria for a single microfluidic chamber. *, P < 0.05. Error bars represent means ± SEM. AU, arbitrary unit. Bars, 5 µm.
Mentions: It has been reported that damaged mitochondria move retrograde toward the soma, presumably, to undergo mitophagy there (Miller and Sheetz, 2004; Cai et al., 2012). However, our observations of mitochondrial motility arrest (Wang et al., 2011) and the accumulation of aged mitochondrial proteins in distal processes (Ferree et al., 2013) argue against this model. In addition, a recent study has shown autophagosome formation in distal regions of undamaged neurons in culture (Maday et al., 2012). To determine whether damaged mitochondria in distal axons could locally recruit autophagosomes, we expressed the autophagosome marker GFP-LC3 along with mt-KR in hippocampal neurons. GFP-LC3 is a cytosolic protein, but upon induction of autophagy, it gets lipidated and incorporated into autophagosomes forming distinct puncta (Mizushima and Komatsu, 2011). Mitochondrially targeted BFP (mt-BFP) was also expressed to mark mitochondria. After activation of mt-KR, we observed GFP-LC3 accumulation on mitochondria, indicating the formation of mitochondrion-containing autophagosomes (Fig. 3, A and B). In some instances, the autophagosomal mitochondrion eventually disappeared, presumably, as a result of the formation of autolysosomes. Our findings establish that the initial stages of mitophagy, specifically colocalization with LC3-positive autophagosomes, can occur locally in axons.

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