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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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Activation of mt-KR in neuronal axons. (A) mt-KR was expressed in hippocampal neurons and activated locally with a 555-nm laser. (B) Two axons expressing mt-KR and mt-GFP before and after irradiation and concomitant photobleaching of mt-KR in the outlined regions. Mitochondria became rounded and fragmented (arrowheads), and their GFP fluorescence intensity increased. Image acquisition settings were kept constant before and after KillerRed activation. (C) Morphological changes in mitochondria expressing mt-KR or mt-DsRed were quantified. n = 88–109 irradiated and n = 293–448 nonirradiated mitochondria from 12–15 transfections. ****, P < 0.0001. Error bars represent means ± SEM. Bars, 5 µm.
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fig1: Activation of mt-KR in neuronal axons. (A) mt-KR was expressed in hippocampal neurons and activated locally with a 555-nm laser. (B) Two axons expressing mt-KR and mt-GFP before and after irradiation and concomitant photobleaching of mt-KR in the outlined regions. Mitochondria became rounded and fragmented (arrowheads), and their GFP fluorescence intensity increased. Image acquisition settings were kept constant before and after KillerRed activation. (C) Morphological changes in mitochondria expressing mt-KR or mt-DsRed were quantified. n = 88–109 irradiated and n = 293–448 nonirradiated mitochondria from 12–15 transfections. ****, P < 0.0001. Error bars represent means ± SEM. Bars, 5 µm.

Mentions: To study mitophagy in neurons, we set out to induce mitochondrial damage in a spatiotemporally controlled manner, while preserving the overall integrity of the mitochondrial network. To this end, mt-KR, a genetically encoded photosensitizer targeted to mitochondria, was used to selectively damage a subset of axonal mitochondria. Light-induced activation of mt-KR causes local ROS-mediated damage to mitochondria (Bulina et al., 2006; Ertürk et al., 2014) and has been previously used to induce mitophagy in HeLa cells (Yang and Yang, 2011; Wang et al., 2012). We expressed mt-KR in rat hippocampal neurons and, with a 555-nm laser, irradiated a 10-µm section of an axon, which typically included three to four mitochondria, to activate and thereby photobleach mt-KR in those mitochondria, while sparing those in adjacent segments (Fig. 1 A). Mitochondrial damage results in stereotypical changes in mitochondrial morphology, such as swelling and fragmentation (Legros et al., 2002; Kaasik et al., 2007). To observe mitochondrial morphology after mt-KR photobleaching, we coexpressed mitochondrially targeted GFP (mt-GFP). Within 10 min of mt-KR activation, a fragmented or rounded morphology arose in 22% of the laser-illuminated mitochondria compared with 4% of the mitochondria outside the illuminated region (P < 0.0001; Fig. 1, B and C). In contrast, irradiation of mitochondria expressing mitochondrially targeted DsRed (mt-DsRed) did not cause fragmentation or rounding (Fig. S1 A). Activation of mt-KR also increased the fluorescence intensity of mt-GFP within those mitochondria (Fig. 1 B), possibly because of dequenching of the fluorophores in the swollen matrix of damaged mitochondria. Consistent with oxidative damage as the mechanism of mt-KR, we rarely detected changes in mitochondrial morphology when mt-KR was activated in Hibernate E medium, which contains antioxidants.


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)

Activation of mt-KR in neuronal axons. (A) mt-KR was expressed in hippocampal neurons and activated locally with a 555-nm laser. (B) Two axons expressing mt-KR and mt-GFP before and after irradiation and concomitant photobleaching of mt-KR in the outlined regions. Mitochondria became rounded and fragmented (arrowheads), and their GFP fluorescence intensity increased. Image acquisition settings were kept constant before and after KillerRed activation. (C) Morphological changes in mitochondria expressing mt-KR or mt-DsRed were quantified. n = 88–109 irradiated and n = 293–448 nonirradiated mitochondria from 12–15 transfections. ****, P < 0.0001. Error bars represent means ± SEM. Bars, 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4151150&req=5

fig1: Activation of mt-KR in neuronal axons. (A) mt-KR was expressed in hippocampal neurons and activated locally with a 555-nm laser. (B) Two axons expressing mt-KR and mt-GFP before and after irradiation and concomitant photobleaching of mt-KR in the outlined regions. Mitochondria became rounded and fragmented (arrowheads), and their GFP fluorescence intensity increased. Image acquisition settings were kept constant before and after KillerRed activation. (C) Morphological changes in mitochondria expressing mt-KR or mt-DsRed were quantified. n = 88–109 irradiated and n = 293–448 nonirradiated mitochondria from 12–15 transfections. ****, P < 0.0001. Error bars represent means ± SEM. Bars, 5 µm.
Mentions: To study mitophagy in neurons, we set out to induce mitochondrial damage in a spatiotemporally controlled manner, while preserving the overall integrity of the mitochondrial network. To this end, mt-KR, a genetically encoded photosensitizer targeted to mitochondria, was used to selectively damage a subset of axonal mitochondria. Light-induced activation of mt-KR causes local ROS-mediated damage to mitochondria (Bulina et al., 2006; Ertürk et al., 2014) and has been previously used to induce mitophagy in HeLa cells (Yang and Yang, 2011; Wang et al., 2012). We expressed mt-KR in rat hippocampal neurons and, with a 555-nm laser, irradiated a 10-µm section of an axon, which typically included three to four mitochondria, to activate and thereby photobleach mt-KR in those mitochondria, while sparing those in adjacent segments (Fig. 1 A). Mitochondrial damage results in stereotypical changes in mitochondrial morphology, such as swelling and fragmentation (Legros et al., 2002; Kaasik et al., 2007). To observe mitochondrial morphology after mt-KR photobleaching, we coexpressed mitochondrially targeted GFP (mt-GFP). Within 10 min of mt-KR activation, a fragmented or rounded morphology arose in 22% of the laser-illuminated mitochondria compared with 4% of the mitochondria outside the illuminated region (P < 0.0001; Fig. 1, B and C). In contrast, irradiation of mitochondria expressing mitochondrially targeted DsRed (mt-DsRed) did not cause fragmentation or rounding (Fig. S1 A). Activation of mt-KR also increased the fluorescence intensity of mt-GFP within those mitochondria (Fig. 1 B), possibly because of dequenching of the fluorophores in the swollen matrix of damaged mitochondria. Consistent with oxidative damage as the mechanism of mt-KR, we rarely detected changes in mitochondrial morphology when mt-KR was activated in Hibernate E medium, which contains antioxidants.

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