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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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Localized depolarization of axonal mitochondria in microfluidic devices. (A–C) Neurons were plated in the somal chamber (A), and axons grew through two sets of 200-µm microgrooves intersected by a 100-µm-long perfusion channel (B) and enlarged areas in C. Addition of 40 µM Antimycin A (Ant A) to the perfusion channel (box 2) decreased mitochondrial TMRM staining in that chamber, whereas mitochondria in the proximal and distal microgrooves (boxes 1 and 3) remained polarized. (D) Relative TMRM intensity, normalized to t = 0, as a function of time after addition of Antimycin A. The change in TMRM intensity is statistically significant only in the perfusion chamber. ***, P < 0.001 by linear regression analysis; n = 4 microfluidic devices. Error bars represent means ± SEM. Bars: (A and B) 50 µm; (C) 20 µm.
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fig2: Localized depolarization of axonal mitochondria in microfluidic devices. (A–C) Neurons were plated in the somal chamber (A), and axons grew through two sets of 200-µm microgrooves intersected by a 100-µm-long perfusion channel (B) and enlarged areas in C. Addition of 40 µM Antimycin A (Ant A) to the perfusion channel (box 2) decreased mitochondrial TMRM staining in that chamber, whereas mitochondria in the proximal and distal microgrooves (boxes 1 and 3) remained polarized. (D) Relative TMRM intensity, normalized to t = 0, as a function of time after addition of Antimycin A. The change in TMRM intensity is statistically significant only in the perfusion chamber. ***, P < 0.001 by linear regression analysis; n = 4 microfluidic devices. Error bars represent means ± SEM. Bars: (A and B) 50 µm; (C) 20 µm.

Mentions: Although mt-KR can restrict damage to a few mitochondria, quantitative examination of mitophagy required damage to a larger population of axonal mitochondria, while still sparing the majority of mitochondria in a neuron. To this end, we took advantage of microfluidic local perfusion chambers (Taylor et al., 2010). Fluidic isolation of compartments in these devices allows for the selective pharmacological manipulation of a subset of axonal mitochondria. Hippocampal neurons were plated in the somal compartment and extended axons through two sets of 200-µm-long microgrooves that were separated by a 100-µm-long perfusion chamber (Fig. 2 A).


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)

Localized depolarization of axonal mitochondria in microfluidic devices. (A–C) Neurons were plated in the somal chamber (A), and axons grew through two sets of 200-µm microgrooves intersected by a 100-µm-long perfusion channel (B) and enlarged areas in C. Addition of 40 µM Antimycin A (Ant A) to the perfusion channel (box 2) decreased mitochondrial TMRM staining in that chamber, whereas mitochondria in the proximal and distal microgrooves (boxes 1 and 3) remained polarized. (D) Relative TMRM intensity, normalized to t = 0, as a function of time after addition of Antimycin A. The change in TMRM intensity is statistically significant only in the perfusion chamber. ***, P < 0.001 by linear regression analysis; n = 4 microfluidic devices. Error bars represent means ± SEM. Bars: (A and B) 50 µm; (C) 20 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig2: Localized depolarization of axonal mitochondria in microfluidic devices. (A–C) Neurons were plated in the somal chamber (A), and axons grew through two sets of 200-µm microgrooves intersected by a 100-µm-long perfusion channel (B) and enlarged areas in C. Addition of 40 µM Antimycin A (Ant A) to the perfusion channel (box 2) decreased mitochondrial TMRM staining in that chamber, whereas mitochondria in the proximal and distal microgrooves (boxes 1 and 3) remained polarized. (D) Relative TMRM intensity, normalized to t = 0, as a function of time after addition of Antimycin A. The change in TMRM intensity is statistically significant only in the perfusion chamber. ***, P < 0.001 by linear regression analysis; n = 4 microfluidic devices. Error bars represent means ± SEM. Bars: (A and B) 50 µm; (C) 20 µm.
Mentions: Although mt-KR can restrict damage to a few mitochondria, quantitative examination of mitophagy required damage to a larger population of axonal mitochondria, while still sparing the majority of mitochondria in a neuron. To this end, we took advantage of microfluidic local perfusion chambers (Taylor et al., 2010). Fluidic isolation of compartments in these devices allows for the selective pharmacological manipulation of a subset of axonal mitochondria. Hippocampal neurons were plated in the somal compartment and extended axons through two sets of 200-µm-long microgrooves that were separated by a 100-µm-long perfusion chamber (Fig. 2 A).

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