Restoring mitofusin balance prevents axonal degeneration in a Charcot-Marie-Tooth type 2A model
Yueqin Zhou, … , Cathleen M. Lutz, Robert H. Baloh
Yueqin Zhou, … , Cathleen M. Lutz, Robert H. Baloh
Published March 18, 2019
Citation Information: J Clin Invest. 2019;129(4):1756-1771. https://doi.org/10.1172/JCI124194.
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Research Article Neuroscience

Restoring mitofusin balance prevents axonal degeneration in a Charcot-Marie-Tooth type 2A model

Abstract

Mitofusin-2 (MFN2) is a mitochondrial outer-membrane protein that plays a pivotal role in mitochondrial dynamics in most tissues, yet mutations in MFN2, which cause Charcot-Marie-Tooth disease type 2A (CMT2A), primarily affect the nervous system. We generated a transgenic mouse model of CMT2A that developed severe early onset vision loss and neurological deficits, axonal degeneration without cell body loss, and cytoplasmic and axonal accumulations of fragmented mitochondria. While mitochondrial aggregates were labeled for mitophagy, mutant MFN2 did not inhibit Parkin-mediated degradation, but instead had a dominant negative effect on mitochondrial fusion only when MFN1 was at low levels, as occurs in neurons. Finally, using a transgenic approach, we found that augmenting the level of MFN1 in the nervous system in vivo rescued all phenotypes in mutant MFN2R94Q-expressing mice. These data demonstrate that the MFN1/MFN2 ratio is a key determinant of tissue specificity in CMT2A and indicate that augmentation of MFN1 in the nervous system is a viable therapeutic strategy for the disease.

Authors

Yueqin Zhou, Sharon Carmona, A.K.M.G. Muhammad, Shaughn Bell, Jesse Landeros, Michael Vazquez, Ritchie Ho, Antonietta Franco, Bin Lu, Gerald W. Dorn II, Shaomei Wang, Cathleen M. Lutz, Robert H. Baloh

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Figure 3

Dominant negative disruption of mitochondrial fusion, but not mitophagy, by MFN2R94Q.

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Dominant negative disruption of mitochondrial fusion, but not mitophagy,...
(A) Cytoplasmic accumulations of mitochondria in the hippocampus of MFN2R94Q:CFP-COX8A mutant mice, not seen in CFP-COX8A control mice (7-month-old mice). Scale bar: 100 μM. n = 2 mice/genotype. (B) Mitochondrial clustering was also seen in distal peripheral nerve and NMJ of MFN2R94Q:CFP-COX8A mice (quadriceps muscle shown). NMJ was labeled with α-bungarotoxin (BTX, red). Arrowheads indicate mitochondrial clusters in distal axons (7-month-old mice). Scale bar: 50 μM. n = 2 mice/genotype. (C) Mitochondrial accumulations in MFN2R94Q mice were colocalized with p62 protein, indicating they were marked for mitophagy. Anti-Flag staining labels transgenic MFN2. DAPI labels nuclei (5-month-old mice). Scale bars: 50 μM. Numbers of p62 aggregates per ROI (0.01 mm2) are quantified. Data are represented as mean ± SEM. n = 3 mice/genotype. Student’s 2-tailed t test, **P < 0.01. (D) Effects of MFN2R94Q on mitochondrial fusion, membrane potential, and mitophagy in MEFs. MEFs were infected with β-gal (control), MFN2WT, or MFN2R94Q lentivirus. Depolarization was induced by treating MEFs with FCCP (10 μM) for 1 hour. Mitochondria were labeled with MitoTracker (green) and TMRE (red). Scale bar: 10 μM. (E) Quantification of D. Mitochondrial fusion at the basal level is presented as mitochondrial aspect ratio measurement (mitochondrial length/width; n = 9 repeat experiments). Initiation of mitophagy is presented as mitochondrial depolarization ratio (green/green+yellow mitochondria; n = 3). (F) Parkin localization. MEFs were infected with adenovirus expressing RFP-parkin. Scale bars: 10 μM. (G) Quantification of parkin localization in F (n = 3 repeat experiments). (H) Colocalization of mitochondria and lysosome. Mitochondria were labeled with MitoTracker Green, and lysosomes were labeled with LysoTracker Red. Scale bars: 10 μM. (I) Quantification of mitochondria and lysosome colocalization in H (n = 3 repeat experiments). In E, G, and I, data are represented as mean ± SEM. One-way ANOVA with Tukey’s test. **P < 0.01; ****P < 0.0001.