The Journal of Neuroscience, January 10, 2007, ():
Altered Axonal Mitochondrial Transport in the Pathogenesis of Charcot-Marie-Tooth Disease from Mitofusin 2 Mutations
J. Neurosci. Baloh et al.
27: 422
Supplemental Data
Files in this Data Supplement:
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Supplemental Table
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Supplemental Figure 1. Fragmentation of mitochondria from MFN2 mutants is not due to induction of apoptosis. A) DRG neurons were infected with lentiviruses encoding either wild-type (WT) MFN2 or mutants R94Q and P251A, which induced significant fragmentation and aggregation of mitochondria (see Figure 3). Neurons were fixed and immunostained with an antibody to activated caspase-3 (aCasp3), which indicates activation of the apoptotic cascade. As shown in the top panel, significant activated caspase-3 was seen in neurons treated with staurosporine (STS) for 12 hours, however no activated caspase-3 was seen in neurons expressing either wild-type or mutant MFN2 proteins despite the presence of fragmentation of mitochondria. B) Overlay image of a DRG neuron infected with a lentivirus encoding MFN2 disease mutant P251A–IRES–Venus fluorescent protein to mark infected neurons, (shown as green), and mito-DsRed2 (top panel). Despite the presence of mitochondrial aggregates in neurons expressing MFN2 disease mutants, neurons are phase bright and were not overtly abnormal (bottom panel).
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Supplemental Figure 2. Mitochondrial clustering in proximal axons of neurons expressing MFN2 disease mutant proteins. A) Fluorescence images of proximal axonal segments from neurons expressing wild-type MFN2 (WT) or mutant MFN2 (shown are R94Q and P251A), together with mito-DsRed2 to visualize mitochondria. Mitochondria in wild-type MFN2 were evenly distributed throughout the axon. In contrast, mitochondria in mutant MFN2 expressing axons were predominantly found in the proximal axonal segments in a “string of pearls” like appearance. B) Higher power images of axons from DRG neurons expressing wild-type or mutant MFN2 proteins. In contrast to the short tubular appearance of mitochondria in axons of wild-type MFN2 expressing neurons, mitochondria in axons expressing disease mutant MFN2 proteins were present as distinct clusters along the axon.
