Abstract
Mutations in GDAP1, an outer mitochondrial membrane protein responsible for recessive Charcot-Marie-Tooth disease (CMT4A), have also been associated with CMT2K, a dominant form of the disease. The three CMT2K patients we studied carried a novel dominant GDAP1 mutation, C240Y (c.719G > A). Mitochondrial respiratory chain complex I activity in fibroblasts from CMT2K patients was 40% lower than in controls, whereas the tubular mitochondria were 33% larger in diameter and the mitochondrial mass was 20% greater. Thus, besides the regulatory role GDAP1 plays in mitochondrial network dynamics, it may also be involved in energy production and in the control of mitochondrial volume.
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References
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Acknowledgments
We thank C. Wetterwald, S. Geraci and the technicians of the laboratory for technical assistance and Kanaya Malkani for the critical reading and comments on the manuscript. This work was supported by INSERM, the University Hospital of Angers, the University of Angers, France, and by grants from Retina France and the “Ouvrir les yeux” patients’ associations (AC, PAB, DB, and PR).
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Julien Cassereau and Arnaud Chevrollier contributed equally to this work.
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Figure 1S
GDAP1 is expressed in fibroblasts from patient and controls. (a): The expression of GDAP1 mRNA in human skin fibroblasts as visualized by gel electrophoresis of PCR-amplified products obtained using selective oligonucleotide primers. Bands of the expected size, i.e., 221 and 174 base pairs (bp) were obtained in wells where samples were subjected to reverse transcriptase (RT). The set of primers did not amplify genomic DNA. Beta 2 microblobulin pair of primers (in the exon) was chosen as a control of cDNA and DNA amplification. b GDAP1 protein is expressed in skin fibroblasts. Fifteen micrograms of protein lysate from the patients and controls was analyzed by Western blotting. GDAP1 was detected at the predicted size of 42 kDa. α-Tubulin was used as loading control. (JPG 238 KB)
Figure 2S
Mitochondrial quantification process. Z-stack images were acquired with a Leica (DMI6000B, Microsystems GmbH, Wetzlar, Germany) and a Roper CoolSnap HQ2 camera. We took 21 plane images along the Z-axis at 0.3-μm increments. Stack images were deconvolved using a Meinel algorithm. On overlay picture (a), the mitochondria labeled with the Mitotracker® probe are shown in red; the contour cells and the nuclei are shown in phase contrast. MetaMorph software (Molecular Devices, CA, USA) was used to analyze the images. The mitochondrial fluorescent signal was thresholded and the image was binarized (b). Length and number were determined using integrated morphometric analysis of regions created around mitochondria (c). At least 50 controls or mutated cells as in this sequence analysis were used to create the bar graph shown in Fig. 3a. We then compared the subgroups of mitochondrial size between controls and patients using a nonparametric Mann–Whitney test. Phase contrast images were used to determine cell contours (green trace) and the corresponding cell areas. Mitochondrial thresholded signals were used to quantify the total mitochondrial area, which was expressed as a fraction of the corresponding cell area. (JPG 347 KB)
Figure 3S
Calculation of mitochondrial diameter in electronic microscopy. Diameters (a or b) are estimated by the average of several measurements on mitochondrial sections (green lines). M1: Measurement 1; M2: Measurement 2. (JPG 173 KB)
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Cassereau, J., Chevrollier, A., Gueguen, N. et al. Mitochondrial complex I deficiency in GDAP1-related autosomal dominant Charcot-Marie-Tooth disease (CMT2K). Neurogenetics 10, 145–150 (2009). https://doi.org/10.1007/s10048-008-0166-9
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DOI: https://doi.org/10.1007/s10048-008-0166-9