Mitochondrial fusion and function in Charcot-Marie-Tooth type 2A patient fibroblasts with mitofusin 2 mutations

Abstract

Charcot-Marie-Tooth Type 2A is a dominantly inherited peripheral neuropathy characterized by axonal degeneration of sensory and motor nerves. The disease is caused by mutations in the mitochondrial fusion gene MFN2. Mfn2 is an integral outer mitochondrial membrane protein composed of a large GTPase domain and two heptad repeat (HR) domains that face the cytoplasm. Mitochondrial membrane fusion and division are balanced processes that are necessary to maintain tubular mitochondrial morphology, respiratory function, and uniform distribution of the organelle throughout the cell. We have utilized primary fibroblasts from CMT2A patients to survey mitochondrial phenotypes associated with heterozygous MFN2 alleles expressed at physiological levels. Our results indicate that, in fibroblasts, mitofusin expression, mitochondrial morphology, ultrastructure, mtDNA content, and respiratory capacity are not affected by the presence of mutant Mfn2 protein. Consistent with a lack of mitochondrial dysfunction, we also show that mitochondrial fusion occurs efficiently in CMT2A patient-derived fibroblasts. Our observations are in agreement with the neuronal specificity of the disease and are consistent with a recent finding that mitochondrial fusion can be maintained in cells that express mutant Mfn2 protein due to complementation by a second mitofusin, Mfn1. We discuss our results and those of others in terms of a comprehensive model for the mechanism(s) by which mutations in MFN2 may lead to CMT2A disease.

Figure 1. Mfn2 domain structure and location of CMT2A mutations

A schematic of the predicted functional and structural domains of Mfn2. The location of each CMT2A-associated amino acid substitution analyzed in this study is indicated. G1–G4 = functional motifs within the GTPase domain, TM = transmembrane domain, HR = heptad repeat or putative coil-coil domain.

Figure 2. Western analysis of mitofusin abundance and Mfn2 localization

(A) Western blot analysis of the mitochondrial proteins Mfn2, porin, and Mfn1 (~86 kDa, 31 kDa, and 84 kDa, respectively) in 10μg protein from whole cell extracts of Control and CMT2A patient-derived fibroblasts. Mfn2 signal was normalized to porin and the ratio for the Control was set equal to 1.0. Bars represent average Mfn2 levels relative to porin and error bars indicate the standard deviation from triplicate analyses. (B) Western blot analysis of Mfn2, porin, and GAPDH (~37 kDa) in mitochondrial pellets (Mito) and post-mitochondrial supernatants (PMS) from Control and Mfn2-mutant CMT2A fibroblasts. The outer mitochondrial membrane proteins Mfn2 and porin are detected primarily in the mitochondrial fractions while cytoplasmic GAPDH is only in PMS fractions. Mfn2 levels appear consistent between samples and both WT and mutant proteins are properly targeted and localized to the mitochondrial fraction.

Figure 3. Mitochondrial morphology in Control and CMT2A patient-derived fibroblasts

Primary fibroblast cultures were stained with 15nM MitoTracker for 6–8 hours and visualized by fluorescence microscopy. (A) Quantification of the percent of each population exhibiting the indicated mitochondrial morphologies. Tubular = interconnected tubular mitochondrial structures forming a network distributed throughout the cell. Mixed = a combination of normal tubular structures intermixed with spherical mitochondrial fragments. Fragmented = completely fragmented or spherical mitochondria. At least 100 individual cells per population were scored per experiment. Bars represent the mean percentage per category and error bars indicate the standard deviation from triplicate experiments. (B) Representative images of MitoTracker stained mitochondria in Control and CMT2A fibroblasts with the indicated Mfn2 amino acid substitutions. Insets are zoomed to highlight distinct tubular structures in the boxed areas.

Figure 4. Mitochondrial ultrastructure in Control and CMT2A patient-derived fibroblasts

Primary fibroblast cultures were fixed and processed for transmission electron microscopy as described in Materials and Methods. Representative electron micrographs show normal gross mitochondrial morphology, quantity, and distribution in Control and the indicated Mfn2 mutant cells. Mitochondria are marked (m) and appear spherical or as longer tubules depending on the sectioning. A visible double membrane surrounds most of the pictured mitochondria and several have visible inner membrane cristae in the plane of focus, confirming normal mitochondrial ultrastructure.

Figure 5. PCR-based mitochondrial DNA analysis

(A) Agarose gels displaying PCR products from muscle and fibroblast DNA samples using primers OLA/D1B and OLB/D1A. An 11kb PCR product generated from OLA/D1B primers is clearly visible in ‘Normal’ muscle and all fibroblast samples. Amplification of DNA isolated from muscle of a KSS patient produced a full-length product of 11kb as well as a deletion product of ~6kb. Amplification of DNA from muscle of a CPEO patient generated a large smear of DNA, indicating multiple deletions of the genome. All products generated from primers OLB/D1A are ~5.6 kb, the expected size for intact mtDNA. Asterisks (*) indicate non-specific amplification products visible in all lanes upon longer exposure. Analysis of DNA isolated from P5 fibroblasts produced results that were identical to those shown here for P11 cells. (B) Results of qRT-PCR analysis of mtDNA in Control and CMT2A fibroblasts. The ratio of mtDNA/β-globin (± standard deviation) was calculated from triplicate experiments to determine the amount of mtDNA relative to a single copy nuclear gene (β-globin) in each sample. The Student’s t-test (unpaired, two-sample equal variance) was applied to the Control (WT) and each MFN2 mutant sample as well as all the mutant samples pooled together (All Mutants). A p value ≤ 0.05 indicates statistical significance.

Figure 6. Respiration rates in control and CMT2A patient-derived fibroblasts

(A) Comparison of oxygen consumption rates for Control and mutant MFN2 (T105M, I213T, and V273G) fibroblast cultures measured under basal and uncoupled conditions. Bars represent the mean respiration rate (nmoles O₂/min/mL) and error bars indicate the standard deviation from four independent measurements. Listed below each set of bars is the fold increase in respiration observed upon uncoupling by DNP addition (ratio of uncoupled/basal) ± standard deviation. (B) Vertical scatter plot indicating the range of basal respiration rates observed from multiple measures for each fibroblast population. (C) Vertical scatter plot indicating the range of uncoupled respiration rates observed from multiple measures for each fibroblast population. Each circle indicates an individual data point and the horizontal bar represents the mean value from replicate measures.

Figure 7. Mitochondrial fusion in cell hybrids formed from Control or CMT2A patient-derived fibroblasts

PEG fusion of Control (A) and I213T mutant (B) fibroblasts expressing mito-GFP and mito-RFP. Cell hybrids contain two or more nuclei visualized with DAPI (blue) and overlapping red and green fluorescence (yellow) indicates successful mitochondrial fusion. The Control cell hybrid in (A) has normal tubular mitochondrial morphology with extensive mitochondrial fusion. The cell hybrid of I213T mutant cells in (B) displays mixed mitochondrial morphology (tubular and spherical mitochondria) with fused tubular mitochondria and some fused spherical mitochondria. (A′) and (B′) are zoomed images of the boxed regions in the ‘Merged’ panels of (A) and (B), respectively. Asterisks (*) indicate unfused mito-GFP spherical mitochondria, carats (^) indicate unfused mito-RFP spherical mitochondria, and arrows (↑) point to fused spherical mitochondria.