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Comparative Study
. 2007 Jan 10;27(2):422-30.
doi: 10.1523/JNEUROSCI.4798-06.2007.

Altered axonal mitochondrial transport in the pathogenesis of Charcot-Marie-Tooth disease from mitofusin 2 mutations

Robert H Baloh  1 , Robert E Schmidt, Alan Pestronk, Jeffrey Milbrandt
Affiliations
Comparative Study

Altered axonal mitochondrial transport in the pathogenesis of Charcot-Marie-Tooth disease from mitofusin 2 mutations

Robert H Baloh et al. J Neurosci. .

Abstract

Mutations in the mitochondrial fusion protein mitofusin 2 (MFN2) are the most commonly identified cause of Charcot-Marie-Tooth type 2 (CMT2), a dominantly inherited disease characterized by degeneration of peripheral sensory and motor axons. However, the mechanism by which mutations in this ubiquitously expressed mitochondrial fusion protein lead to neuropathy has not yet been elucidated. To explore how MFN2 mutations lead to degeneration of peripheral axons, we expressed neuropathy-associated forms of MFN2 in cultured dorsal root ganglion neurons, cells preferentially affected in CMT2. Disease-associated MFN2 mutant proteins induced abnormal clustering of small fragmented mitochondria in both neuronal cell bodies and proximal axons. Interestingly, transport of mitochondria in axons was significantly impaired in neurons expressing disease-mutated forms of MFN2. The diminished axonal mitochondrial transport was not attributable to diminished ATP levels in the neurons, and oxidative respiration was normal in mutant MFN2-expressing cells. Additionally, mitochondrial oxidative enzyme activity was normal in muscle mitochondria from a CMT2 patient with an MFN2 mutation, further supporting that abnormal mitochondrial transport in neurons is independent from an energy production defect. This abnormal mitochondrial trafficking provides a likely explanation for the selective susceptibility of the longest peripheral axons to MFN2 mutations, in which proper localization of mitochondria is critical for axonal and synaptic function.

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Figures

Figure 1.
Figure 1.
Mutant MFN2 proteins have normal half-lives and are properly localized to mitochondria. A, HEK 293T cells were transfected with an expression vector for GFP (CTL) or wild-type human MFN2 (MFN2), and immunoblotting for MFN2 was performed. Lysate from MFN2-transfected cells showed a single band at ∼80 kDa. Longer exposure did reveal a small amount of endogenous MFN2 present in HEK 293T cells (data not shown). B, HEK 293T cells were transfected with either wild-type (WT-MFN2) or the indicated MFN2 mutant. Cells were lysed at the indicated times after addition of 30 μg/ml cycloheximide (CHX) to inhibit new protein synthesis, and immunoblotting was performed using an anti-MFN2 antibody. Acetylated tubulin (AcTub) immunoblotting is shown on the same samples below as a loading control. Disease mutants ran at identical molecular weight and had a comparable stability to wild-type MFN2. Similar results were obtained for all of the mutants examined in this study (data not shown). C, HEK 293T cells were transfected with wild-type or mutant MFN2 constructs, in which GFP was fused to the N terminus. Cells were treated with Mitotracker Red dye to label mitochondria and imaged with fluorescence microscopy. The overlaid image shows that the GFP-fused MFN2 mutant constructs are properly localized to mitochondria.
Figure 2.
Figure 2.
Mitofusin 2 disease mutants have retained GTP-binding activity. Wild-type (WT) and disease mutant MFN2 constructs with GFP fused to the N terminus were transfected into HEK 293T cells, and cell lysates were prepared 24 h later. The lysates were incubated with GTP-agarose beads, and bound GFP-MFN2 protein was measured using a fluorimeter. A, GFP–MFN2 binding to GTP-agarose beads was specific, because there is no background binding of GFP alone, and excess GTP (10 mm) was able to competitively block GFP–MFN2 binding to GTP-agarose beads. B, GTP-agarose binding as above using GFP-MFN2 mutants performed in duplicate demonstrates that all of the MFN2 disease mutants bind GTP to a similar extent.
Figure 3.
Figure 3.
MFN2 mutants induce abnormal mitochondrial clustering and morphology in cell bodies and proximal axons. Embryonic day 15 rat dorsal root ganglion neurons in culture were infected with lentiviruses encoding GFP as a control, wild-type MFN2, or the indicated CMT2 disease mutant followed by an IRES–Venus fluorescent protein to mark infected cells. Subsequently, cells were infected with a second lentivirus encoding a mitochondrially targeted DsRed2, and live cells were imaged. Both GFP infected (GFP) and wild-type MFN2 infected (WT-MFN2) showed a similar pattern of mitochondria in the soma, with a discrete grain-like appearance that was diffuse throughout the cell. In contrast, mutants V69F, L76P, R94Q, P251A, and R280H all led to abnormal clustering of mitochondria, usually in either one or two large clusters, without discrete mitochondria apparent and with cellular regions entirely devoid of mitochondria. This was least pronounced in mutant V69F and not present for mutant W740S. On higher magnification, these appeared as small fragmented mitochondria (see also Figs. 5, 6). Frequently, aggregates of small mitochondria would cluster in the proximal axon (arrow in P251A–MFN2). Electron microscopic images (bottom panels) of HEK 293T cells expressing either wild type or the R94Q mutant MFN2 via lentiviral infection confirmed that the clusters seen in the R94Q mutant represent aggregates of individual mitochondria, many of which appeared misshapen. Scale bars, 2 μm.
Figure 4.
Figure 4.
MFN2 disease mutants induce abnormal axonal mitochondrial mobility. Low-power fluorescence images of mito-DsRed2 fluorescence in neurons expressing wild-type (WT) MFN2 (A) or disease mutants R94Q (B) or P251A (C), taken at the same exposure 96 h after infection with mito-DsRed2 lentivirus. In wild-type MFN2-expressing neurons, mitochondria were distributed evenly throughout the distal axonal segments. In contrast, mitochondria in disease mutant MFN2-expressing neurons were found predominantly in the proximal axonal segments and were not seen in distal axonal regions, consistent with a mitochondrial transport defect. To characterize this further, time-lapse imaging was performed over a 5 min period on 10 axons at 40× magnification for each construct, and kymographs were generated from individual axons. D, Representative axon infected with wild-type MFN2, showing mitochondria with normal short tubular morphology, evenly distributed throughout the axon. E, Representative kymograph of time-lapse movie from a wild-type MFN2-expressing axon shows a large number of mobile mitochondria indicated by sloping lines on the kymograph image. The arrow points to an individual axonal mitochondrion moving during the time-lapse movie. F, Representative axon expressing mutant R94Q MFN2 showing clusters of small mitochondria in the proximal axon (supplemental Fig. 2, available at www.jneurosci.org as supplemental material). G, A representative kymograph of an R94Q mutant MFN2-expressing axon showing mostly stationary mitochondria or clusters of mitochondria, with few moving mitochondria during the time lapse (arrow again indicates a moving mitochondrion). H, Quantitative analysis of mobile mitochondria from kymographs. The average number of moving mitochondria per 100 μm axonal segment was significantly lower in the neurons expressing mutant MFN2 constructs compared with those expressing wild-type MFN2 (Student's paired t test, mutant vs wild-type, *p < 0.0001).
Figure 5.
Figure 5.
Mitochondrial movement is impaired in neuronal cell bodies expressing mutant MFN2 proteins. DRG neurons were infected with lentiviruses encoding wild-type or disease mutant MFN2 constructs, together with a mitochondrially targeted DsRed2 (DsRm) and photoactivatible GFP (PAGFPm). PAGFP is a variant of GFP and has minimal fluorescence at baseline, but its fluorescence increases ∼100-fold when photoactivated with 405 or 413 nm light (Patterson and Lippincott-Schwartz, 2002). After 10 d in culture, mitochondria in approximately half of the cell body were photoactivated using the 405 nm laser (yellow box indicates region of photoexcitation), and individual cells were reimaged 36 h later. Neurons expressing wild-type MFN2 (WT) showed an even redistribution of photoactivated mitochondria throughout the cell body after 36 h. Neurons expressing mutant MFN2 proteins had clustered, fragmented mitochondria. After 36 h, there was very little movement of mitochondria from the region of photoexcitation. The decrement in the post-photoactivation signal suggests that some mitochondrial fusion still took place, although because of the marked mobility defect, this cannot be measured as an independent variable.
Figure 6.
Figure 6.
MFN2 mutants do not alter ATP levels or diminish mitochondrial potential in cultured sensory neurons. A, DRG cultures of neurons expressing either wild-type or mutant MFN2 constructs were collected 10 d after infection, and ATP and protein levels were measured. GFP-infected neurons treated with 2-deoxyglucose and oligomycin (2DG/O) for 12 h showed essentially complete depletion of ATP, whereas ATP levels were normal in cells expressing wild-type or disease mutant MFN2 proteins, despite the marked abnormality in ATP-dependent mitochondrial transport (Figs. 3, 4). B, DRG neurons were infected with lentivirus encoding the indicated wild-type (WT) or mutant MFN2 constructs, stained for 30 min with 25 nm Mitotracker Red, and imaged with confocal microscopy. Mitotracker Red staining of neurons expressing wild-type and disease mutant MFN2 showed similar intensity of staining of mitochondria, indicating that the mitochondrial potential in the aggregated mitochondria is maintained. Staining was diminished in the center of the largest mitochondrial clusters, likely attributable to decreased penetration of the Mitotracker dye in the short staining period. The neuron labeled with an asterisk in panel R94Q was uninfected and is shown for comparison with the marked mitochondrial aggregation seen in the neighboring cell. There was no quantitative difference in fluorescence intensity of the Mitotracker dye between WT and mutant infected cells (data not shown), indicating that the mitochondrial membrane potential was not significantly diminished in neurons expressing mutant MFN2 constructs.
Figure 7.
Figure 7.
Oxidative respiration is normal in cells expressing MFN2 disease mutants despite mitochondrial clustering. A, Representative oxygen consumption graphs for HEK 293T cells expressing wild-type MFN2 (WT) or disease mutants R94Q or W740S. The arrow indicates the addition of 2,4-dinitrophenol, an uncoupling agent to induce maximal oxidative respiration. The slope of the line indicating the rate of respiration is shown above the curves both before (endogenous respiration) and after (maximal respiration) the addition of the uncoupling agent 2,3-dinitrophenol. B, Fluorescence images of HEK 293T cells expressing wild-type (WT) MFN2 or disease mutant R94Q, together with mito-DsRed2 to visualize mitochondria, showed that these cells form similar mitochondrial aggregates to that seen in primary neurons only in the presence of MFN2 disease mutants. C, Despite the mitochondrial clustering induced by mutant MFN2 proteins, both endogenous and maximal respiration rates were similar in cells expressing wild-type or mutant MFN2 proteins, indicating that mutant MFN2 proteins do not alter mitochondrial oxidative respiration.
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