Altered interplay between endoplasmic reticulum and mitochondria in Charcot–Marie–Tooth type 2A neuropathy

CMT2A Tg Mice Display Locomotor and Gait Abnormalities Associated with Slow-Twitch Muscle Denervation.

Previous studies showed that heterozygous (line hMFN2R94QL51, MitoCharc1) and homozygous (line hMFN2R94QL87, MitoCharc2) CMT2A Tg mice develop locomotion impairments in the rotarod test, starting from the age of ∼6 mo (8). As CMT2A patients display symptoms that worsen with age (31), we sought to assess the progression of both motor and sensory dysfunctions in CMT2A Tg mice. To mimic the dominant inheritance of CMT2A we used heterozygous B6;D2-Tg ^(Eno2-MFN2*^R94Q)/− mice [originally named hMFN2R94QL51, MitoCharc1 (8)], hereafter referred as CMT2A Tg mice. We performed a battery of behavioral tests at early and late time points (6 and 12 mo of age). Mouse locomotor function was evaluated using the rotarod and CatWalk gait analysis, whereas their sensory sensitivity to a mechanical stimulus was evaluated using the Von Frey test which is based on evaluation of the mechanical force able to elicit a paw withdrawal response (32). We did not observe any significant effect in sensory function (Fig. 1A). However, the performance on the rotarod was significantly affected in CMT2A Tg animals compared with WT littermates at both 6 and 12 mo of age (Fig. 1B). In the group of CMT2A Tg mice, the latency to fall from the rotarod did not significantly decrease between 6 and 12 mo (Fig. 1B and SI Appendix, Fig. S1A). Furthermore, analysis of the rotarod results according to mouse sex showed that the motor performance was significantly decreased in male but not in female CMT2A Tg mice (SI Appendix, Fig. S1A).

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Fig. 1.

CMT2A Tg mice show locomotor defects and muscle denervation but no detectable motoneuronal death. (A) Evaluation of sensory function with the von Frey test at the age of 6 and 12 mo (WT: n = 11 and CMT2A Tg: n = 12). Statistical analysis: two-way repeated-measures ANOVA (group × time) with Newman–Keuls post hoc test. (B) Evaluation of motor function with rotarod test at the age of 6 and 12 mo (WT: n = 11 and CMT2A Tg: n = 12). Statistical analysis: two-way repeated-measures ANOVA (group × time, significant group effect) with Newman–Keuls post hoc test. (C) Number of motoneurons in lumbar spinal cord of 12-mo-old animals (n = 4 for both WT and CMT2A Tg). Statistical analysis: two-tailed unpaired Student’s t test. (D) Level of innervation of soleus muscle evaluated based on the colocalization of the nerve terminal marker SV2A and acetylcholine receptor marker α-bungarotoxin in 12-mo-old WT (n = 5) and CMT2A Tg (n = 7) mice. Statistical analysis: two-tailed unpaired Student’s t test. (E) Representative photomicrographs of NMJ in WT and CMT2A Tg soleus muscle, stained with anti-SV2A antibodies and α-bungarotoxin. Note the presence of unoccupied NMJ positive for α-bungarotoxin only (indicated by an asterisk) in the CMT2A Tg mice. *P < 0.05.

The CatWalk test revealed that several gait parameters were significantly changed at both 6 and 12 mo in CMT2A Tg mice compared with WT mice. These parameters were classified as indicators of the pressure exerted by each paw, the area of the floor contacted by each paw, gait/posture, and coordination (SI Appendix, Table S1). Intensity variables reflecting paw pressure during contact with the glass walkway floor were reported to be affected by peripheral nerve injury (33). Here, forepaw intensity values were significantly decreased in CMT2A Tg animals at both time points (SI Appendix, Table S1). Print size of the right forepaw was also reduced in 6-mo-old CMT2A Tg mice, suggesting a misplacement of the paw (SI Appendix, Table S1). Dynamic CatWalk variables that evaluate gait/posture as well as coordination were also affected in CMT2A Tg mice. At the age of 6 mo, right forepaw terminal dual stance and left forepaw duty cycle mean were increased, whereas two other variables (left hindpaw stand mean and “support three”) also indicating gait impairment were significantly changed in 12-mo-old CMT2A Tg animals (SI Appendix, Table S1). These observations indicate that CMT2A Tg mice maintain some of their paws longer on the glass plate. Finally, the right forepaw and right hindpaw interpaw coordination reflected by the phase dispersions and couplings parameters were altered in CMT2A Tg mice only at 12 mo (SI Appendix, Table S1).

To assess whether these locomotor defects were linked to muscle weakness, we monitored muscle strength using the grid test (34, 35). No decrease in muscle strength score was observed in CMT2A Tg mice compared with WT at either 6 (WT: 2,308 ± 96.2 and CMT2A Tg: 2,328 ± 144.8) or 12 mo (WT: 1,886 ± 107.7 and CMT2A Tg: 1,852 ± 155.2).

As defects in locomotion can reflect subtler neuromuscular dysfunctions, we evaluated motoneuron survival and neuromuscular junction (NMJ) innervation. We did not observe any loss of motoneurons in CMT2A Tg spinal cord at 12 mo (Fig. 1C). Similarly, there was no significant loss of proximal motor axons in the ventral roots or sensory neurons in the dorsal root (SI Appendix, Fig. S1 B and C). However, we observed a significant decrease in the proportion of fully innervated NMJ at the age of 12 mo in the slow-twitch soleus muscle of CMT2A Tg mice (innervated NMJ WT: 79 ± 5.8%, CMT2A Tg: 48.2 ± 7%) (Fig. 1 D and E). Remarkably, at the same age, NMJ occupancy was not significantly reduced in the fast-twitch tibialis muscle (SI Appendix, Fig. S1D), and electrophysiological recording of the compound muscle action potential (CMAP) did not show any significant decrease in amplitude in CMT2A Tg mice (SI Appendix, Fig. S1E). These results were consistent with preserved muscle strength (dependent on gastrocnemius and tibialis muscles) measured with the grid test in the CMT2A Tg mice until 12 mo. Moreover, NMJ innervation was not affected at 6 mo in the soleus muscle (SI Appendix, Fig. S1F), suggesting that additional mechanisms may underlie the behavioral dysfunctions already observed at this age, and possibly involve other distal muscle groups.

MFN2^R94Q Overexpression Reduces Neurite Length of Primary Motoneurons.

To characterize the mechanisms underlying neuronal dysfunction in CMT2A, we used primary embryonic cultures of either motor or sensory neurons, the two neuronal populations that are typically affected by the disease. To evaluate the effects of mutated CMT2A, we overexpressed either the WT (MFN2^WT) or R94Q mutated form of MFN2 (MFN2^R94Q) using AAV6 vectors (SI Appendix, Fig. S2A). In accordance with our in vivo data, the overexpression of either MFN2^WT or MFN2^R94Q did not reduce the survival of motor or sensory neurons at 4, 6, or 8 d postinfection (dpi) (Fig. 2 A and B). However, the overexpression of MFN2^R94Q led to a significant reduction of neuritic length at 6 dpi. This effect was evident in motoneurons (noninfected: 1,982.6 ± 220.9, MFN2^WT: 2,056.8 ± 175.7 µm, MFN2^R94Q: 1,419.5 ± 110.5 µm) but not in sensory neurons (MFN2^WT: 3,810 ± 648 µm, MFN2^R94Q: 3,547 ± 897 µm) (Fig. 2 C and D). Moreover, in both motor and sensory neurons expressing MFN2^R94Q we observed the presence of axonal swellings and spheroids, as revealed by peripherin staining (Fig. 2D).

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Fig. 2.

Overexpressing MFN2^R94Q induces neurite degeneration in the absence of neuronal death. (A and B) Motor and sensory neurons were infected for 4, 6, and 8 dpi with AAV6-hsyn-hMFN2^WT or AAV6-hsyn-hMFN2^R94Q. Survival of motor and sensory neurons was evaluated following SMI32 and NF-200 staining. Noninfected (NI) neuron cultures were used as a control to evaluate the effect of MFN2 overexpression. Data are expressed as mean percentage ± SEM compared with the NI control cultures (n = 3 independent neuronal cultures). Statistical analysis: two-way ANOVA (group × time), with Tukey’s post hoc test. (C) Neuritic length was quantified at 6 dpi in motor and sensory neurons infected with AAV6-hsyn-hMFN2^WT or AAV6-hsyn-hMFN2^R94Q. Neurons were cotransduced with AAV6-CMV-GFP to label neurites. Values are expressed as mean (micrometers) ± SEM from six (motoneurons) and three (sensory neurons) independent cultures. Statistical analysis: two-way ANOVA (group × time), with Tukey’s post hoc test. (D, Upper) GFP-labeled neurites in motoneurons expressing either MFN2^WT or MFN2^R94Q. (D, Lower) Peripherin staining in the same conditions. Arrowhead indicates a peripherin-positive axonal swelling. *P < 0.05.

MFN2^R94Q Impairs ER–Mitochondria Tethering both in Vitro and in Vivo.

MFN2 has been shown to regulate either positively or negatively ER–mitochondria connections (17, 26⇓–28). We therefore quantified ER–mitochondria contacts in neurons overexpressing either WT or mutated MFN2. We used an in vitro proximity ligation assay (PLA) based on the interaction of the mitochondrial VDAC1 protein and the ER protein IP3R, as previously described (22) (SI Appendix, Fig. S2B). The number of dots reflecting ER–mitochondria contacts was significantly decreased in both motor and sensory neurons overexpressing MFN2^R94Q, compared with both noninfected neurons and neurons overexpressing MFN2^WT (Fig. 3 A and B). To determine if similar defects can be observed in CMT2A pathology, PLA was also performed in CMT2A patient-derived fibroblasts. Compared with control fibroblast cell lines, we again observed a significant decrease in the number of ER–mitochondria contacts (Fig. 3 C and D). As overexpression of MFN2 in cell lines can induce changes in various mitochondrial parameters (36), we investigated if the reduced number of contacts observed in both CMT2A neurons and fibroblasts as well as neuronal neurite shortening could be primarily caused by morphological alterations of mitochondria. Based on localization of myc-MFN2 and myc-MFN1, we did not observe any obvious clustering of mitochondria in neurons overexpressing MFN2^WT or MFN2^R94Q (SI Appendix, Fig. S2 A and E). Furthermore, we evaluated the impact of overexpression of MFN1, which has been reported to effectively complement this CMT2A mutant protein for mitochondrial fusion (37). Motoneuron cultures transduced with an AAV6 vector expressing either GFP or MFN2^R94Q were cotransduced with an AAV6-MFN1 vector (using a total vector dose similar to the previous experiment). MFN1 coexpression did not significantly change the number of ER–mitochondria contacts, which was still decreased in neurons overexpressing MFN2^R94Q (SI Appendix, Fig. S2C). Furthermore, we did not observe any significant effect of MFN1 on neurite shortening in MFN2^R94Q-expressing motoneurons (SI Appendix, Fig. S2D). Similar to neurons, MFN1 expression in patient-derived fibroblasts also did not significantly change the reduced number of ER–mitochondria contacts (SI Appendix, Fig. S2 E and F). These data suggest that the MAM and neuritic defects observed in CMT2A context cannot be rescued by MFN1 overexpression.

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Fig. 3.

MFN2^R94Q affects neuronal ER–mitochondria contacts in vitro and in vivo. (A) Quantification of ER–mitochondria contacts using PLA at 6 dpi in motor (n = 5 independent cultures) and sensory neurons (n = 3), infected with AAV6-hsyn-hMFN2^WT or AAV6-hsyn-hMFN2^R94Q (n = 6 independent cultures). Data are expressed as the mean number of contacts ± SEM per neuron detected by a PLA. Statistical analysis: repeated measures one-way ANOVA with Tukey’s post hoc test (motoneurons) and two-tailed paired Student’s t test (sensory neurons). NI, noninfected motoneurons. (B) Photomicrographs of PLA between the ER protein IP3R and the mitochondrial protein VDAC1 in motor and sensory neurons. Red dots indicate ER–mitochondria proximity. Nuclei are stained with DAPI. (C) Quantification of ER–mitochondria contacts using PLA in fibroblasts from control and CMT2A-R94Q individuals (n = 39 and n = 40 cells, respectively, from two independent cell lines for each condition). Data are expressed as the mean number of contacts ± SEM per cell. Statistical analysis: two-tailed unpaired Student’s t test. (D) Photomicrographs of control and CMT2A human fibroblasts. Intracellular red dots indicate the presence of ER–mitochondria proximity revealed by the PLA. Nuclei are stained with DAPI. (E) Electron microscopy quantification of the percentage of mitochondria with ER contact in motoneuron soma in the lumbar spinal cord of 12-mo-old WT (n = 34 motoneurons from three mice) and CMT2A Tg mice (n = 33 motoneurons from three mice). Statistical analysis: two-tailed unpaired Student’s t test. (F) Analysis of the length of the mitochondria–ER contacts, expressed as the percentage of the mitochondrial perimeter (n = 34 motoneurons from three WT mice and n = 33 motoneurons from three CMT2A Tg mice). Statistical analysis: two-tailed unpaired Student’s t test. (G) Electron microscopy pictures illustrating ER–mitochondria contacts in motoneuron soma of WT and CMT2A Tg mice. ER segments are delineated in green and mitochondria are shown in red. Arrowheads indicate the mitochondria–ER contacts. (H) Neuritic length quantified at 6 dpi in motoneurons overexpressing either MFN2^WT or MFN2^R94Q. Motoneurons treated with a SIGMAR1 agonist (Pre-084, 50 nM) are compared with the untreated condition. Note the significant rescue of neuritic length in MFN2^R94Q motoneurons treated with Pre-084. Mean values are obtained from four independent cultures. Statistical analysis: two-way ANOVA (group × treatment) with Sidak post hoc test. *P < 0.05, ***P < 0.001.

Next, we further evaluated by electron microscopy the contacts between mitochondria and ER, in the motoneuron soma in the lumbar spinal cord of 12-mo-old WT and CMT2A Tg mice. In WT mice, 37.8 ± 2.1% of the mitochondria made at least one contact with the ER, whereas this proportion was significantly decreased in CMT2A Tg motoneurons (31.3 ± 2.1%) (Fig. 3 E and G). Furthermore, the length of these contacts was significantly decreased in CMT2A Tg motoneurons, representing only 6.0 ± 0.3% of mitochondrial perimeter, as compared 9.6 ± 0.4% in their WT counterparts (Fig. 3 F and G). We did not notice any significant change in the level of expression of SIGMAR1 or VAPB, two constituents of MAM, in the spinal cord of CMT2A mice, supporting a specific role of mutated MFN2 in the observed MAM defects (SI Appendix, Fig. S2 G and H). To determine whether the altered contacts between ER and mitochondria played a functional role in neurite shortening following MFN2^R94Q expression, we treated motoneurons with Pre-084 a selective agonist of SIGMAR1, a chaperone protein located at the MAM which controls calcium transfer from the ER toward mitochondria (22). Remarkably, exposure of primary motoneurons to Pre-084 almost completely prevented neurite degeneration induced by MFN2^R94Q, as shown by significant increase in neuritic length compared with untreated neurons (MFN2^R94Q: 1,479 ± 162 µm, MFN2^R94Q + Pre-084: 2,087.5 ± 187.9 µm) (Fig. 3H).

Expression of MFN2^R94Q Leads to Unfolded Protein Response Activation and Intracellular Calcium Defects.

MAM defects have been previously linked to ER malfunction leading to an unfolded protein response (UPR) and calcium homeostasis impairments (22, 38). We therefore analyzed the level of ER stress markers in vitro (phosphorylated eIF2A, P-eIF2α; the ER chaperone PDI). The levels of both P-eIF2α and PDI were significantly increased in motoneurons overexpressing mutated MFN2 relative to MFN2^WT (PDI MFN2^R94Q: +71.2 ± 21.0% compared with MFN2^WT; P-eIF2α MFN2^R94Q: +134 ± 53.4% compared with MFN2^WT) (Fig. 4 A and B). In sensory neurons, there was no significant effect of MFN2^R94Q on the level of both PDI and P-eIF2α (Fig. 4A).

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Fig. 4.

MFN2^R94Q induces ER stress both in vitro and in vivo. (A) Immunoquantification of level of ER stress in motor and sensory neurons infected with AAV6-hsyn-hMFN2^WT or AAV6-hsyn-hMFN2^R94Q at 6 dpi (n = 5 and 3 independent cultures for motor and sensory neurons, respectively). Levels of PDI or P-eIF2α were normalized to the level of either the motoneuronal marker SMI32 or the sensory neuron marker NF-200. Data are expressed as percentage ±SEM relative to MFN2^WT condition. Statistical analysis: one-way ANOVA with Tukey’s post hoc test. NI, noninfected motoneurons. (B) Representative stainings for P-eIF2α and SMI32 in motoneurons expressing MFN2^WT or MFN2^R94Q. (C) Immunoquantification of ER stress markers in motoneurons in the lumbar spinal cord of 12-mo-old WT (n = 6) and CMT2A Tg mice (n = 6). Levels of P-eIF2α and PDI were normalized to the neuronal marker NeuN. Data are expressed as percentage ± SEM relative to WT. Statistical analysis: two-tailed unpaired Student’s t test. (D) The percentage of motoneurons with nuclear vs. cytoplasmic ATF6 was evaluated in the spinal cord of 12-mo-old WT (n = 6) and CMT2A Tg mice (n = 5). Statistical analysis: two-tailed unpaired Student’s t test. (E) Representative photomicrographs of PDI (Left) and ATF6 staining (Right) in WT and CMT2A Tg spinal cord sections. Cytoplasmic and nuclear ATF6 staining is indicated by arrowheads. (F) Neuritic length quantified at 6 dpi in motoneurons overexpressing either MFN2^WT or MFN2^R94Q. Motoneurons were treated with salubrinal (5 µM). Values represent the mean of four independent cultures. Statistical analysis: two-way ANOVA (group × treatment) with Sidak post hoc test. *P < 0.05, **P < 0.01.

To confirm these findings in vivo, we analyzed the level of P-eIF2α, PDI, and activating transcription factor 6 (ATF6) in lumbar motoneurons of 12-mo-old CMT2A Tg animals. Compared with WT littermates, we observed a significant increase in PDI expression similar to the changes seen in vitro (Fig. 4 C and E). Although there was a clear trend toward increased level of P-eIF2α, the difference did not reach statistical significance (Fig. 4C). We also analyzed the nuclear translocation of ATF6 as an additional early marker of ER stress in motoneurons of WT and CMT2A Tg mice. The proportion of motoneurons with a predominant nuclear vs. cytosolic ATF6 staining was significantly higher in the CMT2A Tg lumbar spinal cord (WT: 24 ± 3.2%, CMT2A Tg: 47.4 ± 5.2%) (Fig. 4 D and E).

Interestingly, exposure of primary motoneurons to the ER stress inhibitor salubrinal (22) almost completely prevented neurite degeneration induced by MFN2^R94Q, as shown by a significant increase in neuritic length compared with untreated neurons (MFN2^R94Q: 1,270.7 ± 77.8 µm, MFN2^R94Q + salubrinal: 1,685.5 ± 115.6 µm) (Fig. 4F). These results show that ER stress plays a functional role in neuronal degeneration following MFN2^R94Q expression.

We also monitored intracellular calcium variation in motor and sensory neurons using ratiometric Fura-Red AM calcium dye before, during, and after 25 mM KCl exposure, as previously described (39). In basal conditions, we did not detect any difference in the ratiometric fluorescence of the calcium indicator when comparing motoneurons overexpressing either MFN2^WT or MFN2^R94Q, indicating no difference in basal calcium loading (Fig. 5 A and B). However, we found a lower amplitude in the KCl-evoked calcium rise in MFN2^R94Q motoneurons, compared with cells expressing MFN2^WT (Fig. 5C), while there was no difference in normalized half-time recovery (Fig. 5D). The data from sensory neurons (Fig. 5 E–H) also revealed changes in calcium homeostasis, showing alterations in the basal calcium loading (Fig. 5F), a lower amplitude in the KCl-evoked calcium rise in MFN2^R94Q (Fig. 5G), and an increase in time to return to basal calcium levels (Fig. 5H).

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Fig. 5.

MFN2^R94Q induces changes in calcium homeostasis both in vitro and in vivo. (A–D) Intracellular calcium measurements using Fura-Red ratiometric imaging (F490/F440) after KCl (25 mM) exposure of MFN2^WT (n = 16) and MFN2^R94Q (n = 25) motoneurons. (A) An example of obtained recordings. Basal loading was determined as the average of resting calcium concentration (F490/F440, represented as arbitrary units, a.u.) between 20 and 50 s of measurement. (B) Basal loading levels. (C) Amplitude of the maximal response obtained after 8-s exposure to KCl (25 mM), expressed as the percentage of variation relative to basal fluorescence determined in B. (D) Representation of normalized half-time recovery (50% return to basal level normalized by the amplitude of response). (E–H) Intracellular calcium measurements using Fura-Red ratiometric imaging (F490/F440) after 8-s KCl (25 mM) exposure of MFN2^WT (n = 105) and MFN2^R94Q (n = 81) sensory neurons. (E) An example of obtained recordings. Basal loading was determined as the average of resting calcium concentration (F490/F440, represented as arbitrary units, a.u.) between 20 and 50 s of measurement. (F) Basal loading levels. (G) Amplitude of the maximal response obtained after exposure to KCl (25 mM), expressed as the percentage of variation relative to basal fluorescence. (H) Representation of normalized half-time recovery (50% return to basal level normalized by the amplitude of response). (I–M) Intracellular calcium measurements in control and CMT2A patient-derived fibroblasts evaluated by Fluo-4 AM fluorescent microscopy. Calcium dynamics are expressed as the ratio of fluorescence intensity (F) divided by the mean fluorescence (F¯) averaged over the first 50 frames and represented as a.u. (controls, n = 100 and CMT2A, n = 102). (I) A schematic overview of the experimental setting for J–M. (J) Example of a recording obtained in a fibroblast exposed to extracellular EDTA. (K) Quantification of the drop in intracellular calcium determine as a difference between baseline value and the maximal drop in intracellular calcium induced by presence of extracellular EDTA. (L) Example of a recording obtained in a fibroblast exposed to thapsigargin. (M) Quantification of the change determined as a difference between baseline value and the maximal response in intracellular calcium induced by thapsigargin. Values represent the data from three independent cultures. (N) Quantification of the change determined as a difference between baseline value and the maximal response in intracellular calcium released from mitochondria following exposure to CCCP (2 μM). Values represent the data from three independent cultures. Controls, n = 82 and CMT2A, n = 34. Data are expressed as box-and-whisker plots or as mean ± SEM. Statistical analysis: two-tailed unpaired Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

To further elucidate the possible perturbations in [Ca²⁺]_i homeostasis, we examined the global [Ca²⁺]_i dynamics in CMT2A patient-derived fibroblasts and control fibroblasts (Fig. 5I). To determine whether an impairment in ER handling of [Ca²⁺]_i was present_, we examined the difference in ER Ca²⁺ discharge between control and patient cells by challenging the cells in Ca²⁺-free conditions with thapsigargin (2 µM), a compound known to induce Ca²⁺ release from the ER by inhibiting Ca²⁺ sarco/endoplasmic reticulum Ca²⁺-ATPase pumps. Remarkably, a significant drop in the [Ca²⁺]_i baseline steady state was observed between patient and control cells (0.18 ± 0.017 vs. 0.31 ± 0.021; Fig. 5 J and K) when extracellular Ca²⁺ was removed by EDTA chelation before the application of thapsigargin. Applying thapsigargin resulted in a significantly lower amplitude of the [Ca²⁺]_i response in patient cells compared with controls (0.53 ± 0.033 vs. 0.39 ± 0.019; Fig. 5 L and M).

Mitochondria play a role in the uptake of Ca²⁺ from the cytosol, in particular in presence of high [Ca²⁺]_i levels. We used the protonophore CCCP (carbonyl cyanide m-chlorophenyl hydrazone), which collapses the mitochondrial membrane potential, to release Ca²⁺ from the mitochondria. As seen in Fig. 5N and SI Appendix, Fig. S3A, the level of Ca²⁺ discharged as a result of bath application of CCCP (2 µM) was significantly higher in CMT2A fibroblasts than in their respective controls, which indicates that mitochondria may accumulate abnormally high levels of Ca²⁺ in these cells.

Mitochondrial Transport and Clustering Are Altered in Axons Expressing MFN2^R94Q.

Previous studies reported contradictory results regarding abnormalities of mitochondrial transport following mutated MFN2 expression (10, 12, 15). We monitored axonal mitochondrial transport by time-lapse microscopy in vivo, in the mouse sciatic nerve. Mitochondria were classified according to their velocities, moving at either very slow (<0.3 µm/min), slow (>0.3 and <0.6 µm/min), medium (>0.6 and <0.9 µm/min), or fast (>0.9 µm/min) speed (Fig. 6 A and B and SI Appendix, Fig. S3B). The frequency distribution showed more very-slow-moving and fewer moving mitochondria in axons of the sciatic nerve of 1-mo-old CMT2A Tg mice compared with control mice (SI Appendix, Fig. S3B). In addition, very-slow-moving mitochondria were significantly slower in CMT2A Tg mice, compared with WT littermates (mean speed of very slow mitochondria, WT: 0.24 ± 0.011 µm/min, CMT2A Tg: 0.11 ± 0.01 µm/min) (Fig. 6B). This actually translated into a sharp increase in the proportion of stationary mitochondria (WT: 3.4%, CMT2A Tg: 50.7%). Mitochondrial velocity was not affected toward any specific direction, since there was no difference in the average anterograde or retrograde mitochondrial transport speeds (Fig. 6C). However, the proportion of mitochondria moving anterogradely or retrogradely was different between control and CMT2A Tg mice (WT: 32% retrograde and 68% anterograde moving mitochondria, CMT2A Tg: 55% retrograde and 44% anterograde moving mitochondria). The formation of mitochondria accumulations/clusters has previously been observed in vitro and could potentially be linked to mitochondrial transport defects induced by MFN2^R94Q (14, 16). Therefore, we measured the diameter of mitochondria clusters in axons relative to the average diameter of a single mitochondrion. We noticed a significant overrepresentation of individual mitochondria as well as small mitochondria clusters (size category 0.4–0.6 μm) with a decrease in the numbers of large-size clusters in CMT2A Tg axons compared with WT axons (Fig. 6D). Overall, these observations suggest defects in mitochondrial dynamics following MFN2^R94Q overexpression.

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Fig. 6.

MFN2^R94Q impairs mitochondrial transport and leads to overabundance of small mitochondria clusters in vivo. (A) Examples of kymographs used to determine mitochondrial velocity in sciatic nerve axons of 1-mo-old animals. Colored dots represent different types of mitochondrial movements. (B) mitochondria are classified according to very slow (<0.3 µm/min), slow (>0.3 and <0.6 µm/min), medium (>0.6 and <0.9 µm/min), and fast (>0.9 µm/min) transport velocities. Statistical analysis: two-tailed unpaired Student’s t test. (C) Mitochondrial anterograde and retrograde transport velocities were evaluated in the sciatic nerve of WT (n = 74 mitochondria from seven mice) and CMT2A Tg (n = 29 mitochondria from six mice) mice. (D) Number of mitochondrial clusters according to cluster size in axons of WT (n = 23 axons from 10 mice) and CMT2A Tg (n = 16 axons from six mice) mice. Statistical analysis: two-tailed unpaired Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001.

MFN2^R94Q-Induced Changes in Mitochondrial Morphology Can Be Partially Reverted by Preventing ER Stress.

MFN2 is one of the main proteins controlling mitochondrial fusion (40). Mitochondrial length is reduced in sensory neurons expressing MFN2^R94Q (10). Altered ER–mitochondria contact could be implicated in this phenotype since MAM participate in regulation of mitochondria dynamics (18). We analyzed mitochondria morphology in vitro, by loading motor neurons and sensory neurons with MitoTracker Red, allowing labeling more than 90% of axonal mitochondria (22). We noticed a significant decrease in mitochondria length in both motor and sensory neurons overexpressing MFN2^R94Q (motoneurons: MFN2^WT: 1.76 ± 0.14 µm, MFN2^R94Q: 1.36 ± 0.09 µm; sensory neurons: MFN2^WT: 1.79 ± 0.09 µm, MFN2^R94Q: 1.43 ± 0.09 µm) (Fig. 7 A and B). Mitochondria morphology was also evaluated using in vivo imaging, in axons of the sciatic nerve of 1-mo-old WT and CMT2A Tg mice. In this experiment, axonal mitochondria were labeled with mito-Dsred2 expressed following injection of AAV9-CAG-mitoDsred2 into the spinal cord at postnatal day 1. In CMT2A Tg mice, mitochondrial length was again significantly reduced compared with WT littermates (WT: 3.7 ± 0.1 µm, CMT2A Tg: 3.2 ± 0.12 µm) (Fig. 7 C and D). The diameter of mitochondria was not significantly changed (WT: 0.49 ± 0.13 µm, CMT2A Tg: 0.56 ± 0.1 µm). Furthermore, we also used transmission electron microscopy to assess mitochondrial morphology in the soma of spinal cord motoneurons (SI Appendix, Fig. S3C). In 12-mo-old CMT2A Tg mice, consistent significant decrease in mitochondria length was observed (WT: 0.64 ± 0.014 µm, CMT2A Tg: 0.58 ± 0.02 µm). Together, these results suggest that mutated MFN2 may affect mitochondrial fusion, leading to smaller mitochondria.

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Fig. 7.

Mitochondrial morphology is affected by MFN2^R94Q both in vitro and in vivo. (A and B) Motor and sensory neurons infected with AAV6-hsyn-hMFN2^WT or AAV6-hsyn-hMFN2^R94Q were stained at 6 dpi with MitoTracker Red to assess mitochondria morphology. (A) Mitochondrial length quantified in the proximal part of axons (n = 5 independent motoneuron cultures and n = 3 independent sensory neuron cultures). Statistical analysis: two-tailed unpaired Student’s t test. (B) MitoTracker Red-stained mitochondria in axons of motoneurons expressing either MFN2^WT or MFN2^R94Q. Higher magnifications of MitoTracker Red-stained mitochondria are shown in the middle. (C) Mitochondrial length in sciatic nerve axons of 1-mo-old WT (n = 767 mitochondria from 10 mice) and CMT2A Tg (n = 395 mitochondria from six mice) mice. Statistical analysis: two-tailed unpaired Student’s t test. (D) Representative images of axonal mitochondria stained with AAV9-CAG-mitoDsred2. Images taken with 20× and 63× objectives are from the same area but are time-shifted due to mitochondria movement. (E) Mitochondrial length (visualized with MitoTracker Red) quantified at 6 dpi in motoneurons expressing either MFN2^WT or MFN2^R94Q. Motoneuron cultures were treated with either Pre-084 (50 nM) or salubrinal (5 µM). Results represent n = 3 independent cultures. Statistical analysis: repeated-measure two-way ANOVA with Tukey’s post hoc test. (F) Box-and-whisker plot of mitochondrial size measured by electron microscopy in distal sciatic nerve axons of 6-mo-old WT (n = 291 mitochondria from three mice), CMT2A Tg mice (n = 287 mitochondria from three mice), and CMT2A Tg mice daily treated for 4 wk with 1 mg/kg salubrinal (n = 315 mitochondria from three mice). (G) Box-and-whisker plot of mitochondrial density measured in the same samples by determining the fraction of axoplasm occupied by mitochondria (WT: n = 86 axons; CMT2A Tg: n = 94 axons; CMT2A Tg mice + salubrinal: n = 103 axons). Statistical analysis for F and G: Kruskal–Wallis test with Dunn’s multiple comparison post hoc test. (H) Representative electron microscopy images of axonal mitochondria (red arrowheads) in distal sciatic nerve axons of 6-mo-old WT and CMT2A Tg mice. (I) Evaluation of the effect of 4-wk salubrinal treatment on the rotarod performance of 6-mo-old CMT2A Tg mice compared with saline-treated animals (CMT2A Tg mice + saline, n = 8; CMT2A Tg + salubrinal, n = 9 mice; WT + saline, n = 7). Statistical analysis: two-way repeated-measures ANOVA (group × time, significant group and time effects) with Dunnett’s (group effect) or Sidak (time effect) post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Since perturbations of ER–mitochondria contacts affect mitochondrial dynamics, we sought to explore if treatments with either Pre-084 to reinforce MAM function or salubrinal to prevent ER stress had beneficial effects on mitochondrial morphology in neurons expressing MFN2^R94Q. We measured mitochondria length in primary motoneurons expressing MFN2^R94Q as previously described and found that both treatments restored mitochondrial length to values similar to the control MFN2^WT condition (Fig. 7E). By performing electron microscopy on the distal sciatic nerve of 6-mo-old CMT2A Tg mice, we also measured a significant rescue of mitochondrial size and density after 4 wk of daily salubrinal injections (Fig. 7 F–H). Finally, we also evaluated the motor performance of salubrinal-treated CMT2A Tg mice compared with saline-injected CMT2A Tg and WT animals. In the rotarod test, a significant increase of the latency time until fall was observed only in the salubrinal-treated CMT2A Tg mice, compared with the motor performance measured before treatment (Fig. 7I). Overall, these results demonstrate that both the modulation of MAM and ER stress support proper function of mitochondria and could prevent some defects caused by MFN2^R94Q.