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

Abstract

Mutations in the MFN2 gene encoding Mitofusin 2 lead to the development of Charcot-Marie-Tooth type 2A (CMT2A), a dominant axonal form of peripheral neuropathy. Mitofusin 2 is localized at both the outer membrane of mitochondria and the endoplasmic reticulum and is particularly enriched at specialized contact regions known as mitochondria-associated membranes (MAM). We observed that expression of MFN2^R94Q induces distal axonal degeneration in the absence of overt neuronal death. The presence of mutant protein leads to reduction in endoplasmic reticulum and mitochondria contacts in CMT2A patient-derived fibroblasts, in primary neurons and in vivo, in motoneurons of a mouse model of CMT2A. These changes are concomitant with endoplasmic reticulum stress, calcium handling defects, and changes in the geometry and axonal transport of mitochondria. Importantly, pharmacological treatments reinforcing endoplasmic reticulum-mitochondria cross-talk, or reducing endoplasmic reticulum stress, restore the mitochondria morphology and prevent axonal degeneration. These results highlight defects in MAM as a cellular mechanism contributing to CMT2A pathology mediated by mutated MFN2.

Keywords: CMT2A; endoplasmic reticulum; mitochondria; motoneurons; neuropathy.

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.

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.

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.

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.

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 ($\overline{\text{F}}$) 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.

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.

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.