Synaptic Deficits at Neuromuscular Junctions in Two Mouse Models of Charcot-Marie-Tooth Type 2d

Abstract

Patients with Charcot-Marie-Tooth Type 2D (CMT2D), caused by dominant mutations in Glycl tRNA synthetase (GARS), present with progressive weakness, consistently in the hands, but often in the feet also. Electromyography shows denervation, and patients often report that early symptoms include cramps brought on by cold or exertion. Based on reported clinical observations, and studies of mouse models of CMT2D, we sought to determine whether weakened synaptic transmission at the neuromuscular junction (NMJ) is an aspect of CMT2D. Quantal analysis of NMJs in two different mouse models of CMT2D (Gars(P278KY), Gars(C201R)), found synaptic deficits that correlated with disease severity and progressed with age. Results of voltage-clamp studies revealed presynaptic defects characterized by: (1) decreased frequency of spontaneous release without any change in quantal amplitude (miniature endplate current), (2) reduced amplitude of evoked release (endplate current) and quantal content, (3) age-dependent changes in the extent of depression in response to repetitive stimulation, and (4) release failures at some NMJs with high-frequency, long-duration stimulation. Drugs that modify synaptic efficacy were tested to see whether neuromuscular performance improved. The presynaptic action of 3,4 diaminopyridine was not beneficial, whereas postsynaptic-acting physostigmine did improve performance. Smaller mutant NMJs with correspondingly fewer vesicles and partial denervation that eliminates some release sites also contribute to the reduction of release at a proportion of mutant NMJs. Together, these voltage-clamp data suggest that a number of release processes, while essentially intact, likely operate suboptimally at most NMJs of CMT2D mice.

Significance statement: We have uncovered a previously unrecognized aspect of axonal Charcot-Marie-Tooth disease in mouse models of CMT2D. Synaptic dysfunction contributes to impaired neuromuscular performance and disease progression. This suggests that drugs which improve synaptic efficacy at the NMJ could be considered in treating the pathophysiology of CMT2D patients.

Keywords: 3,4 diaminopyridine; axonal neuropathy; neuromuscular transmission; physostigmine; quantal content; voltage clamp.

Figure 1.

Morphological analysis of LAL muscle in CMT2D mice. Images shown are of LAL terminals in 2 month-old (A, B) wild-type, (C–I) Gars^P278KY and (J–L) 4-month-old Gars^C201R mice. Red shows α-bungarotoxin-stained postsynaptic ACh receptors, green is presynaptic nerve expressing YFP (see Materials and Methods). Innervation analysis (F) included examination of ≥100 terminals from each of three LAL muscles/genotype. Control wild-type (A, B) terminals show typical complex pretzel-like morphology and postsynaptic receptors are entirely apposed by the presynaptic nerve. At low-magnification (20×) it can be seen that (C–E) Gars^P278KY terminals retain some innervation, but when viewed at higher-magnification (63×; G–I) nearly all terminals show some dysmorphology (G–I, upper and lower terminal) and many are only partially innervated (G–I, lower terminal). Occupancy was analyzed, and ∼10% of terminals were denervated but >50% showed some degree of partial denervation muscles. The LAL muscle in Gars^C201R mice showed a similar phenotype to Gars^P278KY muscles, with widespread dysmorphology at most terminals with severity ranging from (J) mild, near-normal to (K) fragmented, or (L) partially denervated. Overall, consistent with previous findings, Gars^C201R LAL muscles had many (F) fewer denervated or partially denervated terminals than seen in Gars^P278KY, but across both alleles at least 80–90% of NMJs retain at least partial innervation.

Figure 2.

Quantal analysis. Voltage-clamp experiments were performed using LAL muscles from 2-month-old Gars^P278KY, (top) and both 2- and 4-month-old Gars^C201R mice (bottom). All recordings were made at a holding potential of −50 mV. At 2 months of age, mutant NMJs in both Gars^P278KY and Gars^C201R mice showed: no change in MEPC amplitude, (A, G) lower-frequency of spontaneous release, (B, H) reduced EPC amplitude, and quantal content (C, I, D, J), and significantly greater depression (10th/1st) in response to 50 Hz stimulation (E, K). At 4 months of age Gars^C201R NMJs show no additional changes in spontaneous release (G′, H′) but a further reduction in EPC amplitude and quantal content (I′, J′) while repetitive stimulation produced less depression compared with 2 month mutant NMJs (K′). F, Increased extracellular calcium successfully increased spontaneous release frequency at 4-month-old mutant Gars^C201R NMJ. LAL muscles of six different mice for each genotype/age were used. In each muscle recordings were made on 3–12 synapses (mean and mode = 8) for a total of between 47 and 58 synapses for each genotype/age. Comparisons made using nested ANOVA. *p < 0.05, mutant versus wild-type; **p < 0.05, 2 versus 4 months Gars^C201R.

Figure 3.

Evoked response to repetitive stimulation. A–C, show plots of averaged normalized EPC amplitude (±SE) for each stimulus of a 10 pulse, 50 Hz stimulus train (average of 20 trains at 0.5 Hz for each NMJ). In contrast to wild-type synapses, mutant NMJs in 2-month-old animals showed reduced or absent potentiation of initial EPCs and somewhat steeper depression. At 4 months of age, initial EPCs showed potentiation similar to wild-type. EPC amplitude was measured for each stimulus and plotted relative to the amplitude of the first pulse. Numbers of animals/synapses are the same as for data shown in Figure 2. Plots in D–G show the first, second, and 10th raw traces from a series of 10 EPC trains recorded in response to a 70 pulse,70 Hz stimulus (1 s duration), delivered every 2 s. Starting quantal content (m) is shown for each of the four NMJs. Mutant NMJs showed marked steadily progressive decrements from the first to 10th train, whereas the wild-type decreased and stabilized. The mutant NMJs are easily identified by comparing response to the 10th stimulus train (lower traces) to wild-type. In addition, clear failures of release were evident (arrows) for NMJs from severe and 2-month-old mild Gars^C201R muscles. Failures were present in at least one train for 5 of 11 mutant NMJs, but were never observed in wild-type. The 1 s duration, 70 Hz trains were recorded in nine different muscles/experiments (n = 5, 6, 3, and 2 NMJs from 4-month-old wild-type, 4M-Gars^C201R, 2M-Gars^C201R, and 2M-Gars^P278KY, respectively).

Figure 4.

Quantification of bassoon-stained release sites in 2-month-old Gars^P278KY LAL muscles. Process of visualization and quantification of bassoon-stained release sites is shown for a wild-type LAL terminal (A–D). Analysis included assessment of: (A) the postsynaptic receptor area, (B) presence of presynaptic nerve, and (C) bassoon-stained puncta. Puncta were identified using a three-step process to render a 3D image (D) for automated counting of release sites (Imaris; see Materials and Methods for details). Typical Gars^P278KY NMJ (E–H) have more diffuse postsynaptic staining (E), apposed by a thinned presynaptic nerve (F), that nonetheless retains bassoon-stained puncta (G) at innervated locations (NB, the areas where YFP appears to not overlie bassoon labeling are due to the faint YFP signal in portions of the axon and the thresholding of the image; at higher gain YFP was detectable in the vicinity of all bassoon-stained puncta). However, as expected, when a portion of the presynaptic nerve vacates, bassoon-stained release sites are no longer evident (merged image H, arrow; expanded in K). Gars^P278KY sample included a total of 34 synapses that included 10 terminals with small areas of evident partial denervation. I, Scatterplot shows NMJ area and counts of bassoon-positive puncta at individual synapses of Gars^P278KY (solid) and wild-type (open) NMJs. Vertical and horizontal lines indicate sample means (Gars^P278KY and wild-type, solid and dashed, respectively). Mean area of LAL NMJs was significantly smaller (p < 0.002) compared with wild-type (solid vs dashed vertical line). Individual puncta counts covered a similar range and neither mean counts (dashed and solid horizontal lines) nor density (J) were significantly different between genotypes (p = 0.4 and 0.17, respectively). Analysis included 5–10 (mean = 7) terminals from each of five LAL muscles of each genotype (n = 34 and 35, Gars^P278KY and wild-type, respectively). Statistical comparisons used a nested ANOVA.

Figure 5.

Electron microscopic analysis of NMJs. Electron micrographs of NMJs from LAL muscles of 2-month-old (A) wild-type and (B) Gars^P278KY mice were captured (5 animals per genotype, 5–10 NMJs (median 8 per muscle). Mutant NMJ shown is representative of a “severely” affected NMJ with a low-vesicle count. Note junctional folds and other synaptic specializations are still evident at mutant synapses. Analysis showed vesicle size was similar for mutant and wild-type terminals (50 vs 54 nm, respectively; data not shown). C, Scatterplot shows that the area of terminal portions analyzed scale with vesicle counts for both genotypes. Note the relative absence of large, high vesicle count terminals (ellipse) and the preponderance of small, low vesicle count NMJs in the mutant (C, lower quadrant; expanded in inset). Counts at some mutant NMJs were lower than values recorded at any wild-type NMJ (inset, ellipse). However, due to large inter-animal variability for both genotypes differences in average vesicle counts for wild-type and Gars^P278KY NMJs (p = 0.08) were not statistically different (see Results). D, The density of the vesicles present in Gars^P278KY terminals was not different from wild-type. Detailed analysis of vesicle location revealed no differences in the number of docked vesicles (within 20 nm), or within 200 nm for either absolute or normalized counts (per micron of membrane). E, Average counts of mitochondria (A, B, arrows) were significantly lower at Gars^P278KY terminals (median14 vs 6, wild-type and Gars^P278KY, respectively).

Figure 6.

Wire-hang performance of CMT2D mice. Performance is expressed as the percentage change in wire-hang time after treatment ([(post-pre)/post]/100; see Materials and Methods for protocol details). A, Gars^C201R mice (1-month-old) were able to perform better with physostigmine (postsynaptic), but showed a decrease in performance after administration of 3,4-DAP (presynaptic) or (B) no change to 3,4 DAP injection at 4 months of age. C, Gars^P278KY mice showed a similar response to physostigmine, but were not tested with 3,4 DAP. Mice were 30- to 40-d-old for testing (1 and 4 month: Gars^C201R, n = 6; +/+, n = 5; Gars^P278KY, n = 5; +/+, n = 4). Wild-type (+/+) mice typically perform the task to completion (60 s max) and showed no drug-related change in performance at the dosages used (physostigmine, 0.1 mg/kg; 3,4 DAP, 2.5 mg/kg), so data are not shown. Pairwise comparisons drug-treated versus NaCl with Student's t test; **(t₍₉₎ = 4.2, p < 0.01; *t₍₈₎ ≥ 2.3, p ≤ 0.047).

Figure 7.

Quantal analysis in the presence of 3.4 DAP. Voltage-clamp experiments were conducted on cohorts of 2- and 4-month-old Gars^C201R and wild-type mice with 3,4 DAP added to the bath (20 μm). Dashed lines are wild-type, solid lines are Gars^C201R, and 2 and 4 month data are shown with circles and triangles, respectively. Control values without DAP are re-plotted from Figure 2. A–E, The addition of 3,4 DAP to prolong presynaptic depolarization caused qualitatively similar changes at mutant and wild-type NMJs. Differences that existed between mutant and wild-type measures without 3,4 DAP (Fig. 2) were eliminated at 4-month-old mutant NMJs when 3,4 DAP was present, although quantal content was still somewhat lower. At 2-month-old mutant NMJs, quantal content remained significantly lower (p = 0.01) compared with wild-type. F, Quantal content calculated for the final (10th) 50 Hz pulse was reduced by 3,4 DAP in both wild-type and mutant.

Figure 8.

NMJ dysfunction in CMT2D mice. A, Plot of individual values for quantal content calculated for NMJs of each of the four experimental groups reveal CMT2D distributions are shifted to lower values compared with combined wild-type sample (white). Release at a proportion of terminals is severely reduced (≤20, left of dashed vertical line), whereas the majority is mildly or moderately affected with quantal content values shifted to the left of the wild-type mean (∼50), whereas the highest wild-type values (60–90) are largely absent for mutant NMJs. Note also, the 2 to 4 month progression at Gars^C201R NMJs (yellow vs red) with the latter overlapping the distribution for severe Gars^P278KY. In B, we show a hypothetical model of how the widespread NMJ dysfunction could produce variable and intermittent muscle weakness in CMT2D. Normal wild-type NMJs have a significant safety factor such that EPCs reliably depolarize the muscle above the threshold for an action potential (AP). In the CMT2D mice, terminals with a mild/moderate reduction in release (mild) would have a reduced safety factor and EPCs may intermittently fail to initiate an AP, whereas EPCs at severely affected terminals (severe) consistently do not reach the AP threshold. Note also, that because EPCs at affected terminals would initiate APs closer to their peak, APs would be slightly delayed (dotted AP) and EMG recordings of an affected muscle would be expected to display significant “jitter,” a clinical measure used to identify transmission failure. C, Repetitive activation increases the extent and variability of transmission failure (Fig. 3). This scenario is depicted by example raw EPC traces of the 50 Hz response of moderately affected mutant terminals and typical wild-type. The horizontal dashed line through these traces indicates the EPC amplitude equivalent to a quantal content of 20 for the wild-type NMJ (equivalent to vertical dashed line in A). This cutoff was selected because all values measured at wild-type NMJs exceeded it, whereas ∼20% of the values for each mutant population were below it. For the 50 Hz trains shown wild-type the amplitude of final EPCs persists well above this level, but depression at mutant NMJs is sufficient to approach this value for the four to five final EPCs (Fig. 3D–G).