Myelin and Axon Pathology in a Long-Term Study ofPMP22-Overexpressing Mice

Abstract

We analyzed clinical and pathological disease in 2 peripheral myelin protein-22 (PMP22) overexpressing mouse models for 1.5 years. C22 mice have 7 and C3-PMP mice have 3 to 4 copies of the human PMP22 gene. C3-PMP mice showed no overt clinical signs at 3 weeks and developed mild neuromuscular impairment; C22 mice showed signs at 3 weeks that progressed to severe impairment. Adult C3-PMP mice had very similar, stable, low nerve conduction velocities similar to adults with human Charcot-Marie-Tooth disease type 1A (CMT1A); velocities were much lower in C22 mice. Myelination was delayed, and normal myelination was not reached in either model but the degree of dysmyelination in C3-PMP mice was considerably less than that in C22 mice; myelination was stable in the adult mice. Numbers of myelinated, fibers were reduced at 3 weeks in both models, suggesting that normal numbers of myelinated fibers are not reached during development in the models. In adult C3-PMP and wild-type mice, there was no detectable loss of myelinated fibers,whereas there was clear loss of myelinated fibers in C22 mice.In C3-PMP mice, there is a balance between myelination status and axonal function early in life, whereas in C22 mice, early reduction of axons is more severe and there is major loss of axons in adulthood. We conclude that C3-PMP mice may be an appropriate model for most CMT1A patients, whereas C22 mice may be more relevant to severely affected patients in the CMT1 spectrum.

PMP22 antisense oligonucleotides reverse Charcot-Marie-Tooth disease type 1A features in rodent models

“…The grip strength and rotarod performance of these mice were measured before treatment began (pretreatment) and at 3, 6, and 9 weeks following ASO treatment. Consistent with previous reports (33), pretreatment/ baseline grip strength (102.5 ± 4.9 g in WT treated with PBS vs. 60.1 ± 2.4 g in C22 treated with PBS, Figure 1C) and the rotarod performance (161.9 ± 10.3 s in WT treated with PBS vs. 85.1 ± 8.0 s in C22 treated with PBS, Figure 1D) of 5-week-old C22 mice were significantly impaired compared with those in WT controls. We observed a significant loss of grip strength over the 9-week testing period in C22 mice treated with PBS (60.1 ± 2.4 g at pretreatment/ baseline vs. 42.2 ± 3.5 g at 9 weeks, P < 0.001).…”

“…While C22 mouse nerves have some onion bulbs, the most conspicuous findings are unmyelinated axons with a diameter of more than 1 micron that are invariably myelinated in WT nerves. These become evident during early myelination and persist in adult C22 mice (32,33). Histological examination of cross-sectioned sciatic nerves revealed significant reduction of unmyelinated axons of more than 1 micron in diameter and onion bulbs, together with a concomitant increase in myelinated axons in C22 mice following 9 weeks of ASO1 treatment ( Figure 1, H and I).…”

“…The C22 mouse model contains 7 copies of a human YAC encompassing the PMP22 gene, resulting in a severe, demyelinating neuropathy that is evident morphologically and behaviorally at early time points (29,32,33,35). To determine whether ASO-mediated lowering of PMP22 mRNA would be beneficial in this model, we screened and identified the most potent ASO (ASO1), which targets the 3′ UTR of the human PMP22 gene (Supplemental Figure 1, A and B; supplemental material available online with this article; https://doi.…”

“…To determine whether ASO1 could ameliorate the neuropathy in C22 mice, mice were treated with weekly subcutaneous injections of saline, a control ASO (50 mg/kg), or ASO1 at 25, 50, or 100 mg/kg per week for 9 weeks. Injections were started in 5-week-old C22 mice (and their WT littermates), an age at which C22 mice exhibit CMT phenotypes including slowed motor nerve conduction velocity (MNCV), reduced compound muscle action potentials (CMAP), and impaired motor performance (33). Similar to the results observed with short-term ASO1 exposure (Supplemental Figure 1), both mouse Pmp22 and human PMP22 mRNA exhibited a dose-dependent decrease relative to PBS following ASO1 treatment, with a 50% reduction of the human (transgenic) PMP22 mRNA at the end of 9 weeks at the highest dose tested ( Figure 1, A and B).…”

“…We therefore performed 3′ mRNA poly-A + sequencing (RNA-seq) on sciatic nerves of a separate cohort of naive 5-week-old C22 versus WT littermates (C22 5-week and WT 5-week) and a cohort of C22 and WT littermates that were treated with either PBS or ASO1 at 100 mg/ kg weekly for 9 weeks (C22 PBS 15-week and C22 ASO1 15-week). Using variance-model-based differential expression analysis, we determined 2,080 differentially expressed genes (DEGs) with a fold change greater than 2 at a false discovery rate (FDR) of 0.00001 or less in 5-week-old C22 versus WT mice and 1,056 DEGs in 15-week-old C22 versus WT mice that had been treated [29][30][31][32][33], ASO-mediated reduction of PMP22 mRNA levels markedly improves and even reverses several neuropathy end points, such as motor, electrophysiology, pathology, and transcriptomic changes. Independent studies of the CMT1A rat model, a well-characterized model of CMT1A (12,34), show that ASOs suppress Pmp22 mRNA levels in several affected nerves and restore myelination and electrophysiological properties of motor axons.…”