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. 2022 Mar;27(1):58-66.
doi: 10.1111/jns.12483. Epub 2022 Feb 13.

SARM1 knockout does not rescue neuromuscular phenotypes in a Charcot-Marie-Tooth disease Type 1A mouse model

Kathryn R Moss  1 , Anna E Johnson  1 , Taylor S Bopp  2 , Andrew T Yu  1 , Ken Perry  1 , Tae Chung  2 , Ahmet Höke  1   3
Affiliations

SARM1 knockout does not rescue neuromuscular phenotypes in a Charcot-Marie-Tooth disease Type 1A mouse model

Kathryn R Moss et al. J Peripher Nerv Syst. 2022 Mar.

Abstract

Charcot-Marie-Tooth disease Type 1A (CMT1A) is caused by duplication of the PMP22 gene and is the most common inherited peripheral neuropathy. Although CMT1A is a dysmyelinating peripheral neuropathy, secondary axon degeneration has been suggested to drive functional deficits in patients. Given that SARM1 knockout is a potent inhibitor of the programmed axon degeneration pathway, we asked whether SARM1 knockout rescues neuromuscular phenotypes in CMT1A model (C3-PMP) mice. CMT1A mice were bred with SARM1 knockout mice to generate CMT1A/SARM1-/- mice. A series of behavioral assays were employed to evaluate motor and sensorimotor function. Electrophysiological and histological studies of the tibial branch of the sciatic nerve were performed. Additionally, gastrocnemius and soleus muscle morphology were evaluated histologically. Although clear behavioral and electrophysiological deficits were observed in CMT1A model mice, genetic deletion of SARM1 conferred no significant improvement. Nerve morphometry revealed predominantly myelin deficits in CMT1A model mice and SARM1 knockout yielded no improvement in all nerve morphometry measures. Similarly, muscle morphometry deficits in CMT1A model mice were not improved by SARM1 knockout. Our findings demonstrate that programmed axon degeneration pathway inhibition does not provide therapeutic benefit in C3-PMP CMT1A model mice. Our results indicate that the clinical phenotypes observed in CMT1A mice are likely caused primarily by prolonged dysmyelination, motivate further investigation into mechanisms of dysmyelination in these mice and necessitate the development of improved CMT1A rodent models that recapitulate the secondary axon degeneration observed in patients.

Keywords: CMT1A; SARM1; behavior; electrophysiology; histology.

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Figures

FIGURE 1:
FIGURE 1:. SARM1 knockout does not rescue CMT1A behavioral deficits.
Three- and six-month-old wildtype, CMT1A and CMT1A/SARM1−/− mice were evaluated by (A) neuromuscular SHIRPA, (B) inverted four limb wire grid holding test, (C) forelimb grip strength test, (D) accelerating rotarod assay and (E) Hargreaves test. n=3–12 animals per condition, one-way ANOVA, Tukey’s post-hoc, *p<0.05.
FIGURE 2:
FIGURE 2:. SARM1 knockout does not rescue CMT1A electrophysiological deficits.
(A) Representative traces of evoked motor responses recorded from tibial nerve-innervated intrinsic foot muscles in three- and six-month-old wildtype, CMT1A and CMT1A/SARM1−/− mice. Quantification of (B) CMAP latency, (C) CMAP amplitude and (D) nerve conduction velocity are displayed. n=5–8 animals per condition, one-way ANOVA, Tukey’s post-hoc, *p<0.05.
FIGURE 3:
FIGURE 3:. SARM1 knockout does not rescue CMT1A nerve morphology deficits.
Sciatic nerves were harvested from three- and six-month-old wildtype, CMT1A and CMT1A/SARM1−/− mice and plastic sections were prepared for light microscopy. (A) Representative images, scale bar 10 μm. Nerve morphometry was performed and average (B) G-Ratios, (C) percent denuded axons, (D) axon diameter and (E) total axons are displayed. n=3–4 animals per condition (>240 axons per animal), one-way ANOVA, Tukey’s post-hoc, *p<0.05.
FIGURE 4:
FIGURE 4:. SARM1 knockout does not improve CMT1A myelin and axon caliber deficits.
Data from Figure 3 were further analyzed by (A, B) plotting G-Ratio against axon diameter and (C, D) creating axon diameter histograms.
FIGURE 5:
FIGURE 5:. SARM1 knockout does not rescue CMT1A muscle morphology deficits.
Gastrocnemius muscles were harvested from three-month-old wildtype, CMT1A and CMT1A/SARM1−/− mice, weighed and processed for immunohistochemistry with an anti-Laminin antibody. (A) Representative images, scale bar 100 μm. Average (B) myofiber cross sectional area, (C) myofiber maximum Feret diameter and (D) muscle mass are displayed. (B, C) n=3–4 animals per condition (>390 myofibers per animal), (D) n=4–6 animals per condition, one-way ANOVA, Tukey’s post-hoc, *p<0.05. Soleus muscles were harvested from three-month-old wildtype, CMT1A and CMT1A/SARM1−/− mice and processed for immunohistochemistry with an anti-β-III-Tubulin (green) antibody and α-Bungarotoxin (red). (E) Representative images, scale bar 10 μm. (F) Neuromuscular junction innervation was scored as innervated, partially denervated and denervated and the percent of total in each group is displayed. n=2–4 animals per condition (>30 NMJs per animal), Kruskal-Wallis one-way ANOVA, Dunn’s post-hoc.
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