Mice carrying an analogous heterozygous Dynamin 2 K562E mutation that causes neuropathy in humans develop predominant characteristics of a primary myopathy.

Some mutations affecting Dynamin 2 (DNM2) can cause dominantly-inherited Charcot-Marie-Tooth (CMT) neuropathy. Here, we describe the analysis of mice carrying the DNM2 K562E mutation which has been associated with dominant-intermediate CMT type B (CMTDIB). Contrary to our expectations, heterozygous DNM2 K562E mutant mice did not develop definitive signs of an axonal or demyelinating neuropathy. Rather, we found a primary myopathy-like phenotype in these mice. A likely interpretation of these results is that the lack of a neuropathy in this mouse model has allowed the unmasking of a primary myopathy due to the DNM2 K562E mutation which might be overshadowed by the neuropathy in humans. Consequently, we hypothesize that a primary myopathy may also contribute to the disease mechanism in some CMTDIB patients. We propose that these findings should be considered in the evaluation of patients, the determination of the underlying disease processes, and the development of tailored potential treatment strategies.

Dnm2 wt/K562E*, and P0Cre Dnm2 fl/K562E*. Control SCs incubated at 4°C defines the baseline of the experiments. A profile of the 4°C condition is shown next to exemplary profiles derived from individual genotypes (right side). All genotypes contained P0Cre and the reporter ROSA eYFP (indicated by an *) to ensure the FACS analysis was exclusive to SCs.
(independent Schwann cell preparations were performed from every mouse, n = 5 mice/genotype). One-Way ANOVA with Tukey´s multiple comparisons test.
6 Supplementary Material Figure S4: Additional evidence for a myopathy in Dnm2 wt/K562E soleus muscles.
(A) Exemplary pictures of Mason's Trichrome-stained soleus muscle cryosections derived from control and Dnm2 wt/K562E mice at 2 months-and 1 year of age. Dnm2 wt/K562E soleus muscles display a wider extracellular-stained area in the mutant compared to control samples.
(n = 5 mice per genotype, at least 3 pictures per mouse were acquired). Scale bar: 50µm, refers to whole panel. (B, C) Exemplary cryosections of soleus muscles from control and Dnm2 wt/K562E mice at 2 months of age probed with antibodies in (B) targeting laminin or embryonic myosin isoform (eMyo), and stained with DAPI. Exemplary eMyo-positive myofibers (white arrows) are highlighted. Scale bar: 200µm for the lower magnification images in the panel, and 50µm for the magnified images in the panel. Quantification of eMyo-positive myofibers (C) shows a significant increase in Dnm2 wt/K562E soleus muscles compared to controls. Bar 7 heights: Mean; error bars: s.e.m. (n = 5 mice/genotype, images from at least 3 sections quantified and averaged per mouse). (D) Analysis of soleus muscle weight (including the calcaneal tendon, used for handling the tissue) of control and Dnm2 wt/K562E sex-specific mice at 2 months-and 1 year of age. The muscles have a lower weight in Dnm2 wt/K562E males and females if compared to the respective controls at 1 year, a feature that is also significant in males at 2 months. Bar heights: Mean; error bars: s.e.m. (females at 2 months: n = 9 control and n = 10 Dnm2 wt/K562E; females at 1 year: n = 5 control and n = 7 Dnm2 wt/K562E; males at 2 months: n = 14 control and n = 12 Dnm2 wt/K562E; males at 1 year : n = 13 control and n = 9 Dnm2 wt/K562E). (E) Analysis of centronucleated fibers (CNF) in control and Dnm2 wt/K562E soleus muscle cryosections derived from 1 year-old mice reveals no significant changes between both genotypes. Bar heights: Mean; error bars: s.e.m. (n = 5 mice/genotype, images from 3 sections quantified and averaged per mouse). Two-tailed unpaired Student's t-test. Significance was set at *p < 0.05, **p < 0.01, ***p < 0.001. (A, C) Exemplary images from whole-mount soleus muscle fibers with labelled neuromuscular junctions (NMJs) post-synaptic (a-bungarotoxin) and pre-synaptic (synaptophysin) terminals derived from mice at 2 months (A) and 1 year of age (C). Examples of post-synaptic signals that are partially (white arrowhead) or completely (white arrow) not overlaid by the respective pre-synaptic signals are highlighted. Scale bar: 50µm, refers to whole panel. (B, D) Quantification of the NMJ endplates that are fully innervated, partially denervated or completely denervated at 2 months (B) and 1 year of age (D) shows no significant changes between control and Dnm2 wt/K562E mice in the number of fully denervated structures at both ages.
The number of fully innervated NMJs are mildly reduced in mutants at both ages, and the partially denervated ones show a trend towards an increase in 2 months-old Dnm2 wt/K562E animals, which reaches statistical significance by 1 year of age. Bar heights: Mean; error bars: Figure S6: Checkerboard pattern of fiber-type distribution is preserved in Dnm2 wt/K562E soleus muscle.

Supplementary Material
(A) Exemplary pictures from immunolabelled cryosections of control and Dnm2 wt/K562E soleus muscles derived from 2 months-and 1 year-old mice, probed with isotype-specific antibodies recognizing myosin heavy chains characteristic of type I, or type IIA, or type IIB myofibers. Myofibers not labelled by any of the three antibodies used are likely type IIX. Note that the red signal is also not-specifically present around fibers of all types. The checkerboard random pattern of fiber-type distribution is not disrupted in Dnm2 wt/K562E samples. (B) Quantification of the percental abundance of type I and type II (includes the type IIA and IIB) myofibers from control and Dnm2 wt/K562E soleus muscle sections (shown in A) reveals only marginal changes in the numbers of both type I and type II fibers between both genotypes. Bar heights: Mean; error bars: s.e.m. (n = 5 mice/genotype at 2 months and 1 year, images from at least 3 sections per mouse were quantified and averaged). Two-Way ANOVA with Sidak´s multiple comparisons test. Significance was set at *p < 0.05. IPA Ingenuity analysis of canonical pathways reveals that oxidative phosphorylation and mitochondrial dysfunction are the most significantly represented categories in the dataset (FDR < 0.05 and log2 fold change at least ± 0.58). Heatmap representing the transcripts associated by Ingenuity with each of these 2 canonical pathways confirms they are dominantly downregulated in Dnm2 wt/K562E compared to control soleus muscles (n = 4 mice/genotype). Figure S8: Statistics Summary