The Capillary Morphogenesis Gene 2 Triggers the Intracellular Hallmarks of Collagen VI-Related Muscular Dystrophy

Abstract

Collagen VI-related disorders (COL6-RD) represent a severe form of congenital disease for which there is no treatment. Dominant-negative pathogenic variants in the genes encoding α chains of collagen VI are the main cause of COL6-RD. Here we report that patient-derived fibroblasts carrying a common single nucleotide variant mutation are unable to build the extracellular collagen VI network. This correlates with the intracellular accumulation of endosomes and lysosomes triggered by the increased phosphorylation of the collagen VI receptor CMG2. Notably, using a CRISPR-Cas9 gene-editing tool to silence the dominant-negative mutation in patients' cells, we rescued the normal extracellular collagen VI network, CMG2 phosphorylation levels, and the accumulation of endosomes and lysosomes. Our findings reveal an unanticipated role of CMG2 in regulating endosomal and lysosomal homeostasis and suggest that mutated collagen VI dysregulates the intracellular environment in fibroblasts in collagen VI-related muscular dystrophy.

Keywords: CMG2; collagen VI; muscular dystrophy; patient-derived fibroblasts; super-resolution microscopy.

Figure 1

Altered extracellular matrix in cultured COL6-RD patient-derived fibroblasts. (A) Representative 3D surface plot generated from the STED images (Figure S1) of the extracellular collagen VI matrix in untreated (upper panel) and collagenase-treated (lower panel) controls, patients, and CRISPR-treated fibroblasts showing the COL6 fluorescence levels. Fluorescence intensity analysis was also quantified in untreated (B) and collagenase-treated (C) samples. Data are expressed as the mean ± SEM; n = 3; independent primary cell preparations (30 images of 8–10 stacks for each CONTROL, COL6-RD, and CRISPR samples) were used for all panels. * p < 0.05, ** p < 0.01; one-way ANOVA/Bonferroni’s multiple comparison test. Scale bar 5 µm.

Figure 2

Altered abundance of mitochondria, endo/lysosomal-like vesicles, and multivesicular bodies in COL6-RD patients’ cells. (A)Three-dimensional reconstruction of whole-cell volumes of control-, andCOL6-RD patient-derived fibroblasts and CRISPR-treated fibroblasts. Threshold-based isosurface segmentation of the surface boundaries identifies the different organelles present in the cells: nucleus in yellow, mitochondria in light blue, endo/lysosomal-like vesicles in violet, and multivesicular bodies in pink. (B) Quantification of the percentage of cytoplasm occupied by the organelles analysed. Data are expressed as the mean ± SEM; n = 10; different cells (10 CONTROL cells, 10 COL6-RD cells, and 10 CRISPR samples) were used for all panels. ** p < 0.01, *** p < 0.001; Two-way ANOVA/Bonferroni’s multiple comparison test. Scale bar 500 nm.

Figure 3

The endo-lysosomal compartments are altered in COL6-RD patient-derived fibroblasts. (A) Representative STED images of Lamp1- and (B) EEA1-positive organelles labelled in control- and COL6-RD patient-derived fibroblasts and in CRISPR-treated COL6-RD patient-derived fibroblasts. (C) Lamp1- and (E) EEA1-positive organelles density, together with the area of (D) Lamp1- and (F) EEA1-positive organelles was quantified. Data are expressed as the mean ± SEM; n = 3; independent primary cell preparations were used for all panels. * p < 0.05; one-way ANOVA/Bonferroni’s multiple comparison test. Scale bar 5 µm.

Figure 4

Biochemical confirmation of the intracellular phenotypes observed with super-resolution microscopy. (A) Primary fibroblast homogenates from control and COL6-RD patients and CRISPR-treated COL6-RD patient-derived fibroblasts were subjected to Western blotting using antibodies against Lamp1, EEA1, and Tom20 as shown. The GAPDH marker was used as the loading control. (B) Quantification of Lamp1 from the three phenotypes. (C) Total quantification of EEA1 from the three phenotypes. (D) Total quantification of Tom20 from all the samples. Data are expressed as the mean ± SEM; n = 3; independent primary cell preparations were used for all panels. * p < 0.05; one-way ANOVA/Bonferroni’s multiple comparison test.

Figure 5

CMG2 phosphorylation levels are increased in COL6-RD patient-derived fibroblasts. (A) Western blotting for CMG2 receptor and its tyrosine-phosphorylated form after immunoprecipitation (IP) of control, COL6-RD patient, and CRISPR-treated COL6-RD patient-derived fibroblasts. Blots of equally loaded protein extracts prior to IP (input) are also shown. The marker GAPDH is used as the loading control. (B) Total quantification of the IP phosphorylated form of CMG2 versus IP CMG2 from the three phenotypes is shown. Data are expressed as the mean ± SEM; n = 3; independent primary cell preparations were used for all panels. * p < 0.05, ** p < 0.01; one-way ANOVA/Bonferroni’s multiple comparison test.