by Judith R. Reinhard, Shuo Lin, Karen K

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Linker proteins restore basement membrane and correct LAMA2-related muscular dystrophy in mice by Judith R. Reinhard, Shuo Lin, Karen K. McKee, Sarina Meinen, Stephanie C. Crosson, Maurizio Sury, Samantha Hobbs, Geraldine Maier, Peter D. Yurchenco, and Markus A. Rüegg Sci Transl Med Volume 9(396):eaal4649 June 28, 2017 Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Fig. 1. Muscles of LAMA2 MD patients and dyW/dyW mice contain high amounts of laminin-α4 and show deficits in BM. Muscles of LAMA2 MD patients and dyW/dyW mice contain high amounts of laminin-α4 and show deficits in BM. (A) Representative immunofluorescence images (magnified images in insets) of human muscle biopsy cross sections stained for laminin-α4 and laminin-β1γ1. n = 3 LAMA2 MD patients or healthy controls. (B) Western blot analysis and quantification of laminin chains in human muscle biopsies. α-Actinin was used as loading control. n = 4 controls, n = 3 LAMA2 MD patients. (C) Representative immunofluorescence images (magnified images in insets) of triceps muscle from 8-week-old mice stained for laminin-α4 and laminin-β1γ1. n = 4 mice per genotype. (D) Western blot analysis and quantification of laminin-α4 (left graph), laminin-β1γ1 (middle graph), and Lama4 transcripts (right graph) from triceps muscle of 8-week-old mice. n = 3 mice per genotype (Western blot) and n = 4 mice per genotype (quantitative reverse transcription polymerase chain reaction). (E) Schematic of subcellular fractionation. (F) Western blot analysis of S1 to S4 of a muscle biopsy from a control patient. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Na+/K+-ATPase (sodium-potassium adenosine triphosphatase) were used as markers for cytosolic and membrane proteins, respectively. (G) Western blot analysis of S3 and S4 fractions from control and LAMA2 MD patient muscle biopsies probed for the indicated proteins. Right: Ratio of the laminin-β1γ1 intensity in S3 and S4 fraction. n = 4 healthy controls, n = 3 LAMA2 MD patients. Data are means ± SEM. *P < 0.05; **P < 0.01; n.s. (not significant), P > 0.05 (for exact P values, see table S3), Student’s t test. Scale bars, 100 μm (A and C). Judith R. Reinhard et al., Sci Transl Med 2017;9:eaal4649 Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Fig. 2. Binding of recombinant full-length Lm-411 to muscle receptors, myotubes, and self-polymerization is enhanced by mag and αLNNd. Binding of recombinant full-length Lm-411 to muscle receptors, myotubes, and self-polymerization is enhanced by mag and αLNNd. (A) Schematic of Lm-111, Lm-211, and Lm-411 and the linker molecules mag and αLNNd. Binding interactions of mag, αLNNd, laminins, integrins, and dystroglycan are indicated. (B) Binding curves of integrin α7X2β1 to Lm-111 and Lm-411. (C) Binding curves of Lm-111 and Lm-411 to purified αDG. (D) Binding curves of Lm-411 and Lm-411 plus an equimolar concentration of mag to purified αDG. Data in (B) to (D) are means ± SEM with n = 3 per condition. Curve fitting used the equation Y = Bmax*X/(Kd + X). (E) Polymerization assay for Lm-111, Lm-411, and an equimolar mixture of Lm-411 and αLNNd. Polymer formation was monitored by SDS–polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining after centrifugation. r2 = 0.998 (Lm-111), r2 = 0.941 (Lm-411 and αLNNd), and r2 = 0.0013 (Lm-411) using linear regression. (F) Confluent layer of cultured C2C12 myotubes after incubation with no Lm, recombinant Lm-211, Lm-411, or Lm-411 in the presence of an equimolar concentration of αLNNd and/or mag. C2C12-bound Lm-211 or Lm-411 was visualized by staining with antibodies to laminin-γ1. Representative immunofluorescence images (left) and quantification (right) of laminin-γ1 immunofluorescence (IF). Scale bar, 200 μm. n = 9 cultures (Lm-411 and mag), n = 6 cultures (Lm-411, Lm-411 + αLNNd, Lm-411 + αLNNd + mag, and Lm-211). Data are means ± SEM. ***P < 0.001; n.s., P > 0.05 (for exact P values, see table S3), one-way analysis of variance (ANOVA) with Bonferroni post hoc test. Judith R. Reinhard et al., Sci Transl Med 2017;9:eaal4649 Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Fig. 3. Transgenic expression of mag and αLNNd in dyW/dyW mice. Transgenic expression of mag and αLNNd in dyW/dyW mice. (A) Western blot analysis to detect αLNNd and mag in lysates from triceps muscle from 8-week-old mice. GAPDH was used as loading control. The αLNNd-specific band is indicated by an arrow because the antibodies also detect nonspecific bands at higher Mr; mag runs on SDS-PAGE as two bands because of its cleavage by neurotrypsin (see fig. S5, F and G, for details). (B) Representative immunofluorescence (magnified images in insets) images of triceps cross sections from 8-week-old mice stained for mag, αLNNd, and laminin-γ1. n = 4 mice per genotype. Scale bar, 100 μm. (C) Western blot analysis of immunoprecipitates using anti–laminin-α4 antibodies or immunoglobulin Y (IgY) (as control) and lysates of triceps muscle from 8-week-old dyW/dyW DT or dyW/dyW mice. (D) Western blot analysis of laminin-α4 and laminin-β1γ1 in lysates of triceps muscles from 8-week-old mice. α-Actinin was used as loading control. (E) Protein quantification in lysates from the different mice. n = 3 mice per genotype. Data are means ± SEM. ***P < 0.001; n.s., P > 0.05 (for exact P values, see table S3), one-way ANOVA with Bonferroni post hoc test. Judith R. Reinhard et al., Sci Transl Med 2017;9:eaal4649 Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Fig. 4. Expression of mag and αLNNd in dyW/dyW mice improves muscle BM. Expression of mag and αLNNd in dyW/dyW mice improves muscle BM. (A and B) Western blot analysis (top) and quantification (bottom) of laminin-α4 (A) and laminin-β1γ1 (B) in the fractions obtained by differential extraction of triceps muscles of 8-week-old mice (see scheme in Fig. 1E). GAPDH and Na+/K+-ATPase were used as markers for the soluble (S1) and the membrane proteins (S2), respectively. n = 4 mice (dyW/dyW), n = 6 mice (dyW/dyW mag, dyW/dyW αLNNd, and dyW/dyW DT), n = 5 mice (wild type). Data are means ± SEM. *P < 0.05; **P < 0.01; n.s., P > 0.05 (for exact P values, see table S3), one-way ANOVA with Bonferroni post hoc test. (C) Transmission electron micrographs of triceps muscle from 8-week-old mice. White arrow, sarcolemma; white arrowhead, BM. Scale bar, 100 nm. Judith R. Reinhard et al., Sci Transl Med 2017;9:eaal4649 Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Fig. 5. Transgenic expression of αLNNd and mag in dyW/dyW mice improves muscle histology in 8-week-old mice. Transgenic expression of αLNNd and mag in dyW/dyW mice improves muscle histology in 8-week-old mice. (A) Representative images (magnified images in insets) of H&E-stained cross sections from triceps muscle. An example of a muscle fiber with centralized myonuclei is indicated with an arrow. n = 5 mice per genotype. (B) Representative images (magnified images in insets) of Sirius Red–stained cross sections (collagen in red) from triceps muscle. n = 3 mice per genotype. (C) Relative hydroxyproline content in triceps indicative of collagen content. Values are normalized to controls. n = 4 mice (dyW/dyW mag, dyW/dyW αLNNd, and dyW/dyW DT), n = 5 mice (dyW/dyW), n = 6 mice (control). (D) Distribution of the muscle fiber diameters (left), the mean of median fiber diameter (middle), and the total fiber number (right) in 8-week-old triceps muscle. n = 4 male mice (dyW/dyW and control), n = 5 male mice (dyW/dyW αLNNd and dyW/dyW DT), n = 6 male mice (dyW/dyW mag). Statistical evaluation of fiber size distribution is shown in table S1. (E) Expression of the inflammatory marker Adgre1 (encoding F4/80). n = 4 mice (dyW/dyW, dyW/dyW mag, dyW/dyW αLNNd, and control), n = 5 mice (dyW/dyW DT). (F) Expression of transcripts encoding the matricellular protein tenascin-C in triceps muscle. n = 4 mice (dyW/dyW mag, dyW/dyW αLNNd, and control), n = 5 mice (dyW/dyW and dyW/dyW DT). (G) Quantification of CNFs in triceps muscle. n = 3 mice per genotype. (H) Quantification of relative hydroxyproline content in TA from 8-week-old mice. n = 3 mice (dyW/dyW DT), n = 4 mice (dyW/dyW mag and dyW/dyW αLNNd), n = 5 mice (dyW/dyW), n = 6 mice (control). (I) Quantification of fibrotic area (visualized by Sirius Red staining) in diaphragm cross sections. n = 4 mice (dyW/dyW and dyW/dyW mag), n = 7 mice (dyW/dyW αLNNd, dyW/dyW DT, and control). (J) Creatine kinase activity in blood. n = 7 mice (dyW/dyW and dyW/dyW DT), n = 15 mice (control). Data are means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., P > 0.05 (for exact P values, see table S3), one-way ANOVA with Bonferroni post hoc test. Controls are wild-type or dyW/+ littermates. Scale bars, 100 μm. Judith R. Reinhard et al., Sci Transl Med 2017;9:eaal4649 Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Fig. 6. Transgenic expression of αLNNd and mag in dyW/dyW mice improves muscle function, increases body weight, and prolongs life span. Transgenic expression of αLNNd and mag in dyW/dyW mice improves muscle function, increases body weight, and prolongs life span. (A) Picture of a representative dyW/dyW, dyW/dyW DT, and control mouse (8 weeks old). Note that dyW/dyW mice appear small because of the severe kyphosis, and dyW/dyW DT mice are leaner than controls. n = 10 mice per genotype. (B) Quantification of grip strength of 8-week-old mice. Grip strength was measured by hang time on a vertical grid. Test was stopped after 180 s (dotted line). n = 4 mice (dyW/dyW), n = 6 mice (dyW/dyW mag), n = 7 mice (dyW/dyW αLNNd), n = 9 mice (dyW/dyW DT). (C) Peak tetanic force of EDL muscle from 8-week-old mice. n = 9 mice (dyW/dyW), n = 10 mice (dyW/dyW mag), n = 12 mice (dyW/dyW αLNNd), n = 13 mice (dyW/dyW DT), n = 14 mice (control). (D) Peak tetanic force of EDL from 16-week-old mice. n = 4 mice (dyW/dyW αLNNd), n = 5 mice (dyW/dyW mag and control), n = 6 mice (dyW/dyW DT). Data in (B) and (C) are means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., P > 0.05 (for exact P values, see table S3), one-way ANOVA with Bonferroni post hoc test. (E) Body weight of 5- to 15-week-old male mice. Mice that died between 5 and 15 weeks of age were excluded from analysis. n = 7 mice (dyW/dyW), n = 6 (dyW/dyW mag and dyW/dyW αLNNd), n = 11 mice (dyW/dyW DT), n = 8 mice (control). Data are means ± SEM. *P < 0.05, **P < 0.01 (for exact P values, see table S3), two-way ANOVA with Bonferroni post hoc test. (F) Survival curves for dyW/dyW (n = 19), dyW/dyW mag (n = 16), dyW/dyW αLNNd (n = 16), and dyW/dyW DT (n = 17) mice. Marks indicate mice that were still alive at the end of the study. **P < 0.01 (dyW/dyW mag versus dyW/dyW DT), ***P < 0.001 (dyW/dyW versus dyW/dyW DT or dyW/dyW versus dyW/dyW mag) (for exact P values, see table S3), log-rank test. Controls are wild-type or dyW/+ littermates. Judith R. Reinhard et al., Sci Transl Med 2017;9:eaal4649 Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.