Neuroscience

A Novel Mouse Model for LAMA2-Related Muscular Dystrophy: Analysis of Molecular Pathogenesis and Clinical Phenotype

Dandan Tan
Yidan Liu
Huaxia Luo
Qiang Shen
Xingbo Long
Luzheng Xu
Jieyu Liu
Nanbert Zhong author has email address
Hong Zhang author has email address
Hui Xiong author has email address

Department of Pediatrics, Peking University First Hospital, Beijing, 100034, China
Department of Neurology, the First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, 330006, China
Institute of Cardiovascular Sciences and Key Laboratory of Molecular Cardiovascular Sciences, Peking University Health Science Center, Beijing, 100191, China
Department of Urology, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, 510060, China
Medical and Health Analysis Center, Peking University, Beijing, 100191, China
New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314, USA
Beijing Key Laboratory of Molecular Diagnosis and Study on Pediatric Genetic Diseases, Beijing 100034, China

https://doi.org/10.7554/eLife.94288.1

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Figures and data

Generation of a mouse model with deletion of exon 3 (ΔExon 3) in the Lama2 gene.
(A) Strategy of generation of ΔExon 3 mice (dy^H/dy^H mice) by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9. The sequences of the Lama2 gene marked with blue indicated sgRNAs targeting sequences, ones marked with brown indicated the location of polymerase chain reaction (PCR) primers (Lama2-F, Lama2-R1, and Lama2-R2), respectively. (B) PCR analysis for genotype identification of the F2 mice. c, DNA sequencing of reverse transcription (RT)-PCR products from dy^H/dy^H muscle validated the deletion of exon 3, which resulted in a frameshift downstream sequence of Lama2 gene.

General phenotype and muscle MRI of dy^H/dy^H mice.
(A) General phenotype of dy^H/dy^H mice. There was no significant difference in survival, body weight, muscle function (four-limb grip and the number of electric shocks on the treadmill) and serum CK levels between the wild-type (WT) and heterozygous mice at each point. Kaplan–Meier survival analysis revealed that the median survival of dy^H/dy^H mice was 21 days (range 12-35 days), and none of the wild-type and heterozygous mice died during the observation period. Significant difference in body weight (g) between the WT and dy^H/dy^H mice was marked at P7 (3.74 vs 3.16, t-test, p < 0.001), P10 (5.58 vs 4.09, t-test, p < 0.001), P14 (6.81 vs 4.41, t-test, p < 0.001), P17 (7.66 vs 5.04, t-test, p < 0.001), P21 (8.34 vs 4.09, t-test, p< 0.001), and P24 (10.41 vs 3.73, t-test, p < 0.001). Two-week-old dy^H/dy^H mice could be easily identified due to their smaller size. Significant differences in the mean relative four-limb grip (force per gram body weight) between the WT and dy^H/dy^H mice were marked at P10 (3.16 vs 2.46, t-test, p < 0.001), P14 (4.65 vs 3.37, t-test, p < 0.001), P17 (5.05 vs 3.58, t-test, p = 0.001), P21 (7.41 vs 5.12, t-test, p< 0.001), and P24 (7.53 vs 4.73, t-test, p = 0.003). The mean number of electric shocks on the treadmill significantly increased number of the electric shocks in dy^H/dy^H mice at P18 (t-test, p = 0.010), P19 (t-test, p = 0.012), P21 (t-test, p = 0.003), P22 (t-test, p < 0.001), P23 (t-test, p = 0.017), and P24 (t-test, p = 0.010). Approximately 9-10 times higher CK levels were detected in dy^H/dy^H mice than in WT mice at P14 and P21. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001, ns (p ≥ 0.05), blue ‘ns’ for wild-type vs heterozygous mice, black ‘ns’ for vs dy^H/dy^H mice at two ages, and orange asterisks or ‘ns’ for WT vs dy^H/dy^H mice. (B) Muscle MRI in dy^H/dy^H mice. Pelvic and hindlimb muscle MRI showed significantly smaller muscle volumes on T1-weighted MRI and significantly increased hyperintense regions on T2-weighted muscle MRI in dy^H/dy^H mice than in WT mice at P14 and P21 (****p < 0.001).

Muscle pathology with age in the biceps femoris of dy^H/dy^H mice.
H&E and Sirius Red staining of the biceps femoris were compared between wild-type and dy^H/dy^H mice at P1, P4, P7, P14 and P21. H&E staining showed a noticeable inequality muscle fiber size in P7 dy^H/dy^H mice, the most extreme dystrophic changes (inequality muscle fiber size, degeneration, necrosis and regeneration of muscle fibers), and connective tissue penetration and inflammation in P14 dy^H/dy^H mice. Sirius Red staining showed increased collagen content and fibrosis in dy^H/dy^H mice from P7.

Extensive involvement with muscle pathology and laminin α2 deficiency in dy^H/dy^H mice.
(A) H&E staining showed dystrophic changes in the quadriceps femoris, gastrocnemius, triceps brachii, diaphragm and tongue muscles, but not as much as in the biceps femoris, while the heart and intestinal muscles were spared. (B) The 300 kDa fragment of laminin α2 chain and 700 kDa laminin-α2β1γ1 (LM-211) complex were not detected in the biceps femoris of dy^H/dy^H mice (n = 3) by Western blot analysis. (C) Colocalization of laminin α2 chain (green fluorescence, yellow arrow) and collagen VI (red fluorescence, blue arrow) showed that they were normally located and expressed in the basement membrane in WT muscle, but laminin α2 deficiency along with increased collagen VI were observed in dy^H/dy^H muscle.

scRNA-seq analysis of brains from dy^H/dy^H and WT mice.
(A) UMAP visualization of all the cells colored by sample. (B) UMAP visualization of all the cells from dy^H/dy^H and WT brains colored by cluster identity. (C) Cell composition proportion of every annotated cell cluster in dy^H/dy^H and WT separately. (D) Violin plots of the expression of Lama2 gene in WT and dy^H/dy^H in each annotated cell cluster. (E) Immunofluorescence of laminin α2, GFAP (astrocytes), and PLP1 on P14 mouse brain slices. DAPI was for nuclei staining. White arrows indicated the cortical surface. (F) GO enrichment of differentially expressed genes in vascular and leptomeningeal fibroblasts, vascular smooth muscle cells and astrocytes. (G) Cell-cell communications among cell clusters. Total number of inferred cell-cell interaction counts (left) and strength (right) in dy^H/dy^H and WT, respectively. Heatmap of ligand-receptor interaction counts in all pairwise cell clusters in WT and dy^H/dy^H brains.

Laminin pathway interactions and hypothesis for cortical dysplasia.
(A) The ligand-receptor pairs in laminin pathway. (B) Violin plots showed the expression of Mbp, Mobp, Plp1, Slc1a2, Mt3, and Slc6a13 in every annotated cell cluster in dy^H/dy^H and WT separately. *** for p-value< 0.001, ** for p-value<0.01, * for p-value<0.05. (C) Hypothesis that cortical dysplasia (cobblestone malformation) and neuronal over migration in the cortex due to a defective gliovascular basal lamina of the blood-brain barrier caused by the lack of laminin α2 in LAMA2-MD.

The transcriptional landscape of dy^H/dy^H (KO) mouse muscles in response to wild-type (WT) mouse muscles.
(A) Principal component analysis for dimension reduction of differentially expressed genes (DEGs) in the biceps femoris of dy^H/dy^H and dy^H/+ mice in response to WT mice. (B) DEGs in dy^H/dy^H muscles relative to WT muscles were enriched in adhesion/extracellular matrix, cytoskeletal proteins and muscle development, ion channel and cell membrane transport, inflammation, mitochondrial metabolism, and apoptosis. (C) Protein-protein interactions among Lama2 and integrins-related genes including Itgal, Itgax, Itgam, Itgb2, and Itgb7. (D) The neuron death related signaling pathways were enriched in dy^H/dy^H muscles relative to WT muscles by GO pathway analyses.

Changes of muscle cytoskeleton and development proteins in dy^H/dy^H mice.
(A) Immunofluorescence staining for MYHC, desmin, β-tubulin, MYOG, MYOD1 and MYF-5 in WT and dy^H/dy^H muscles at P14. MYHC, MYOG, MYOD1 and MYF-5 were focally increased, while desmin and β-tubulin showed mild decrease in the cytoplasm of hypertrophic muscle fibers as well as focal increase in developmental muscle fibers in dy^H/dy^H mice. Wheat-germ agglutinin (WGA; red) was used to visualize the muscle fibers and connective tissue. White asterisk indicated focal regions with changes. (B) F-actin, α-actin, MYH2, MYHC, desmin, β-tubulin, MYOG and MYOD1 were detected by Western blot in P14 WT (n = 3) and dy^H/dy^H (n = 3) muscles. The levels of F-actin, MYOG and MYOD1 were significantly decreased in dy^H/dy^H muscles (p < 0.05), MYH2 and MYHC were significantly increased in dy^H/dy^H muscles (p < 0.05), while α-actin, desmin and β-tubulin showed no significant difference between WT (n = 3) and dy^H/dy^H mice.

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