Deletion of murine SMN exon 7 directed to skeletal muscle leads to severe muscular dystrophy

Abstract

Spinal muscular atrophy (SMA) is characterized by degeneration of motor neurons of the spinal cord associated with muscle paralysis and caused by mutations of the survival motor neuron gene (SMN). To determine whether SMN gene defect in skeletal muscle might have a role in SMA pathogenesis, deletion of murine SMN exon 7, the most frequent mutation found in SMA, has been restricted to skeletal muscle by using the Cre-loxP system. Mutant mice display ongoing muscle necrosis with a dystrophic phenotype leading to muscle paralysis and death. The dystrophic phenotype is associated with elevated levels of creatine kinase activity, Evans blue dye uptake into muscle fibers, reduced amount of dystrophin and upregulation of utrophin expression suggesting a destabilization of the sarcolemma components. The mutant mice will be a valuable model for elucidating the underlying mechanism. Moreover, our results suggest a primary involvement of skeletal muscle in human SMA, which may contribute to motor defect in addition to muscle denervation caused by the motor neuron degeneration. These data may have important implications for the development of therapeutic strategies in SMA.

Figure 1

Motor defect and skeletal muscle morphology of control and (SMN^{F7/ Δ7} , HSA-Cre) mice. (A) Control littermate. (B) Note the marked paralysis with abnormal posture of the limbs and cyphosis of 4-wk-old (SMN^{F7/ Δ7} , HSA-Cre) mice. (C–E) Hematoxylin and eosin staining of transverse sections of gastrocnemius from control littermate (C), 3- (D), and 4- (E) wk-old (SMN^{F7/ Δ7} , HSA-Cre) mice. Before the onset of muscle paralysis (D), muscle histology is similar to control skeletal muscle except the presence of rare necrotic muscle fibers surrounded by mononuclear cells (indicated by arrow). After the onset of muscle paralysis (E), skeletal muscle histology revealed necrotic fibers (filled arrow), regenerating myocytes with central nucleus (open arrow), variation in fiber size and infiltration of connective tissue. Bar, 35 μm.

Figure 3

Motor neuron morphology and labeling of the neuromuscular junctions in control (A, C, E, and E′) and (SMN^{F7/ Δ7} , HSA-Cre) mice (B, D, F, and F′). Toluidine blue staining of transverse semithin sections of spinal cord (A and B) does not reveal any morphological changes of motor neurons of mutant mice (B) compared with control (A). Labeling of AChR using rhodamine-conjugated α-bungarotoxin on transverse sections of skeletal muscle of control (C) and mutant mice (D). AChRs are concentrated at the neuromucular junctions with their characteristic curved staining in both control and mutant mice. In toto immunostaining of neuromuscular junctions on whole mount preparations of teased muscle fibers from control (E and E′) and mutant mice (F and F′). Any changes of presynaptic terminals labeled with neurofilament antibody or postsynaptic folds stained with rhodamine-conjugated α-bungarotoxin were observed in mutant mice. Bars: (A, B, and E–F′) 25 μm; (C and D) 50 μm.

Figure 2

Analysis of SMN and components of the DGC. (A) Semiquantitative RT-PCR amplification of SMN transcripts from skeletal muscle using primers flanking exon 7 reveals transcripts containing (SMN FL) or lacking exon 7 (SMNΔ7, first panel). Primers ex5sou2 and ex7sou2 were used to amplify SMN transcripts containing exon 7 only (SMN FL, second panel). Aldolase A cDNA was coamplified and used as internal control (third panel). RT-PCR analysis reveals SMN FL transcript only in control (+/+, lane 2). In addition to the full-length RT-PCR product (SMN FL), a shorter product corresponding to SMNΔ7 transcript is detected in +/SMN^Δ7 tissue (lane 1). In mutant mice (MM, lanes 3–7), note the dramatic reduction of full-length product in skeletal muscle from 15 d of age (15 d., lane 5–7) whereas the SMN ^Δ7 is the predominant form. Note the slight decrease of SMN transcript during the postnatal period of control mice (+/+, fourth panel). (B) Tissue specific deletion of the SMN^F7 allele was demonstrated by PCR analysis of DNA extracted from a variety of tissues of mice carrying the (SMN^{F7/ +}, HSA-Cre) genotype using primers PHR5 and GS8. A 450-bp fragment was successfully amplified in skeletal muscles but not in the other tissues indicating the presence of Cre recombinase activity restricted to skeletal muscle (upper panel). PCR amplification analysis using primers flanking SMN exon 4 was used as internal positive control (630 bp, lower panel; Frugier et al. 2000). (C) Western blot analysis of proteins extracted from skeletal muscle using a monoclonal antibody directed against the NH₂ terminus of SMN. Note the marked reduction of SMN levels from 15-d-old mutant mice (MM, 15 d., lane 8–10) compared with control (+/+, lanes 1–5). Incubation with monoclonal anti-actin antibody was used as internal control. (D and E) Western blot analysis of proteins extracted from skeletal muscle using a monoclonal anti-dystrophin antibody (MANDRA1, D) and polyclonal antibodies against utrophin (E). Note the reduction of dystrophin in 4-wk-old mice (SMN^{F7/ Δ7}, HSA-Cre, lanes 1–4) compared with control (+/+) associated with an upregulation of utrophin expression (E). Actin was used as internal control. Relative quantitation was achieved by densitometric scanning and the amount is indicated. MM, mutant mice; +/+, wild type; SMNFL, full-length SMN transcript; SMNΔ7, SMN transcript lacking exon 7; d., postnatal day.

Figure 6

Immunofluorescent staining of α-chain of laminin (A and B), collagen IV (C and D), α-sarcoglycan (E and F), β-sarcoglycan (G and H), and β-dystroglycan (I and J) on transverse sections of skeletal muscle from control (A, C, E, G, and I) and (SMN^{F7/ Δ7}, HSA-Cre) mice (B, D, F, H, and J). The immunofluorescent staining of laminin and collagen IV is similar to that of control whereas the labeling of α-sarcoglycan, β-sarcoglycan, or β-dystroglycan is lacking in rare muscle fibers of mutant mice (arrows). Bar, 50 μm.

Figure 4

Vital staining with EBD and immunofluorescence analysis of dystrophin in skeletal muscle of control (A, A′, and A′′) and mutant mice at 3 (B, B′, and B′′) and 4 wk of age (C, C′, and C′′). Dye inclusion was not detected in control muscle (A). Vital staining with EBD reveals muscle fibers in intercostal muscles of 3- or 4-wk-old mutant mice associated with the lack of dystrophin staining at the sarcolemma (arrows). Note the uptake of the fluorescein-conjugated anti–mouse IgG antibody in EBD positive muscle fibers (B′). (A′′, B′′, and C′′), phase contrast. Bar, 35 μm.

Figure 5

Immunofluorescent staining of dystrophin (A and B) and utrophin (C and D) on transverse frozen sections of skeletal muscle from control (A and C) and (SMN^{F7/ Δ7}, HSA-Cre) mice (B and D). The MANDRA1 anti-dystrophin antibody stains the sarcolemma of muscle fibers from control mouse tissue (A) although it fails to detect the sarcolemma of some muscle fibers from mutant mice (B). In control mouse tissue, the polyclonal antiutrophin antibodies stain the neuromuscular junction only (C, arrow), although a marked extrajunctional labeling of sarcolemma is observed in mutant mice (D). Double immunostaining experiment of dystrophin (E and F) and utrophin (E′ and F′) on transverse frozen sections of skeletal muscle from control (E, E′, and E′′), and (SMN^{F7/ Δ7} , HSA-Cre) mice (F, F′, and F′′). In mutant mouse, some muscle fibers lacking dystrophin sarcolemmal staining (F, filled arrow) display an upregulation of the sarcolemmal staining of utrophin (F′), whereas in other muscle fibers the utrophin sarcolemmal staining is observed despite the expression of dystrophin at the plasma membrane (open arrow). (E′′ and F′′) merged images. Bars: (A–D) 50 μm; (E–F′′) 35 μm.