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

Cifuentes-Diaz C, Frugier T, Tiziano FD, Lacène E, Roblot N, Joshi V, Moreau MH, Melki J - J. Cell Biol. (2001)

Bottom Line: 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.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.These data may have important implications for the development of therapeutic strategies in SMA.

View Article: PubMed Central - PubMed

Affiliation: Molecular Neurogenetics Laboratory, Institut National de la Santé et de la Recherche Médicale (INSERM), Université d'Evry, EMI-9913, Genopole, 91057 Evry, France.

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.

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Related in: MedlinePlus

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 (SMNF7/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.
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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 (SMNF7/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.

Mentions: These data led to examine the components of the dystrophin-glycoprotein complex (DGC) in (SMNF7/Δ7, HSA-Cre) mutant mice. The immunofluorescence staining of dystrophin was either patchy or lacking on transverse sections of skeletal muscle from mutant mice using antibodies directed against either the NH2 or the COOH terminus of dystrophin (Fig. 5). Muscle fibers lacking dystrophin staining were scattered and observed in different muscles including gastrocnemius, intercostal or biceps brachii. EBD accumulation into muscle fibers correlated with the lack of dystrophin staining (Fig. 4). Nevertheless, numerous muscle fibers lacking dystrophin did not display accumulation of EBD (Fig. 4). Dystrophin staining on sections of heart from mutant mice appeared similar to that of control (data not shown). Immunofluorescence analysis of utrophin, the autosomal homologue of dystrophin, was performed in control and mutant mice. In 4-wk-old control mice, utrophin is concentrated at the neuromuscular junction, while a marked extra-junctional labeling was observed in mutant mice of the same age (Fig. 5). This observation could be related to the lack of dystrophin in muscle fibers. To test this hypothesis, double labeling experiment of utrophin and dystrophin on transverse sections of skeletal muscle from control or mutant mice was performed. In mutant mice, sarcolemmal staining of utrophin was observed outside the neuromuscular junctions of muscle fibers expressing or lacking dystrophin (Fig. 5). These data suggest that muscle fibers displaying an abnormal expression pattern of utrophin correspond to regenerated fibers, which would be expected to express higher levels of utrophin. In mutant mice, α- and β-sarcoglycan and β-dystroglycan staining was comparable to that of control levels although immunofluorescent labeling was lacking in some muscle fibers (Fig. 6). Finally, α-2 chain of laminin and collagen IV, components of the basal lamina (Sanes 1982), were present in muscle fibers of mutant mice with an immunofluorescent staining similar to that of control muscle (Fig. 6). To further examine the expression of the DGC components, immunoblot analysis was performed on muscle proteins prepared from control or mutant mice. Although dystrophin was present with a size similar to that of control, the level of dystrophin was reduced in SMN mutant mice associated with an higher amount of utrophin in accordance with the immunofluorescence staining (Fig. 2). The amount of γ-sarcoglycan was slightly reduced while α-sarcoglycan is similar to control levels (data not shown).


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

Cifuentes-Diaz C, Frugier T, Tiziano FD, Lacène E, Roblot N, Joshi V, Moreau MH, Melki J - J. Cell Biol. (2001)

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 (SMNF7/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.
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Related In: Results  -  Collection

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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 (SMNF7/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.
Mentions: These data led to examine the components of the dystrophin-glycoprotein complex (DGC) in (SMNF7/Δ7, HSA-Cre) mutant mice. The immunofluorescence staining of dystrophin was either patchy or lacking on transverse sections of skeletal muscle from mutant mice using antibodies directed against either the NH2 or the COOH terminus of dystrophin (Fig. 5). Muscle fibers lacking dystrophin staining were scattered and observed in different muscles including gastrocnemius, intercostal or biceps brachii. EBD accumulation into muscle fibers correlated with the lack of dystrophin staining (Fig. 4). Nevertheless, numerous muscle fibers lacking dystrophin did not display accumulation of EBD (Fig. 4). Dystrophin staining on sections of heart from mutant mice appeared similar to that of control (data not shown). Immunofluorescence analysis of utrophin, the autosomal homologue of dystrophin, was performed in control and mutant mice. In 4-wk-old control mice, utrophin is concentrated at the neuromuscular junction, while a marked extra-junctional labeling was observed in mutant mice of the same age (Fig. 5). This observation could be related to the lack of dystrophin in muscle fibers. To test this hypothesis, double labeling experiment of utrophin and dystrophin on transverse sections of skeletal muscle from control or mutant mice was performed. In mutant mice, sarcolemmal staining of utrophin was observed outside the neuromuscular junctions of muscle fibers expressing or lacking dystrophin (Fig. 5). These data suggest that muscle fibers displaying an abnormal expression pattern of utrophin correspond to regenerated fibers, which would be expected to express higher levels of utrophin. In mutant mice, α- and β-sarcoglycan and β-dystroglycan staining was comparable to that of control levels although immunofluorescent labeling was lacking in some muscle fibers (Fig. 6). Finally, α-2 chain of laminin and collagen IV, components of the basal lamina (Sanes 1982), were present in muscle fibers of mutant mice with an immunofluorescent staining similar to that of control muscle (Fig. 6). To further examine the expression of the DGC components, immunoblot analysis was performed on muscle proteins prepared from control or mutant mice. Although dystrophin was present with a size similar to that of control, the level of dystrophin was reduced in SMN mutant mice associated with an higher amount of utrophin in accordance with the immunofluorescence staining (Fig. 2). The amount of γ-sarcoglycan was slightly reduced while α-sarcoglycan is similar to control levels (data not shown).

Bottom Line: 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.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.These data may have important implications for the development of therapeutic strategies in SMA.

View Article: PubMed Central - PubMed

Affiliation: Molecular Neurogenetics Laboratory, Institut National de la Santé et de la Recherche Médicale (INSERM), Université d'Evry, EMI-9913, Genopole, 91057 Evry, France.

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.

Show MeSH
Related in: MedlinePlus