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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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Motor defect and skeletal muscle morphology of control and (SMNF7/Δ7, HSA-Cre) mice. (A) Control littermate. (B) Note the marked paralysis with abnormal posture of the limbs and cyphosis of 4-wk-old (SMNF7/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 (SMNF7/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.
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Figure 1: Motor defect and skeletal muscle morphology of control and (SMNF7/Δ7, HSA-Cre) mice. (A) Control littermate. (B) Note the marked paralysis with abnormal posture of the limbs and cyphosis of 4-wk-old (SMNF7/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 (SMNF7/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.

Mentions: Through homologous recombination in embryonic stem cells, mouse line carrying two loxP sites flanking SMN exon 7 (SMNF7) has been previously generated and a strain homozygous for the SMNF7 allele has been established (SMNF7/F7; Frugier et al. 2000). To direct deletion of SMN exon 7 to skeletal muscle, transgenic mouse line expressing the Cre recombinase gene driven by the promoter of the human α-skeletal actin gene has been used (HSA-Cre79; Miniou et al. 1999). Mice homozygous for the SMNF7 allele (SMNF7/F7) were crossed to mice carrying an heterozygous deletion of SMN exon 7 (SMNΔ7; Frugier et al. 2000) and expressing the HSA-Cre79 transgene. 37 of 140 mice (26.4%) carried both HSA-Cre79 recombinase transgene and the SMNF7/Δ7 genotype (termed SMNF7/Δ7, HSA-Cre), as expected. Mutant mice carrying this genotype were indistinguishable from their control littermates up to 3 wk of age. However, after 3 wk of age, (SMNF7/Δ7, HSA-Cre) mutant mice of both sexes develop severe muscle paralysis with reduced spontaneous and induced motor activity and severe cyphosis (Fig. 1). Mutant mice exhibit an extremely reduced life expectancy dying at a mean age of 33 d (28–37, n = 18).


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)

Motor defect and skeletal muscle morphology of control and (SMNF7/Δ7, HSA-Cre) mice. (A) Control littermate. (B) Note the marked paralysis with abnormal posture of the limbs and cyphosis of 4-wk-old (SMNF7/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 (SMNF7/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.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198815&req=5

Figure 1: Motor defect and skeletal muscle morphology of control and (SMNF7/Δ7, HSA-Cre) mice. (A) Control littermate. (B) Note the marked paralysis with abnormal posture of the limbs and cyphosis of 4-wk-old (SMNF7/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 (SMNF7/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.
Mentions: Through homologous recombination in embryonic stem cells, mouse line carrying two loxP sites flanking SMN exon 7 (SMNF7) has been previously generated and a strain homozygous for the SMNF7 allele has been established (SMNF7/F7; Frugier et al. 2000). To direct deletion of SMN exon 7 to skeletal muscle, transgenic mouse line expressing the Cre recombinase gene driven by the promoter of the human α-skeletal actin gene has been used (HSA-Cre79; Miniou et al. 1999). Mice homozygous for the SMNF7 allele (SMNF7/F7) were crossed to mice carrying an heterozygous deletion of SMN exon 7 (SMNΔ7; Frugier et al. 2000) and expressing the HSA-Cre79 transgene. 37 of 140 mice (26.4%) carried both HSA-Cre79 recombinase transgene and the SMNF7/Δ7 genotype (termed SMNF7/Δ7, HSA-Cre), as expected. Mutant mice carrying this genotype were indistinguishable from their control littermates up to 3 wk of age. However, after 3 wk of age, (SMNF7/Δ7, HSA-Cre) mutant mice of both sexes develop severe muscle paralysis with reduced spontaneous and induced motor activity and severe cyphosis (Fig. 1). Mutant mice exhibit an extremely reduced life expectancy dying at a mean age of 33 d (28–37, n = 18).

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