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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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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.
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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.

Mentions: To test whether the muscular dystrophic phenotype was associated with membrane damage of skeletal muscle, the release of muscle enzyme into the circulating blood was evaluated in mutant mice. (SMNF7/Δ7, HSA-Cre) mutant mice exhibited serum CK activity approximately six times higher (3,455 U/liter, n = 11) than control littermates of the same age (SMNF7/+, 590 U/liter, n = 15). Sarcolemma integrity was further examined by intraperitoneal injection of EBD, a membrane impermeant molecule, in control and mutant mice. No uptake of EBD into skeletal muscle fibers of control mice was detected (Fig. 4). In contrast, red EBD autofluorescence was observed in 4-wk-old mutant mice. Fluorescence microscopic analysis revealed EBD uptake into 1.9 and 4.4% of skeletal muscle fibers from gastrocnemius and intercostal, respectively (Fig. 4). Interestingly, EBD-positive fibers were also detected in muscles of 3-wk-old mutant mice indicating that plasma membrane defect occured before muscle paralysis and marked histological changes (Fig. 4).


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)

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.
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Related In: Results  -  Collection

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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.
Mentions: To test whether the muscular dystrophic phenotype was associated with membrane damage of skeletal muscle, the release of muscle enzyme into the circulating blood was evaluated in mutant mice. (SMNF7/Δ7, HSA-Cre) mutant mice exhibited serum CK activity approximately six times higher (3,455 U/liter, n = 11) than control littermates of the same age (SMNF7/+, 590 U/liter, n = 15). Sarcolemma integrity was further examined by intraperitoneal injection of EBD, a membrane impermeant molecule, in control and mutant mice. No uptake of EBD into skeletal muscle fibers of control mice was detected (Fig. 4). In contrast, red EBD autofluorescence was observed in 4-wk-old mutant mice. Fluorescence microscopic analysis revealed EBD uptake into 1.9 and 4.4% of skeletal muscle fibers from gastrocnemius and intercostal, respectively (Fig. 4). Interestingly, EBD-positive fibers were also detected in muscles of 3-wk-old mutant mice indicating that plasma membrane defect occured before muscle paralysis and marked histological changes (Fig. 4).

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