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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 neuron morphology and labeling of the neuromuscular junctions in control (A, C, E, and E′) and (SMNF7/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.
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Figure 3: Motor neuron morphology and labeling of the neuromuscular junctions in control (A, C, E, and E′) and (SMNF7/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.

Mentions: To determine whether the muscular changes were associated with or due to a neurogenic process, the neuromuscular system was examined. Rhodamine-conjugated α-bungarotoxin was used to label the AChR at the neuromuscular junction on transverse sections of skeletal muscle. In 4-wk-old mutant mice, AChR staining was concentrated at the neuromuscular junction, an aspect similar to that of control mice and no extrajunctional labeling was observed (Fig. 3). Moreover, motor end plates labeled on whole mount preparations of teased muscle fibers did not reveal any change in presynaptic terminals, postsynaptic folds, or in the number of neuromuscular junctions of mutant mice (Fig. 3 and data not shown). A morphological analysis on transverse semi-thin sections of spinal cord was performed on 4-wk-old control and mutant mice using toluidine blue staining. In mutant mice, the morphology of motor neurons was similar to that of control mice (Fig. 3). Finally, quantification of motor neuron number was performed at the lumbar level of the spinal cord using double labeling of nuclei and cytoplasm by DAPI and antibody specific to choline acetyl transferase, an enzyme specific to cholinergic neurons, respectively. At 4 wk of age, no significant loss of motor neurons of the anterior horns was detected in mutant mice (data not shown). These data strongly suggest that the muscular changes found in mutant mice are not caused by or do not lead to motor neuron changes.


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 neuron morphology and labeling of the neuromuscular junctions in control (A, C, E, and E′) and (SMNF7/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.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Motor neuron morphology and labeling of the neuromuscular junctions in control (A, C, E, and E′) and (SMNF7/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.
Mentions: To determine whether the muscular changes were associated with or due to a neurogenic process, the neuromuscular system was examined. Rhodamine-conjugated α-bungarotoxin was used to label the AChR at the neuromuscular junction on transverse sections of skeletal muscle. In 4-wk-old mutant mice, AChR staining was concentrated at the neuromuscular junction, an aspect similar to that of control mice and no extrajunctional labeling was observed (Fig. 3). Moreover, motor end plates labeled on whole mount preparations of teased muscle fibers did not reveal any change in presynaptic terminals, postsynaptic folds, or in the number of neuromuscular junctions of mutant mice (Fig. 3 and data not shown). A morphological analysis on transverse semi-thin sections of spinal cord was performed on 4-wk-old control and mutant mice using toluidine blue staining. In mutant mice, the morphology of motor neurons was similar to that of control mice (Fig. 3). Finally, quantification of motor neuron number was performed at the lumbar level of the spinal cord using double labeling of nuclei and cytoplasm by DAPI and antibody specific to choline acetyl transferase, an enzyme specific to cholinergic neurons, respectively. At 4 wk of age, no significant loss of motor neurons of the anterior horns was detected in mutant mice (data not shown). These data strongly suggest that the muscular changes found in mutant mice are not caused by or do not lead to motor neuron changes.

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