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A transgene carrying an A2G missense mutation in the SMN gene modulates phenotypic severity in mice with severe (type I) spinal muscular atrophy.

Monani UR, Pastore MT, Gavrilina TO, Jablonka S, Le TT, Andreassi C, DiCocco JM, Lorson C, Androphy EJ, Sendtner M, Podell M, Burghes AH - J. Cell Biol. (2003)

Bottom Line: We suggest that only in the presence of low levels of full-length SMN is the A2G transgene able to form partially functional higher order SMN complexes essential for its functions.Animals homozygous for the mutant transgene are less severely affected than heterozygotes.This demonstrates the importance of SMN levels in SMA even if the protein is expressed from a mutant allele.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Ohio State University, Columbus, OH 43210, USA. monani.2@osu.edu

ABSTRACT
5q spinal muscular atrophy (SMA) is a common autosomal recessive disorder in humans and the leading genetic cause of infantile death. Patients lack a functional survival of motor neurons (SMN1) gene, but carry one or more copies of the highly homologous SMN2 gene. A homozygous knockout of the single murine Smn gene is embryonic lethal. Here we report that in the absence of the SMN2 gene, a mutant SMN A2G transgene is unable to rescue the embryonic lethality. In its presence, the A2G transgene delays the onset of motor neuron loss, resulting in mice with mild SMA. We suggest that only in the presence of low levels of full-length SMN is the A2G transgene able to form partially functional higher order SMN complexes essential for its functions. Mild SMA mice exhibit motor neuron degeneration, muscle atrophy, and abnormal EMGs. Animals homozygous for the mutant transgene are less severely affected than heterozygotes. This demonstrates the importance of SMN levels in SMA even if the protein is expressed from a mutant allele. Our mild SMA mice will be useful in (a) determining the effect of missense mutations in vivo and in motor neurons and (b) testing potential therapies in SMA.

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Electromyography and axonal sprouting in mild SMN mice. EMG recordings in (A) cranial tibial and (B) suprascapular muscle of 4-mo-old type III SMA mice. Spontaneous abnormal electrical activity was recorded in the form of fibrillation potentials (dark arrows) and occasional biphasic positive sharp waves (red arrow). (C) Quantification of MNCVs and the amplitudes of compound muscle action potentials in type III SMA mice and normal (Smn+/+) littermates. At least four mice from each group were analyzed. Data are given as mean ± SD. Although the amplitude was reduced in SMA mice (P < 0.05, t test), the MNCVs did not significantly change. Axonal sprouting in the (D) gastrocnemius and (E) triceps of type III SMA mice is a further sign of denervation. The former is an example of nodal sprouting whereas the latter depicts terminal sprouts (red arrows). Arrow in white indicates main axonal branch. Motor axons are silver stained in black while neuromuscular junctions stain blue. Normal gastrocnemius (F) shows at least three different motor axons, each innervating a separate muscle fiber (running diagonally from lower left to upper right; magnification 400×). Graphical representation (G) of the extent of sprouting (terminal and nodal) in type III SMA mice and unaffected littermates. Data are given as mean ± SD.
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fig5: Electromyography and axonal sprouting in mild SMN mice. EMG recordings in (A) cranial tibial and (B) suprascapular muscle of 4-mo-old type III SMA mice. Spontaneous abnormal electrical activity was recorded in the form of fibrillation potentials (dark arrows) and occasional biphasic positive sharp waves (red arrow). (C) Quantification of MNCVs and the amplitudes of compound muscle action potentials in type III SMA mice and normal (Smn+/+) littermates. At least four mice from each group were analyzed. Data are given as mean ± SD. Although the amplitude was reduced in SMA mice (P < 0.05, t test), the MNCVs did not significantly change. Axonal sprouting in the (D) gastrocnemius and (E) triceps of type III SMA mice is a further sign of denervation. The former is an example of nodal sprouting whereas the latter depicts terminal sprouts (red arrows). Arrow in white indicates main axonal branch. Motor axons are silver stained in black while neuromuscular junctions stain blue. Normal gastrocnemius (F) shows at least three different motor axons, each innervating a separate muscle fiber (running diagonally from lower left to upper right; magnification 400×). Graphical representation (G) of the extent of sprouting (terminal and nodal) in type III SMA mice and unaffected littermates. Data are given as mean ± SD.

Mentions: To further characterize the muscle weakness in our type III SMA mice, EMG recordings were made on 4–6-mo-old SMN2;Smn−/− animals heterozygous for the mutant SMN A2G transgene. Recordings were made on resting muscle as it is difficult to obtain controlled, voluntary muscle contraction in mice. Normal muscle is electrically silent at rest and no spontaneous activity is detected after cessation of movement of the recording needle. In both of the type III (1SMN A2G;SMN2;Smn−/−) animals we tested, we found abnormal spontaneous activity of single muscle fibers (fibrillation potentials) and of motor units (fasciculation potentials) accompanied occasionally by biphasic positive sharp waves (Fig. 5, A and B). These observations made on multiple pelvic (cranial tibial, vastus lateralis, and gastrocnemius; see Fig. S2, available online at http://www.jcb.org/cgi/content/full/jcb.200208079/DC1) and thoracic (suprascapular) muscles are a clear indication of denervation and provide a simple diagnosis of this process, as previously demonstrated in human patients (Dubowitz, 1995). We did not see abnormal electrical activity in age-matched type III SMA animals homozygous for the A2G transgene, indicating that increased levels of SMN from the A2G transgene correct this abnormality. This is consistent with data from some very mildly affected type III SMA patients (Hausmanowa-Petrusewicz and Karwanska, 1986). To assess the nature of the muscle weakness in mild SMA mice, we analyzed evoked compound action potentials and motor nerve conduction velocities (MNCVs) of the tibial nerve (Fig. 5 C). Although nerve conduction velocity tests on type III SMA (30.1 ± 5.6; n = 4) mice did not show a significant difference from those on age-matched controls (31.6 ± 3.3; n = 4), the amplitudes of the evoked muscle potentials were reduced (SMA, 4.3 ± 5.5, n = 4; normals, 15.07 ± 8.9, n = 4). These results indicate that demyelination of nerves is not a feature of SMA. Instead, it is likely that defects in neuronal axons contribute to SMA pathology in mildly affected mice.


A transgene carrying an A2G missense mutation in the SMN gene modulates phenotypic severity in mice with severe (type I) spinal muscular atrophy.

Monani UR, Pastore MT, Gavrilina TO, Jablonka S, Le TT, Andreassi C, DiCocco JM, Lorson C, Androphy EJ, Sendtner M, Podell M, Burghes AH - J. Cell Biol. (2003)

Electromyography and axonal sprouting in mild SMN mice. EMG recordings in (A) cranial tibial and (B) suprascapular muscle of 4-mo-old type III SMA mice. Spontaneous abnormal electrical activity was recorded in the form of fibrillation potentials (dark arrows) and occasional biphasic positive sharp waves (red arrow). (C) Quantification of MNCVs and the amplitudes of compound muscle action potentials in type III SMA mice and normal (Smn+/+) littermates. At least four mice from each group were analyzed. Data are given as mean ± SD. Although the amplitude was reduced in SMA mice (P < 0.05, t test), the MNCVs did not significantly change. Axonal sprouting in the (D) gastrocnemius and (E) triceps of type III SMA mice is a further sign of denervation. The former is an example of nodal sprouting whereas the latter depicts terminal sprouts (red arrows). Arrow in white indicates main axonal branch. Motor axons are silver stained in black while neuromuscular junctions stain blue. Normal gastrocnemius (F) shows at least three different motor axons, each innervating a separate muscle fiber (running diagonally from lower left to upper right; magnification 400×). Graphical representation (G) of the extent of sprouting (terminal and nodal) in type III SMA mice and unaffected littermates. Data are given as mean ± SD.
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Related In: Results  -  Collection

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fig5: Electromyography and axonal sprouting in mild SMN mice. EMG recordings in (A) cranial tibial and (B) suprascapular muscle of 4-mo-old type III SMA mice. Spontaneous abnormal electrical activity was recorded in the form of fibrillation potentials (dark arrows) and occasional biphasic positive sharp waves (red arrow). (C) Quantification of MNCVs and the amplitudes of compound muscle action potentials in type III SMA mice and normal (Smn+/+) littermates. At least four mice from each group were analyzed. Data are given as mean ± SD. Although the amplitude was reduced in SMA mice (P < 0.05, t test), the MNCVs did not significantly change. Axonal sprouting in the (D) gastrocnemius and (E) triceps of type III SMA mice is a further sign of denervation. The former is an example of nodal sprouting whereas the latter depicts terminal sprouts (red arrows). Arrow in white indicates main axonal branch. Motor axons are silver stained in black while neuromuscular junctions stain blue. Normal gastrocnemius (F) shows at least three different motor axons, each innervating a separate muscle fiber (running diagonally from lower left to upper right; magnification 400×). Graphical representation (G) of the extent of sprouting (terminal and nodal) in type III SMA mice and unaffected littermates. Data are given as mean ± SD.
Mentions: To further characterize the muscle weakness in our type III SMA mice, EMG recordings were made on 4–6-mo-old SMN2;Smn−/− animals heterozygous for the mutant SMN A2G transgene. Recordings were made on resting muscle as it is difficult to obtain controlled, voluntary muscle contraction in mice. Normal muscle is electrically silent at rest and no spontaneous activity is detected after cessation of movement of the recording needle. In both of the type III (1SMN A2G;SMN2;Smn−/−) animals we tested, we found abnormal spontaneous activity of single muscle fibers (fibrillation potentials) and of motor units (fasciculation potentials) accompanied occasionally by biphasic positive sharp waves (Fig. 5, A and B). These observations made on multiple pelvic (cranial tibial, vastus lateralis, and gastrocnemius; see Fig. S2, available online at http://www.jcb.org/cgi/content/full/jcb.200208079/DC1) and thoracic (suprascapular) muscles are a clear indication of denervation and provide a simple diagnosis of this process, as previously demonstrated in human patients (Dubowitz, 1995). We did not see abnormal electrical activity in age-matched type III SMA animals homozygous for the A2G transgene, indicating that increased levels of SMN from the A2G transgene correct this abnormality. This is consistent with data from some very mildly affected type III SMA patients (Hausmanowa-Petrusewicz and Karwanska, 1986). To assess the nature of the muscle weakness in mild SMA mice, we analyzed evoked compound action potentials and motor nerve conduction velocities (MNCVs) of the tibial nerve (Fig. 5 C). Although nerve conduction velocity tests on type III SMA (30.1 ± 5.6; n = 4) mice did not show a significant difference from those on age-matched controls (31.6 ± 3.3; n = 4), the amplitudes of the evoked muscle potentials were reduced (SMA, 4.3 ± 5.5, n = 4; normals, 15.07 ± 8.9, n = 4). These results indicate that demyelination of nerves is not a feature of SMA. Instead, it is likely that defects in neuronal axons contribute to SMA pathology in mildly affected mice.

Bottom Line: We suggest that only in the presence of low levels of full-length SMN is the A2G transgene able to form partially functional higher order SMN complexes essential for its functions.Animals homozygous for the mutant transgene are less severely affected than heterozygotes.This demonstrates the importance of SMN levels in SMA even if the protein is expressed from a mutant allele.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Ohio State University, Columbus, OH 43210, USA. monani.2@osu.edu

ABSTRACT
5q spinal muscular atrophy (SMA) is a common autosomal recessive disorder in humans and the leading genetic cause of infantile death. Patients lack a functional survival of motor neurons (SMN1) gene, but carry one or more copies of the highly homologous SMN2 gene. A homozygous knockout of the single murine Smn gene is embryonic lethal. Here we report that in the absence of the SMN2 gene, a mutant SMN A2G transgene is unable to rescue the embryonic lethality. In its presence, the A2G transgene delays the onset of motor neuron loss, resulting in mice with mild SMA. We suggest that only in the presence of low levels of full-length SMN is the A2G transgene able to form partially functional higher order SMN complexes essential for its functions. Mild SMA mice exhibit motor neuron degeneration, muscle atrophy, and abnormal EMGs. Animals homozygous for the mutant transgene are less severely affected than heterozygotes. This demonstrates the importance of SMN levels in SMA even if the protein is expressed from a mutant allele. Our mild SMA mice will be useful in (a) determining the effect of missense mutations in vivo and in motor neurons and (b) testing potential therapies in SMA.

Show MeSH
Related in: MedlinePlus