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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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Motor neuron cell bodies and ventral roots in mild SMA mice. Motor neuron degeneration in the (A) facial nucleus and (C) lumbar spinal cord of a 3.5-mo-old type III SMA mouse showing fewer cells with intact nuclei compared with the normal Smn+/− control in B and D, respectively. An examination of the motor axons in the ventral roots of (E) type III SMA mice by toluidine blue staining shows a myelin ovoid and numerous axons undergoing Wallerian degeneration (arrows). F represents an age-matched normal control (magnification 600×).
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fig6: Motor neuron cell bodies and ventral roots in mild SMA mice. Motor neuron degeneration in the (A) facial nucleus and (C) lumbar spinal cord of a 3.5-mo-old type III SMA mouse showing fewer cells with intact nuclei compared with the normal Smn+/− control in B and D, respectively. An examination of the motor axons in the ventral roots of (E) type III SMA mice by toluidine blue staining shows a myelin ovoid and numerous axons undergoing Wallerian degeneration (arrows). F represents an age-matched normal control (magnification 600×).

Mentions: To assess motor neuron degeneration in 3.5-mo-old SMN2;Smn−/− type III SMA mice heterozygous for the A2G transgene, transverse sections of the spinal lumbar region (unpublished data) and coronal sections of the facial nucleus (Fig. 6, A and B) were stained with cresyl echt violet and the morphology and numbers of the cells determined. The most obvious difference between the SMA mice and age-matched controls is a significant decrease in the numbers of motor neurons in the former. In the lumbar spinal cord, SMA mice have 29% fewer motor neurons than age-matched Smn+/− controls, while in the facial nucleus there is a ∼19% loss of motor neurons (Table I; Fig. 6, A and B). Previously, we showed a significant loss of motor neurons in the spinal cord and brain stem of 5-d-old severe SMA mice (Monani et al., 2000). To determine whether motor neuron loss occurs this early in type III SMA mice, 5-d-old SMN2;Smn−/− animals heterozygous for the A2G transgene were killed and motor neuron cell bodies in the facial nucleus counted. We found no evidence of motor neuron loss at this age in type III SMA mice (unpublished data). This is consistent with our previous finding that motor neuron loss is a late event in SMA. To further examine motor neuron degeneration, we also conducted ventral root counts on our type III SMA mice. Mice perfused with 4% paraformaldehyde, 1% glutaraldehyde were used to isolate the ventral roots from the L1–L5 lumbar spinal cord region. Semi-thin sections were cut, stained with toluidene blue (Fig. 6, C and D), and at least four different fields examined to quantitatively determine the number of motor axons in the roots of diseased mice and an age-matched control. Two striking differences were observed. First, type III SMA mice have fewer myelinated axons (Table II). The loss in motor axons correlates with the loss of motor neuron cell bodies in the spinal cord. Second, many of the remaining axons in the diseased mice are shriveled and exhibit signs of Wallerian degeneration (Fig. 6 D).


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)

Motor neuron cell bodies and ventral roots in mild SMA mice. Motor neuron degeneration in the (A) facial nucleus and (C) lumbar spinal cord of a 3.5-mo-old type III SMA mouse showing fewer cells with intact nuclei compared with the normal Smn+/− control in B and D, respectively. An examination of the motor axons in the ventral roots of (E) type III SMA mice by toluidine blue staining shows a myelin ovoid and numerous axons undergoing Wallerian degeneration (arrows). F represents an age-matched normal control (magnification 600×).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172739&req=5

fig6: Motor neuron cell bodies and ventral roots in mild SMA mice. Motor neuron degeneration in the (A) facial nucleus and (C) lumbar spinal cord of a 3.5-mo-old type III SMA mouse showing fewer cells with intact nuclei compared with the normal Smn+/− control in B and D, respectively. An examination of the motor axons in the ventral roots of (E) type III SMA mice by toluidine blue staining shows a myelin ovoid and numerous axons undergoing Wallerian degeneration (arrows). F represents an age-matched normal control (magnification 600×).
Mentions: To assess motor neuron degeneration in 3.5-mo-old SMN2;Smn−/− type III SMA mice heterozygous for the A2G transgene, transverse sections of the spinal lumbar region (unpublished data) and coronal sections of the facial nucleus (Fig. 6, A and B) were stained with cresyl echt violet and the morphology and numbers of the cells determined. The most obvious difference between the SMA mice and age-matched controls is a significant decrease in the numbers of motor neurons in the former. In the lumbar spinal cord, SMA mice have 29% fewer motor neurons than age-matched Smn+/− controls, while in the facial nucleus there is a ∼19% loss of motor neurons (Table I; Fig. 6, A and B). Previously, we showed a significant loss of motor neurons in the spinal cord and brain stem of 5-d-old severe SMA mice (Monani et al., 2000). To determine whether motor neuron loss occurs this early in type III SMA mice, 5-d-old SMN2;Smn−/− animals heterozygous for the A2G transgene were killed and motor neuron cell bodies in the facial nucleus counted. We found no evidence of motor neuron loss at this age in type III SMA mice (unpublished data). This is consistent with our previous finding that motor neuron loss is a late event in SMA. To further examine motor neuron degeneration, we also conducted ventral root counts on our type III SMA mice. Mice perfused with 4% paraformaldehyde, 1% glutaraldehyde were used to isolate the ventral roots from the L1–L5 lumbar spinal cord region. Semi-thin sections were cut, stained with toluidene blue (Fig. 6, C and D), and at least four different fields examined to quantitatively determine the number of motor axons in the roots of diseased mice and an age-matched control. Two striking differences were observed. First, type III SMA mice have fewer myelinated axons (Table II). The loss in motor axons correlates with the loss of motor neuron cell bodies in the spinal cord. Second, many of the remaining axons in the diseased mice are shriveled and exhibit signs of Wallerian degeneration (Fig. 6 D).

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