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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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Expression analysis of the mutant SMN A2G transgene in transgenic mice. (A) RT-PCR on RNA from indicated tissues of an SMN A2G;Smn+/+ animal was performed as described in the Materials and methods. The β-actin gene was simultaneously amplified as a control. (B) Western blot analysis on 5-d-old type I SMA, type III SMA, and an Smn+/− mouse was performed using the anti-SMN monoclonal antibody, MANSMA2. The blot was subsequently stripped and probed with an anti–β-actin antibody to control for loading amounts. Type I SMA mice produce very little SMN, which is visible only after longer exposures. (C) Western blot analysis on 3.5-mo-old type III SMA mice (heterozygous for the A2G transgene, Het.), type III SMA mice (homozygous for the A2G transgene, Hom.), and normal mice (Smn+/−) using the anti-SMN monoclonal antibody, MANSMA2. β-actin or β-tubulin serve as loading controls.
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fig3: Expression analysis of the mutant SMN A2G transgene in transgenic mice. (A) RT-PCR on RNA from indicated tissues of an SMN A2G;Smn+/+ animal was performed as described in the Materials and methods. The β-actin gene was simultaneously amplified as a control. (B) Western blot analysis on 5-d-old type I SMA, type III SMA, and an Smn+/− mouse was performed using the anti-SMN monoclonal antibody, MANSMA2. The blot was subsequently stripped and probed with an anti–β-actin antibody to control for loading amounts. Type I SMA mice produce very little SMN, which is visible only after longer exposures. (C) Western blot analysis on 3.5-mo-old type III SMA mice (heterozygous for the A2G transgene, Het.), type III SMA mice (homozygous for the A2G transgene, Hom.), and normal mice (Smn+/−) using the anti-SMN monoclonal antibody, MANSMA2. β-actin or β-tubulin serve as loading controls.

Mentions: To assess the expression of the SMN A2G transgene, RT-PCR studies (Fig. 3 A) on A2G animals lacking SMN2 and Western blot analyses on mild SMA animals (Figs. 3, B and C) were performed. Our data show that the transgene is expressed in all of the tissues examined. This is similar to the expression of the endogenous gene, suggesting that the 3.4-kb promoter fragment used here contains the necessary elements for ubiquitous expression. Western blot analysis performed on SMN A2G;SMN2;Smn−/− animals shows that mild SMA mice express significantly higher SMN levels than do age-matched type I SMA mice, but not as much as normal littermates (Fig. 3 B). Furthermore, type III SMA animals homozygous for the A2G transgene express higher levels of the SMN protein than do heterozygous animals (Fig. 3 C). This is consistent with previous data showing that a tight correlation exists between SMN levels and phenotypic severity in SMA.


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)

Expression analysis of the mutant SMN A2G transgene in transgenic mice. (A) RT-PCR on RNA from indicated tissues of an SMN A2G;Smn+/+ animal was performed as described in the Materials and methods. The β-actin gene was simultaneously amplified as a control. (B) Western blot analysis on 5-d-old type I SMA, type III SMA, and an Smn+/− mouse was performed using the anti-SMN monoclonal antibody, MANSMA2. The blot was subsequently stripped and probed with an anti–β-actin antibody to control for loading amounts. Type I SMA mice produce very little SMN, which is visible only after longer exposures. (C) Western blot analysis on 3.5-mo-old type III SMA mice (heterozygous for the A2G transgene, Het.), type III SMA mice (homozygous for the A2G transgene, Hom.), and normal mice (Smn+/−) using the anti-SMN monoclonal antibody, MANSMA2. β-actin or β-tubulin serve as loading controls.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Expression analysis of the mutant SMN A2G transgene in transgenic mice. (A) RT-PCR on RNA from indicated tissues of an SMN A2G;Smn+/+ animal was performed as described in the Materials and methods. The β-actin gene was simultaneously amplified as a control. (B) Western blot analysis on 5-d-old type I SMA, type III SMA, and an Smn+/− mouse was performed using the anti-SMN monoclonal antibody, MANSMA2. The blot was subsequently stripped and probed with an anti–β-actin antibody to control for loading amounts. Type I SMA mice produce very little SMN, which is visible only after longer exposures. (C) Western blot analysis on 3.5-mo-old type III SMA mice (heterozygous for the A2G transgene, Het.), type III SMA mice (homozygous for the A2G transgene, Hom.), and normal mice (Smn+/−) using the anti-SMN monoclonal antibody, MANSMA2. β-actin or β-tubulin serve as loading controls.
Mentions: To assess the expression of the SMN A2G transgene, RT-PCR studies (Fig. 3 A) on A2G animals lacking SMN2 and Western blot analyses on mild SMA animals (Figs. 3, B and C) were performed. Our data show that the transgene is expressed in all of the tissues examined. This is similar to the expression of the endogenous gene, suggesting that the 3.4-kb promoter fragment used here contains the necessary elements for ubiquitous expression. Western blot analysis performed on SMN A2G;SMN2;Smn−/− animals shows that mild SMA mice express significantly higher SMN levels than do age-matched type I SMA mice, but not as much as normal littermates (Fig. 3 B). Furthermore, type III SMA animals homozygous for the A2G transgene express higher levels of the SMN protein than do heterozygous animals (Fig. 3 C). This is consistent with previous data showing that a tight correlation exists between SMN levels and phenotypic severity in SMA.

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