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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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In vitro binding studies on SMN A2G. (A) Self-association studies between 35S-labeled SMN proteins and bound GST–SMN fusion proteins. (B) The interaction of SMN A2G, FL-SMN, and Δ7 SMN each with FL-SMN. (C) Binding of FL-SMN, SMN A2G, or Δ7 SMN (as indicated) with the Sm protein SmN. In all three studies, binding values were calculated as a % of 35S-labeled SMN A2G, FL-SMN, or Δ7 SMN.
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fig2: In vitro binding studies on SMN A2G. (A) Self-association studies between 35S-labeled SMN proteins and bound GST–SMN fusion proteins. (B) The interaction of SMN A2G, FL-SMN, and Δ7 SMN each with FL-SMN. (C) Binding of FL-SMN, SMN A2G, or Δ7 SMN (as indicated) with the Sm protein SmN. In all three studies, binding values were calculated as a % of 35S-labeled SMN A2G, FL-SMN, or Δ7 SMN.

Mentions: SMN self-associates using domains encoded by exons 2b and 6 (Lorson et al., 1998; Young et al., 2001). We investigated the effect of the missense A2G mutation on self-association. A GST–SMN A2G fusion protein was immobilized on glutathione-linked agarose beads and then incubated with 35S-labeled SMN A2G protein. Bound fractions were washed extensively and resolved by SDS-PAGE. As controls, similar self-association studies were performed with full-length SMN and Δ7 SMN. Our results show that SMN A2G molecules self-associate with an efficiency between that of full-length SMN and Δ7 SMN (Fig. 2 A) as do other mild SMA mutations (Lorson et al., 1998). This is consistent with the mild SMA phenotype we see in patients carrying this mutation and our SMN A2G;SMN2;Smn−/− mice. To determine the effect of the A2G mutation on the ability of the mutant protein to bind native full-length (FL) SMN and Sm proteins, GST–SMN and GST–SmN fusion proteins immobilized on agarose beads were incubated with labeled SMN A2G. Bound fractions were washed as described above. As a comparison, the ability of Δ7 SMN and FL-SMN each to bind to SmN and native SMN was assessed. As expected, SMN A2G binds FL-SMN with a higher affinity than does Δ7 SMN but with a lower affinity than does native SMN (Fig. 2 B). Interestingly, the ability of SMN A2G to bind the Sm protein SmN was greatly decreased even though the mutation does not lie in the Sm binding domain (Fig. 2 C).


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)

In vitro binding studies on SMN A2G. (A) Self-association studies between 35S-labeled SMN proteins and bound GST–SMN fusion proteins. (B) The interaction of SMN A2G, FL-SMN, and Δ7 SMN each with FL-SMN. (C) Binding of FL-SMN, SMN A2G, or Δ7 SMN (as indicated) with the Sm protein SmN. In all three studies, binding values were calculated as a % of 35S-labeled SMN A2G, FL-SMN, or Δ7 SMN.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: In vitro binding studies on SMN A2G. (A) Self-association studies between 35S-labeled SMN proteins and bound GST–SMN fusion proteins. (B) The interaction of SMN A2G, FL-SMN, and Δ7 SMN each with FL-SMN. (C) Binding of FL-SMN, SMN A2G, or Δ7 SMN (as indicated) with the Sm protein SmN. In all three studies, binding values were calculated as a % of 35S-labeled SMN A2G, FL-SMN, or Δ7 SMN.
Mentions: SMN self-associates using domains encoded by exons 2b and 6 (Lorson et al., 1998; Young et al., 2001). We investigated the effect of the missense A2G mutation on self-association. A GST–SMN A2G fusion protein was immobilized on glutathione-linked agarose beads and then incubated with 35S-labeled SMN A2G protein. Bound fractions were washed extensively and resolved by SDS-PAGE. As controls, similar self-association studies were performed with full-length SMN and Δ7 SMN. Our results show that SMN A2G molecules self-associate with an efficiency between that of full-length SMN and Δ7 SMN (Fig. 2 A) as do other mild SMA mutations (Lorson et al., 1998). This is consistent with the mild SMA phenotype we see in patients carrying this mutation and our SMN A2G;SMN2;Smn−/− mice. To determine the effect of the A2G mutation on the ability of the mutant protein to bind native full-length (FL) SMN and Sm proteins, GST–SMN and GST–SmN fusion proteins immobilized on agarose beads were incubated with labeled SMN A2G. Bound fractions were washed as described above. As a comparison, the ability of Δ7 SMN and FL-SMN each to bind to SmN and native SMN was assessed. As expected, SMN A2G binds FL-SMN with a higher affinity than does Δ7 SMN but with a lower affinity than does native SMN (Fig. 2 B). Interestingly, the ability of SMN A2G to bind the Sm protein SmN was greatly decreased even though the mutation does not lie in the Sm binding domain (Fig. 2 C).

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