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Characterization of three SMN missense mutations using mouse models of Spinal Muscular Atrophy

Madabusi, Narasimhan Kandaye

Abstract Details

2012, Master of Science, Ohio State University, Molecular, Cellular and Developmental Biology.

Spinal Muscular Atrophy (SMA) is an autosomal recessive neurodegenerative disease characterized by loss of lower motor neurons and muscle atrophy. It is caused due to reduced levels of the ubiquitously expressed Survival Motor Neuron (SMN) protein. Humans have two SMN genes – SMN1 which produces full length SMN and SMN2 which mainly produces a truncated unstable protein and a small amount of full length SMN (~10%). SMN is a multi-functional protein with a known role in snRNP assembly. To date, it is still not known why the lower motor neurons are specifically affected in SMA although SMN is expressed in all tissues.

Several missense mutations of SMN1 have been studied to understand the mechanism of SMA. One such synthetic mutation which affects the first glutamine (Q) of the QNQKE motif present in exon 7 is Q282A. A previous study has shown that this mutant construct cannot rescue the axonal defects in a zebrafish model of SMA. Since in vitro assays have suggested that SMN-Q282A can function in snRNP assembly, the authors proposed that restoring snRNP assembly function of SMN was not sufficient to rescue SMA.

To analyze this mutant further, we generated transgenic mice carrying SMN-Q282A and crossed them to generate SMA mice with the following genotype: SMN-Q282A;SMN2;mSmn-/-. Currently, we have 25 such rescue mice and they show no physical or behavioral abnormality when compared to normal mice. This indicates that SMN-Q282A acts to complement the full length SMN produced by SMN2. However, as in previous studies with SMN missense mutations, the SMN-Q282A could not rescue a mSmn-/- mice. This is to be expected since no single mutant form of SMN has been able to rescue a mSmn-/- mouse by itself. These results suggest that snRNP assembly plays an important role in SMA and that the axonal defects in fish do not correlate with the critical function of SMN in SMA. We are currently performing snRNP assembly assays on brain and spinal cord lysates of these rescue mice. This will confirm our hypothesis that restoring snRNP assembly is sufficient to rescue SMA mice.

Similar experiments were done with SMN-I116F, a mutation seen in SMA Type I patients. Initial results show that this mutant construct can provide only minimal complementation to SMN2. SMA mice of genotype SMN-I116F;SMN2+/+;mSmn-/- could survive only for 16 days, similar to the SMND7 SMA mice. The effect of another mutation SMN-D44V, seen in a Type III patient, was studied using scAAV9 vector. The viral vector carrying the SMN-D44V mutant was injected via the facial vein into SMA neonate pups at day 1. Partial recovery was seen with survival of the mice extended to 35±2 days. Transgenic mice having the SMN-D44V mutant have been generated and we expect them to fully complement SMN2, being a milder allele. Further experiments are being carried out to characterize these two transgenes, which will give us more input as to how different mutations affect the functioning of the SMN complex and its composition.

Arthur Burghes, Dr (Advisor)
Brian Kaspar, Dr (Committee Member)
Denis Guttridge, Dr (Committee Member)

Recommended Citations

Citations

  • Madabusi, N. K. (2012). Characterization of three SMN missense mutations using mouse models of Spinal Muscular Atrophy [Master's thesis, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1339442849

    APA Style (7th edition)

  • Madabusi, Narasimhan. Characterization of three SMN missense mutations using mouse models of Spinal Muscular Atrophy. 2012. Ohio State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1339442849.

    MLA Style (8th edition)

  • Madabusi, Narasimhan. "Characterization of three SMN missense mutations using mouse models of Spinal Muscular Atrophy." Master's thesis, Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1339442849

    Chicago Manual of Style (17th edition)