Characterization of mutant SMN and development of mutant SMN transgenic mice.

摘要

Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disease. Loss of the survival motor neuron (SMN1) gene, in the presence of the SMN2 gene causes SMA. Ninety-five percent of SMA patients have a loss of SMN1 and in the majority of the remaining cases, a small mutation in the SMN1 gene occurs. SMN functions in snRNP assembly in all cell types, however, it is unclear how this function results in specifically motor neuron cell death. In this thesis, the properties of various mutations of SMN are investigated in both in vitro studies and in transgenic mouse models to determine the function of SMN important in SMA.;GST-fusion binding assays were used to determine the ability of each mutant SMN to bind itself, full-length SMN or Sm proteins, which form the core of snRNP complexes. All of these properties are necessary for SMN to efficiently perform snRNP biogenesis. The ability of each mutation to rescue axonal defects in zebrafish embryos that occur with knockdown of endogenous smn with morpholinos was investigated. Of the mutations studied, two were striking: SMN(A111G) and SMN(VDQNQKE). SMN(A111G) was able to bind itself and Sm proteins, thus making it able to form snRNP complexes; however, this mutation failed to rescue axonal defects in zebrafish. SMN(VDQNQKE), in contrast, was unable to bind to itself or Sm proteins, but yet was able to rescue axonal defects. These data seemed to indicate that snRNP assembly and the SMA phenotype were independent of each other.;To further investigate these two mutations, transgenic mice were developed. Lack of endogenous mouse SMN (Smn) in mice results in embryonic lethality. Introduction of 2 copies of human SMN2 results in a mouse with severe SMA, while 1 copy of SMN2 is insufficient to overcome embryonic lethality. In this thesis, it is shown that SMN(A111G), an allele capable of snRNP assembly, can rescue mice that lack Smn and contain either one or two copies of SMN2 (SMA mice). In contrast, SMN(VDQNQKE) did not result in sufficient SMN protein and did not rescue SMA animals. The correction of SMA in these animals was directly correlated with snRNP assembly activity in spinal cord, as was correction of snRNA levels. Also, re-investigation of these mutations in the zebrafish assay revealed that increased amounts of SMN(A111G) could rescue axonal defects; whereas, decreased amounts of SMN(VDQNQKE) resulted in fish with increased axonal defects. These data support snRNP assembly as being the critical function affected in SMA and suggests that the levels of snRNPs are, indeed, critical to motor neurons.;Furthermore, SMN(A111G) cannot rescue Smn-/- mice without SMN2 suggesting that both SMN(A111G) and SMN2 undergo intragenic complementation in vivo to function in heteromeric complexes that have greater function than either allele alone. The oligomer composed of limiting full-length SMN and SMN(A111G) has substantial snRNP assembly activity. Also, the SMN(A2G) and SMN(A111G) alleles in vivo did not complement each other leading to the possibility that these mutations could affect the same function. Lastly, transgenic mice containing the SMN(I116F) missense mutation were developed. SMN(I1116F) is capable of binding itself, but has lowered ability to bind Sm proteins and thus, does not form snRNP complexes efficiently. It is expected that this mutation will not be able to rescue mice and further support the connection of snRNP assembly and SMA.