Genetic Suppression of SMN Mutations in Spinal Muscular Atrophy

脊髓性肌萎缩症是一种由SMN蛋白不足引起的破坏性神经肌肉疾病。 SMA是由SMN1基因的缺失或突变以及SMN2基因的保留引起的。SMN2基因是一种 表型修饰符，轻度SMA患者有更多的SMN2拷贝。很少有SMA患者有 SMN1基因的错义突变。我们可以利用这些突变和它们破坏的蛋白质结构域 研究SMN蛋白的功能。我们已经证明了SMA错义突变是不起作用的 但可以在某些全长野生型SMN蛋白存在的情况下发挥作用。此外，我们还拥有 研究表明，N端和C端SMN错义突变可以相互补充，挽救SnRNP组装 在完全缺乏小鼠全长野生型SMN蛋白的情况下。我们已经开发出一种细胞系 有条件地去除功能性SMN，以允许我们在培养中测试SMN错义突变。我们还使用了 对该细胞系进行SMNE134K突变抑制物检测。我们已经在SMF中发现了一个抑制者 完全修复因SMN E134K突变而丢失的SnRNP组装的蛋白质。我们现在有了一个系统来 筛选SMN错义突变的抑制子。在这个提案中，我们将测试我们已有的SMF抑制器 在两种不同的SMN E134K小鼠模型中发现，并确定该突变是否挽救了SMA表型 以及SMA小鼠的存活。因此，我们可以从以下方面研究SMN在SnRNP组装中的单独功能 SMN在轴突中的作用。我们将使用其他SMN患者来源的基因来筛选其他抑制子 突变以检测SMN的其他功能结构域。我们将研究SMN在轴突非依赖性中的作用 通过scAAV9将HUD和截短形式的SMN导入SMA小鼠，从而获得Sm组装。我们有 在细胞中显示SMN外显子2B不是细胞生存所必需的。我们将在SMA小鼠身上测试ScAAV9-SMN∆2以 证实这一发现，挽救SMA表型。最后，我们将测试Profilin在轴突功能中的作用 使用SMNS230L突变的SMA小鼠。利用基因突变，我们可以剖析SMN的功能 在剪接和轴突中，以解决SMN蛋白减少导致SMA的潜在机制。

Spinal Muscular Atrophy is a devastating neuromuscular disease caused by insufficient amounts of SMN protein. SMA is caused by loss or mutation of the SMN1 gene and retention of the SMN2 gene. The SMN2 gene is a modifier of phenotype where milder SMA cases having more copies of SMN2. Rarely SMA patients have a missense mutation in the SMN1 gene. We can use these mutations and the protein domains they disrupt to study the function of the SMN protein. We have shown that SMA missense mutations are not functional by themselves but can function in the presence of some full-length wild-type SMN protein. Furthermore, we have shown that N and C-terminal SMN missense mutations can complement each other and rescue snRNP assembly in the complete absence of full-length wild-type SMN protein in mice. We have developed cell line that conditionally removes functional SMN to allow us test SMN missense mutations in culture. We have also used this cell line to test for suppressors of the SMNE134K mutation. We have identified a suppressor in the SmF protein that fully restores snRNP assembly lost due to the SMN E134K mutation. We now have a system to screen for suppressors of SMN missense mutations. In this proposal we will test the SmF suppressor we have found in two different SMN E134K mouse models and determine if this mutation rescues the SMA phenotype and survival of the SMA mouse. Thus, we can study the separate functions of SMN in snRNP assembly from the function of SMN in the axon. We will screen for additional suppressors using other SMN patient derived mutations to test other functional domains of SMN. We will investigate the role of SMN in the axon independent of Sm assembly by introducing HuD and truncated forms of SMN into the SMA mice via scAAV9. We have shown in cells that Smn exon2B is not required for cell survival. We will test scAAV9-Smn∆2 in SMA mice to confirm this finding and rescue the SMA phenotype. Finally we will test the role of profilin in axonal function in the SMA mouse using the SMNS230L mutation. Using genetic mutations we can dissect the functions of SMN in splicing and in the axon to resolve the underlying mechanism by which reduced SMN protein causes SMA.