Genetic Suppression of SMN Mutations in Spinal Muscular Atrophy

脊髓性肌萎缩症是一种毁灭性的神经肌肉疾病，由 SMN 蛋白不足引起。 SMA 是由 SMN1 基因缺失或突变以及 SMN2 基因保留引起的。 SMN2 基因是 表型修饰剂，其中较轻的 SMA 病例具有更多 SMN2 拷贝。 SMA 患者很少有 SMN1 基因错义突变。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 SMA 错义突变不起作用 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，我们还有 表明 N 和 C 端 SMN 错义突变可以相互补充并挽救 snRNP 组装 小鼠体内完全缺乏全长野生型 SMN 蛋白。我们开发了细胞系 有条件地去除功能性 SMN，以便我们在培养物中测试 SMN 错义突变。我们也用过 该细胞系用于测试 SMNE134K 突变的抑制因子。我们在 SmF 中发现了一个抑制器 完全恢复因 SMN E134K 突变而丢失的 snRNP 组装的蛋白质。我们现在有一个系统可以 筛选 SMN 错义突变的抑制子。在本提案中，我们将测试我们拥有的 SmF 抑制器 在两种不同的 SMN E134K 小鼠模型中发现，并确定该突变是否可以挽救 SMA 表型 和 SMA 小鼠的存活率。因此，我们可以从snRNP组装中研究SMN的单独功能 SMN 在轴突中的功能。我们将使用其他 SMN 患者衍生的其他抑制器进行筛选 突变来测试 SMN 的其他功能域。我们将研究 SMN 在轴突独立中的作用 通过 scAAV9 将 HuD 和截短形式的 SMN 引入 SMA 小鼠中，从而实现 Sm 组装。我们有 在细胞中显示 Smn 外显子 2B 不是细胞存活所必需的。我们将在 SMA 小鼠中测试 scAAV9-SmnΔ2 证实这一发现并挽救 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 剪接和轴突，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.