The Spinal Muscular Atrophy NMJ phenotype: mechanisms and molecular mediators

脊髓性肌萎缩症是一种常见的，reckless遗传，儿科神经肌肉疾病引起的， 运动神经元存活1（SMN 1）基因的突变和SMN蛋白的缺陷。SMN是 普遍表达，并报告在RNA加工中发挥关键作用，通过协调生物合成， 剪接体小核核糖核蛋白（snRNP）颗粒。这些粒子的聚集是严重的 在SMA模型小鼠中受损。恢复SMN的突变体不仅纠正了这一缺陷， 挽救SMA表型。然而，SMN在snRNP组装中的作用，这是所有人都需要的。 细胞，一直难以与SMA的选择性神经肌肉疾病表型特征相一致。 解释这一难题的一种方法是认为，在一个或多个细胞中选择性表达的转录本， 神经肌肉系统由于SMN的管家功能的缺陷而不能被适当地处理。 或者，选择性SMA表型可能源于运动单位中的新型SMN功能。在这 我们希望解决每一个可能性的项目。在该项目的目标1中，我们将确定神经元聚集蛋白， 在SMA运动神经元中发现了错误剪接，推测是snRNP缺陷的结果 生物发生，是SMA表型的真正介质。已知神经元聚集蛋白对于神经元的生长和发育是重要的。 神经肌肉突触的发育，这些结构在SMA中受到深刻影响。为了测试 为了研究聚集蛋白和SMA表型之间的联系，我们将转基因选择性地将蛋白质恢复到马达， SMA模型小鼠的神经元。然后，我们将评估聚集蛋白在小鼠中的补充的结果， 分子、细胞和表型水平。在项目的目标2中，我们将识别转录/剪接改变 在SMA运动神经元中，在定义神经肌肉突触成熟的关键时间窗口期间。这 实验利用了一种新的他莫昔芬诱导的SMN敲低小鼠，我们已经 开发，并利用新的发现表明，SMN蛋白的需求是最大的，当 神经肌肉突触成熟。在SMN急性耗竭之前或之后立即 神经肌肉突触成熟后，我们将对突变体中运动神经元基因表达的变化进行编目， 对照这种方法补充了目标1，但对任何一个基因都是无偏的，它将揭示 这些分子在神经肌肉突触的成熟中是重要的， SMA。其中一些分子改变最终可能指向新的，疾病相关的和表型- 蛋白质的特殊功能。该项目的集体结果将导致对疾病的新见解， 其最佳治疗方法尚未开发，其表型继续困扰科学家， 根据目前对SMN蛋白的了解。

Spinal muscular atrophy is a common, recessively inherited, pediatric neuromuscular disorder caused by mutations in the Survival of Motor Neuron 1 (SMN1) gene and a deficiency of the SMN protein. SMN is ubiquitously expressed and reported to play a critical role in RNA processing, by orchestrating the biogenesis of spliceosomal small nuclear ribonucleoprotein (snRNP) particles. The assembly of these particles is severely compromised in SMA model mice. Restoring SMN to the mutants not only corrects this defect but also fully rescues the SMA phenotype. Nevertheless, SMN’s role in snRNP assembly, which is a requirement of all cells, has been difficult to reconcile with the selective neuromuscular disease phenotype characteristic of SMA. One way to explain this conundrum is to suggest that transcripts selectively expressed in one or more cells of the neuromuscular system fail to be properly processed owing to defects in SMN’s housekeeping function. Alternatively, the selective SMA phenotype could stem from novel SMN functions in the motor unit. In this project we wish to address each possibility. In aim 1 of the project we will determine if neuronal agrin, which was found to be mis-spliced in SMA motor neurons, presumably as a consequence of defects in snRNP biogenesis, is a true mediator of the SMA phenotype. Neuronal agrin is known to be important for the development of neuromuscular synapses, structures that are profoundly affected in SMA. To test possible links between agrin and the SMA phenotype, we will transgenically restore the protein selectively to the motor neurons of SMA model mice. We will then assess the consequences of agrin repletion in the mice at the molecular, cellular and phenotypic levels. In aim 2 of the project we will identify transcriptional/splice alterations in SMA motor neurons during a critical window of time that defines neuromuscular synapse maturation. This experiment takes advantage of a novel line of tamoxifen-induced SMN knockdown mice that we have developed, and exploits new findings suggesting that the requirements for the SMN protein are greatest when neuromuscular synapses mature. Following acute depletion of SMN prior to or immediately after neuromuscular synapses mature, we will catalogue motor neuronal gene expression changes in mutants and controls. This approach which complements Aim 1, but is unbiased with respect to any one gene, will uncover molecules that are important in the maturation of the neuromuscular synapses, a process that is disrupted in SMA. Some of these molecular alterations may eventually point to novel, disease-relevant and phenotype- specific functions of the protein. The collective results of the project will lead to new insights into a disease for which an optimal treatment has yet to be developed, and whose phenotype continues to puzzle scientists in light of what is currently known about the SMN protein.