The Spinal Muscular Atrophy NMJ phenotype: mechanisms and molecular mediators

脊髓性肌萎缩症是一种常见的、隐性遗传的儿科神经肌肉疾病，由 运动神经元存活1(SMN1)基因突变和SMN蛋白缺陷。SMN IS 无处不在地表达并被报道通过协调生物发生在RNA加工中发挥关键作用 剪接体小核糖核蛋白(SnRNP)颗粒。这些粒子的聚集严重地 在SMA模型小鼠中出现了妥协。将SMN恢复到突变体不仅纠正了这一缺陷，而且完全 挽救SMA表型。然而，SMN在SnRNP组装中的作用，这是所有人的要求 细胞，已经很难与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转录本在一个或多个细胞中选择性地表达 由于SMN的家务管理功能缺陷，神经肌肉系统无法得到适当的处理。 或者，选择性的SMA表型可能源于运动单位中新的SMN功能。在这 我们希望解决每一种可能性的项目。在项目的目标1中，我们将确定神经元集聚蛋白，即 被发现在SMA运动神经元中错误拼接，推测是由于SnRNP的缺陷造成的 生物发生，是SMA表型的真正中介。已知神经元集聚蛋白对 神经肌肉突触的发展，这是在SMA中受到深刻影响的结构。为了测试可能性 在集聚蛋白和SMA表型之间的联系，我们将有选择地将转基因蛋白质恢复到马达 SMA模型小鼠的神经元。然后，我们将评估聚集素在小鼠体内补充的后果 分子、细胞和表型水平。在项目的目标2中，我们将确定转录/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中。这 实验利用了我们现有的一种新的三苯氧胺诱导的SMN基因敲除小鼠 开发并利用新的发现，表明对SMN蛋白的需求在以下情况下最大 神经肌肉突触成熟。在SMN急性耗尽之前或之后 神经肌肉突触成熟，我们将记录突变体和突变体运动神经元基因表达的变化 控制。这种方法补充了目标1，但对任何一个基因都是公正的，将揭示 在神经肌肉突触成熟过程中至关重要的分子，这一过程在 SMA。其中一些分子变化最终可能指向新的、与疾病相关的表型- 蛋白质的特定功能。该项目的集体成果将导致对一种疾病的新见解 它的最佳治疗方法尚未开发出来，其表型继续困扰着科学家 了解目前已知的SMN蛋白。

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.