RNA Processing Defects in SMA and Their Contribution to the Disease Phenotype

 描述(申请人提供)：脊髓性肌萎缩症(SMA)是一种毁灭性的神经退行性疾病，是导致婴儿死亡的最常见的遗传原因。SMA是由于运动神经元(SMN)蛋白功能存活水平降低，导致神经肌肉连接处细胞自主缺陷、轴突变性和脊髓运动神经元丢失所致。广泛表达的SMN蛋白在所有组织中的剪接体小核核糖核蛋白(SnRNPs)的组装中具有重要的功能，但目前尚不清楚mRNA前剪接缺陷在多大程度上与SMA有关。这是该领域的一个中心问题，为什么低水平的SMN蛋白对脊髓运动神经元的影响比其他类型的细胞更严重。我们和其他人已经证明，SMN也存在于高度流动的多蛋白复合体中，这些复合体在培养神经元的轴突中沿着微管和肌动蛋白细丝活跃地运输。最近，我们发现培养的SMN缺陷运动神经元的轴突损害了特定的mRNA结合蛋白(MRBPs)和mRNAs在轴突中的定位，这些蛋白和mRNAs已知在轴突生长中发挥作用。这些发现使我们假设SMN在为轴突生长和维持服务的神经元过程中信使核糖核蛋白(MRNPs)的组装和运输中发挥关键作用。然而，这些SMN依赖的过程中的缺陷如何可能有助于SMA的发病机制仍不清楚。为了揭示SMA中的mRNA加工缺陷及其对疾病表型的贡献，我们提出了两个具体的目标：在目标1中，我们将通过详细而全面地分析轴突转录组和蛋白质组的差异来揭示疾病特异性的分子轴突缺陷。使用新的SMA患者干细胞来源的运动神经元和分区培养将使我们第一次能够全面地分类SMA在mRNA处理中的特定缺陷及其对轴突发育的影响，并确定修复这些缺陷的方法。这些研究将深入了解SMA患者干细胞来源的运动神经元中的mRNA处理缺陷，以及它们如何在体外导致轴突缺陷。在目标2中，我们将使用核糖体与核糖体的细胞类型特异性标记系统来彻底表征SMA模型小鼠脊髓运动神经元核糖体相关转录组的差异。我们将确定已知SMN靶标的轴突定位，并通过基于AAV9的病毒转导增强轴突mRNP定位的基因来挽救SMA小鼠的这些轴突缺陷。这些实验将首次能够评估SMA小鼠模型中脊髓运动神经元的mRNA处理缺陷的频谱，以及它们如何在体内对疾病表型做出贡献。这一建议有望增加我们对神经肌肉系统中人类病理学的理解，并促进针对SMA运动神经元和相关神经肌肉疾病的mRNA处理缺陷的治疗方法的开发。

 DESCRIPTION (provided by applicant): Spinal muscular atrophy (SMA) is a devastating neurodegenerative disease that represents the most common genetic cause of infant death. SMA is caused by reduced levels of functional survival of motor neuron (SMN) protein, leading to cell autonomous defects at the neuromuscular junctions, axon degeneration, and loss of motor neurons in the spinal cord. The ubiquitously expressed SMN protein has a well characterized essential function in the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs) in all tissues, but it is still unclear to what extent pre-mRNA splicing defects contribute to SMA. It is a central question in the field why spinal motor neurons are more severely affected by low SMN protein levels than other cell types. We and others have shown that SMN is also present in highly mobile multi-protein complexes that are actively transported along microtubules and actin filaments in axons of cultured neurons. More recently, we have discovered that axons of cultured SMN-deficient motor neurons have impaired localization of specific mRNA binding proteins (mRBPs) and mRNAs in axons that are known to play roles in axon growth. These findings have led us to hypothesize that SMN plays a critical role in the assembly and trafficking of messenger ribonucleoproteins (mRNPs) in neuronal processes that serve axonal growth and maintenance. However, how defects in these SMN-dependent processes may contribute to the SMA pathomechanism is still unknown. With the goal to reveal mRNA processing defects in SMA and their contribution to the disease phenotype, we propose two specific aims: in Aim 1, we will uncover disease-specific molecular axonal defects by a de- tailed and comprehensive analysis of differences in the axonal transcriptome and proteome. The use of novel SMA patient stem cell-derived motor neurons and compartmentalized cultures will allow us for the first time to comprehensively catalogue SMA-specific defects in mRNA processing and their consequences on axon development, and identify ways to rescue these defects. These studies will provide insight into mRNA processing defects in SMA patient stem cell-derived motor neurons and how they contribute to axonal defects in vitro. In Aim 2, we will use cell type-specific tagging of ribosomes with the RiboTag system to thoroughly characterize differences in the ribosome-associated transcriptome in spinal cord motor neurons of SMA mouse models. We will characterize axonal localization of known SMN targets, and rescue these axonal defects in SMA mice via AAV9-based viral transduction of genes that enhance axonal mRNP localization. These experiments will allow for the first time the assessment of the spectrum of mRNA processing defects in spinal motor neurons from an SMA mouse model and how they contribute to the disease phenotype in vivo. This proposal is expected to both increase our understanding of human pathology in the neuromuscular sys- tem, and to facilitate the development of therapies that are specifically targeted at mRNA processing defects in motor neurons in SMA and related neuromuscular diseases.