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

SMA 中的 RNA 加工缺陷及其对疾病表型的贡献

• 批准号: 9098856
• 负责人: Wilfried Rossoll
• 金额: $ 39.65万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9098856
• 关键词: Affect; Animal Model; Axon; Binding Proteins; Biological; Cataloging; Catalogs; Cells; Defect; Denervation; Development; Disease; Functional disorder; Genes; Genetic; Goals; Growth Cones; Housekeeping; Human Pathology; Impairment; In Vitro; Intervention; Knowledge; Link; Maintenance; Mediating; Messenger RNA; Microfilaments; Microtubules; Modeling; Molecular; Molecular Chaperones; Molecular and Cellular Biology; Motor; Motor Neuron Disease; Motor Neurons; Mus; Muscular Atrophy; Mutation; Nerve; Nerve Degeneration; Neurodegenerative Disorders; Neuromuscular Diseases; Neuromuscular Junction; Neurons; Outcome; Pathology; Pathway interactions; Patients; Play; Process; Proteins; Proteome; Proteomics; Public Health; RNA; RNA Processing; RNA Splicing; Research; Ribonucleoproteins; Ribosomes; Role; SMN protein (spinal muscular atrophy); SMN1 gene; Small Nuclear Ribonucleoproteins; Spinal; Spinal Cord; Spinal Muscular Atrophy; Stem cells; Synapses; System; Tail; Testing; Time; Tissues; Transcript; Transduction Gene; Translations; Up-Regulation; Vertebrates; Viral; Work; axon growth; axonal degeneration; base; cell type; disease phenotype; in vivo; infant death; insight; mRNA Precursor; messenger ribonucleoprotein; motor neuron degeneration; mouse model; neurodevelopment; neuromuscular; neuromuscular system; new therapeutic target; novel; novel therapeutics; protein complex; public health relevance; research study; therapy development; trafficking; transcriptome; transcriptome sequencing

 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.

 描述（由申请人提供）：脊髓性肌萎缩症（SMA）是一种毁灭性的神经退行性疾病，是婴儿死亡的最常见遗传原因。SMA是由运动神经元（SMN）蛋白的功能性存活水平降低引起的，导致神经肌肉接头处的细胞自主缺陷、轴突变性和脊髓中运动神经元的丧失。广泛表达的SMN蛋白在所有组织中剪接体小核核糖核蛋白（snRNP）的组装中具有良好的基本功能，但目前仍不清楚前mRNA剪接缺陷在多大程度上有助于SMA。为什么脊髓运动神经元比其他细胞类型更严重地受到低SMN蛋白水平的影响，这是该领域的一个中心问题。 我们和其他人已经表明，SMN也存在于高度移动的多蛋白复合物中，这些复合物在培养的神经元轴突中沿着微管和肌动蛋白丝被积极转运。最近，我们已经发现，培养的SMN缺陷的运动神经元的轴突受损的特定mRNA结合蛋白（mRBP）和mRNA的轴突中，已知在轴突生长中发挥作用的本地化。这些发现使我们假设SMN在神经元过程中的信使核糖核蛋白（mRNP）的组装和运输中起着关键作用，这些过程为轴突的生长和维持提供服务。然而，这些SMN依赖性过程中的缺陷如何导致SMA病理机制仍然未知。 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目标：在目标1中，我们将通过对轴突转录组和蛋白质组差异的详细和全面分析来揭示疾病特异性分子轴突缺陷。使用新型SMA患者干细胞衍生的运动神经元和区室化培养物将使我们能够首次全面分类SMA特异性mRNA加工缺陷及其对轴突发育的影响，并确定挽救这些缺陷的方法。这些研究将深入了解SMA患者干细胞衍生的运动神经元中的mRNA加工缺陷以及它们如何在体外促成轴突缺陷。在目标2中，我们将使用RiboTag系统对核糖体进行细胞类型特异性标记，以彻底表征SMA小鼠模型脊髓运动神经元中核糖体相关转录组的差异。我们将表征已知SMN靶标的轴突定位，并通过基于AAV9的增强轴突mRNP定位的基因的病毒转导来挽救SMA小鼠中的这些轴突缺陷。这些实验将首次评估SMA小鼠模型脊髓运动神经元中mRNA加工缺陷的谱以及它们如何促成体内疾病表型。预计该提案将增加我们对神经肌肉系统中人类病理学的理解，并促进开发专门针对SMA和相关神经肌肉疾病中运动神经元mRNA加工缺陷的疗法。