Survival Motor Neuron (SMN) function in motoneuron development

项目摘要 了解运动神经元功能障碍的机制基础及其在运动神经元疾病中的作用将填补一个 神经科学的一个重大空白，并推动了治疗肌萎缩性疾病等毁灭性疾病的新方法 脊髓侧索硬化症（ALS）、遗传性运动神经病和脊髓性肌萎缩症（SMA）。这些疾病折磨着 在美国，每年有超过10万的成人、婴儿和儿童。ALS和SMA特别是 通常在诊断后的几年内导致瘫痪和死亡的毁灭性疾病。这些基因 疾病的研究表明，运动神经元特别容易受到负责关键RNA的蛋白质缺陷的影响。 处理功能。然而，为什么运动神经元容易受到RNA加工缺陷的影响， 明白这个项目的科学原理是从机械上阐明 RNA可以破坏运动神经元的功能并导致运动神经元死亡。阐明运动神经元特异性 由这些突变引起的RNA加工缺陷对于理解正常人和正常人的运动神经元是必不可少的。 和疾病状况，并将指导急需的治疗方法。为了解决这个问题，我们把重点放在 广泛表达的运动神经元存活（SMN）蛋白和运动神经元疾病SMA。SMA是一 影响婴儿/儿童的运动神经元疾病，由运动神经元存活（SMN）蛋白水平低引起。 SMN在RNA代谢的许多方面发挥作用。然而，SMN的关键RNA处理功能， 运动神经元尚未解决。证据支持SMN与各种神经元RNA结合蛋白相互作用 在发育过程中稳定和/或运输RNA到轴突和树突的RBP。利用独特的斑马鱼 模型，我们已经产生，我们已经表明，SMN是所需的正常脊椎动物运动神经元 发育包括树突形成和运动轴突生长和树枝状化。这是一个关键的发现， 提示SMA并非退行性病变，但运动神经元功能障碍是由运动神经元功能低下引起的 导致神经元衰竭的发展。我们假设SMN与神经元RBP及其相关性有关。 货物RNA以发育调节的方式指导运动神经元发育，包括轴突 外生长和分支、树突形成和突触形成。为了验证这一点，我们将回答三个 基本问题：什么样的SMN：RBP复合物在运动神经元的发育中？这些限制性商业惯例中的缺陷 影响运动神经元的发育这些复合物中有哪些RNA，当SMN或 RBPs缺失或减少了？我们所有的实验都将在运动神经元中进行，相关的 细胞类型，并使用广泛的实验方法，如生物化学，质谱分光光度法， RNAseq，单神经元成像和遗传学。这些实验的数据将对以下方面产生广泛影响： 了解RNA参与正常运动神经元发育，SMA和其他运动神经元疾病， 就像ALS。此外，我们的方法将严格测试SMN的重要性：RBP复合物及其相关的 揭示运动神经元生物学基本分子机制的RNA。

Project Summary Understanding the mechanistic basis of motoneuron dysfunction and its role in motoneuron diseases would fill a major gap in neuroscience and advance new approaches for treating devastating diseases such as amyotrophic lateral sclerosis (ALS), hereditary motor neuropathy and spinal muscular atrophy (SMA). These diseases afflict over one hundred thousand adults, infants, and children per year in the US. ALS and SMA are particularly devastating diseases resulting in paralysis and death often within a few years of diagnosis. The genetics of these diseases indicates that motoneurons are particularly vulnerable to defects in proteins tasked with critical RNA processing functions. However, exactly why motoneurons are vulnerable to RNA processing defects is not understood. The scientific rationale for this project is to elucidate mechanistically how mishanding of RNAs can disrupt motoneuron function and lead to motoneuron death. Elucidating the motoneuron-specific RNA processing defects caused by these mutations is essential for understanding motoneurons in both normal and diseased conditions and will direct critically needed therapeutics. To tackle this issue, we focus on the ubiquitously expressed survival motor neuron (SMN) protein and the motoneuron disease SMA. SMA is a motoneuron disease that affects infants/children and is caused by low survival motor neuron (SMN) protein levels. SMN functions in many aspects of RNA metabolism. However, the critical RNA handling function of SMN in motoneurons is unresolved. Evidence supports that SMN interacts with various neuronal RNA binding proteins (RBPs) that stabilize and/or transport RNAs to axons and dendrites during development. Using unique zebrafish models that we have generated, we have shown that SMN is required for normal vertebrate motoneuron development including dendrite formation and motor axon outgrowth and arborization. This is a key finding and reveals that SMA is not a degenerative defect, but the motoneuron dysfunction is caused by poor motoneuron development leading to neuronal failure. We hypothesize that SMN associates with neuronal RBPs and their cargo RNAs in a developmentally regulated manner to direct motoneuron development including axon out growth and branching, dendrite formation, and synapse formation. To test this we will answer three essential questions: What SMN:RBP complexes are in developing motoneurons? How do defects in these RBPs affect motoneuron development? What RNAs are in these complexes, and how are they affected when SMN or the RBPs are missing or decreased? All of our experiments will be performed in vivo in motoneurons, the relevant cell type and use a broad range of experimental approaches such as biochemistry, mass spectrophotometry, RNAseq, single neuron imaging and genetics. Data from these experiments will have broad implications for understanding RNA involvement in normal motoneuron development, SMA, and other motoneuron diseases such as ALS. In addition, our approach will rigorously test the importance of SMN:RBP complexes and their associated RNAs revealing a fundamental molecular mechanism in motoneuron biology.