Mechanistic studies of ALS-causative mutations and RNP-focussed drug discovery using in vitro reconstitution of RNP complexes

肌萎缩性侧索硬化症（ALS）是运动神经元疾病（也称为Lou Gehrig病）的最常见形式，是一种致命的神经系统疾病。肌萎缩侧索硬化症可以遗传或没有已知的遗传原因，但所有患者都会出现非常相似的情况。在所有病例中，主要位于脊髓的运动神经元神经细胞都会受到影响并最终死亡。这会导致肌肉失去支撑，导致肌肉萎缩、无力和瘫痪。目前批准用于治疗ALS的两种药物可以将患者的寿命延长几个月，但无法治愈患者。因此，很明显需要新的药物来治疗ALS。ALS的特点是神经元中由蛋白质和核糖核酸（rna）组成的分子复合物代谢异常，称为核糖核蛋白（RNP）复合物。突变、异常定位或其他失调的蛋白质获得异常的rna结合特性，并在结构上扰乱RNP复合物，导致ALS病理。同样，疾病相关RNA可以建立未经许可的RNA-蛋白质相互作用，对细胞RNA代谢产生深远的影响。我们发现，一种名为NEAT1_2的特异RNA诱导形成RNP复合物（称为副斑），可能在ALS发病机制中起重要作用。尽管健康个体的神经元中不存在这种RNA，但这种RNA会在ALS运动神经元中积累。此外，许多调控NEAT1_2/副斑的蛋白也受到ALS突变的影响。我们相信它有保护作用，帮助神经元存活。然而，目前还没有针对neat12的药物可用。传统的药物发现工作集中在蛋白质上，但最近已经有可能识别作用于RNA及其与蛋白质的复合物的药物。更具体地说，目前正在对儿童致命性疾病脊髓性肌萎缩症（SMA）患者评估影响RNPs的药物。最近，我们开发了一种系统，通过将一部分NEAT1_2 RNA附着在多孔测试板的微珠上，可以组装与NEAT1_2/paraspeckles组成相似的复合物。该系统可用于使用超快显微成像分析快速分析蛋白质与这些复合物的结合。在目前的研究中，我们的目标是更好地了解NEAT1_2/paraspeckles与ALS之间的联系，并发现可以调节这些复合物的新型小分子，这些小分子有可能用于ALS的研究和药物发现。此外，我们将利用C9ORF72基因产生的主要als致病RNA对该系统进行修改，使其适用于寻找任何als相关RNA分子的小分子。我们的团队在RNA生物学，ALS病理生物学和药物发现以及所有相关的实验室工具方面具有重要的背景。因此，我们处于独特的位置，可以使用我们开发的上述最先进的方法，将我们对ALS病理生理学的理解的最新突破转化为治疗开发，并最终影响患者。

Amyotrophic lateral sclerosis (ALS), the most common form of motor neuron disease (also known as Lou Gehrig disease), is a fatal disease of the nervous system. ALS can be inherited or have no known genetic cause, yet all patients will develop a very similar condition. In all patents, nervous cells called motor neurons, mainly located in the spinal cord, become affected and eventually die. This leads to loss of support for muscles, causing their wasting, weakness and paralysis. Two drugs currently approved for ALS extend the lifespan by a few months and are not able to cure patients. Hence, there is a clear need for new drugs to treat ALS. ALS is characterised by abnormal metabolism of the molecular complexes in neurons composed of proteins and ribonucleic acids (RNAs), called ribonucleoprotein (RNP) complexes. Mutant, abnormally localised, or otherwise dysregulated proteins gain abnormal RNA-binding properties and structurally perturb RNP complexes leading to ALS pathology. Similarly, disease-relevant RNAs can establish unlicensed RNA-protein interactions with profound consequences for cellular RNA metabolism. We found that a specific RNA called NEAT1_2 that induces formation of RNP complexes known as paraspeckles might be an important player in ALS pathogenesis. Although absent from the neurons of healthy individuals, this RNA becomes accumulated in ALS motor neurons. In addition, many proteins that regulate NEAT1_2/paraspeckles are affected by ALS mutations. We believe it has a protective effect and helps neurons survive. However currently no NEAT1_2 targeted drugs are available.Traditional drug discovery efforts have focussed on the protein but it has recently become possible to identify drugs which act on RNA and its complexes with proteins. More specifically, drugs which affect RNPs are currently being evaluated in patients with the fatal childhood disease spinal muscular atrophy (SMA). Recently, we have developed a system that will allow us assembling complexes similar in composition to NEAT1_2/paraspeckles, by attaching a portion of NEAT1_2 RNA to micro-beads in a multi-well test plate. This system can be used for rapid analysis of protein binding to these complexes using an ultrafast microscopic imaging analysis. In the current proposal, we aim to better understand the link between NEAT1_2/paraspeckles and ALS, and find novel small molecules that can modulate these complexes with a potential to be used in ALS research and drug discovery. Further, we will modify this system to make it suitable for finding small molecules for any ALS-relevant RNA molecule using a major ALS-causative RNA produced from C9ORF72 gene. Our teams have significant background in RNA biology, ALS pathobiology and drug discovery as well as all the relevant laboratory tools. Therefore we are uniquely placed to use the above state-of-the-art approach we developed, to translate recent breakthroughs in our understanding of ALS pathophysiology into therapies development and ultimately, patient impact.