Targeting Dysregulated RNA Splicing in Neurodegenerative Diseases

拉格尔-图雷恩·沃德·布莱尼-摘要 额颞叶痴呆(FTD)和肌萎缩侧索硬化症(ALS)是两个相互关联且无法治愈的疾病 神经退行性疾病。TDP-43和FUS等RNA结合蛋白(RBPs)突变导致家族性 ALS/FTD的形式以及这些蛋白的错误定位是疾病的病理特征。功能丧失 限制性商业惯例的发生与它们的错误定位同时发生，并可能导致前-mRNA的严重错误剪接 转录本，如隐蔽外显子的表达。这种错误拼接经常导致表达减少 受影响的基因和下游功能对神经生物学的影响。我们和其他人有 鉴定和鉴定TDP-43相关的FTD/ALS中出现的几个错接基因，如 作为STMN 2和UNC13A。然而，我们很难理解不同疾病之间的功能关系- 相关限制性商业惯例或不同细胞类型的病理性错误拼接的多样性。我们也不知道 是什么导致限制性商业惯例在FTD/ALS中错误定位，我们也不知道如何逆转病理性错误剪接 一旦它发生了。在这项提案中，由Clotilde Lagier-Tourenne、Michael Ward和Paul领导的三个研究小组 布莱尼将把互补的背景和技术结合起来，以解决这些突出的问题 问题。使用基于IPSC的六种疾病相关细胞类型和尖端长读RNA的细胞模型 测序方法，我们将描述剪接是如何因五个FTD/ALS功能的丧失或获得而改变的- 相关限制性商业惯例(目标1)。然后，我们将分析与其中两个错误本地化相关的剪接更改 来自FTD/ALS患者大脑的神经元、星形胶质细胞和小胶质细胞中的RBPs、TDP-43和FUS(目标2)。接下来，我们 将在IPSC神经元、星形胶质细胞和 小胶质细胞，以确定病理性剪接的调节器，以及RBP错误定位和 功能障碍(目标3)。最后，我们将针对上游监管机构逆转病理性剪接，随后 功能分析确定FTD/ALS iPSC来源的新剪接调节子与疾病的关系 细胞模型(目标4)。总的来说，这些研究将揭示病理性疾病的基本机制。 拼接FTD/ALS，为研究社区生成基础数据集，并从治疗角度进行识别 病理性剪接的靶向调节因子和RBP功能障碍的上游驱动因子。

LAGIER-TOURENNE WARD BLAINEY – ABSTRACT Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are two interrelated and uncurable neurodegenerative disorders. Mutations in RNA binding proteins (RBPs) such as TDP-43 and FUS cause familial forms of ALS/FTD, and mislocalization of these proteins are pathological hallmarks of disease. Loss of function of RBPs occurs concurrently with their mislocalization, and can result in profound mis-splicing of pre-mRNA transcripts such as the expression of cryptic exons. This mis-splicing frequently leads to reduced expression of the impacted genes and downstream functional consequences on neuronal biology. We and others have identified and functionally characterized several mis-spliced genes that occur in TDP-43-related FTD/ALS, such as STMN2 and UNC13A. However, we poorly understand the functional relationships between different disease- associated RBPs or the diversity of pathological mis-splicing across different cell types. We also do not know what causes mis-localization of RBPs in FTD/ALS, nor do we know how to reverse pathological mis-splicing once it occurs. In this proposal, three research teams led by Clotilde Lagier-Tourenne, Michael Ward, and Paul Blainey will bring together complementary backgrounds and technologies to address these outstanding questions. Using iPSC-based cellular models of six disease-relevant cell types and cutting-edge long read RNA sequencing methods, we will characterize how splicing is altered by the loss or gain of function of five FTD/ALS- associated RBPs (Aim 1). We will then analyze splicing changes associated with mislocalization of two of these RBPs, TDP-43 and FUS, in neurons, astrocytes, and microglia from FTD/ALS patient brains (Aim 2). Next, we will perform FACS-based CRISPRi and optical pooled genetic screens in iPSC neurons, astrocytes, and microglia to identify modulators of pathological splicing, and upstream drivers of RBP mislocalization and dysfunction (Aim 3). Finally, we will target upstream regulators to reverse pathological splicing followed by functional assays to determine the relationship of new splice modulators to disease in FTD/ALS iPSC-derived cellular models (Aim 4). Collectively, these studies will reveal fundamental mechanisms underlying pathological splicing in FTD/ALS, generate foundational datasets for the research community, and identify therapeutically targetable modulators of pathologic splicing and upstream drivers of RBP dysfunction.