Targeting Dysregulated RNA Splicing in Neurodegenerative Diseases

靶向神经退行性疾病中失调的 RNA 剪接

基本信息

批准号：
10729566
负责人：
Paul Clark Blainey
金额：
$ 207.29万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-01 至 2028-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10729566
关键词：
ALS patients Address Amyotrophic Lateral Sclerosis Astrocytes Autopsy Biological Biological Assay Biology Brain CRISPR screen Cell model Cells Cellular Assay Clustered Regularly Interspaced Short Palindromic Repeats Collaborations Communities Custom DNA Damage Data Set Defect Dementia Disease Event Exons Fluorescent in Situ Hybridization Functional disorder Genes Genetic Genetic Screening Genomic approach Genomics Induced pluripotent stem cell derived neurons Knowledge Libraries Link Maps Methods Microglia Microscopy Morphology Motor Neurons Mutate Mutation Nerve Degeneration Neurodegenerative Disorders Neuroglia Neuronal Dysfunction Neurons Nuclear Optics Pathologic Pathology Pathway interactions Patients Phenotype Process Protein Isoforms Proteins RNA Processing RNA Splicing RNA metabolism RNA-Binding Proteins Reaction Research Resources Ribonuclease H Seminal Skeletal Muscle Spliced Genes Technology Therapeutic Therapeutically Targetable Time Tissues Transcript Work axon regeneration cell type frontotemporal lobar dementia amyotrophic lateral sclerosis functional genomics gain of function genome wide screen genome-wide in situ sequencing induced pluripotent stem cell inhibitory neuron innovation knock-down loss of function mRNA Precursor new therapeutic target novel therapeutics nucleocytoplasmic transport protein TDP-43 screening stem cell biology transcriptome sequencing transcriptomics ward

项目摘要

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.

Lagier-Tourenne Ward Blainey - 摘要额颞痴呆（FTD）和肌萎缩性侧索硬化症（ALS）是两个相互关联的神经退行性疾病。 RNA结合蛋白（RBP）的突变，例如TDP-43和FUS引起家族 ALS/FTD的形式以及这些蛋白质的错误定位是疾病的病理标志。功能丧失 Rbps的不定位同时发生，并可能导致前MRNA的严重错误转录本，例如加密外显子的表达。这种错误的拼写经常导致降低的表达受影响的基因和下游功能对神经元生物学的影响。我们和其他人有在与TDP-43相关的FTD/ALS中发生的几个遗漏基因鉴定并在功能上表征了几个毫无疑问的基因如STMN2和UNC13A。但是，我们对不同疾病之间的功能关系很糟糕 - 相关的RBP或不同细胞类型的病理错误分解的多样性。我们也不知道是什么导致FTD/ALS中RBP的错误定位，我们也不知道如何逆转病理误解一旦发生。在该提案中，由Clotilde Lagier-Tourenne，Michael Ward和Paul领导的三个研究团队 Blainey将汇集互补的背景和技术来解决这些杰出的问题。使用基于IPSC的六种相关细胞类型的基于IPSC的细胞模型和尖端的长读RNA 测序方法，我们将表征如何通过五个ftd/als-的功能损失或增益来改变剪接相关的RBP（AIM 1）。然后，我们将分析与其中两个的错误定位相关的剪接变化来自FTD/ALS患者大脑的神经元，星形胶质细胞和小胶质细胞中的RBP，TDP-43和FUS（AIM 2）。接下来，我们将在IPSC神经元，星形细胞和小胶质细胞鉴定病理剪接的调节因子，以及RBP错误定位和上游驱动因素功能障碍（目标3）。最后，我们将针对上游调节器逆转病理剪接，然后功能测定以确定新剪接调节剂与FTD/ALS IPSC衍生的疾病的关系蜂窝模型（AIM 4）。总的来说，这些研究将揭示病理学基础的基本机制在FTD/ALS中进行拼接，为研究社区生成基础数据集，并识别治疗 RBP功能障碍的病理剪接和上游驱动因素的目标调节剂。