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.

拉吉尔-图伦·沃德·布莱尼 – 摘要额颞叶痴呆 (FTD) 和肌萎缩侧索硬化症 (ALS) 是两种相互关联且无法治愈的疾病 TDP-43 和 FUS 等 RNA 结合蛋白 (RBP) 突变会导致家族性神经退行性疾病。 ALS/FTD 的多种形式以及这些蛋白质的错误定位是疾病功能丧失的病理标志。 RBP 的错误定位与它们的错误定位同时发生，并可能导致前 mRNA 的严重错误剪接这种错误剪接经常导致转录本的表达减少。我们和其他人已经了解了受影响的基因和下游功能对神经元生物学的影响。鉴定并功能表征了 TDP-43 相关 FTD/ALS 中发生的几个错误剪接基因，例如然而，我们对不同疾病之间的功能关系知之甚少。我们也不知道相关的 RBP 或不同细胞类型之间病理性错误剪接的多样性。是什么导致 FTD/ALS 中 RBP 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，由 Clotilde Lagier-Tourenne、Michael Ward 和 Paul 领导的三个研究小组。 Blainey 将汇集互补的背景和技术来解决这些突出问题使用基于 iPSC 的六种疾病相关细胞类型和尖端长读长 RNA 的细胞模型。测序方法中，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然后我们将分析与其中两个错误定位相关的剪接变化。 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（目标 2）。将在 iPSC 神经元、星形胶质细胞和小胶质细胞识别病理剪接的调节剂以及 RBP 错误定位的上游驱动因素最后，我们将针对上游调节因子来逆转病理剪接。功能测定以确定新剪接调节剂与 FTD/ALS iPSC 衍生疾病的关系总的来说，这些研究将揭示病理学的基本机制。 FTD/ALS 中的剪接，为研究界生成基础数据集，并确定治疗方法病理剪接的靶向调节剂和 RBP 功能障碍的上游驱动因素。