Targeting Dysregulated RNA Splicing in Neurodegenerative Diseases

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.