Intersection Between RNA Methylation and TDP43-Mediated Toxicity in ALS

Abstract

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease characterized by progressive loss of motor neurons that results in muscle loss, paralysis, and respiratory failure. ALS diagnosis is complicated by its heterogeneous biochemical, genetic, and clinical features. In addition, >90% of ALS cases arise without previous family history or genetic cause of disease, while only <10% result from an inherited mutation in a known ALS causing gene. However, the vast majority of ALS cases present with dysregulation of a key RNA binding protein TDP43. A primarily nuclear protein, TDP43 is mislocalized to the cytoplasm in ALS, accumulating in cytoplasmic inclusions. TDP43 is an essential protein involved with many levels of RNA processing such as alternative splicing and RNA transport, largely influencing RNA stability. Previous studies uncovered widespread RNA destabilization in models of ALS, implicating dysregulation of RNA stability in disease. RNA stability can be modified by many of the over 150 known RNA modifications. The most common internal RNA modification, methylation of RNA at the N6 position of adenosine (m6A), significantly contributes to RNA stability as well as RNA transport and translation. m6A is co-transcriptionally deposited across many species of RNA at specific motifs by methyltransferases (“writers”) and removed by demethylases (“erasers”). m6A alters the fate of RNA depending on which RNA binding protein (“reader”) binds the methylated RNA, thereby influencing the transport, translation, and stability of that RNA. In this dissertation, I determine whether and how m6A-modified RNA influences TDP43 RNA substrate recognition and processing. Chapter 1 begins with a review of TDP43, focusing on its structure and functions in RNA processing then characterizing its role in ALS. Chapter 1 also introduces m6A, methods to measure m6A, the function of pathway components, and ties to neurodegenerative disease. Chapter 2 explores the hypothesis that TDP43 recognizes m6A methylated RNA, identifying methylation sites on TDP43 substrates and increased methylation levels in patient tissue. Additionally, removal of an m6A site in the TARDBP transcript reduced TDP43s ability to autoregulate and modulation of m6A components can alter TDP43-associated toxicity. Of these, knockout of the m6A reader YTHDF2 rescued toxicity in rodent and human neuronal models of ALS. YTHDF2 is upregulated in patient spinal cord offering a novel therapeutic target. Chapter 3 summarizes key findings from this research and discusses open questions surrounding the overlap between TDP43 and m6A. Finally, Appendix A describes an improved method for CRISPR sgRNA cloning and creating multiplexed vectors as a method to create genome edited cell lines.