UC Irvine

Huntington’s disease (HD) is a devastating neurodegenerative disease that affects movement, psychiatric well-being and cognition (Group. et al., 1993; Huntington, 2003; Saudou & Humbert, 2016). Neuropathology includes morphological atrophy of the cortex and degeneration of the striatum, with selective loss of medium spiny neurons (MSNs). HD is caused by a CAG repeat expansion to ≥40 repeats in the first exon of the Huntingtin gene (HTT) which translates to form a mutant protein (mHTT) containing an expanded polyglutamine tract (Group. et al., 1993). Currently, there are no approved disease modifying treatments. Changes in RNA expression is an early, reproducible, and progressive feature of disease (Feyeux et al., 2012). However, to date underlying causal mechanisms of RNA dysregulation in HD remain elusive. HTT interactor studies have revealed interactions of HTT with RNA-binding proteins (RBPs) that regulate cellular processes such as RNA processing including RNA splicing, RNA localization, RNA Stability, and stress granule (SG) formation (Kaltenbach et al., 2007a; Ratovitski et al., 2012a; Shirasaki et al., 2012). A potential disruption of RBP interactions with HTT or dysregulation of RBP expression and localization due to the presence of the mutation may contribute to the transcriptional dysregulation observed in HD. To date, the field lacks a dedicated study into how HTT’s interaction with RBPs contributes to HD progression. My thesis focuses on understanding how HTT’s interaction with RBPs can regulate post-transcriptional RNA-processing. This work has targeted multiple HTT interactors and multiple RNA processes, in addition to developing a new molecular tool to study these HTT-dependent events. Together, this thesis established two mHTT dependent RBP phenotypes (Chapters 1 & 3), and developed a method to study HTT’s RBP interactors and their target RNA (Chapter 2).Chapter 1: Stress granules (SGs) have been established as a neuropathological feature of neurodegenerative diseases including Amyotrophic Lateral Sclerosis (ALS) and Alzheimer’s disease (AD) (Japtok et al., 2015; Wolozin & Ivanov, 2019a). SGs form during cellular stress and are a type of RNA granule comprised of RBPs and RNAs. The main SG protein G3BP1 has been shown to interact with HTT (Ratovitski et al., 2012b). Here, I helped study and characterize SGs in HD and our results identified increased and persistent SG formation in the HD mouse model R6/2 brain and human HD brain tissue. Chapter 2: Previous HTT interactor studies have been performed in yeast, mouse models, and non-neuronal cell types, and have not focused on RNA-binding proteins and the RNA transcripts they regulate. Here, I developed and optimized a system to identify RBP interactors of HTT and RNA that indirectly bind to HTT. This method uses mass-spectrometry combined with RNA-sequencing, and I have identified ~3000 HTT interactors along with differential binding preferences between HTT and mHTT in a susceptible neuronal population in HD. Chapter 3: My analysis of RNA sequencing data from the R6/2 model revealed upregulation of RNA-processing and identified the TAR DNA-binding protein (TARDBP/TDP-43) and METTL3 as contributing to the HD dysregulated transcriptome. Using RBP eCLIP-sequencing and m6A eCLIP-sequencing I have established a potential role for these two proteins in HD progression involving the regulation of alternative splicing and RNA stability. In addition, I identified the translocation of nuclear TDP-43 to the disease-associated cytoplasmic, phosphorylated TDP-43 in both the R6/2 HD mouse and HD patient brain tissues.