Dissecting the Molecular Determinants of Cytoplasmic Gain of Function Pathomechanisms in TDP-43-linked Amyotrophic Lateral Sclerosis

点击翻译按钮获取中文摘要

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease that leads to motor neuron loss. In vast majority of the cases (~95%), the RNA-binding protein TDP-43 is mislocalised and accumulates into cytoplasmic aggregates, a hallmark pathology of ALS (Neumann et al., 2006; Ling et al., 2013). As under physiological conditions TDP-43 is found in the nucleus and plays a significant role RNA metabolism, it's loss of function in the nucleus has been associated with significant alterations in RNA splicing and its liquid-liquid phase separation (LLPS) function (Polymenidou et al., 2011; Tollervey et al., 2011). On the other hand, evidence also supports cytoplasmic gain-of-function pathomechanisms for TDP-43 in disease (Suk and Rousseaux, 2020).For example, ALS-linked TDP-43 mutants can cause motor neuron degeneration in mice without aggregation or loss of nuclear TDP-43. Although studies have begun to unveil the molecular basis for TDP-43 RNA-binding and LLPS in cytoplasm, the pathomolecular determinants of cytoplasmic gain of functions in TDP-43-linked neurodegeneration have yet to be determined (Schmidt et al., 2019; Conicella et al., 2020; Hallegger et al., 2021). We propose generating three different novel mouse embryonic stem (mES) cell models to study the contribution of different disease associated properties of human cytoplasmic TDP-43, expressed in physiological levels.Aim of the investigation (up to 150 words)State primary research question and where appropriate the primary hypotheses being testedTDP-43 cytoplasmic aggregation is a key hallmark pathology of ALS. However, the contribution of different mechanisms by which mislocalised TDP-43 contributes to neurodegeneration is yet to be clearly elucidated. In this project we aim to dissect the roles of RNA-binding, LLPS and oligomerisation in TDP-43-linked ALS using novel TDP-43 mutant mouse embryonic stem (mES) cell models. In further studies, we aim to use these cells to develop in vivo mouse models to dissect neurodegeneration in a full animal context.Aim 1: Characterising novel CRISPR-edited mES cells to study TDP-43 linked ALSAim 2: Dissecting the cytoplasmic gain-of function TDP-43 pathomechanisms in novel CRISPR-edited mES cell derived neuronsAim 3: Determining cytoplasmic gain-of function TDP-43 pathomechanisms in novel and physiologically relevant in-vivo models for TDP-43 linked ALS