Transcriptomic characterization of in vitro human iPSC-derived motor neuron systems for the exploration of cell autonomous mechanisms and modifiers of TDP-43 perturbation associated neurotoxicity
Transactive Response DNA-binding protein 43 (TDP-43) is a multifunctional nuclear protein ubiquitously expressed in all tissues. Although it is primarily nuclear, Amyotrophic Lateral Sclerosis (ALS) patients frequently present with cytoplasmic aggregates of TDP-43 in motor neurons on post-mortem examination. Although only about 5% of all ALS patients have a TARDBP mutation (Ghasemi and Brown 2018; Ingre et al. 2015), which is known to cause these insoluble cytoplasmic aggregates, 97% of ALS patients will develop cytoplasmic, insoluble TDP-43 aggregates regardless of whether they have a mutation in TARDBP or not (Ling et al. 2013). Overexpression of wild-type TDP-43 has previously been shown to be a valid model of inducing neurotoxicity as well as a limited amount of cytoplasmic re-localization and aggregation characteristic of ALS (Elden et al. 2010; Wils et al. 2010). In the search for modifiers of toxicity as conferred by TDP-43 overexpression in these models, knockout of Ataxin-2 (ATXN2) was proposed as a protective intervention in the context of TDP-43 perturbation in non-human model systems (Elden et al. 2010, Becker et al. 2017). This promising finding had not previously been validated in a human motor neuron system, nor had a thorough investigation been conducted to determine the mechanism of neuroprotection that ATXN2 knockout employs to drive the therapeutic effect. This sequencing study examines both ATXN2 intact and ATXN2 knockout motor neurons in the context of TDP-43 perturbation to explore global transcriptomic changes that arise from these changes in cellular state that are associated with progression of ALS (ATXN2 intact) and what pathways could be implicated in protection from ALS-associated neurodegeneration (ATXN2 Knockout). Overall design: To investiage global transcriptomic changes resulting from TDP-43 perturbation via overexpression, we performed bulk RNA sequencing on isogenic motor neuron lines with and without induced TDP-43 perturbation at the same time point. Samples compared are an Empty Vector Control and a YFP expression only control as negative controls, wild-type TDP-43 overexpression, and cytoplasmically locked TDP-43 expression (TDP-43?NLS). The cytoplasmic TDP-43 condition was included because it is represenative of the end physiological state of ALS patients. There are four biological replicates of each sample present in this study. This study also examines the role of Ataxin-2 (ATXN2), a proposed modifier of TDP-43 associated neurodegeneration, by including isogenic motor neuron lines (from the same background as the above mentioned TDP-43 perturbed lines) harboring Ataxin-2 knockout (conferred via CRISPR). This allows for comparison and discovery of factors that are exclusively modulated when Ataxin-2 is knocked out and whether those have an effect on the set of factors perturbed when TDP-43 is overexpressed or cytoplasmically constrained. Ataxin-2 knockout samples are available in this data set also in the Empty Vector, YFP only, TDP-43 overexpression, and TDP-43?NLS conditions. These samples are also presented as the results of four biological replicates each.