Elucidating mechanisms of ATXN2 interaction with TDP-43 in human motor neuron models of Amyotrophic Lateral Sclerosis

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Project Summary/Abstract Amyotrophic Lateral Sclerosis is an incurable, rapidly progressing motor neuron degenerative disease with few treatment options. Although the cause of ALS remains poorly understood, one unifying phenotype that arises in approximately 97% of ALS patients, regardless of the presence of an associated genetic lesion, is the formation of insoluble protein aggregates containing Transactive Response DNA-binding protein 43 (TDP-43) in the cytoplasm of degenerating motor neurons. In yeast, primary neurons, and human cell models, TDP-43 overexpression has been shown to be sufficient to recapitulate similar TDP-43 aggregation and subsequent cellular apoptosis. Previous screening studies have identified Ataxin-2 (ATXN2) as a dose-dependent modifier of this TDP-43 overexpression-mediated toxicity. In support of this finding, our preliminary data suggest that knockout of ATXN2 by CRISPR is sufficient to protect iPSCs and differentiating motor neuron cultures from TDP- 43 overexpression-related toxicity. However, no published study to date has deeply examined the mechanism of why this protection occurs upon ATXN2 knockout. This proposal seeks to identify a mechanism by which ATXN2 loss mitigates TDP-43 proteinopathy-mediated toxicity in human motor neurons. Our group and others have reported that both TDP-43 and ATXN2 preferentially interact with proteins involved in mRNA processing and metabolism, including each other. Thus, we firstly hypothesize that ATXN2 loss provides protection from TDP-43 associated toxicity by altering mRNA metabolism and processing of TDP-43. Aim 1 will test how TDP-43 metabolism is changed upon ATXN2 knockout compared to baseline. In particular, we will examine the effect of ATXN2 loss on TDP-43 mRNA levels, mRNA stability, protein levels, and protein stability in the context of baseline TDP-43 levels and TDP-43 overexpression. Secondly, we hypothesize that ATXN2 loss provides protection from TDP-43 associated toxicity by altering interactions between TDP-43 and its target RNAs. In Aim 2, we will explore how ATXN2 loss may provide neuroprotection by changing how TDP-43 interacts with its RNA substrates. We will accomplish this goal in two parts. First (Aim 2.1), we will examine the changes in mRNAs that TDP-43 interacts with in the nucleus and cytoplasm of human motor neurons in the context of ATXN2 loss compared to baseline using Fractionation iCLIP sequencing. Second (Aim 2.2), because both ATXN2 and TDP-43 are recruited to stress granules where mRNAs and RNA binding proteins are transiently sequestered, we will evaluate changes in motor neuron stress granule dynamics and localization that occur in the context of ATXN2 knockout compared to baseline. We predict that these mechanisms shield neurons from detrimental TDP-43 gain of function-related effects. The goal of this proposed project is to provide insight into the role ATXN2 plays in the metabolism of TDP-43 and TDP-43 targets so we may learn more about the underlying biology of TDP-43 associated neurodegeneration that affects nearly all ALS patients. We hope that our findings inform future studies in evaluating actionable therapeutic mechanisms for treating ALS.