Assessing the impact of ALS Mutations on mRNA Translation in hiPSC-derived Neurons and Neuromuscular Models

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Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease which results in the gradual deterioration of upper and lower motor neurons (reviewed in Masrori and Van Damme, 2020). Molecularly, it is characterised partly by progressive RNA deregulation related to disrupted protein-RNA complexes that culminate in the formation of aggregates. Such deregulation can be initiated by mutations in several RNA-binding proteins (RBPs), such as TDP-43 (Chia et al., 2018; Taylor et al., 2016) and FUS (reviewed in Deng et al., 2014). Many of these RBPs form biomolecular condensates when bound to RNAs, which can selectively contribute to RNA regulation (Hallegger et al, 2021). Many ALS-causing mutations in RBPs have been shown to change their condensation propensity, which can increase the probability of transitioning into potentially toxic aggregated forms (reviewed in Cestra et al., 2017, Taylor et al., 2016; Wiedner and Giudice, 2021). Previous work done by the Ule lab has focused on the physiological roles of TDP-43 condensation, showing that it allows specific types of RNA binding, especially the capacity to assemble on long 3'UTR regions in a highly multivalent manner (Halleger et al., 2021). Formation of such "binding-region condensates" was shown to steer the capacity of TDP-43 to regulate 3' end mRNA polyadenylation. ALS-linked mutations subtly disrupt this condensation propensity and it was shown that this also subtly impacts 3'UTR processing, but the impact on mRNA translation has not been examined. Given the importance of 3'UTRs in mRNA translation, it will be crucial to understand how it is impacted by perturbed ribonucleoprotein (RNP) condensation, and how such direct RNA deregulation contributes to the early disease stages. Additionally, a recent analysis of ribosome profiling data from the Ule lab detected the usage of alternative open reading frames (ORFs) in ALS. Alternative ORFs can increase or decrease the synthesis of the canonical protein from a transcript, alter its length, or create a new protein altogether, serving therefore as an important yet often overlooked layer of translational regulation (reviewed in Orr et al., 2020). Understanding the impact of RBP dysfunction in ALS on translation on translation could enable the development of new therapeutic approaches to stop disease progression in its early stages.We will investigate how ALS-causing mutations in RBPs directly affect mRNA translation in a cellular model of the human neuromuscular system. The first aim of the project will be to contribute to the phenotypic characterisation of human iPSC-derived neuron models with mutations in TDP-43 and Matr3 established by the Lieberam and Ule labs and to assess their transcriptome wide translational status with Ribo-seq. We will then use individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) on the same models and run bioinformatic analyses to identify mechanisms of translational deregulation, specifically to understand how mutant TDP-43 and Matr3 change their interactions with translationally deregulated RNAs. We will use the regulatory elements from representative RNAs to develop new bichromatic fluorescent reporters to follow translational deregulation with live imaging. These will allow us to investigate translational deregulation in a co-culture model for neuromuscular circuits.