Dottorato in SCIENZE DELLA VITA

Titolo della tesi: RNA-binding protein network alteration causes aberrant axon branching and growth phenotypes in FUS-ALS mutant motor neurons.

Mutations in RNA-binding proteins (RBPs) have been genetically associated with the motoneuron disease Amyotrophic Lateral Sclerosis (ALS), suggesting a causative link between dysregulation of RNA metabolism and this neurodegenerative disease. However, the lack of mechanistic insights has hampered the development of therapeutic approaches. In this regard, my work aimed to evaluate the toxic functions gained by the ALS-linked gene FUS, with a specific mechanistic focus on the aberrant crosstalk between mutant FUS and other RBPs. To achieve this goal, a novel protocol for obtaining functional spinal and cranial motor neurons (MNs) from human induced pluripotent stem cells (iPSCs) based on ectopic transcription factor modules was developed. By programming rapid cell differentiation into a pure population with high efficiency and reproducibility, this method overcomes significant issues of converting iPSCs to specific disease-relevant cell types (Garone et al., 2019; Garone e Rosa, 2021). iPSC-derived MNs were used to study the effects of ALS mutant FUS on the human motoneuronal proteome. This analysis revealed that, in FUS mutant MNs, proteins involved in catabolic processes and oxidation-reduction are upregulated, whereas cytoskeletal proteins and factors driving neuron projection are downregulated. Interestingly, proteome alteration does not correlate with transcriptome changes. Rather, a strong correlation with selective binding was observed by mutant FUS to mRNAs in their 3’UTR (Garone et al., 2020). To gain mechanistic insights, a relevant FUS target, the neural RBP HuD (encoded by the ELAVL4 gene), was investigated. In both iPSC-derived MNs and FUS mouse model, mutant FUS leads to upregulation of HuD protein levels through competition for HuD 3’UTR binding with FMRP, an RBP encoded by the Fragile-X gene FMR1. Indeed, FMRP was found as a novel repressor of HuD translation, and mutant FUS disrupts this function. In turn, increased HuD levels overly stabilize the transcript of two key targets, NRN1 and GAP43. Both factors are involved in axon growth and, accordingly, axon branching and growth upon injury were increased in FUS mutant MNs. Notably, such phenotypes could be rescued by dampening NRN1 levels (Garone et al., 2021). Since similar axonal phenotypes have been previously described in SOD1 and TDP-43 mutant models, aberrant axonal growth and branching might represent broad early events in the pathogenesis of ALS. Thus, based on this work, novel therapeutic approaches targeting HuD and/or NRN1 and GAP43 might be pursued for ALS in the future. Moreover, evidence of cross-regulation between HuD and FMRP, two important neural RBPs playing multiple roles in nervous system development and neurological diseases, has implications that may extend beyond the ALS field.