Thesis title: Targeting the RNA binding protein HuD to control ALS disease
HuD is an RNA-binding protein (RBP) encoded by the ELAVL4 gene, expressed in the nervous system and playing a crucial role during nervous system's development. However, recent evidence suggests its potential involvement in neurodegenerative diseases, including Amyotrophic Lateral Sclerosis (ALS), which is characterized by the degeneration and death of upper and lower Motor Neurons (MNs). Notably, upregulation of HuD at both mRNA and protein levels has been observed in MNs derived from human induced pluripotent stem cells (hiPSCs) carrying the FUS P525L ALS pathogenic variant and in Fus-D14 knock-in mouse models, affecting MNs’ transcriptome and phenotype. Indeed, FUS-mutant MNs exhibit increased axonal branching compared to FUS wild type (WT) MNs. Moreover, HuD’s gain-of-function effect has been shown to phenocopy cell-autonomous defects caused by the severe FUS P525L variant, leading to impairment in Neuromuscular Junctions (NMJs)’ formation and apoptotic phenomena, evident in co-cultures of hiPSC-derived MNs and Skeletal Muscle (SKM). Consistently, neuronal overexpression of ELAV, the Drosophila Melanogaster ortholog of ELAVL4, has been shown to impair motor performance in flies. Recent evidence also indicates a potential role for HuD in sporadic ALS. HuD expression is selectively upregulated in response to oxidative stress in hiPSC-derived WT MNs and in post-mortem brain samples from sporadic ALS patients, who exhibit a distinctive oxidative stress molecular signature. Based on these findings and as part of the Spoke 3 – Neurodegeneration of the National Center for RNA & Gene Therapy project, we set out to design and evaluate RNA-based therapeutic molecules, specifically siRNAs, miRNA mimics and Antisense Oligonucleotides (ASOs) gapmers, targeting HuD mRNA to lower its expression levels. RNA-based therapeutics represent a rapidly advancing class of medicines, offering the unique ability to directly modulate gene expression and intervene at the root of neurological disease mechanisms, by regulating multiple aspects of RNA metabolism, including mRNA degradation. Most of these molecules have already been approved and commercialized, including Nusinersen for Spinal Muscular Atrophy (SMA) and Tofersen for ALS. Moreover, both ASOs and miRNA mimics are currently being investigated in numerous ongoing clinical trials. By testing them in human in vitro cellular models, our aim is to provide the first proof of principle of their effectiveness towards the development of a future RNA-based therapeutic strategy for ALS.