PhD student Sapienza University of Rome Sapienza University of Rome, Lazio, Italy
Abstract: HuD is an RNA-binding protein encoded by the ELAVL4 gene, expressed in the nervous system and playing a crucial role in neurodevelopment. Recent evidence suggests its involvement in Amyotrophic Lateral Sclerosis (ALS), a neurodegenerative disease characterized by death of Motor Neurons (MNs). Notably, an upregulation of HuD mRNA and protein has been observed in MNs derived from human-induced pluripotent stem cells (hiPSCs) carrying FUS P525L mutation, which is responsible for a severe familial form of ALS. This alteration leads to changes in transcriptome and phenotype of MNs and demonstrated to exacerbate cell-autonomous effects of the FUS P525L variant, leading to defects in neuromuscular junctions’ establishment and apoptosis phenomena in co-cultures of hiPSC-derived MNs and skeletal muscle (SKM), with a rescue of the phenotypes after siRNA against HuD treatment. However, very little is still known about the role of HuD in MNs and how its deregulation contributes to these phenotypes. To further investigate the role of HuD in FUS P525L ALS and with the intention of downregulate the target using possible therapeutic molecules, we designed RNA-based molecules such as miRNA mimics and Antisense Oligonucleotides (ASO) Gapmers specific for HuD. These molecules exhibit high biodistribution and tolerability and are increasingly being tested in clinical trials, with some already commercialized. As a model system, hiPSCs offer a closer approximation to human physiology. We tested a few Gapmers candidates in SK-N-BE neuroblastoma cell line, evaluating different concentrations and optimizing the transfection method. Next, we moved to the model system of hiPSCs-derived P525L MNs, and we tested the selected Gapmers using different transfection protocols, reagents, and molecule concentrations. The same approach has been applied for miRNA mimic 375, which has the sequence of the endogenous miR-375, that targets HuD and is downregulated in FUS P525L MNs. These molecules will be transfected into mutant MNs and wild-type SKM cocultures to assess phenotypic rescue and validate their efficacy in prospective of deeper investigations on the role of HuD. The use of RNA-based molecules, studied and applied for therapeutic purposes, may represent a forward-looking approach for modulating the expression of HuD in ALS.
Funding Source: CAMPIONE NAZIONALE 3 finanziato dal PNRR Missione 4 su PNRR MISSIONE 4 – COMPONENTE 2 - INVESTIMENTO 1.4
