A Novel Approach to Investigating the TARDBP p.G376D Mutation in an Italian Familial ALS Case: Molecular Analysis in Fibroblasts and iPSC-Derived Motor Neuron Models

Background. Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease that affects motor neurons and leads patients to progressive paralysis and death. ALS can be sporadic ALS (sALS), which affects approximately 90% of cases, or familial (fALS), which affects approximately 10% of cases. To date, more than 40 ALS-related genes have been identified, including TARDBP (Transactive DNA-Binding Protein), which encodes for TDP-43 protein, involved in RNA metabolism. Recently, a novel TARDBP mutation, pG376D, was described in a large Italian family, causing an aggressive and rapid ALS. To date, this mutation is unique among Caucasians.Aim of the study. To better understand the molecular pathological mechanisms underlying ALS in subjects carrying the G376D TDP-43 mutation, 4 objectives were set:1) Study of the expression and sub-cellular localization of TDP-43 in patient-derived fibroblasts under both basal and chronic stress conditions2) Generation of induced pluripotent stem cells (iPSCs) from patient fibroblasts and transfection with NIL and NIP transposons using the PiggyBac system3) Differentiation and characterization of MNs4) Study of the expression and sub-cellular localization of TDP-43 in the MNs under both basal and stress conditions (acute and chronic).Results. Patient-derived fibroblasts from symptomatic TDP-43 G376D mutation carrier (s.G376D), an asymptomatic TDP-43 G376D mutation carrier (a.G376D), and a healthy control (WT) were analysed under basal and chronic stress conditions. Under basal conditions, TDP-43 exhibited its expected nuclear localization, consistent with its physiological role. However, upon chronic stress induction with Sodium Arsenite, the s.G376D fibroblasts showed a reduced ability to form stress granules and a more rapid disassembly of these granules during recovery.iPSCs were successfully generated from fibroblasts and transfected with NIL constructs using the PiggyBac system. The iPSCs differentiated in MNs – using an established protocol with transcription factors –showed the typical morphology of MNs and expressed canonical markers – ChAT, Hb9, βIII- tubulin and others – validating the robustness of the differentiation protocol. Few challenges were encountered while establishing this human-based ALS model – particularly due to the presence of differentiated cells – therefore, protocols refinements were carried out to enhance the culture quality and viability of MNs population, including improved passaging methods (use of ReLeSR), a selection of post-transfected colonies, and the use of Floxuridine to suppress proliferation of the differentiated cells.TDP-43 remained mostly nuclear in MNs, with some diffusion in the neurites, under basal conditions, which aligns with aligns with its physiological roles and suggests that pathological mislocalization may occur at later stages of disease progression, highlighting the need for further investigations in mature MNs.Conclusions. This study provides important insights into the inefficient cellular responses of TDP-43 in symptomatic G376D carriers and offers valuable improvements to iPSC culture and differentiation protocols for ALS modelling. While further refinements are needed to improve the quality of differentiated MNs and reduce the presence of “flat cells”, the results presented here lay the groundwork for using iPSC-derived models to investigate ALS pathogenesis and potential therapeutic interventions targeting G376D TDP-43.