VCP autophagic modulation rescues C9ORF72 pathological features in ALS-neuronal models

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder characterized by motoneuronal death. The loss of motoneurons is caused by various pathological mechanisms, including the accumulation of toxic misfolded protein aggregates, altered autophagy and disruptions in lysosome functionality and stability. Some familial ALS cases are associated with the repeat expansion of a hexanucleotide sequence (G4C2) within the C9ORF72 gene, resulting in an unconventional translation of five different dipeptide repeat proteins (DPRs). These DPRs tend to aggregate, forming intracellular inclusions that are detrimental to cells. Furthermore, DPRs are implicated in organelle alterations and malfunctioning of protein quality control (PQC) system. Valosin-containing protein (VCP), an AAA ATPase, plays a crucial role in the PQC system, extracting altered proteins and facilitating their clearance through the ubiquitin-proteasome system (UPS) and autophagy. Our study focused on analyzing the biochemical behavior of individual DPRs in immortalized motoneurons and the role of VCP in their clearance. We confirmed their accumulation and localization, observing their impact on lysosome stability. We demonstrated that all DPRs induce damage to lysosome membranes and alterations in lysosome activity. Notably, DPRs prevented the activation of autophagy, normally triggered to eliminate damaged lysosomes, by impeding the nuclearization of transcription factors TFEB and TFE3, crucial for autophagy and lysosome biogenesis. Additionally, overexpressing VCP in these models rescued the accumulation of the most toxic DPRs. Our data revealed an enhancement in clearance mediated by VCP through both UPS and autophagy. VCP overexpression was also associated with a reduction in lysosome alterations, possibly linked to the clearance of DPRs aggregates facilitated by VCP. Collectively, these findings propose the modulation of VCP as a potential target to alleviate DPRs-mediated toxicity and preserve motoneuron viability in ALS. Fondazione Telethon; Kennedy’s disease association; Fondazione Cariplo, Italy; Fondazione AriSLA, Italy; Bando Straordinario per Italian Ministry of University and Research (MIUR), PRIN—Progetti di ricerca di interesse nazionale ; Italian Ministry of University and Research (Progetto Dipartimenti di Eccellenza). National Center for Gene Therapy and Drugs based on RNA Technology – PNRR – CN3.