VCP ALS-mutants induce lysosomal damage and autophagy activation

Valosin Containing Protein (VCP) is an ATPase protein of AAA+ family. VCP has a key role in many cellular pathways that maintain cellular homeostasis including control in protein misfolding, damaged organelle degradation and membranes formation. VCP mutations have been correlated to IBMPDF, a multisystem proteinopathy and different neurodegenerative diseases including ALS. VCP-mutants have been associated with signs of alteration of the Protein Quality Control System as the presence of ubiquitin inclusions, TDP-43 mis-localization and aggregation, and the presence of vacuoles. To date, the VCP-mutants mechanisms that lead to cell toxicity and death are still not defined. In this study, we aim to identify VCP-mutants pathological mechanisms in an ALS-model. We overexpress VCP WT, VCP R155H and VCP R191Q in NSC-34, a motor neuron mouse immortalized cell line. In first instance, we found that both VCP mutants induce lysosomal alteration in size, morphology, activity and membrane breakage. Literature data show that lysosomal damage is correlated with the activation of the autophagic flux to remove altered toxic lysosomes, thus we analyzed if VCP-mutants were correlated with a positive regulation of autophagy. Our findings demonstrate that VCP-mutants induce the autophagic flux: indeed, we found an increase in LC3-II conversion in presence of VCP-mutants that further increases with NH4Cl treatment, a late inhibitor of autophagy. In support to this, we also found an increased trend in p62 levels. Moreover, we determined that VCP-mutants positively regulate autophagic flux by specifically activating the transcription factor TFE3. Results also determined that TFE3 VCP-mutants activation is mediated by calcineurin, a phosphatase Ca2+ dependent. In parallel, we excluded the involvement of TFEB in this pathway. These data suggest that lysosomal damage and leakage induced by VCP-mutants activate calcineurin which in turn mediates TFE3 dephosphorylation and nuclear translocation inducing autophagy. In support to this we found that VCP mutants enhance insoluble protein-aggregates with a specific dependency from the autophagic pathway.