Valosin Containing Protein (VCP) is an AAA+ protein that has a key role in several cellular pathways maintaining homeostasis. In fact, VCP misfunctioning and mutations have been correlated to IBMPFD, a multisystem proteinopathy, and different neurodegenerative diseases including ALS. VCP mutants are correlated with TDP-43 mislocalization and aggregation, the presence of ubiquitin inclusions and of vacuoles that suggest an alteration in the Protein Quality Control system. To date, the mechanisms by which VCP mutants lead to cell toxicity and death are still not clear.
In this study, we mean to identify VCP mutants pathological mechanisms in an ALS-model. To this aim we overexpress VCP WT, VCP R155H and VCP R191Q in a motor neuron mouse immortalized cell line, NSC-34. In first instance, we found that in this neuronal model VCP-mutants form insoluble aggregates and induce lysosomal alteration in size, morphology and membrane breakage.
It is known that lysosomal damage is correlated with activation of the autophagic pathway that removes damaged toxic lysosomes. Thus, we investigated if VCP-mutants were associated with a positive regulation of autophagy. Our results demonstrate that VCP-mutants induce autophagy. Indeed, we found that VCP-mutants expression increases LC3-II conversion which further increases inhibiting autophagy with NH4Cl. In these conditions, we also found an increased trend in p62 levels. Furthermore, we determined that VCP-mutants autophagy positive regulation is specifically associated with TFE3 activation as a result of a process that involves the activation of calcineurin, a phosphatase Ca2+ dependent. At the same time, we excluded the involvement of TFEB in this pathway. Our findings suggest that lysosomal membrane rupture and leakage caused by VCP mutants, trigger calcineurin activation which mediates TFE3 dephosphorylation and nuclear translocation to induce autophagy. In support to this, we found that VCP mutants induce insoluble protein-aggregates clearance with a specific dependency from the autophagic pathway.