VALOSIN CONTAINING PROTEIN IN AMYOTROPHIC LATERAL SCLEROSIS: NEW INSIGHT IN PATHOLOGICAL MECHANISMS

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by the progressive death of upper and lower motor neurons (MNs). ALS can be classified in familiar forms (fALS), occurring in 10% of the cases, and sporadic form (sALS), occurring in the remaining 90%. Many pathways have been found to concur with MNs death, among which has a great importance alteration in proteostasis. Indeed, all ALS forms are characterized by the presence of protein aggregates and vacuoles in the brain tissue of effected patients. These are signs of an altered protein quality control (PQC) system. Protein aggregates can be either triggered by the mutations of genes that express unstable proteins that misfold and aggregate, or by mutations of genes that express proteins involved in the degradation pathways as ubiquitin-proteasome system (UPS) and autophagy. One of these genes associated to ALS is VCP that encodes for Valosin Containing Protein (VCP), a protein involved in many steps of the PQC system. One of VCP roles is to disassemble protein aggregates and concur to their degradation. Thus, in the first part of my thesis, I focused on VCP contribute in the removal of protein aggregates positive to ALS-associated SOD1-mutant (SOD1 G93A). The aim of this part was to define if the modulation of VCP expression could improve altered proteostasis, ameliorating the pathological condition. Moreover, I also studied two VCP-mutants associated to ALS (VCP R155H and VCP R191Q) comparing to VCP WT, their contribution in this pathway. To study VCP role I overexpressed VCP WT and VCP-mutants in NSC34 cells, an immortalized motor neuron cell line, that transiently expressed SOD1 G934A. My work demonstrated that VCP WT overexpression enhanced SOD1-mutant clearance through the UPS. Surprisingly also VCP-mutants increased SOD1-mutants clearance. VCP-mutants contribute in SOD1-mutants clearance differed from VCP WT as it resulted dependent from the autophagic pathway. In the second part of my thesis, I focused on VCP-mutants to better understand their pathological mechanisms. In particular, I studied their effects on lysosomal damage and their impact on the autophagic flux. To define VCP-mutants pathological contribute in an ALS-model I overexpressed VCP R155H and VCP R191Q in NSC34 cells. Firstly, I determined that VCP-mutants aggregated in this model. Moreover, I found that VCP-mutants expression was associated to lysosome alteration in content, size, morphology, and membrane breakage. Lysosomal damaged induced by VCP-mutants led to the specific activation of TFE3, a transcription factor regulator of autophagy and lysosome biogenesis. In fact, by studying autophagic markers, I determined that the presence of VCP-mutants was associated to the activation of the autophagic flux. Finally, in this part, I found that VCP-mutants prevented damaged lysosome clearance when it was induced by chemical compounds, as trehalose, or by SOD1-mutant overexpression. Conversely, VCP WT overexpression enhanced lysosomal damage clearance in both of these conditions. Overall with my work, I determined new pathological mechanisms of VCP-mutants ALS-associated. Moreover, I found that VCP modulation could ameliorate ALS-pathological conditions through different pathways.