Valosin Containing Protein (VCP) is AAA+ protein involved in many cellular pathways that maintain cellular homeostasis. Indeed, VCP misfunctioning and mutations have been correlated to IBMPFD, a multisystem proteinopathy, and different neurodegenerative diseases including ALS. VCP mutants are correlated with the presence of ubiquitin inclusions, TDP-43 mislocalization and aggregation, as well as with the presence of vacuoles suggesting an alteration in Protein Quality Control System. However, the mechanisms by which VCP mutants 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. To this goal 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 model both VCP mutants form insoluble aggregates and induce lysosomal alteration in size, morphology and membrane breakage. As lysosomal damage is generally correlated with activation of autophagy to remove altered toxic lysosomes, we investigated if VCP-mutants were correlated with a positive regulation of autophagy. From our findings, we could determine that VCP-mutants induce the autophagic flux. Indeed, we found an increase in LC3-II conversion in presence of VCP-mutants that further increases in the condition in which autophagy is inhibited with NH4Cl. In these conditions, we also found an increased trend in p62 levels. Moreover, we demonstrated that VCP-mutants autophagy activation is specifically associated with TFE3 activation, in a process that involves the activation of calcineurin, a phosphatase Ca2+ dependent. In parallel, we excluded the involvement of TFEB in this pathway. This suggests that lysosomal damage and leakage induced by VCP mutants activates calcineurin which mediates TFE3 dephosphorylation and nuclear translocation to induce autophagy. In support to this we found that VCP mutants enhance insoluble protein-aggregates clearance with a specific dependency from the autophagic pathway.

VCP mutants induce lysosomal damage and autophagy activation in amyotrophic lateral sclerosis (ALS) / V. Ferrari, M.E. Cicardi, P. Rusmini, V. Crippa, R.M. Cristofani, B. Tedesco, E. Casarotto, M. Chierichetti, M. Cozzi, M. Piccolella, M. Galbiati, A. Poletti. ((Intervento presentato al convegno WORLDSymposium 2021 tenutosi a virtuale nel 2021.

VCP mutants induce lysosomal damage and autophagy activation in amyotrophic lateral sclerosis (ALS)

V. Ferrari;M.E. Cicardi;P. Rusmini;V. Crippa;R.M. Cristofani;B. Tedesco;E. Casarotto;M. Chierichetti;M. Cozzi;M. Piccolella;M. Galbiati;A. Poletti
2021

Abstract

Valosin Containing Protein (VCP) is AAA+ protein involved in many cellular pathways that maintain cellular homeostasis. Indeed, VCP misfunctioning and mutations have been correlated to IBMPFD, a multisystem proteinopathy, and different neurodegenerative diseases including ALS. VCP mutants are correlated with the presence of ubiquitin inclusions, TDP-43 mislocalization and aggregation, as well as with the presence of vacuoles suggesting an alteration in Protein Quality Control System. However, the mechanisms by which VCP mutants 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. To this goal 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 model both VCP mutants form insoluble aggregates and induce lysosomal alteration in size, morphology and membrane breakage. As lysosomal damage is generally correlated with activation of autophagy to remove altered toxic lysosomes, we investigated if VCP-mutants were correlated with a positive regulation of autophagy. From our findings, we could determine that VCP-mutants induce the autophagic flux. Indeed, we found an increase in LC3-II conversion in presence of VCP-mutants that further increases in the condition in which autophagy is inhibited with NH4Cl. In these conditions, we also found an increased trend in p62 levels. Moreover, we demonstrated that VCP-mutants autophagy activation is specifically associated with TFE3 activation, in a process that involves the activation of calcineurin, a phosphatase Ca2+ dependent. In parallel, we excluded the involvement of TFEB in this pathway. This suggests that lysosomal damage and leakage induced by VCP mutants activates calcineurin which mediates TFE3 dephosphorylation and nuclear translocation to induce autophagy. In support to this we found that VCP mutants enhance insoluble protein-aggregates clearance with a specific dependency from the autophagic pathway.
8-feb-2021
Settore BIO/13 - Biologia Applicata
Settore BIO/09 - Fisiologia
VCP mutants induce lysosomal damage and autophagy activation in amyotrophic lateral sclerosis (ALS) / V. Ferrari, M.E. Cicardi, P. Rusmini, V. Crippa, R.M. Cristofani, B. Tedesco, E. Casarotto, M. Chierichetti, M. Cozzi, M. Piccolella, M. Galbiati, A. Poletti. ((Intervento presentato al convegno WORLDSymposium 2021 tenutosi a virtuale nel 2021.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/860139
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