Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that causes death of motor-neurons. The 90% of all ALS cases can be classified as sporadic, and the 10% can be classified as familial forms (fALS). There are many different forms of fALS correlated to different mutations. Every mutation is deleterious by different mechanisms, but they are all correlated to presence of aggregates in the brain tissue of affected patients. All aggregates can be removed by the Protein Quality Control (PQC) system, which is composed by chaperone, the Ubiquitin-Proteasome System (UPS) and the autophagic pathway. Lysosome is an important organelle involved in autophagy. The alteration of lysosome is deleterious for cell firstly for its key role in the PQC system and secondly lysosome-damage leads to massive lysosomal leakage that causes an increase in cytosolic acidity, uncontrolled breakdown of cell components and cell death by necrosis. There are some mechanisms that can be activated to maintain the lysosomal activity. The most studied mechanism is lysophagy. Here we focused our attention on Valosin Containing Protein (VCP), an ATPase with many roles in cellular proteinostasis. Recent studies demonstrated that VCP has a key role in lysophagy. Mutations of this protein are found in fALS, as well as in IBMPDF, AD, PD. We previously demonstrated that the overexpression of VCP wild type and fALS mutations (VCP R155H and VCP R191Q) decrease the levels of insoluble species of a SOD1-mutant (SOD1 G93A). SOD1 mutations are correlated to 30% of fALS and they encode proteins that misfold and form intracellular aggregates. In this work we study lysosomal-damage response in presence of overexpressed VCP wt, and its mutants VCP R155H and VCP R191Q, in NSC-34 cell line. To study their contribute in the clearance of damaged lysosomes we used trehalose treatment that induces lysosome damage. We observed that overexpressed VCP wt reduces lysosomal damage when it is induced by trehalose. We also observed that VCP R155H induces lysosomal damage in basal conditions and prevents the clearance of damaged lysosomes when the damage is induced. VCP R191Q also induces lysosome damage in basal conditions but lower level compared to VCP R155H. When the damage is induced we observed that VCP R191Q can partially reduce the lysosomal damage, but it significantly loses its functionality compared to overexpressed VCP wt. Moreover, the overexpression of the VCP R155H leads to the conversion of LC3I into LC3II that increases when cells are treated with NH4Cl, a inhibitior of autophagy. This suggest an activation of autophagy in the presence of VCP-mutant. The activation of the autophagic pathway due to this mechanism could explain the decrease of insoluble aggregates when VCP-mutants are overexpressed as we have previously demonstrated.

ALS-associeted VCP-mutants lead to lysosome damage / V. Ferrari, M.E. Cicardi, P. Rusmini, V. Crippa, R. Cristofani, B. Tedesco, M. Galbiati, E. Messi, M. Piccolella, A. Poletti. ((Intervento presentato al convegno National Meeting of PhD Students in Neuroscience tenutosi a Napoli nel 2019.

ALS-associeted VCP-mutants lead to lysosome damage

V. Ferrari;P. Rusmini;V. Crippa;R. Cristofani;B. Tedesco;M. Galbiati;E. Messi;M. Piccolella;A. Poletti
2019

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that causes death of motor-neurons. The 90% of all ALS cases can be classified as sporadic, and the 10% can be classified as familial forms (fALS). There are many different forms of fALS correlated to different mutations. Every mutation is deleterious by different mechanisms, but they are all correlated to presence of aggregates in the brain tissue of affected patients. All aggregates can be removed by the Protein Quality Control (PQC) system, which is composed by chaperone, the Ubiquitin-Proteasome System (UPS) and the autophagic pathway. Lysosome is an important organelle involved in autophagy. The alteration of lysosome is deleterious for cell firstly for its key role in the PQC system and secondly lysosome-damage leads to massive lysosomal leakage that causes an increase in cytosolic acidity, uncontrolled breakdown of cell components and cell death by necrosis. There are some mechanisms that can be activated to maintain the lysosomal activity. The most studied mechanism is lysophagy. Here we focused our attention on Valosin Containing Protein (VCP), an ATPase with many roles in cellular proteinostasis. Recent studies demonstrated that VCP has a key role in lysophagy. Mutations of this protein are found in fALS, as well as in IBMPDF, AD, PD. We previously demonstrated that the overexpression of VCP wild type and fALS mutations (VCP R155H and VCP R191Q) decrease the levels of insoluble species of a SOD1-mutant (SOD1 G93A). SOD1 mutations are correlated to 30% of fALS and they encode proteins that misfold and form intracellular aggregates. In this work we study lysosomal-damage response in presence of overexpressed VCP wt, and its mutants VCP R155H and VCP R191Q, in NSC-34 cell line. To study their contribute in the clearance of damaged lysosomes we used trehalose treatment that induces lysosome damage. We observed that overexpressed VCP wt reduces lysosomal damage when it is induced by trehalose. We also observed that VCP R155H induces lysosomal damage in basal conditions and prevents the clearance of damaged lysosomes when the damage is induced. VCP R191Q also induces lysosome damage in basal conditions but lower level compared to VCP R155H. When the damage is induced we observed that VCP R191Q can partially reduce the lysosomal damage, but it significantly loses its functionality compared to overexpressed VCP wt. Moreover, the overexpression of the VCP R155H leads to the conversion of LC3I into LC3II that increases when cells are treated with NH4Cl, a inhibitior of autophagy. This suggest an activation of autophagy in the presence of VCP-mutant. The activation of the autophagic pathway due to this mechanism could explain the decrease of insoluble aggregates when VCP-mutants are overexpressed as we have previously demonstrated.
1-mar-2019
Settore BIO/13 - Biologia Applicata
Settore BIO/09 - Fisiologia
ALS-associeted VCP-mutants lead to lysosome damage / V. Ferrari, M.E. Cicardi, P. Rusmini, V. Crippa, R. Cristofani, B. Tedesco, M. Galbiati, E. Messi, M. Piccolella, A. Poletti. ((Intervento presentato al convegno National Meeting of PhD Students in Neuroscience tenutosi a Napoli nel 2019.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/637758
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