Amyotrophic Lateral Sclerosis is a neurodegenerative disease that affects motoneurons. The alteration in proteostasis is an hallmark of ALS. The Chaperone Assisted Selective Autophagy (CASA) system belongs to the proteostasis network and it has been demonstrated that ALS-related proteins can be degraded through this pathway. CASA system consists of a protein complex formed by the small heat shock protein B8 (HSPB8), the co-chaperone BAG3, HSP70 and the E3 ubiquitin ligase CHIP, which recognizes misfolded proteins, routing them to autophagic degradation. It has been demonstrated that HSPB8 overexpression or induction enhance the clearance of ALS related proteins, counteracting their aggregation. Mutations in HSPB8 (K141E/N) are causative of diseases that affect neurons. Two new mutations of HSPB8 (S181C and P41S) have been found in two ALS patients by Dr. Ticozzi and colleagues (Istituto Auxologico Italiano). Here, we demonstrate that these two new mutants are not prone to misfold and aggregate in NSC-34 motoneuronal cell model, as shown in western blot, filter retardation assays and immunofluorescence. Moreover, they do not have an impact on autophagic markers p62 and LC-3 I/II protein levels in WB. Interestingly, the S181C mutant shows an altered migration on SDS-PAGE under not reducing conditions. In ALS cell model of disease overexpressing SOD1-G93A, P41S and S181C mutants have a detrimental effect on SOD1 clearance in respect to the wild-type HSPB8 and to the known mutant carrying K141E mutation, as shown in IF and FTA assay. In a sporadic model of disease overexpressing TDP-43, HSPB8 S181C mutant shows a detrimental function in the clearance of insoluble species of TDP-43. In summary, these data give new insights in HSPB8 role and mutations that could have an impact in ALS disease.

New HSPB8 mutations related to Amyotrophic Lateral Sclerosis : characterization and impact in cell models of disease / B. Tedesco, V. Crippa, P. Rusmini, R. Cristofani, M.E. Cicardi, V. Ferrari, G. Vezzoli, M. Meroni, E. Messi, M. Piccolella, R. Galbiati, A. Poletti. ((Intervento presentato al convegno ABCD - National Ph.D. Meeting tenutosi a Salerno nel 2018.

New HSPB8 mutations related to Amyotrophic Lateral Sclerosis : characterization and impact in cell models of disease

B. Tedesco;V. Crippa;P. Rusmini;R. Cristofani;M.E. Cicardi;V. Ferrari;M. Meroni;E. Messi;M. Piccolella;R. Galbiati;A. Poletti
2018

Abstract

Amyotrophic Lateral Sclerosis is a neurodegenerative disease that affects motoneurons. The alteration in proteostasis is an hallmark of ALS. The Chaperone Assisted Selective Autophagy (CASA) system belongs to the proteostasis network and it has been demonstrated that ALS-related proteins can be degraded through this pathway. CASA system consists of a protein complex formed by the small heat shock protein B8 (HSPB8), the co-chaperone BAG3, HSP70 and the E3 ubiquitin ligase CHIP, which recognizes misfolded proteins, routing them to autophagic degradation. It has been demonstrated that HSPB8 overexpression or induction enhance the clearance of ALS related proteins, counteracting their aggregation. Mutations in HSPB8 (K141E/N) are causative of diseases that affect neurons. Two new mutations of HSPB8 (S181C and P41S) have been found in two ALS patients by Dr. Ticozzi and colleagues (Istituto Auxologico Italiano). Here, we demonstrate that these two new mutants are not prone to misfold and aggregate in NSC-34 motoneuronal cell model, as shown in western blot, filter retardation assays and immunofluorescence. Moreover, they do not have an impact on autophagic markers p62 and LC-3 I/II protein levels in WB. Interestingly, the S181C mutant shows an altered migration on SDS-PAGE under not reducing conditions. In ALS cell model of disease overexpressing SOD1-G93A, P41S and S181C mutants have a detrimental effect on SOD1 clearance in respect to the wild-type HSPB8 and to the known mutant carrying K141E mutation, as shown in IF and FTA assay. In a sporadic model of disease overexpressing TDP-43, HSPB8 S181C mutant shows a detrimental function in the clearance of insoluble species of TDP-43. In summary, these data give new insights in HSPB8 role and mutations that could have an impact in ALS disease.
Settore BIO/13 - Biologia Applicata
Settore BIO/09 - Fisiologia
New HSPB8 mutations related to Amyotrophic Lateral Sclerosis : characterization and impact in cell models of disease / B. Tedesco, V. Crippa, P. Rusmini, R. Cristofani, M.E. Cicardi, V. Ferrari, G. Vezzoli, M. Meroni, E. Messi, M. Piccolella, R. Galbiati, A. Poletti. ((Intervento presentato al convegno ABCD - National Ph.D. Meeting tenutosi a Salerno nel 2018.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/813966
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