ALS and FTLD are characterized by the presence of pathological ubiquitinated and phosphorilated inclusions in the cytosol of affected cells. The main components of these inclusions are the TAR DNA-binding protein of 43 KDa (TDP-43) and its C-terminal fragments of 35 (TDP-35) and 25 KDa (TDP-25). TDP-inclusions are toxic for the cells. Indeed, cells use different mechanisms to eliminate inclusions: the protein quality control (PQC) system, which is the main mechanism responsible for the maintenance of proteostasis, the unconventional secretion of free proteins and the extracellular vesicles (EVs). We focused on EVs and their possible interplay with PQC, in the removal of TDP-species. In particular, we compared large (LVs) with small vesicles (SVs) for their TDP-content and the presence of some members of the PQC, both in basal condition and after PQC blockage. We isolated EVs produced by immortalized motoneuronal cells, untreated or treated with MG132 and/or NH4Cl (proteasome and autophagy inhibitors), using the differential ultracentrifugation method. Then, we analysed EVs for size, count and morphology through the Nanoparticle Tracking Analysis and the transmission electron microscopy, and for their protein content through western blot analysis. We observed that both TDP-43 and its C-terminal fragments (especially TDP-35) are secreted in EVs. Interestingly, in cells TDPs are mainly in soluble forms, while in EVs TDPs species are mainly insoluble. Finally, we found that many PQC-components are secreted in EVs and that PQC modulation determines an increase of EVs release, especially with the blockage of both the proteasome and the autophagy. In conclusion EVs may cooperate with PQC, having a protective role in the clearance of insoluble TDPs species. However, they could also contribute to the prion-like distribution of TDP-neurotoxic species in neighbouring and more distant cells. Funding Fondazione Cariplo, Italy n. 2017-0747, Italian Ministry of University and Research (MIUR), PRIN n. 2017F2A2C5

Extracellular vesicles and the secretion of TDP species in Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD) / E. Casarotto, D. Sproviero, S. Gagliardi, F. Fabbiano, M.C. Gagliani, M. Cozzi, B. Tedesco, R. Cristofani, V. Ferrari, M. Chierichetti, P. Rusmini, M. Galbiati, V.G. D’Agostino, K. Cortese, C. Cereda, A. Poletti, V. Crippa. ((Intervento presentato al 1. convegno ESN Virtual Conference: Future perspectives for European neurochemistry : a young scientists conference tenutosi a online nel 2021.

Extracellular vesicles and the secretion of TDP species in Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD)

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

Abstract

ALS and FTLD are characterized by the presence of pathological ubiquitinated and phosphorilated inclusions in the cytosol of affected cells. The main components of these inclusions are the TAR DNA-binding protein of 43 KDa (TDP-43) and its C-terminal fragments of 35 (TDP-35) and 25 KDa (TDP-25). TDP-inclusions are toxic for the cells. Indeed, cells use different mechanisms to eliminate inclusions: the protein quality control (PQC) system, which is the main mechanism responsible for the maintenance of proteostasis, the unconventional secretion of free proteins and the extracellular vesicles (EVs). We focused on EVs and their possible interplay with PQC, in the removal of TDP-species. In particular, we compared large (LVs) with small vesicles (SVs) for their TDP-content and the presence of some members of the PQC, both in basal condition and after PQC blockage. We isolated EVs produced by immortalized motoneuronal cells, untreated or treated with MG132 and/or NH4Cl (proteasome and autophagy inhibitors), using the differential ultracentrifugation method. Then, we analysed EVs for size, count and morphology through the Nanoparticle Tracking Analysis and the transmission electron microscopy, and for their protein content through western blot analysis. We observed that both TDP-43 and its C-terminal fragments (especially TDP-35) are secreted in EVs. Interestingly, in cells TDPs are mainly in soluble forms, while in EVs TDPs species are mainly insoluble. Finally, we found that many PQC-components are secreted in EVs and that PQC modulation determines an increase of EVs release, especially with the blockage of both the proteasome and the autophagy. In conclusion EVs may cooperate with PQC, having a protective role in the clearance of insoluble TDPs species. However, they could also contribute to the prion-like distribution of TDP-neurotoxic species in neighbouring and more distant cells. Funding Fondazione Cariplo, Italy n. 2017-0747, Italian Ministry of University and Research (MIUR), PRIN n. 2017F2A2C5
25-mag-2021
Settore BIO/13 - Biologia Applicata
European Society for Neurochemistry-Young Scientist Steering Committee
Extracellular vesicles and the secretion of TDP species in Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD) / E. Casarotto, D. Sproviero, S. Gagliardi, F. Fabbiano, M.C. Gagliani, M. Cozzi, B. Tedesco, R. Cristofani, V. Ferrari, M. Chierichetti, P. Rusmini, M. Galbiati, V.G. D’Agostino, K. Cortese, C. Cereda, A. Poletti, V. Crippa. ((Intervento presentato al 1. convegno ESN Virtual Conference: Future perspectives for European neurochemistry : a young scientists conference tenutosi a online nel 2021.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/846859
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