Introduction ALS and FTLD are neurodegenerative diseases characterized by pathological ubiquitinated and phosphorilated inclusions in the cytosol of affected cells. In 98% of ALS and in the majority of Tau-negative FTLD cases the main component is the TAR DNA-binding protein of 43 KDa (TDP-43) together with its C-terminal fragments of 35 (TDP-35) and 25 KDa (TDP-25). TDP-inclusions are mainly removed from cells via the protein quality control (PQC) system, but they could also be secreted within extracellular vesicles (EVs). In our work we first analysed the TDP-content of the EVs, by comparing large (LVs) with small vesicles (SVs); then, we evaluated the presence of some PQC-members. Finally, we investigated the effect of PQC blockage on EVs secretion and content. Methods We isolated EVs produced by NSC34 cells untreated or treated with MG132 or NH4Cl (proteasome and autophagy inhibitors). To isolate EVs we used the differential ultracentrifugation method. We analysed EVs size, count and morphology through the Nanoparticle Tracking Analysis and the transmission electron microscopy, and their protein content through western blot analysis. Results We showed that both TDP-43 and its C-terminal fragments (especially TDP-35) are secreted in EVs, mainly in LVs. Interestingly, in cells TDPs are present as soluble forms, instead the secreted TDPs are mainly insoluble. We found that many PQC-components are secreted in EVs and PQC modulation resulted in a significant increase in EVs numbers, that is paralleled by a slight increase in TDP-content. Conclusions EVs may positively contribute to the clearance of insoluble TDPs species by cooperating with PQC, having a protective role for affected cells. However, they may also contribute to the prion-like distribution of TDP-neurotoxic forms in neighboring and more distant cells. Funding Fondazione Cariplo, Italy n. 2017-0747, Italian Ministry of University and Research (MIUR), PRIN n. 2017F2A2C5
The role of Extracellular Vesicles (EVs) in Amyotrophic Lateral Sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) / E. Casarotto, D. Sproviero, S. Gagliardi, E. Corridori, 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 10. convegno ISEV Annual Meeting tenutosi a online nel 2021.
The role of Extracellular Vesicles (EVs) in Amyotrophic Lateral Sclerosis (ALS) and frontotemporal lobar degeneration (FTLD)
E. Casarotto;E. Corridori;M. Cozzi;B. Tedesco;R. Cristofani;V. Ferrari;M. Chierichetti;P. Rusmini;M. Galbiati;A. Poletti;V. Crippa
2021
Abstract
Introduction ALS and FTLD are neurodegenerative diseases characterized by pathological ubiquitinated and phosphorilated inclusions in the cytosol of affected cells. In 98% of ALS and in the majority of Tau-negative FTLD cases the main component is the TAR DNA-binding protein of 43 KDa (TDP-43) together with its C-terminal fragments of 35 (TDP-35) and 25 KDa (TDP-25). TDP-inclusions are mainly removed from cells via the protein quality control (PQC) system, but they could also be secreted within extracellular vesicles (EVs). In our work we first analysed the TDP-content of the EVs, by comparing large (LVs) with small vesicles (SVs); then, we evaluated the presence of some PQC-members. Finally, we investigated the effect of PQC blockage on EVs secretion and content. Methods We isolated EVs produced by NSC34 cells untreated or treated with MG132 or NH4Cl (proteasome and autophagy inhibitors). To isolate EVs we used the differential ultracentrifugation method. We analysed EVs size, count and morphology through the Nanoparticle Tracking Analysis and the transmission electron microscopy, and their protein content through western blot analysis. Results We showed that both TDP-43 and its C-terminal fragments (especially TDP-35) are secreted in EVs, mainly in LVs. Interestingly, in cells TDPs are present as soluble forms, instead the secreted TDPs are mainly insoluble. We found that many PQC-components are secreted in EVs and PQC modulation resulted in a significant increase in EVs numbers, that is paralleled by a slight increase in TDP-content. Conclusions EVs may positively contribute to the clearance of insoluble TDPs species by cooperating with PQC, having a protective role for affected cells. However, they may also contribute to the prion-like distribution of TDP-neurotoxic forms in neighboring and more distant cells. Funding Fondazione Cariplo, Italy n. 2017-0747, Italian Ministry of University and Research (MIUR), PRIN n. 2017F2A2C5
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