Extracellular vesicles (EVs) are membrane-enclosed particles released from all eukaryotic cells that carry proteins, lipids, RNA and DNA. They are classified in two main types of EVs: large vesicles (LVs) and small vesicles (SVs). In our previous studies we observed that both LVs and SVs play a role in the disposal of neurotoxic aggregates of the TAR DNA-binding protein 43 (TDP-43) and its C-terminal fragments of 35 (TDP-35) and 25 KDa (TDP-25), associated with Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD).This release increased when the protein quality control (PQC) system [i.e. chaperone proteins, the ubiquitin proteasome system (UPS) and the autophagic pathway] was impaired, a common condition observed both in ALS and FTD. Since TDP-43 is an RNA-binding protein and it is involved in miRNA biogenesis, we wondered whether PQC impairment could also affect miRNA content in EVs. LVs and SVs were isolated from medium of NSC-34 cells, treated or not with the UPS inhibitor MG132 or with the autophagy inhibitor NH4Cl, by differential centrifugation and characterized by Nanosight. MicroRNA libraries were generated using Small RNA-Seq Library Prep Kit (Lexogen) and sequenced on a NextSeq 500/550 (Illumina). Interaction prediction was carried out on TarBase v.8 database. We found a total of 91 Differentially Expressed (DE) (log Fold Change (FC) >1 and <-1) microRNAs in treated-EVs compared to untreated EVs. No DE miRNA were found in NH4Cl-LVs, only 7 miRNA were DE in MG132-LVs and of the 82 miRNAs in MG132-SVs and 66 in NH4Cl-SVs, 43 were in common. Interestingly, the most enriched pathway targeted by commonly DE SVs-miRNAs is the prion disease. In conclusion our observation suggests that, in disease condition, EVs are enriched in both toxic TDP-43 species and potentially harmful miRNA, and thus they may contribute to the propagation of the disease from affected to healthy cells.

How PQC inhibition modulates miRNA loading in large and small extracellular vesicles / E. Casarotto, M. Garofalo, L. Messa, D. Sproviero, S. Carelli, M. Cozzi, M. Chierichetti, R. Cristofani, V. Ferrari, M. Galbiati, F. Mina, M. Piccolella, P. Rusmini, B. Tedesco, P. Pramaggiore, C. Cereda, S. Gagliardi, A. Poletti, V. Crippa. ((Intervento presentato al convegno Inflammation and proteinopathy in ALS FTD spectrum disorder tenutosi a Rijeka : June 30 - July 03 nel 2022.

How PQC inhibition modulates miRNA loading in large and small extracellular vesicles

E. Casarotto
;
S. Carelli;M. Cozzi;M. Chierichetti;R. Cristofani;V. Ferrari;M. Galbiati;F. Mina;M. Piccolella;P. Rusmini;B. Tedesco;P. Pramaggiore;A. Poletti;V. Crippa
2022

Abstract

Extracellular vesicles (EVs) are membrane-enclosed particles released from all eukaryotic cells that carry proteins, lipids, RNA and DNA. They are classified in two main types of EVs: large vesicles (LVs) and small vesicles (SVs). In our previous studies we observed that both LVs and SVs play a role in the disposal of neurotoxic aggregates of the TAR DNA-binding protein 43 (TDP-43) and its C-terminal fragments of 35 (TDP-35) and 25 KDa (TDP-25), associated with Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD).This release increased when the protein quality control (PQC) system [i.e. chaperone proteins, the ubiquitin proteasome system (UPS) and the autophagic pathway] was impaired, a common condition observed both in ALS and FTD. Since TDP-43 is an RNA-binding protein and it is involved in miRNA biogenesis, we wondered whether PQC impairment could also affect miRNA content in EVs. LVs and SVs were isolated from medium of NSC-34 cells, treated or not with the UPS inhibitor MG132 or with the autophagy inhibitor NH4Cl, by differential centrifugation and characterized by Nanosight. MicroRNA libraries were generated using Small RNA-Seq Library Prep Kit (Lexogen) and sequenced on a NextSeq 500/550 (Illumina). Interaction prediction was carried out on TarBase v.8 database. We found a total of 91 Differentially Expressed (DE) (log Fold Change (FC) >1 and <-1) microRNAs in treated-EVs compared to untreated EVs. No DE miRNA were found in NH4Cl-LVs, only 7 miRNA were DE in MG132-LVs and of the 82 miRNAs in MG132-SVs and 66 in NH4Cl-SVs, 43 were in common. Interestingly, the most enriched pathway targeted by commonly DE SVs-miRNAs is the prion disease. In conclusion our observation suggests that, in disease condition, EVs are enriched in both toxic TDP-43 species and potentially harmful miRNA, and thus they may contribute to the propagation of the disease from affected to healthy cells.
Settore BIO/13 - Biologia Applicata
How PQC inhibition modulates miRNA loading in large and small extracellular vesicles / E. Casarotto, M. Garofalo, L. Messa, D. Sproviero, S. Carelli, M. Cozzi, M. Chierichetti, R. Cristofani, V. Ferrari, M. Galbiati, F. Mina, M. Piccolella, P. Rusmini, B. Tedesco, P. Pramaggiore, C. Cereda, S. Gagliardi, A. Poletti, V. Crippa. ((Intervento presentato al convegno Inflammation and proteinopathy in ALS FTD spectrum disorder tenutosi a Rijeka : June 30 - July 03 nel 2022.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/950335
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