INTRODUCTION Secretion of extracellular vesicles (EVs) and EV-mediated exchange of molecules among brain cells, under physiological conditions, has been described years ago. At the same time, ability of EVs to transfer proteins and nucleic acids to the surrounding cells, thus modifying their phenotypes, has been clearly demonstrated for tumor cells of different origins, including brain cancers. More recently, involvement of EVs in pathological processes affecting the Central Nervous System (CNS), and their capacity of transferring damaging cargoes have been also suggested. To explore these functions, we exposed primary cultures of astrocytes to EVs isolated from cerebral spinal fluid (CSF) of patients affected by multiple sclerosis (MS), in order to study the potential ability of these EVs to induce in receiving cells morphological and functional changes, such as modifications of proliferation rate, apoptosis or cell death. METHODS Our study was carried out on 10 individuals who, after clinical diagnosis, were separated into two different groups: controls and MS patients. The diagnosis has always been confirmed by a neurologist and the patients gave informed consent to participate in a molecular study on MS. EV population is actually composed of two kind of vesicles: (a) exosomes (Es), which originate from the endosomal compartment and are smaller, and larger membrane vesicles (MVs), which directly bud from the plasma membrane. To separate Es from MVs, before ultracentrifugation, CSF was filtered through a 0.2-μm filter and centrifuged at 10,000 × g for 30 min. The pellet containing MVs was saved, while the supernatant was further centrifuged at 105,000 × g for 90 min to obtain Es. Pelleted vesicles were suspended with phosphate-buffered saline, pH 7.5 (PBS) and used to treat astrocytes for 24 hours. RESULTS MVs from MS patients induced a higher degree of cell death than those prepared from the CSF of control group (99,35% dead cells vs 89,33), but the difference was not significant (p value=0,142). A dramatic effect was instead noticed when treating astrocytes with Es from patients, which were able to induce a highly significant (p value=0,0001) degree of cell death respect to Es from controls (94,38% vs 71,21). CONCLUSIONS Our study suggests that EVs present in the CSF contain molecules which can be toxic to astrocytes and this is probably the reason why brain cells discard such molecules via different kinds of EVs. However, EVs, and in particular Es, present in CSF of MS patients are even more toxic for cells than EVs present in the CSF of healthy controls. Further studies are now necessary to identify the active molecules present in EVs.
Ragonese, P., Salemi, G., Schiera, G., Realmuto, S., Di Liegro, C., Di Liegro, I., et al. (2017). Effect of extracellular vesicles from CSF of multiple sclerosis patients and healthy controls on astrocytes in culture, Volume: 24, Supplement 1(Volume: 24, Supplement: 1), 654-654.
Effect of extracellular vesicles from CSF of multiple sclerosis patients and healthy controls on astrocytes in culture
Ragonese, P;Salemi, G;Schiera, G;Realmuto, S;Di Liegro, CM;Di Liegro, I;Proia, P.
2017-01-01
Abstract
INTRODUCTION Secretion of extracellular vesicles (EVs) and EV-mediated exchange of molecules among brain cells, under physiological conditions, has been described years ago. At the same time, ability of EVs to transfer proteins and nucleic acids to the surrounding cells, thus modifying their phenotypes, has been clearly demonstrated for tumor cells of different origins, including brain cancers. More recently, involvement of EVs in pathological processes affecting the Central Nervous System (CNS), and their capacity of transferring damaging cargoes have been also suggested. To explore these functions, we exposed primary cultures of astrocytes to EVs isolated from cerebral spinal fluid (CSF) of patients affected by multiple sclerosis (MS), in order to study the potential ability of these EVs to induce in receiving cells morphological and functional changes, such as modifications of proliferation rate, apoptosis or cell death. METHODS Our study was carried out on 10 individuals who, after clinical diagnosis, were separated into two different groups: controls and MS patients. The diagnosis has always been confirmed by a neurologist and the patients gave informed consent to participate in a molecular study on MS. EV population is actually composed of two kind of vesicles: (a) exosomes (Es), which originate from the endosomal compartment and are smaller, and larger membrane vesicles (MVs), which directly bud from the plasma membrane. To separate Es from MVs, before ultracentrifugation, CSF was filtered through a 0.2-μm filter and centrifuged at 10,000 × g for 30 min. The pellet containing MVs was saved, while the supernatant was further centrifuged at 105,000 × g for 90 min to obtain Es. Pelleted vesicles were suspended with phosphate-buffered saline, pH 7.5 (PBS) and used to treat astrocytes for 24 hours. RESULTS MVs from MS patients induced a higher degree of cell death than those prepared from the CSF of control group (99,35% dead cells vs 89,33), but the difference was not significant (p value=0,142). A dramatic effect was instead noticed when treating astrocytes with Es from patients, which were able to induce a highly significant (p value=0,0001) degree of cell death respect to Es from controls (94,38% vs 71,21). CONCLUSIONS Our study suggests that EVs present in the CSF contain molecules which can be toxic to astrocytes and this is probably the reason why brain cells discard such molecules via different kinds of EVs. However, EVs, and in particular Es, present in CSF of MS patients are even more toxic for cells than EVs present in the CSF of healthy controls. Further studies are now necessary to identify the active molecules present in EVs.File | Dimensione | Formato | |
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