Effects of miRNAs in exosomes derived from α-synuclein overexpressing SH-SY5Y cells on autophagy and inflammation of microglia.

Abstract Our previous study has revealed that exosomes derived from GFP-α-synuclein overexpressing SH-SY5Y cells (GFP-SNCA Exo) decrease autophagy in microglia via their load of miRNAs. However, it is unclear whether GFP-SNCA Exo can affect microglial inflammation via modulation of autophagy. The aim of this study was to investigate the effects of miRNAs carried by GFP-SNCA Exo on autophagy and inflammation of microglia. We transfected SH­SY5Y cells with lentivirus expressing α-synuclein and collected their exosomes. Western blot and laser confocal images showed that α-synuclein transferred between SH-SY5Y cells and microglia through exosomes. Microarray analysis showed that there were differentially expressed miRNAs between exosomes derived from α-synuclein overexpressing SH-SY5Y and vector cells. Bioinformatics analysis revealed that the target genes of the differentially expressed miRNAs were enriched in the MAPK and autophagy-associated signaling pathway. The expression of P62, p-JNK/JNK, and p-ERK/ERK and the release of IL-6 significantly increased whereas LC3 II/I decreased in microglia exposed to GFP-SNCA Exo for 48 h when compared to the control group. But rapamycin could reverse the increasing expression of p-JNK/JNK, p-ERK/ERK and the release of IL-6 induced by GFP-SNCA Exo. Dual immunofluorescence staining for LC3B and LAMP1 showed that the fluorescence density of LC3B decreased and the fluorescence of LC3B and LAMP1 were not co-located in microglia after 48 h co-culture with GFP-SNCA Exo compared with that in the control group, which indicated that these exosomes decreased autophagy and impaired the autophagy flux in recipient microglia. Taken together, our results indicate that exosomes derived from GFP-α-synuclein overexpressing SH-SY5Y cells activate the MAPK signaling pathway and inflammation by decreasing autophagy in microglia.