Fig. 7

Drp1 inhibition reduces protein aggregation induced by exosomes from PFF-treated microglia. a Experimental scheme: In the presence or absence of mdivi-1, mouse primary microglia were treated with PFF for 24 h, and further cultured for 36 h after PFF withdrawal. To activate microglia, cells were treated with LPS (1 μg/mL) for 3 h followed by 15 min of ATP (5 mM) before harvesting. Conditioned media (CM) were collected for exosome isolation, and cells were lysed for western blot quantification: b. Equal volume of CM were loaded into each well and quantified for exosome levels using Alix and Tsg101 as markers. Data represent the mean ± SEM, (n = 3). c Equal amount of exosomes were loaded into each well to quantify for α-syn content. Data represent the mean ± SEM, (n = 4). d SH-SY5Y cells were incubated with extracted exosomes from PFF + LPS treated microglia for 4 days and immunostained for α-syn. Scale bar: 20 μm. e Experimental scheme: BV-2 microglia were transfected with siRNA-Drp1 prior to PFF treatment, and cells were treated with PFF (2 μg/ml) for 24 h and further cultured for 36 h after PFF withdraw, during which time cell were activated with LPS (1 μg/ml) and ATP (5 mM) for 3 h and 15 min, correspondingly. Then the conditional media were collected for exosome isolation, and cells were lysed for western blot quantification. f Same amount of EF was loaded for western blot analysis confirming the reduction of α-syn in exosomes via Drp1 silencing. Data represent the mean ± SEM, (n = 4). g Equal volume of CM were loaded into each well for western blot quantification of exosome markers Alix and Tsg101, and Drp1 silencing reduced exosome release from BV2 cells. Data represent the mean ± SEM, (n = 6). h SH-SY5Y cells treated with EF from BV2 cells were fixed after 24 h and stained for α-syn. Representative ICC images showing Drp1 knockdown in donor cells (BV2) significantly reduced α-syn aggregation formation in recipient cells (SH-SY5Y). Scale bar: 20 μm Data were analyzed using one-way ANOVA followed by Newman–Keuls post-hoc testing. * p < 0.05