CD11b+ exosomes from different protocols were added to main neurons in 24-well plates and incubated for 4 days. Transmission electron microscopy Exosome AGN 194310 pellets were resuspended in sterile water after purification. inducers of neuroinflammation. How and to what extent microglia and their exosomes impact -synuclein pathology has not been well delineated. We statement here that when treated with human -synuclein preformed fibrils, exosomes made up of -synuclein released by microglia are fully capable of inducing protein aggregation in the recipient neurons. Additionally, when combined with microglial proinflammatory cytokines, these exosomes further increased protein aggregation in neurons. Inhibition of exosome synthesis in microglia reduced -synuclein transmission. The significance of these exosomes was exhibited by stereotaxic injection of exosomes isolated from -synuclein preformed fibrils treated microglia into the mouse striatum. Phosphorylated -synuclein was observed in multiple brain regions consistent with their neuronal connectivity. These animals also exhibited neurodegeneration in the nigrostriatal pathway in a time-dependent manner. Depleting microglia dramatically suppressed the transmission of -synuclein after stereotaxic injection of preformed fibrils. Mechanistically, we statement here that -synuclein preformed AGN 194310 fibrils impaired autophagy flux by upregulating PELI1, which in turn, resulted in degradation of LAMP2 in activated microglia. More importantly, by purifying microglia/macrophage derived exosomes in the CSF of Parkinsons disease patients, we confirmed the presence of -synuclein oligomer in CD11b+ exosomes, which were able to induce -synuclein aggregation in neurons, further supporting the translational aspect of this study. Taken together, our study supports the view that microglial exosomes contribute to the progression of -synuclein pathology and therefore, they may serve as a encouraging AGN 194310 therapeutic target for Parkinsons disease. Day 7. Cell purity ( 95%) was confirmed by immunofluorescence using Map-2 as a marker MSH6 for neurons (Supplementary Fig. 11A). Main microglia cultures Main cultured mouse microglia were prepared from post-natal Day 0 (P0) newborn C57BL/6 pups as explained previously (Floden Experiments (Appear) guidelines. Preformed fibril injection C57BL/6 mice between 2 and 3 months of age were anaesthetized with ketamine hydrochloride (100 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) and stereotaxically injected in the right hemisphere with 5 g AGN 194310 PFF (2 g/l). Sham animals received sterile D-PBS. Dorsal neostriatum (relative to bregma: ?0.2 mm caudal, 2 mm lateral, ?2.6 mm ventral from dura) injections were performed using a 10 l syringe (Hamilton) at a rate of 0.1 l per min (2.5 l total per site) with the needle in place for 5 min after the end of the injection at each site. PLX3397 treatment PLX3397 (#S7818, Selleck Chemicals) was formulated in standard chow (Research Diets Inc.) at 290 mg/kg. Mice were fed PLX3397 or control chow for 1 month before and after the PFF injection and then sacrificed (at room heat for 15 min. The resultant supernatant was mixed with 4 ml of ExoQuick-TC PLUS? Exosome Purification Kit (#WQPL10TC-1, SBI System Biosciences) according to the manufacturers instructions. Exosome-free conditioned medium was prepared by centrifuging conditioned medium at 3000followed by 100?000for 1?h. The supernatant was then collected as exosome-free conditioned medium. Exosome pellets were resuspended in culture medium and added to normal neurons (70% confluence, 35 mm plate) for 4 days. For the exosomes from patients, CD11b+ exosomes from 3 ml CSF were purified by EasySep? Release PE Positive Kit (Stemcell, #17654) with a PE-conjugated CD11b antibody (BioLegend, #101207). Then CD11b+ exosomes were resuspended in 200 l culture medium and added to main neurons in 24-well plates and incubated for 4 days. To demonstrate that this exosomes pulled were of microglial origin, we performed the following two approaches: first, we used an alternative PE-conjugated CD11b antibody, which has no reactivity to human (anti-rat, BioLegend, #201807). Second, we incubated the CD11b antibody with its blocking peptide (MyBioSource, #MBS425396) at a ratio of 10:1 (blocking peptide: CD11b antibody) for 1 h at room temperature to block the antigen binding sites of CD11b antibody before the exosomes were isolated. CD11b+ exosomes from different protocols were added to main neurons in 24-well plates and incubated for 4 days. Transmission electron microscopy Exosome pellets were resuspended in sterile water after purification. Exosomes were.