Neuronal exosomes facilitate synaptic pruning by up-regulating complement factors in microglia

Abstract

Selective elimination of synaptic connections is a common phenomenon which occurs during both developmental and pathological conditions. Glial cells have a central role in the pruning of synapses by specifically engulfing the degenerating neurites of inappropriate connections, but its regulatory mechanisms have been largely unknown. To identify mediators of this process, we established an in vitro cell culture assay for the synapse elimination. Neuronal differentiation and synapse formation of PC12 cells were induced by culturing the cells with nerve growth factor (NGF) in a serum-free medium. To trigger synapse elimination, the NGF-containing medium was replaced with DMEM containing 10% FBS, and the neurites of PC12 cells degenerated within two days. Co-culturing with MG6 cells, a mouse microglial cell line, accelerated the removal of degenerating neurites of PC12 cells by phagocytosis. When MG6 cells were pre-incubated with exosomes secreted from the differentiated PC12 cells after depolarization, the removal was further accelerated by increasing the expression levels of complement component 3 in the MG6 cells. These results define a role for exosomes as a regulator of synapse elimination and clarify a novel mechanism whereby active synapses promote the pruning of inactive ones by stimulating microglial phagocytosis with exosomes.

Figure 1. An in vitro assay for synaptic development and elimination.

(a) Differentiation and neurite outgrowth of PC12 cells were induced by culturing the cells in sf-DMEM containing 100 ng/ml NGF for 7 days. The cells were stained with antibody against drebrin A (green) and their staining profile was merged with the phase contrast image in the third panel. Scale bar, 10 μm. (b) The differentiated PC12 cells were further cultured for 2 days after replacing the culture medium with sf-DMEM in the presence (+) or absence (−) of NGF, or with NGF(−) DMEM/10F. In the third panel, Q-VD (2 μM), a potent apoptosis inhibitor, was added to NGF(−) sf-DMEM. Scale bar, 40 μm. (c) Quantification of neurite length of PC12 cells. Number of neurites longer than one cell body diameter were counted and their length were measured by using FV10i-LIV confocal microscope and FLUOVIEW software, Ver. 4.1 (Olympus, Japan). Arrows indicate representative cells that were quantified in the lower table. (d) Cells with more than one neurite were defined as neurite (+) cells and the percentage of neurite (+) cells per total number of cells was determined. (e) The average neurite length per cell was calculated as the sum of total neurite length divided by the number of neurites from a cell. 100 cells were quantified in each condition (cells were selected from 10 different fields in three independent experiments), and the average values were plotted with standard deviations (SD). *p = 0.005, **p = 0.0014; n.s., not significant, ANOVA with Bonferroni correction for multiple comparison.

Figure 2. Microglia promote removal of degenerating neurites of PC12 cells.

(a) After culturing with NGF for 7 days, the differentiated PC12 cells were labelled with CMFDA (green). The PC12 cells were further cultured either in NGF(+) sf-DMEM or NGF(−) DMEM/10F in the absence or presence of MG6 cells for 8 h. PC12 cells and their neurites were visualized by confocal microscopy. Phase contrast images are shown in the lower panels, where MG6 cells are pointed by red arrows. Scale bar, 20 μm. The percentage of PC12 cells that retain neurites (b) and the average length of neurites (c) were quantified from 100 cells in each condition (cells were selected from 10 different fields in three independent experiments), and the average values were plotted with SD. *p = 0.0027, **p < 0.001; n.s., not significant. Student's t test. (d) Merged images of the phase contrast and CD68 staining (red) of MG6 cells that were co-cultured with PC12 cells either in NGF(+) sf-DMEM or NGF(−) DMEM/10F. Scale bar, 20 μm. (e) CMFDA-labelled PC12 cells (green) were co-cultured with PKH26-labelled MG6 cells (red) in NGF(−) DMEM/10F. Time-lapse images after replacing the culture medium were taken by using a confocal microscope equipped with culture chamber kept at 37°C and 5% CO₂. Arrows indicate MG6 cells engulfing the degenerating neurites. Scale bar, 10 μm.

Figure 3. Synaptic pruning is not mediated by PS.

(a) After differentiation in NGF(+) sf-DMEM for 7 days, PC12 cells were labelled with CMFDA (green) and further cultured in NGF(−) DMEM/10F for 8 h in the absence or presence of 3T3/Tim4 or MG6 cells. When indicated, recombinant D89E mutant of MFG-E8 (rD89E, 3 μg/ml) that masks PS was added into the culture medium during co-culture. Phase contrast images are shown in the lower panels. Scale bar, 20 μm. The percentage of PC12 cells that retain neurites (b) and the average length of neurites (c) were quantified from 100 cells in each condition (cells were selected from 10 different fields in three independent experiments), and the average values were plotted with SD. *p < 0.001; n.s., not significant. ANOVA with Bonferroni correction for multiple comparison.

Figure 4. Exosomes promote synaptic pruning by microglia.

(a) Exosomes were collected by ultra-centrifugation from the culture medium of differentiated PC12 cells that were cultured in sf-DMEM with or without 25 mM KCl for 3 h. Nanoparticle tracking analysis was performed to determine the density (left graph) and size distribution (right panel) of the exosomes in the medium. The experiments were performed in triplicate, and the average values with SD were plotted. *p = 0.0015, Student's t test. (b) Exosomes secreted from PC12 cells after depolarization were collected by ultra-centrifugation and analysed by western blot using antibodies against MFG-E8 and flotillin-2, exosomal markers. (c) Phagocytosis of pHrodo Red E.coli BioParticles by MG6 cells that were exposed (solid lines) or not exposed (dot lines) to the exosomes from differentiated PC12 cells after depolarization was allowed to proceed for 2 h. The efficiency of phagocytosis was compared by FACS analysis. (d) Differentiated PC12 cells were cultured in NGF(−) DMEM/10F for 8 h in the absence or presence of MG6 cells or MG6 cells that have engulfed the exosomes from PC12 cells (red). After labelling with PKH26, the exosomes were incubated with MG6 cells for 16 h. Scale bar, 40 μm. (e) The percentage of PC12 cells that retain neurites and the average length of neurites were quantified from 100 cells in each condition (cells were selected from 10 different fields in three independent experiments). The average values were plotted with SD. *p < 0.001. ANOVA with Bonferroni correction for multiple comparison. (f) The same assay was performed with MG6 cells that have engulfed the equal amounts of exosomes (exo) from PC12 cells or NIH3T3 cells. *p = 0.0013, **p = 0.003, ***p < 0.001; n.s., not significant. ANOVA with Bonferroni correction for multiple comparison.

Figure 5. Exosomes up-regulate the expression of complement factors in microglia.

(a) A screening for differentially expressed genes (DEGs) in MG6 cells exposed vs. not exposed to exosomes secreted from differentiated PC12 cells was performed using microarray analysis. Two arrays per condition were used and DEGs were selected as probes detected in at least one array and with an absolute log₂ fold change (M) greater than 3 (i.e. × 8 expression change). This approach resulted in 275 probes differentially expressed, which mapped to 183 known genes. (b) Functional enrichment of KEGG pathways was performed to identify the biological processes altered in microglia exposed to exosomes. Enrichment analysis resulted in a number of immune related processes significantly enriched (p < 0.05), including “Phagosome” (H2-M2, Marco, C3, Olr1, Tlr2) and “Complement and coagulation cascades” (C3, Cfb). (c) qPCR analyses were performed to validate the microarray results using gene-specific primers and RNA samples extracted from MG6 cells incubated 16 h with (black bar) or without (white bar) exosomes. Analysis was done with delta Ct method. Graphs represent gene expression levels relative to GAPDH. The experiments were performed in triplicate, and the average values with SD were plotted. p < 0.001, Student's t test. (d) The gel photograph of the qPCR products is shown. (e) qPCR analysis was performed to compare C3 mRNA levels between total purified exosomes and MG6 cells that have engulfed the same amount of the exosomes. Graphs represent gene expression levels relative to GAPDH. N.D. stands for not detectable. *p < 0.001, Student's t test. (f) Differentiated PC12 cells were cultured in NGF(−) DMEM/10F for 8 h with MG6 cells or MG6 cells that have engulfed the exosomes (red). Neutralizing anti-C3a/C3 antibody (+ α-C3 Ab) or IgG isotype control antibody (+ Ctrl. Ab) was added into the culture medium at the concentration of 20 μg/ml during co-culture. Scale bar, 40 μm. (g) The percentage of PC12 cells that retain neurites and the average length of neurites were quantified from 100 cells in each condition (cells were selected from 10 different fields in three independent experiments). The average values were plotted with SD. *p < 0.001; n.s., not significant. ANOVA with Bonferroni correction for multiple comparison.