Neural Progenitor Cell- and Developing Neuron-Derived Extracellular Vesicles Differentially Modulate Microglial Activation

Abstract

The developmental processes of microglia follow a general pattern, from immature amoeboid (activated) cells to fully ramified (inactivated) surveilling microglia. However, little is known about the mechanisms controlling the transition of microglia from an activated to an inactivated state during brain development. Due to the complexity of microenvironmentally dynamic changes during neuronal differentiation, interactions between developing nerve cells and microglia might be involved in this process. Extracellular vesicles (EVs) are cell-released particles that serve as mediators of cellular crosstalk and regulation. Using neural progenitor cells (NPCs) and a long-term neuron culture system, we found that EVs derived from NPCs or developing neurons possessed differential capacity on the induction of microglial activation. The exposure of microglia to NPC- or immature neuron (DIV7)-derived EVs resulted in the higher expression of protein and mRNA of multiple inflammatory cytokines (e.g., TNF-α, IL-1β, and IL-6), when compared with mature neuron-derived EVs. Exploration of the intracellular signaling pathways revealed that MAPK signaling, IκBα phosphorylation/degradation, and NF-κB p65 nuclear translocation were strongly induced in microglia treated with NPC- or immature neuron-derived EVs. Using a pharmacological approach, we further demonstrate that Toll-like receptor (TLR) 7-mediated activation of NF-κB and MAPK signaling cascades contribute to EV-elicited microglial activation. Additionally, the application of conditioned media derived from microglia treated with NPC- or immature neuron-derived EVs is found to promote the survival of late-developing dopaminergic neurons. Thus, our results highlight a novel mechanism used by NPCs and developing neurons to modulate the developmental phases and functions of microglia through EV secretion.

Keywords: MAPKs; NF-κB; Toll-like receptor 7; developing neurons; extracellular vesicles; microglia; neural progenitor cells.

Figure 1

Characterization of developing neurons and EVs. (A) Molecular maturation of mesencephalic neurons in culture. Mesencephalic neurons were cultured for the indicated times. Cells were lysed and equal amounts of proteins were subjected to Western blotting. Specific antibodies for the different synaptic proteins were used, with β-actin as the loading control. (B) Western blot analysis of EV and non-EV marker proteins, including Alix, TSG101, flotillin-1, CD9, and calnexin (ER marker) in the cell lysates and EVs. (C) Size distribution of EVs derived from NPCs and developing neurons evaluated by nanoparticle tracking analysis. Estimated mean particle sizes are represented in the profiles.

Figure 2

Effects of NPC- and developing neuron-derived EVs on microglial activation. (A) EVs were labeled with the dye PKH67 (green) and then added to microglia. After 1 h of incubation, cells were fixed, stained with Iba1 antibody (red) and DAPI (blue), and analyzed by confocal microscopy. Scale bar = 20 μm. Microglia were treated with PBS or EVs secreted from NPCs or mesencephalic neurons of different culture age for 4 h (C), 10 h ((B), TNF-α), or 24 h ((B), IL-1β and IL-6). The released TNF-α, IL-1β, and IL-6 levels (B) were measured by ELISA. Expression of cytokine mRNA (C) was quantified by real-time RT-PCR. Data are presented as mean ± SEM for three independent experiments. Data were analyzed by one-way ANOVA followed by Scheffe’s post hoc test. * p < 0.05; ** p < 0.01 compared with control. ## p < 0.01 compared with NPC EVs.

Figure 3

NPC- and developing neuron-derived EVs differentially induce activation of MAPK signaling in microglia. (A) Microglia were treated with PBS or EVs derived from NPCs or mesencephalic neurons for 1 h. Whole-cell extracts were prepared. Western analysis was used to determine EV-induced p38, ERK, and JNK phosphorylation. Data are presented as mean ± SEM for three independent experiments. Data were analyzed by one-way ANOVA followed by Scheffe’s post hoc test. * p < 0.05; ** p < 0.01 compared with control. ## p < 0.01 compared with NPC EVs. (B) Microglia were pretreated with SB203580 (SB, 10 μM), SP600125 (SP, 10 μM), or U0126 (10 μM) for 1 h, followed by exposure to NPC or immature neuron (DIV7) EVs for another 10 h (TNF-α) or 24 h (IL-1β and IL-6). Released cytokines were measured by ELISA. Data are presented as mean ± SEM for three independent experiments. Data were analyzed by one-way ANOVA followed by Scheffe’s post hoc test. ** p < 0.01 compared with respective control.

Figure 4

Discrepant activation of NF-κB signaling pathway following EV treatments. Microglia were treated with PBS or EVs derived from NPCs or mesencephalic neurons for 1 h (A,C) or 2 h (B). Whole-cell lysates were subjected to Western blotting using antibodies specific for total/phosphorylated (Ser 32) IκBα (A) or total/phosphorylated (Ser 468 and 536) NF-κB p65 (C). (B) After EV treatments, microglia were fixed and immunostained using anti-p65 antibody followed by counterstaining with DAPI. Representative confocal images are shown. The p65 fluorescence intensities in the nucleus were determined by image analysis. Scale bar = 10 μm. Data are presented as mean ± SEM for three independent experiments. Data were analyzed by one-way ANOVA followed by Scheffe’s post hoc test. * p < 0.05; ** p < 0.01 compared with control. # p < 0.05; ## p < 0.01 compared with NPC EVs. (D) Microglia were pretreated with BAY 11-7082 (an IκBα phosphorylation inhibitor, 2 μM) for 1 h prior to stimulation with NPC- or immature neuron-derived EVs for another 10 h (TNF-α) or 24 h (IL-1β and IL-6). Released cytokines were measured by ELISA. Data are presented as mean ± SEM for three independent experiments. Data were analyzed by paired t-test. ** p < 0.01 compared with control.

Figure 5

EV-mediated microglial activation involves TLR7-dependent signaling pathway. Microglia were preincubated with the TLR7 inhibitor chloroquine (10 μM) for 1 h before stimulation with NPC- (A) or immature neuron (B) -derived EVs or LPS (10 ng/mL) (C) for another 10 h (TNF-α) or 24 h (IL-1β and IL-6). Released cytokines were measured by ELISA. Data are presented as mean ± SEM for three independent experiments. Data were analyzed by paired t-test. * p < 0.05; ** p < 0.01 compared with control. (D,E) Microglia were pretreated with 10 μM chloroquine (CQ) for 1 h, then stimulated with NPC-derived EVs for 1 h. The levels of phosphorylated and total IκBα (D), p38, ERK, and JNK (E) proteins were detected by Western blotting. Data are presented as mean ± SEM for three independent experiments. Data were analyzed by paired t-test. * p < 0.05; ** p < 0.01 compared with EVs alone.

Figure 6

NPC- and immature neuron-derived EV-activated microglia promote the survival of differentiating dopaminergic neurons. Ventral midbrain precursor cultures were treated with unconditioned medium (control) or conditioned medium (35%) from PBS or EV-stimulated microglia for 3 days (A) or 6 days (B). TH immunocytochemical analysis was carried out. Neurons immunostained with TH were counted in the entire surface area of each culture well (i.e., 1.9 cm²). Data are presented as mean ± SEM for three independent experiments. Data were analyzed by one-way ANOVA followed by Scheffe’s post hoc test. ** p < 0.01 compared with unconditioned medium. ## p < 0.01 compared with PBS-treated conditioned medium. (C) TH immunostaining shows an increase in the number of TH-immunoreactive cells at 6 days after treatment with conditioned medium from NPC- or immature neuron-derived EV-stimulated microglia. Scale bar = 100 μm.