β-amyloid binds to microglia Dectin-1 to induce inflammatory response in the pathogenesis of Alzheimer's disease

Abstract

Microglia-mediated neuroinflammation is closely related to the development of Alzheimer's disease (AD). In the early stages of the inflammation response, pattern recognition receptors (PRRs) play a key role in clearing damaged cells and defending against infection by recognizing endogenous and exogenous ligands. However, the regulation of pathogenic microglial activation and its role in AD pathology remains poorly understood. Here we showed that a pattern recognition receptor called Dectin-1, expressed on microglia, mediates the pro-inflammatory responses of beta-amyloid (Aβ). Knockout of Dectin-1 reduced Aβ1-42 (Aβ₄₂)-induced microglial activation, inflammatory responses, and synaptic and cognitive deficits in Aβ₄₂-infused AD mice. Similar results were obtained in the BV2 cell model. Mechanistically, we showed that Aβ₄₂ could directly bind to Dectin-1, causing Dectin-1 homodimerization and activating downstream spleen tyrosine kinase (Syk)/nuclear factor-κB (NF-κB) signaling pathway to induce the expression of inflammatory factors and, in turn, AD pathology. These results suggest the important role of microglia Dectin-1 as a new direct receptor for Aβ₄₂ in microglial activation and AD pathology and provide a potential therapeutic strategy for neuroinflammation in AD.

Keywords: Alzheimer's disease; Dectin-1; Microglia; Neuroinflammation; β-amyloid.

Figure 1

Dectin-1 is up-regulated in AD model mice. (A) RNA-seq analyses using hippocampus from the brains of C57 following DMSO infusion or C57 following Aβ₄₂ infusion for two weeks. N=6, 3 mice per group. (B) Bioinformatic analysis of RNA-seq. (C) Protein levels of Dectin-1 in the hippocampal tissue following Aβ₄₂ infusion. (D) Protein levels of Dectin-1 in the hippocampal tissues of APP/PS1 mice. (E) Representative dual-immunofluorescence staining of Microglia marker Iba1 (green), Astrocyte marker GFAP (green), Neuron Marker (NeuN) and Dectin-1 (red) in the hippocampal tissue of Aβ₄₂ infused mice. Sections were counterstained with DAPI (blue) [scale bar = 10 μm]. (F) Quantification analysis of Dectin-1 in E. (G) Western blot level of Dectin-1 in mouse bone marrow-derived macrophages (PC12), microglia cell line (BV2).

Figure 2

Dectin-1 knockout restore cognitive functions of Aβ₄₂ infusion model mice. (A) Representative average escape latency curves during place navigation test (four trials per day, during four consecutive days) and probe tests on day 5 without platform. N=40, N=10 mice per group. (B) Time course of escape latency, defined as the time taken to find the hidden platform. (C) Escape latency of all four groups on day 4. (D) The number of platform crossings. (E) The time in target quadrant. (F) Average swimming speed of mice in each group. (G) Total swimming distance of mice in each group. (H) Representative curves during novel object recognition test in each group. (I) Number of approaches in each group. (J) Total during of approaches in each group. (K) Total latency of all four groups.

Figure 3

Dectin-1 knockout improves neuropathology of Aβ₄₂ infusion mice. (A) Representative immunofluorescence staining of MAP2 (green) in brain tissues of WT and Aβ₄₂ infusion mice. Sections were counterstained with DAPI (blue) [scale bar = 50 μm]. (B) Quantification of MAP2 staining. (C)Representative western blot analysis of MAP2 and PSD95 in hippocampal tissue lysates from WT and Aβ₄₂ infusion mice. (D) Quantification and statistical analysis of MAP2 in C. (E) Quantification and statistical analysis of PSD95 in C. (F) IF of NeuN staining: Hippo [scale bar = 200 μm], CA1[scale bar = 100 μm] in brain tissues of WT and Aβ₄₂ infusion mice. (G) Quantification of NeuN staining.

Figure 4

Dectin-1 mediates microglia activation and inflammatory response in brain tissue. (A) Venn diagram depicting expressed genes in WT-vs- Aβ₄₂ and D1KO-vs- Aβ₄₂. (B) Heatmap shows four groups of expressed genes that expressed differentially between 8-week-old WT, Aβ₄₂, D1KO, and D1KO+ Aβ₄₂ mice (P < 0.05). (C) Heatmap of microglia markers, markers in activated microglia, NF-κB signaling, and cytokines from these four groups of mice. (D) Representative immunofluorescence staining of Iba1(green) in brain tissues of WT and Model (Aβ₄₂ infusion) mice. Sections were counterstained with DAPI (blue) [scale bar = 50 μm]. (E) Representative double-immunofluorescence staining of TNFα (red) and IL-1β (green). Slides were counterstained with DAPI (blue) [scale bar = 10 μm]. (F) Representative immunofluorescence staining of NFκB (green) in brain tissues of WT and Aβ₄₂ infusion mice. Sections were counterstained with DAPI (blue) [scale bar = 10 μm]. (G) Quantification of Iba1 staining in D. (H-I) Quantification of IL-1β and TNFα staining in E. (J) Quantification of NFκB staining in F. (K-N) Representative western blot analysis of Iba1, TNFα, and IL-1β in hippocampus tissue lysates. (O-Q) Representative western blot analysis of p-Syk, Syk, P-NFκB65, and NFκB65 in mouse brain tissue lysates.

Figure 5

Dectin-1 interference prevents Aβ₄₂-induced inflammatory response in BV2 cells. (A) Time-course of Dectin-1-Syk interaction. BV2 cells were exposed to 20 µM Aβ₄₂ for indicated times and co-immunoprecipitation using Dectin-1 antibody to probe for Syk was performed. (B) HEK-293T cells were transfected with Flag-tagged Dectin-1 (Flag-D1) and HA-tagged Dectin-1 (HA-D1). Time-course of Dectin-1 dimerization (Flag-HA interaction) assessed following exposure of cell to 20 µM Aβ₄₂ for indicated times. (C) Time-course of Syk phosphorylation. BV2 cells are exposed to 20 µM Aβ₄₂ for indicated times. Total proteins were extracted and probed for p-Syk and Syk levels. GAPDH was used as loading control. (D) Dose-course of Syk phosphorylation. BV2 cells were exposed to increasing levels of Aβ₄₂ for 45 mins. Total proteins were used to measure p-Syk and Syk levels. GAPDH was used as loading control. (E) Determination of Dectin-1 interference efficiency in BV2 cells. Total proteins were extracted and probed for p-Syk and Syk levels. GAPDH was used as loading control. (F) Representative western blot analysis of p-Syk, Syk, P-NFκB65, and NFκB65 in BV2 cells. GAPDH was used as loading control. (G) Representative immunofluorescence staining of NFκB65 (green) in BV2 cells transfected with or without siD1 [scale bar = 10 μm]. (H) TNFα release in cell supernatant measured by ELISA. (I) IL-1β release in in cell supernatant measured by ELISA. (J) mRNA levels of Tnfa, Il1b, Il6, Inos, and Cox2 in the hippocampus tissues. Transcript levels were normalized to GAPDH.

Figure 6

Aβ₄₂ activates the Dectin-1-Syk signaling pathway by binding to Dectin-1 and inducing its homodimerization. (A) BV2 cells are treated with 20 µM Bio- Aβ₄₂ or free biotin for 45 min, and cells are double-stained for biotin (green) and Dectin-1 (red) or Trem 2 (red), respectively. Nuclei are counterstained with DAPI (blue) [scale bar = 5 μm]. (B) Bio-Aβ₄₂ is added to streptavidin-agarose beads and incubated. Biotin alone was used as control. Lysates prepared from BV2 cells were added to beads. The eluent was loaded onto a polyacrylamide gel for western blot analysis. Total lysates were used as input controls. Trem2 was used as a positive control. (C) The Dectin-1-APP/ β-amyloid interaction is analyzed by co-immunoprecipitation in the brains of WT and model mice. (D) Surface plasmon resonance (SPR) analysis showing a direct interaction between Aβ₄₂ and rhDectin-1. Mean KD constant derived from five separate experiments. (E) 3D binding model of dectin-1 and amyloid beta peptide. The backbones of dectin-1 and amyloid beta peptide are shown as cartoons. The residues in dectin-1 are shown as cyan sticks, whereas the residues in amyloid beta-peptide are shown as wheat sticks. The hydrogen bonds are depicted as red dashed lines and the salt bridges are depicted as blue dashed lines.

Figure 7

Dectin-1 interference prevents Aβ₄₂-induced neuronal damage. (A) BV2 cells pretreated with siD1 for 24 h are stimulated with/without 20 µM Aβ₄₂ for another 24 h, and the conditioned culture supernatant is collected. (B) PC12 cells are plated into four groups: CTL, Aβ₄₂, siD1, and siD1+Aβ_42; BV2 serum is added to stimulate the PC12 cells for 24 h; and cell viability is measured by MTT assay. (C) LDH assay is used to test cell membrane damage. (D) TUNEL staining and Ros staining are used to test the apoptosis and intracellular ROS level [scale bar = 100 μm]. (E) Quantification of apoptotic cell in TUNEL staining in C. (F) Caspase 3 activity is detected using caspase 3 assay. (G) Quantification of ROS staining in D.