Key Aging-Associated Alterations in Primary Microglia Response to Beta-Amyloid Stimulation

Abstract

Alzheimer's disease (AD) is characterized by a progressive cognitive decline and believed to be driven by the self-aggregation of amyloid-β (Aβ) peptide into oligomers and fibrils that accumulate as senile plaques. It is widely accepted that microglia-mediated inflammation is a significant contributor to disease pathogenesis; however, different microglia phenotypes were identified along AD progression and excessive Aβ production was shown to dysregulate cell function. As so, the contribution of microglia to AD pathogenesis remains to be elucidated. In this study, we wondered if isolated microglia cultured for 16 days in vitro (DIV) would react differentially from the 2 DIV cells upon treatment with 1000 nM Aβ_1-42 for 24 h. No changes in cell viability were observed and morphometric alterations associated to microglia activation, such as volume increase and process shortening, were obvious in 2 DIV microglia, but less evident in 16 DIV cells. These cells showed lower phagocytic, migration and autophagic properties after Aβ treatment than the 2 DIV cultured microglia. Reduced phagocytosis may derive from increased CD33 expression, reduced triggering receptor expressed on myeloid cells 2 (TREM2) and milk fat globule-EGF factor 8 protein (MFG-E8) levels, which were mainly observed in 16 DIV cells. Activation of inflammatory mediators, such as high mobility group box 1 (HMGB1) and pro-inflammatory cytokines, as well as increased expression of Toll-like receptor 2 (TLR2), TLR4 and fractalkine/CX3C chemokine receptor 1 (CX3CR1) cell surface receptors were prominent in 2 DIV microglia, while elevation of matrix metalloproteinase 9 (MMP9) was marked in 16 DIV cells. Increased senescence-associated β-galactosidase (SA-β-gal) and upregulated miR-146a expression that were observed in 16 DIV cells showed to increase by Aβ in 2 DIV microglia. Additionally, Aβ downregulated miR-155 and miR-124, and reduced the CD11b+ subpopulation in 2 DIV microglia, while increased the number of CD86+ cells in 16 DIV microglia. Simultaneous M1 and M2 markers were found after Aβ treatment, but at lower expression in the in vitro aged microglia. Data show key-aging associated responses by microglia when incubated with Aβ, with a loss of reactivity from the 2 DIV to the 16 DIV cells, which course with a reduced phagocytosis, migration and lower expression of inflammatory miRNAs. These findings help to improve our understanding on the heterogeneous responses that microglia can have along the progression of AD disease and imply that therapeutic approaches may differ from early to late stages.

Keywords: Alzheimer’s disease; CD11b; CD86; M1/M2 microglia subtypes; aged-cultured microglia; amyloid-β peptide; inflammatory-microRNAs; neuroinflammation.

Figure 1

Reactive and aged cultured microglia display soma enlargement and amoeboid morphology after treatment with amyloid-β (Aβ) peptide. Microglia that were kept in culture for 2 and 16 days in vitro (DIV) were treated with 1000 nM Aβ for 24 h. Cells were immunostained for Iba1 and characterized for their morphometric features. (A) Representative images show increased ramification by age, which was counteracted by Aβ exposure. Microglia perimeter (B), Feret’s diameter (C), area (D) and circularity values (E) were measured using ImageJ software and expressed in graph bars as mean ± SEM. Cultures, n = 4 per group. Two-way analysis of variance (ANOVA; Post hoc Bonferroni test): *p < 0.05 and **p < 0.01 vs. respective non-treated Control; ^†p < 0.05 vs. 2 DIV cells; (B) DIV × Aβ interaction F_(4.74), p < 0.05; (C) DIV × Aβ interaction F_(5.27), p < 0.05; (E) DIV × Aβ interaction F_(5.14), p < 0.05. Scale bar equals 50 μm.

Figure 2

Release of matrix metalloproteinase 2 (MMP2) and MMP9 is differently induced by amyloid-β (Aβ) peptide in reactive and aged cultured microglia. Microglia that were kept in culture for 2 and 16 days in vitro (DIV) were treated with 1000 nM Aβ for 24 h. Activities of MMP2 and MMP9 were evaluated by gelatin zymography. (A) Representative images of zymography gels. (B) Results are expressed in graph bars as mean ± SEM. Cultures, n = 4 per group. Two-way ANOVA (Post hoc Bonferroni test): *p < 0.05 and **p < 0.01 vs. respective non-treated Control; ^††p < 0.01 vs. 2 DIV; (B) DIV × Aβ interaction F_(4.39), p < 0.05.

Figure 3

Autophagy is differently promoted by amyloid-β (Aβ) peptide in reactive and aged cultured microglia. Microglia that were kept in culture for 2 and 16 days in vitro (DIV) were treated with 1000 nM Aβ for 24 h. Total cell lysates were analyzed for the presence of Beclin 1. (A) Representative images of Beclin 1 protein expression. (B) Results of densitometric analysis of Beclin 1 blots are expressed in graph bars as mean ± SEM. Microtubule-associated- protein-light-chain-3 (LC3)-positive puncta cells were detected by immunostaining for LC3. (C) Representative images of immunocytochemistry for LC3 (green) and nuclei staining (blue). Scale bar equals 50 μm. (D) Percentage of cells showing LC3-positive puncta are expressed in graph bars as mean ± SEM. Cultures, n = 4 per group. Two-way ANOVA (Post hoc Bonferroni test): *p < 0.05 vs. respective non-treated Control; ^†p < 0.05 and ^††p < 0.01 vs. 2 DIV.

Figure 4

Amyloid-β (Aβ) peptide promotes cell senescence in reactive cultured microglia. Microglia that were kept in culture for 2 and 16 days in vitro (DIV) were treated with 1000 nM Aβ for 24 h. Activity of senescence-associated β-galactosidase (SA-β-gal) was determined using a commercial kit, and SA-β-gal-positive cells were counted. (A) Representative images of cells showing blue turquoise SA-β-gal staining. Scale bar equals 20 μm. (B) Percentage of cells showing SA-β-gal-positive staining are expressed in graph bars as mean ± SEM. (C) MicroRNA (miR)-146a expression was evaluated by Real-Time PCR. Results are expressed in graph bars as mean ± SEM. Cultures, n = 4 per group. Two-way ANOVA (Post hoc Bonferroni test): *p < 0.05 and **p < 0.01 vs. respective non-treated Control; ^††p < 0.01 vs. 2 DIV cells; (B) DIV × Aβ interaction F_(28.1), p < 0.01.

Figure 5

Reactive cultured microglia show increased ability to migrate towards amyloid-β (Aβ) peptide and ATP, while aged microglia are immotile and unresponsive to such chemoattractants. Microglia were kept in culture for 2 and 16 days in vitro (DIV) and cellular chemotactic migration to 1000 nM Aβ and 10 μM ATP (positive chemotactic control) was evaluated after 6 h incubation using the Boyden chamber method. (A) Representative images of cells that have migrated to Aβ and ATP. Scale bar equals 50 μm. (B) Number of migrated cells was counted and results are expressed in graph bars as mean ± SEM. Cultures, n = 4 per group. Two-way ANOVA (Post hoc Bonferroni test): **p < 0.01 vs. respective non-treated Control; ^††p < 0.01 vs. 2 DIV cells; (B) DIV × Aβ interaction F_(4.36), p < 0.05.

Figure 6

Ability of microglia to phagocytose is reduced by amyloid-β (Aβ) peptide mainly in the reactive cultured cells. Microglia that were kept in culture for 2 and 16 days in vitro (DIV) were treated with 1000 nM Aβ for 24 h. Phagocytic capacity was assessed after 75 min incubation with fluorescent latex beads. (A) Representative images of microglia immunostained for Iba1 (red) and stained with Hoechst for nuclei staining (blue) containing phagocytosed fluorescent latex beads (green). Scale bar equals 50 μm. (B) Number of phagocytosed beads per cell and (C) number of microglial cells phagocytosing less than 5, 5–10 and more than 10 beads was counted. (D) Expression of milk fat globule-EGF factor 8 protein (MFG-E8), triggering receptor expressed on myeloid cells 2 (TREM2) and CD33, associated to microglial phagocytosis, was evaluated by Real-Time PCR. Results are expressed in graph bars as mean ± SEM. Cultures, n = 4 per group. Two-way ANOVA (Post hoc Bonferroni test): *p < 0.05 and **p < 0.01 vs. respective non-treated Control; ^†p < 0.05 and ^††p < 0.01 vs. 2 DIV; (D) MFG-E8: DIV × Aβ interaction F_(13.9), p < 0.01.

Figure 7

Amyloid-β (Aβ) peptide decreases the expression of miRNA (miR)-155 and miR-124 in the reactive cultured microglia toward that of 16 days in vitro (DIV) cells. Microglia that were kept in culture for 2 and 16 DIV were treated with 1000 nM Aβ for 24 h. (A) Expression of M1/pro-inflammatory-related miR-155 and of (B) M2/anti-inflammatory-related miR-124 was evaluated by Real-Time PCR. Results are expressed in graph bars as mean ± SEM. Cultures, n = 4 per group. Two-way ANOVA (Post hoc Bonferroni test); *p < 0.05 vs. respective non-treated Control; ^†p < 0.05 and ^††p < 0.01 vs. 2 DIV cells.

Figure 8

Increased expression of inflammatory mediators in microglia treated with amyloid-β (Aβ) peptide is more evident in the reactive cultured cells. Microglia that were kept in culture for 2 and 16 days in vitro (DIV) were treated with 1000 nM Aβ for 24 h. (A) Expression of inflammatory cytokines [e.g., tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6] and of (B) inflammasome-related proteins [e.g., high-mobility group protein B1 (HMGB1), IL-18 and NOD-like receptor family pyrin domain containing 3 (NLRP3)] were evaluated by Real-Time PCR. Results are expressed in graph bars as mean ± SEM. Cultures, n = 4 per group. Two-way ANOVA (Post hoc Bonferroni test): *p < 0.05 vs. respective non-treated Control; ^††p < 0.01 vs. 2 DIV cells.

Figure 9

Amyloid-β (Aβ) peptide upregulates the expression of Toll-like receptor 2 (TLR2), TLR4 and fractalkine/CX3C chemokine receptor 1 (CX3CR1) in the reactive cultured microglia, but not in aged cells. Microglia that were kept in culture for 2 and 16 days in vitro (DIV) were treated with 1000 nM Aβ for 24 h. Expression of TLR2 (A), TLR4 (B) and CX3CR1 (C) was evaluated by Real-Time PCR. Results are expressed in graph bars as mean ± SEM. Cultures, n = 4 per group. Two-way ANOVA (Post hoc Bonferroni test): *p < 0.05 vs. respective non-treated Control; ^††p < 0.01 vs. 2 DIV cells; (A) DIV × Aβ interaction F_(5.29), p < 0.05.

Figure 10

Mixed representation of M1/pro-inflammatory and M2/anti-inflammatory polarization markers in 2 and 16 days in vitro (DIV) microglia treated with amyloid-β (Aβ) peptide suggests the presence of different cell subsets. Microglia that were kept in culture for 2 and 16 DIV were treated with 1000 nM Aβ for 24 h. (A) Expression of M1/pro-inflammatory [e.g., inducible nitric oxide synthase (iNOS) and major histocompatibility (MHC) class II] and of (B) M2/anti-inflammatory [e.g., Arginase and transforming growth factor β (TGFβ)] was evaluated by Real-Time PCR. Results are expressed in graph bars as mean ± SEM. Cultures, n = 4 per group. Two-way ANOVA (Post hoc Bonferroni test): *p < 0.05 and **p < 0.01 vs. respective non-treated Control; ^†p < 0.05 and ^††p < 0.01 vs. 2 DIV cells; (A) MHC class II: DIV × Aβ interaction F_(4.53), p < 0.05.

Figure 11

Cell aging and treatment with amyloid-β (Aβ) peptide decrease the population of microglia CD11b+ cells, while increase the number of CD86+ cells. Microglia that were kept in culture for 2 and 16 days in vitro (DIV)were treated with 1000 nM Aβ for 24 h. The population of CD11b+ and CD86+ cells was detected by flow cytometry. (A) Analysis of microglia expressing CD11b. Results are expressed in graph bars as mean ± SEM. Cultures, n = 4 per group. Two-way ANOVA (Post hoc Bonferroni test): *p < 0.05 and **p < 0.01 vs. respective non-treated Control; ^†p < 0.05 vs. 2 DIV cells. (B) Representative flow cytogram of CD11b+ and CD86+ cells in reactive (2 DIV) and aged (16 DIV) cultured microglia. (C) Results are expressed in 2D pie graphs as mean. Cultures, n = 4 per group. Two-way ANOVA (Post hoc Bonferroni test): *p < 0.05 and **p < 0.01 vs. respective non-treated Control; ^†p < 0.05 and ^††p < 0.01 vs. 2 DIV cells.