Naturally-aged microglia exhibit phagocytic dysfunction accompanied by gene expression changes reflective of underlying neurologic disease

Abstract

Age-associated microglial dysfunction contributes to the accumulation of amyloid-β (Aβ) plaques in Alzheimer's disease. Although several studies have shown age-related declines in the phagocytic capacity of myeloid cells, relatively few have examined phagocytosis of normally aged microglia. Furthermore, much of the existing data on aging microglial function have been generated in accelerated genetic models of Alzheimer's disease. Here we found that naturally aged microglia phagocytosed less Aβ over time. To gain a better understanding of such dysfunction, we assessed differences in gene expression between young and old microglia that either did or did not phagocytose Aβ. Young microglia had both phagocytic and neuronal maintenance signatures indicative of normal microglial responses, whereas, old microglia, regardless of phagocytic status, exhibit signs of broad dysfunction reflective of underlying neurologic disease states. We also found downregulation of many phagocytic receptors on old microglia, including TREM2, an Aβ phagocytic receptor. TREM2 protein expression was diminished in old microglia and loss of TREM2⁺ microglia was correlated with impaired Aβ uptake, suggesting a mechanism for phagocytic dysfunction in old microglia. Combined, our work reveals that normally aged microglia have broad changes in gene expression, including defects in Aβ phagocytosis that likely underlies the progression to neurologic disease.

Figure 1

Aged microglia have reduced phagocytic capacity. Brains from female young (2 months) and old (19 months) mice (n = 3) were processed and live CD45 intermediate CD11b⁺ cells (microglia) were assessed for phagocytic capacity. (a) Cells were incubated with 0.5 μM of fluorescent fibril Aβ42 or medium (control) for 1 h and phagocytosis was assessed by flow cytometry, images made using ©BioRender - biorender.com. (b–c) Representative flow plots of 1 replicate of 4 are shown and mean ± s.e.m % of microglia showing phagocytosis of fluorescent Aβ42 are graphed *p ≤ 0.05, Student’s t Test.

Figure 2

Aged microglia have less uptake capacity of Aβ overtime than their younger counterparts. Microglia from 3 young (2 months) or old (22 months) female mice were isolated, combined with their age-matched counterparts, and divided into 5 time points where they were incubated with 2.5 μM of Aβ42 for 1, 3, 6, and 12 h. Microglia were analyzed via flow cytometry to measure phagocytosis. (a) Representative flow plots of 1 replicate showing uptake of Aβ42 by microglia over time. (b) % of microglia that are Aβ42 positive over time. Dashed lines indicate line of best fit. (c) Geometric mean of Aβ42 in Aβ42⁺ microglia. Dashed lines indicate line of best fit. Simple linear regression was used to determine differences between the lines of best fit.

Figure 3

Pathway analysis of young and old microglia that either did or did not take up peptide showed deficiencies of old microglia. Young (2 months) and old (18 months) microglia (3 male mice were combined to make one replicate; this was repeated for a total of 4 replicates/group) were incubated with 2.5 μM of Aβ42 for 2 h and then were FACSorted based on Aβ42 uptake. Sorted microglia’s RNA was isolated and used to run bulk RNAseq analysis. (a) Schematic of phagocytosis assay and flow sorting, images made using ©BioRender - biorender.com. (b) Principal component analysis plot of the four different microglia groups (Young Aβ42⁺, Young Aβ42⁻, Old Aβ42⁺, Old Aβ42⁻) with 4 replicates each. Pathway analysis of DEGs in both the young and old (c) Aβ42⁺ and (d) Aβ42⁻ microglia graphed based on their p value. The number of DEGs in each pathway are indicated in parentheses.

Figure 4

Expression of TREM2, an Aβ42 receptor, is decreased in aging microglia. (a) Heatmap representation of phagocytic receptor gene expression levels for Young Aβ42⁺, Young Aβ42⁻, Old Aβ42⁺, and Old Aβ42⁻. The scale represents the row Z-score from 2 (highest expression) to − 2 (lowest expression). Young (2 months) and aged (21 months) microglia were incubated with 0.5 μM Aβ42 for 1 h and stained for microglial markers and TREM2 (n = 3/group; replicated 4 times with both male and female mice) (b) Representative flow plots (n = 3/group) of young and aged microglia TREM2 expression and graph of % of young and old microglia that are TREM2⁺. (c) Representative flow plots of young and aged microglia TREM2 expression and Aβ42 uptake. (d) Graphs showing the average percentage of TREM2 expression in young (black) and old (red) Aβ42⁺ microglia and (e) Average percentage of Aβ fluorescence in young (black) and old (red) TREM2⁺ microglia. (f) Geometric mean fluorescence of Aβ42 in TREM2⁺ and TREM2⁻ Aβ42⁺ microglia. *p ≤ 0.05, **p ≤ 0.01, mean ± s.e.m, Student’s t Test.