Dual roles of Aβ in proliferative processes in an amyloidogenic model of Alzheimer's disease

Abstract

Alzheimer's disease is a major neurodegenerative disorder that leads to severe cognitive deficits in the elderly population. Over the past two decades, multiple studies have focused on elucidating the causative factors underlying memory defects in Alzheimer's patients. In this regard, new evidence linking Alzheimer's disease-related pathology and neuronal stem cells suggests that hippocampal neurogenesis impairment is an important factor underlying these cognitive deficits. However, because of conflicting results, the impact of Aβ pathology on neurogenesis/gliogenesis remains unclear. Here, we investigated the effect of Aβ on neuronal and glial proliferation by using an APP/PS1 transgenic model and in vitro assays. Specifically, we showed that neurogenesis is affected early in the APP/PS1 hippocampus, as evidenced by a significant decrease in the proliferative activity due to a reduced number of both radial glia-like neural stem cells (type-1 cells) and intermediate progenitor cells (type-2 cells). Moreover, we demonstrated that soluble Aβ from APP/PS1 mice impairs neuronal cell proliferation using neurosphere cultures. On the other hand, we showed that oligomeric Aβ stimulates microglial proliferation, whereas no effect was observed on astrocytes. These findings indicate that Aβ has a differential effect on hippocampal proliferative cells by inhibiting neuronal proliferation and triggering the formation of microglial cells.

Figure 1

Neurogenesis is severely affected in the SGZ of APP/PS1 mice. (A and B) Western-blot analysis shows a significant decrease (n = 5/genotype/age; ANOVA F(5,22) = 88.33; Tukey’s post hoc p < 0.05) in the level of DCX at 4 and 6 months of age in APP/PS1 mice compared with age-matched WT mice. (C) Light microscopic images of DCX immunolabeling in the dentate gyrus of WT (c1–c3), and APP/PS1 (c4–c6) mice from 2 to 6 months of age. Significant reduction in DCX-positive cells was observed in APP/PS1 mice compared to WT mice during aging; g: granular cell layer; h: hilus. Scale bars: 100 μm (c1–c6).

Figure 2

Cell proliferation is significantly reduced in the hippocampus of APP/PS1 mice at 6 months of age. (A) Light microscopic images of double immunolabeled cells, BrdU (blue)-DCX (brown), in the DG of 2- and 6-months-old WT (a1 and a2), and APP/PS1 (a3 and a4) mice. (B) The total number of BrdU/DCX-positive cells was significantly reduced in APP/PS1 mice compared to WT mice at 6 months of age (n = 4/age/genotype); ANOVA F(3,11) = 15.57; Tukey’s post hoc test *p < 0.05, ***p < 0.001. Scale bars: 100 μm (a1–a4).

Figure 3

BLBP-positive stem cells in the SGZ in WT and APP/PS1 mice. (A) Light microscopic images of the dentate gyrus of WT (a1–a3) and APP/PS1 (a4–a6) mice immunostained for BLBP. (B) Double confocal images of GFAP (b1–3) and BLBP (b4–6) markers demonstrate the presence of radial glial-like stem cell type-1 progenitors in both WT and APP/PS1 mice as shown in the co-localization images (b7–9). g: granular cell layer; h: hilus; m: molecular cell layer. Scale bars: 25 μm (a1–a6 and b1–b9).

Figure 4

BLBP-positive stem cells are reduced in aging and in APP/PS1 mice. (A) Light microscopic images of the dentate gyrus of WT (a1–a3) and APP/PS1 (a4–a6) mice immunostained for BLBP. (B) Stereological quantification demonstrates an age-depend decrease in BLBP-positive cells (n = 4/genotype/age; ANOVA F(2,8) = 38.93, Tukey’s post hoc test, ***p < 0.001). (C) Stereological quantification shows a significant decrease in the number of BLBP-cells in APP/PS1 mice compared to age-matched WT mice (n = 4/age/genotype; t-test **p < 0.01). Red arrows: BLBP-positive stem cells with broccoli-like cell endings. g: granular cell layer; h: hilus; m: molecular cell layer. Scale bars: 100 μm (a1–a6), 25 μm for inserts.

Figure 5

Thin and thick BLBP-positive stem cells are differentially affected in APP/PS1 mice. (A) Representative images of BLBP-positive cells in the dentate gyrus of 6-month-old APP/PS1 mice; radial glia-like stem cells (type-1 cells) display a thin (green arrows) or thick (blue arrows) long radial process that extends from the cell body in the SGZ and traverses the granule cell layer; BLBP-positive non-stem cells exhibit a cell body located out of SGZ and many branched processes (red arrows) resembling mature astrocytes. (B) Stereological quantifications show a significant decrease in the number of BLBP-stem cells extending thin apical process in the APP/PS1 mice compared to WT mice at 6 months of age. However, a significant increase in the number of BLB-stem cells with a thick process is observed in these APP/PS1 mice compared to WT mice (n = 4/genotype/age; ANOVA F(3,12) = 85.545; Tukey’s post hoc test, *p < 0.05, ***p < 0.001). (C) Percentage of thin and thick BLBP-positive stem cells in WT and APP/PS1 at 4- and 6-month-old. g: granular cell layer; h: hilus; m: molecular cell layer. Scale bars: 25 μm.

Figure 6

Soluble APP/PS1-derived Aβ inhibits neurospheres growth and proliferation. (A) Light microscopic images of the dentate gyrus of APP/PS1 mice showing an age-dependent increase in the extracellular Aβ accumulation (a1–a3); quantification of Aβ plaque loading shows a significant increase from 4 months of age up to 12 months (n = 5/age; ANOVA; F(2,8) = 54.29; p = 000.1; Tukey’s post hoc test, *p < 0.05, ***p < 0.001) (a4). (B) Western-blot analysis (b1) shows a significant age-dependent increase (b2) in the level of monomeric Aβ in APP/PS1 mice (ANOVA; F(2,9) = 119.3; P = 0.0001; Tukey’s post hoc test p < 0.001). (C) Soluble proteins (S1) from APP/PS1 mice inhibit neurospheres growth. (D) Aβ-immunodepleted soluble proteins from APP/PS1 mice hippocampus demonstrate that Aβ is a key molecular factor inhibiting neurospheres growth (Kruskal Wallis p = 0.0001, Dunn test p < 0.05). g: granular cell layer; h: hilus. Scale bars: 100 μm (a1–a3); 200 μm (c1–c3).

Figure 7

The microglial population increases in the dentate gyrus of APP/PS1 mice. (A) Double immunofluorescence confocal images for tomato lectin/BrdU (a1–a6) in 6-month-old APP/PS1 mice. The corresponding merged images showed that the BrdU-cells (arrows) observed in the hilus corresponded to microglial cells. (B) Quantitative analysis shows a significant increase of tomato lectin/BrdU double-labeled cells in APP/PS1 mice compared to WT mice (n = 6/genotype; t-test ***p < 0.001). (C) Bv2 proliferation assay shows an increase of proportion of microglia cells in S phase after Aβ treatment. (D) Proliferation index ratio shows a significant increase (showing in the graph) in Bv2 cells treatment with soluble oligomeric Aβ compared to control. Scale bars: 10 μm.