Amyloid-beta 1-40 is associated with alterations in NG2+ pericyte population ex vivo and in vitro

Abstract

The population of brain pericytes, a cell type important for vessel stability and blood brain barrier function, has recently been shown altered in patients with Alzheimer's disease (AD). The underlying reason for this alteration is not fully understood, but progressive accumulation of the AD characteristic peptide amyloid-beta (Aβ) has been suggested as a potential culprit. In the current study, we show reduced number of hippocampal NG2+ pericytes and an association between NG2+ pericyte numbers and Aβ1-40 levels in AD patients. We further demonstrate, using in vitro studies, an aggregation-dependent impact of Aβ1-40 on human NG2+ pericytes. Fibril-EP Aβ1-40 exposure reduced pericyte viability and proliferation and increased caspase 3/7 activity. Monomer Aβ1-40 had quite the opposite effect: increased pericyte viability and proliferation and reduced caspase 3/7 activity. Oligomer-EP Aβ1-40 had no impact on either of the cellular events. Our findings add to the growing number of studies suggesting a significant impact on pericytes in the brains of AD patients and suggest different aggregation forms of Aβ1-40 as potential key regulators of the brain pericyte population size.

Keywords: Alzheimer's disease; amyloid-beta 1-40; hippocampus; pericytes.

Figure 1

Column scatter plots in (a and c) demonstrate decreased number of NG2+ pericytes/μm² (a) and number of NG2+ pericytes/vessel length (b) in brains from patients with Alzheimer's disease (AD) compared to nondemented controls (NC). Column scatter plots in (c and d) demonstrate unaltered number of laminin+ pericytes/μm² (c) and number of laminin+ pericytes/vessel length (d) in brains from patients with AD compared to NC. Data are analysed using Student's t‐test, results are presented as means ± standard deviations. Significant difference at *p < .05. Images in (e and f) show hippocampal pericytes stained against NG2 (indicated by arrows) in nondemented controls (NC) (e) and patients with Alzheimer's disease (AD) (f). Image in (g) shows a representative staining against laminin, where pericytes are indicated by arrows. The area in which pericytes were counted, that is the molecular layer (ML) of hippocampus, is outlined in image (h). Scale bar (e–g) 20 μm. Scale bar (h) 1,000 μm

Figure 2

Scatter plots with linear regression lines demonstrating correlations between Aβ1‐40 levels in formic acid‐treated hippocampus sections and number of pericytes/μm² in the molecular layer of hippocampus. Scatter plot in (a) demonstrates the correlation between Aβ1‐40 levels and pericyte numbers/μm² within the AD patient group. Scatter plot in (b) shows the correlation between Aβ1‐40 levels and number of pericytes/vessel length within the AD patient group. Data are analysed using Spearman correlation test

Figure 3

Bar graphs demonstrating alterations in cytotoxicity, measured by LDH assay, in cell culture supernatants from HBVPs after exposure to 10 μm monomer, oligomer‐ or fibril‐EP Aβ1‐40 and Aβ1‐42 for 96 hr. Graph in (a) shows significantly increased LDH activity (i.e. increased cell death) in HBVP exposed to fibril‐EP Aβ1‐40, oligomer‐EP Aβ1‐42 and fibril‐EP Aβ1‐42 compared to Ctrl O/F. Graph in (b) shows the significantly decreased LDH activity (i.e. increased viability) in HBVP exposed to monomer Aβ1‐40 and monomer Aβ1‐42 compared to Ctrl M. Data were analysed using one‐way analysis of variance (ANOVA), followed by Dunnett's post hoc correction (n = 4 comparisons) (oligomer‐ and fibril‐EP Aβ1‐40 and Aβ1‐42) or Student's t‐test (monomers Aβ1‐40 and Aβ1‐42). Results are presented as means ± standard deviations. Significant difference at **p < .01 and ***p < .001

Figure 4

Bar graphs demonstrating alterations in cytotoxicity, measured by caspase 3/7 activity, in HBVPs after exposure to 10 μm monomer, oligomer‐ or fibril‐EP Aβ1‐40 and Aβ1‐42 for 24 hr. Graph in (a) shows the significantly increased relative caspase 3/7 activity in HBVP after exposure of fibril‐EP of Aβ1‐40 compared to Ctrl O/F. Graph in (b) demonstrates the significant increased relative caspase 3/7 activity in HBVP after exposure to monomer Aβ1‐40 compared to Ctrl M. Data were analysed using one‐way analysis of variance (ANOVA), followed by Dunnett's post hoc correction (n = 4 comparisons) (oligomer‐ and fibril‐EP of Aβ1‐40 and Aβ1‐42) or Student's t‐test (monomer preparations of Aβ1‐40 and Aβ1‐42). Results are presented as means ± standard deviations. Significant difference at *p < .05 and ***p < .001

Figure 5

Bar graphs demonstrating alterations in Ki67+ cells, indicative of cell proliferation, in HBVPs after exposure to 10 μm monomer, oligomer‐ or fibril‐EP Aβ1‐40 for 24 hr. Image in (a) shows untreated HBVP stained against Ki67 and DAPI. Image in (b) demonstrates Aβ1‐40 monomer‐exposed HBVP stained against Ki67 and DAPI. Image in (c) demonstrates HBVP stained against Ki67 and DAPI after exposure to oligomer‐EP Aβ1‐40. Image in (d) shows fibril‐EP Aβ1‐40‐exposed HBVP stained against Ki67 and DAPI. Graph in e) shows the significantly decreased number of Ki67+ HBVPs after exposure of fibril‐EP of Aβ1‐40 compared to Ctrl O/F. Graph in (f) demonstrates the significantly increased number of Ki67+ HBVPs after exposure of monomer EP of Aβ1‐40 compared to Ctrl M. Data were analysed using one‐way analysis of variance (ANOVA), followed by Dunnett's post hoc correction (n = 2 comparisons) (oligomer‐ and fibril‐EP of Aβ1‐40) or Student t‐test (monomer preparations of Aβ1‐40). Results are presented as means ± standard deviations. Significant difference at *p < .05, **p < .01