APOE4 impairs autophagy and Aβ clearance by microglial cells

Abstract

Alzheimer's disease (AD) is a predominant form of dementia in elderly. In sporadic AD and in families with higher risk of AD, correlation with apolipoprotein E4 (APOE) allele expression has been found. How APOE4 induces its pathological effects is still unclear. Several studies indicate that autophagy, a major degradation pathway trough the lysosome, may be compromised in AD. Here we studied, the effects of APOE isoforms expression in microglia cells. By using an in-situ model, the clearance of Aβ plaques from brain sections of transgenic 5xFAD mice by the APOE expressing microglia was examined. The results show that APOE4 microglia has Impairment In clearance of insoluble Aβ plaques as compared to APOE3 and APOE2 microglia. Furthermore, APOE4 affect the uptake of soluble Aβ. We found that microglia expressing APOE4 exhibit reduced autophagic flux as compared to those expressing APOE3. The autophagy inhibitor chloroquine also blocked Aβ plaque uptake in APOE3 expressing cells. Furthermore, we found that APOE4 expressing microglia have altered mitochondrial dynamics protein expression, mitochondrial morphology and mitochondrial activity compared to those expressing APOE2, and APOE3. Rapamycin treatment corrected Mitochondrial Membrane Potential in APOE4-expressing cells. Taken together, these findings suggest that APOE4 impairs the activation of autophagy, mitophagy, and Aβ clearance and that autophagy-inducing treatments, such as rapamycin, can enhance autophagy and mitochondrial functions in APOE4 expressing microglia. Our results reveal a direct link between APOE4 to autophagy activity in microglia, suggesting that the pathological effects of APOE4 could be counteracted by pharmacological treatments inducing autophagy, such as rapamycin.

Keywords: Alzheimer's disease (AD); Amyloid β; Apolipoprotein E4 (apoE4); Autophagy.

Fig. 1

Impaired amyloid plaque clearance by APOE4 microglia cells. Brain section from 5XFAD mice sections were incubated for 48 h with microglia cells expressing different type of human APOE: APOE2, APOE3 and APOEE4. Media without cells was used as a negative control. Following 48 h the brain sections were stained with anti- Aβ antibody against Aβ 1–42. Amyloid plaques clearance in the hippocampus region was assessed by microscope and ImageJ. Scale bar is 500 µM (A). Representative images of amyloid plaque-containing hippocampi following incubation with microglia. (B) Analysis of the % hippocampus area covered by Aβ plaques from each microglia type or (C) average plaques size vs. normalized to control (n = 4–5). Statistical analysis was performed using one way ANOVA; * p = 0.01, ** p < 0.0001

Fig. 2

Uptake and degradation of Aβ by APOE4 compared to APOE3 microglia. APOE3 and APOE4 expressing microglial cells were cultured in a 12-wells plates. Cells were treated for 2 h with 0.1 µM HiLyteTM 488-labeled Aβ 1–42 in the presence or absence of 10 µM chloroquine (CQ). Images of the cells were taken every 15 min, (four fields for each well). To consider the effect of the number of cells in each well, near-infrared (NIR) values were normalized to phase scans. A The changes in Aβ intensity inside the cells were measured using Incucyte machine, indicating Aβ uptake. B At the indicated time point, cells were washed twice with PBS and incubated with Aβ free medium for further 2 h, in 37◦C. The changes in Aβ intensity inside the cells were measured, indicating Aβ degradation. Right panel depicts representative images in the absence or in the presence of chloroquine (CQ), (scale bars is 400 µM). Left panels depict densitometric analysis of the results which are presented as fold induction of the controls (Mean ± SEM). Statistical analysis was performed using two tailed unpaired student t-test; *** p < 0.001, **** p < 0.0001 (n ≥ 4)

Fig. 3

EBSS induced autophagy in APOE3 and APOE4 expressing N9 microglia. APOE4 and APOE3 microglia were starved (incubation in EBSS) for the indicated times. The levels of p62 and LC3-I and LC3-II were measured by western blot analysis. In B, C and D, the densitometric analyses are presented as fold induction compared to the controls at time = 0 of each of the cell lines (mean ± SE; *p < 0.05, **p < 0.01, *** p < 0.001 treated compared to untreated cells; within group; #p < 0.05, ## p < 0.01 between groups (APOE3 compared to APOE4)

Fig. 4

Rapamycin induced autophagy in APOE3 and APOE4 expressing N9 microglia. APOE3 and APOE4 microglia were treated for 24 h with rapamycin (Rapa) at the indicated concentrations. The autophagic markers p62 and LC3 were measured by western blot analysis. Lower panels depict densitometric analysis of the results which are presented as fold induction compared to the untreated control of each cell line, (mean ± SE; *p < 0.05, **p < 0.01,***p < 0.001).

Fig. 5

Autophagic flux in APOE3 and APOE4 expressing N9 microglia. Cells were starved (incubation in EBSS) for 1 h, with or without 10 µM chloroquine (CQ). The markers of autophagy, p62 (B) and LC3 (C) were measured by western blot analysis. Lower panel depicts densitometric quantification of the results which are presented as fold induction compared to APOE3 control (mean ± SE; ^p < 0.05 and, ^^p < 0.01,^^^p < 0.001, (EBSS/CQ treated compared to untreated cells); *p < 0.05 **p < 0.001,ApoE3 compared to APOE4 cells (white bars, APOE3 and black bars, APOE4)

Fig. 6

Rapamycin induced autophagy in APOE3 and APOE4 N9 microglia. APOE3 and APOE4 expressing microglia were transiently transfected with LC3-EGFP-mRFP expression vector. 48 h later, cells were treated or untreated with 150nM rapamycin for further 24 h. Eventually, the cells were (A) photographed using LeicaSP8 laser confocal microscopy are shown in the representative images (Scale bars is 10 μm). B Quantification of the results was performed using Image pro 10 analysis of the LC3-EGFP-mRFP (yellow) or LC3-mRFP (red) positive puncta area (%); (data are the means ± SE; ^^p < 0.01, ^^^p < 0.001); treated compared to untreated cells. Means ± SE (****p < 0.001 and *p < 0.05); APOE3- compared with APOE4. n ≥ 50 cells/treatment

Fig. 7

Levels of mitochondrial dynamics proteins in APOE3 and APOE4 expressing N9 miroglia. APOE2, APOE3 and APOE4 microglia were subjected to Immunoblot, using the indicated antibodies. Upper panels, representative results; lower panels, densitometric analysis of APOE4 or APOE3 cells as fold of APOE2. The results are the means ± SE; *p < 0.05 and **p < 0.01 of at least four experiments

Fig. 8

Mitochondrial morphology in APOE isoforms expressing microglia. APOE2, APOE3 and APOE4 microglia were incubated with MitoTracker Deep Red (MTDR). Upper panels, representative images and morphological skeleton analysis as generated by the MiNa tool (Scale bars, 10 μm) (A) Lower panels, quantification of branches per network (mean network size) and individual mitochondria relative to the mitochondrial network footprint (B-C). (n ≥ 50 cells) results are presented as the mean ± SE (***p < 0.001 and **** p < 0.0001). D. Cells were stained with MitoTracker Red (MTR) and fluorescence was measured (APOE3 compared to APOE4; *p < 0.05 and APOE2 compared to APOE3 or APOE4; ^^^p < 0.001)

Fig. 9

Mitochondrial function in APOE2, APOE3 and APOE4 N9 microglia. APOE2, APOE3 and APOE4 microglia were incubated for 4 h with CCCP at the indicated concentrations. Cells were subjected to MTT and Methylene blue (MB) assays. A represent cell viability (MB) and B represent MTT assay normalized to MB (viability). The results are presented fold of the untreated controls (mean ± SEM). Statistical analysis was performed using 2way-ANOVA. * p < 0.05, between groups, comparison between APOE2, APOE3 or APOE4 expressing N9 cells, (n ≥ 3). (C) Cells were treated for 4 h with 15 µM CCCP. Levels of LC3 were determined by immunoblot upper panel, lower panel depicts densitometric analysis of the results which are presented as fold induction compared to the untreated control of each cell line (mean ± SE, ***p < 0.001), treated compared to untreated cells. DAPOE2, APOE3, and APOE4 expressing microglia were incubated with 150 nM rapamycin for 24 h. Then cells were stained with MitoTracker Red (MTR). Fluorescence intensities were measured using flow cytometry. Results are presented as the fold induction of the mean fluorescence of APOE3 control. (mean ± SE, ^p < 0.05, ^^^^p < 0.0001. APOE3 compared to APOE4 cells; ***p < 0.001. APOE4 treated compared to APOE4 untreated **p < 0.01). n ≥ 4