Inflammatory Stress on Autophagy in Peripheral Blood Mononuclear Cells from Patients with Alzheimer's Disease during 24 Months of Follow-Up

Abstract

Recent findings indicate that microglia in Alzheimer's disease (AD) is senescent whereas peripheral blood mononuclear cells (PBMCs) could infiltrate the brain to phagocyte amyloid deposits. However, the molecular mechanisms involved in the amyloid peptide clearance remain unknown. Autophagy is a physiological degradation of proteins and organelles and can be controlled by pro-inflammatory cytokines. The purpose of this study was to evaluate the impact of inflammation on autophagy in PBMCs from AD patients at baseline, 12 and 24 months of follow-up. Furthermore, PBMCs from healthy patients were also included and treated with 20 μM amyloid peptide 1-42 to mimic AD environment. For each patient, PBMCs were stimulated with the mitogenic factor, phytohaemagglutin (PHA), and treated with either 1 μM C16 as an anti-inflammatory drug or its vehicle. Autophagic markers (Beclin-1, p62/sequestosome 1 and microtubule-associated protein-light chain 3: LC3) were quantified by western blot and cytokines (Interleukin (IL)-1β, Tumor necrosis Factor (TNF)-α and IL-6) by Luminex X-MAP® technology. Beclin-1 and TNF-α levels were inversely correlated in AD PBMCs at 12 months post-inclusion. In addition, Beclin-1 and p62 increased in the low inflammatory environment induced by C16. Only LC3-I levels were inversely correlated with cognitive decline at baseline. For the first time, this study describes longitudinal changes in autophagic markers in PBMCs of AD patients under an inflammatory environment. Inflammation would induce autophagy in the PBMCs of AD patients while an anti-inflammatory environment could inhibit their autophagic response. However, this positive response could be altered in a highly aggressive environment.

Fig 1. Longitudinal monitoring of Beclin-1 expression in PBMCs from AD patients.

Representative immunoblots (panel A) showed the immunoreactivity of Beclin-1 in PBMCs of AD patients at the Day of inclusion (D0, panel B) and after a follow-up at 12 months (M12, panel C) and 24 months (M24, panel D). PBMCs were isolated from blood and cultured either with 1 μM C16 or its vehicle (0.8% DMSO) for 48hr as described in method section. Semi-quantitative analysis of immunoblot was performed using Gene Tools software (Syngene, Ozyme France). The immunoreactivity of protein was normalized to β-actin immunoreactivity. The line on the graphs (B to D) represents the mean of 36, 33 and 27 patients at D0, M12 and M24, respectively. The mean expression of Beclin-1 in PBMCs at D0, M12 and M24 are shown in the panels E and F, without or with the C16 treatment, respectively. *p < 0.05 compared to vehicle-treated PBMCs at M12 by Wilcoxon matched-pairs signed rank test; ^$p < 0.05 compared to PBMCs at M12 by ANOVA for repeated measures and post-hoc Sheffe’s test. Friedman test: p = 0.0019 for Beclin-1 in vehicle condition and p = 0.0002 in C16 condition.

Fig 2. Longitudinal monitoring of p62 and LC3 expression in PBMCs from AD patients.

Representative immunoblots showed the immunoreactivity of p62 (A, C, D and E), LC3 I (B, F, G and H) and LC3 II (B, I, J and K) in PBMCs of AD patients at the Day of inclusion (D0) and after a follow-up at 12 months (M12) and 24 months (M24). PBMCs were isolated from blood and cultured either with 1 μM C16 or its vehicle (0.8% DMSO) for 48hr as described in method section. Semi-quantitative analysis of immunoblot was performed using Gene Tools software (Syngene, Ozyme France). The immunoreactivity of protein was normalized to β-actin immunoreactivity. The line on the graphs represents the mean of 34, 27 and 23 patients for p62, mean of 21, 17 and 14 patients for LC3 I and mean of 21, 17 and 13 patients for LC3 II at D0, M12 and M24, respectively. Some signals were not analyzed on the blot due to a very low or absent signal. *p < 0.05, **p < 0.005 compared to respective vehicle-treated PBMCs by Wilcoxon matched-pairs signed rank test. Friedman test was not significant for these three autophagic markers.

Fig 3. Expression of autophagic markers in PBMCs from healthy patients.

Representative immunoblots showed the immunoreactivity of Beclin-1 (panel A), p62 (panel B), LC3 I and LC3 II (panel C) in PBMCs of healthy patients cultured in three experimental culture conditions (serum medium, serum free medium and 20μM Aβ42 in serum free medium) with or without 1 μM C16 or its vehicle (0.8% DMSO) for 48hrs as described in method section. Semi-quantitative analysis of immunoblot was performed using Gene Tools software (Syngene, Ozyme France). The immunoreactivity of protein was normalized to β-actin immunoreactivity. The line on each graph represents the mean. *p < 0.05 compared to DMSO-treated PBMCs in serum medium, ^$p < 0.05 compared to Aβ42-treated PBMCs in serum free medium with DMSO by Wilcoxon matched-pairs signed rank test.