Severe chronic cerebral hypoperfusion induces microglial dysfunction leading to memory loss in APPswe/PS1 mice

Abstract

Cerebral vasculature plays a key role in controlling brain homeostasis. Cerebral vasculature dysfunction, associated to irregularities in cerebral blood perfusion, has been proposed to directly contribute to Alzheimer's disease (AD) pathogenesis. More precisely, chronic cerebral hypoperfusion, which impairs brain homeostasis, was demonstrated to take place even before cognitive decline. However, the mechanisms underlying the implication of chronic cerebral hypoperfusion in AD pathogenesis remain elusive. Therefore, this study aims at investigating the role of severe chronic cerebral hypoperfusion (SCCH) in AD pathogenesis. For this purpose, SCCH was induced in young APPswe/PS1 in order to evaluate the progression of AD-like pathology in these mice. We observed that SCCH accelerated the cognitive decline of young APPswe/PS1 mice, which was associated with an increased amyloid plaque number in brain parenchyma. In addition, SCCH reduced the activity of extracellular signal-regulated kinases 1/2 (ERK1/2), which has been shown to play an important role in the adaptive responses of neurons. Importantly, SCCH impaired the function of microglial cells, which are implicated in amyloid-β (Aβ) elimination. In vitro approaches underlined the ability of a low-glucose microenvironment to decrease the general activity and phagocytic capacity of microglia. By using a new model of SCCH, our study unravels new insights into the implication of severe chronic cerebral hypoperfusion in AD pathogenesis, mainly by altering microglial cell activity and consequently Aβ clearance.

Keywords: Alzheimer’s disease; Gerotarget; cerebral hypoperfusion; microglia.

Figure 1. SCCH aggravates APP_swe/PS1 cognitive deficits

The surgery has no effect on animal weight A.. Old APPswe/PS1 mice were used as positive control for cognitive decline. Water t-maze B.,C. and novel object recognition tests D. were used to assess mouse cognitive function. SCCH slightly increases the number of trails B. and significantly increases the latency C. of mice in the water t-maze test, which were comparable to those of old mice. SCCH significantly reduces mouse capacity in recognizing the novel object during the NOR test, which also were comparable to those of old mice. Data are means ± SEM (n = 6-8 animals per group) * P < 0.05 compared to sham group; #P < 0.05 compared with sham object 1. Pre-op: Pre-operatory.

Figure 2. Number of Aβ plaques increases following SCCH without affecting soluble Aβ levels

Aβ plaque number and size, and soluble Aβ peptide levels in the brain of APP_swe/PS1 were assessed by immunofluorescence staining A.-C. and ELISA (D., E.), respectively. SCCH increases the number of Aβ plaques in the brain of mice A.,B. without affecting plaque load C.. The levels of soluble Aβ_1-40 D. and soluble Aβ_1-42 E. are not affected by SCCH. Data are means ± SEM (n = 6-8 animals per group, 3 sections per animal's brain for immunofluorescence staining) *** P < 0.005 compared with sham. Images were acquired with a 4X objective. Scale bar = 500μm.

Figure 3. SCCH induces slightly increases patrolling monocyte subset frequency in blood circulation

Frequency of total A., inflammatory C. and patrolling monocytes D. were analyzed by flow cytometry in blood. Using a gating strategy based on Ly6C expression level B., inflammatory and patrolling monocyte subsets were discriminated. SCCH does not affect the frequency of total monocyte frequency A. and inflammatory monocyte subset (Ly6C^High) C., in the blood of mice 14 weeks following surgery. SCCH slightly (P = 0.0779) increases the frequency of patrolling monocyte subset (Ly6C^Low) in the blood of mice D.. Data are means ± SEM (n = 6-8 animals per group). Data were analyzed with standard two-tailed unpaired t- test's.

Figure 4. SCCH alters microglial cell function in APP_swe/PS1

Microglial cell function was assessed by using immunofluorescence staining A.-E.. SCCH significantly decreases microglial coverage (Iba⁺) of Aβ plaques A., B., without affecting overall microglial activation (CD68⁺) A., C.. SCCH significantly decreases the number of activated microglia associated to Aβ plaques D., and slightly decreases (P = 0.0559) the area of Aβ plaques covered by activated microglia E.. Data are means ± SEM (n = 6-8 animals per group, 3 section per animal's brain for immunofluorescence staining). * P < 0.05, ** P < 0.01 compared with sham group. Images were acquired with 4X objective. Scale bar = 500μm.

Figure 5. Low-glucose microenvironment alters microglial cell activity and phagocytic capacity

Viability, global microglial cell activity and phagocytic capacity were investigated by using, XTT assay A., Griess assay B. and flow cytometry C., D., respectively. Low-glucose microenvironment (i.e. DMEM-Low), maintained for 24 hours, does not alter BV2 cell (i.e. immortalized murine microglial cell) viability A.. BV2 cells exposed for 24 hours to 750 μM of H₂O₂ were used as a positive control for cell death A.. Low-glucose medium (i.e. DMEM-Low) significantly decreases global microglial cell activity B., activation C. and phagocytic capacity D.. BV2 cells incubated on ice were used as negative control for the phagocytosis assay D.. DMEM-High (4500mg/L glucose) and DMEM-Low (1000mg/L glucose) medium were used. Data are means ± S.D. (n = 5 experiments per condition, 4 replicates per experiment for viability assay; n = 6 experiments per condition, 3 replicates per experiment for Griess assay; n = 5-6 samples per condition for flow cytometry). * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.0001 compared with DMEM High group; ^#### P < 0.0001 compared with control.

Figure 6. SCCH decreases ERK1/2 pathway activation

Western blot analysis was used to assess total A. and phosphorylated B. ERK1/2, which were corrected with β-actin expression level. SCCH does not affect total ERK1/2 expression A., but decreases ERK1/2 phosphorylation (i.e. activation) B.. Data are means ± SEM (n = 5-7 animals per group). * P < 0.05 compared with sham.