Neuro-inflammation induced by lipopolysaccharide causes cognitive impairment through enhancement of beta-amyloid generation

Abstract

Background: Alzheimer's disease (AD) is characterized by extensive loss of neurons in the brain of AD patients. Intracellular accumulation of beta-amyloid peptide (Abeta) has also shown to occur in AD. Neuro-inflammation has been known to play a role in the pathogenesis of AD.

Methods: In this study, we investigated neuro-inflammation and amyloidogenesis and memory impairment following the systemic inflammation generated by lipopolysaccharide (LPS) using immunohistochemistry, ELISA, behavioral tests and Western blotting.

Results: Intraperitoneal injection of LPS, (250 microg/kg) induced memory impairment determined by passive avoidance and water maze tests in mice. Repeated injection of LPS (250 microg/kg, 3 or 7 times) resulted in an accumulation of Abeta1-42 in the hippocampus and cerebralcortex of mice brains through increased beta- and gamma-secretase activities accompanied with the increased expression of amyloid precursor protein (APP), 99-residue carboxy-terminal fragment of APP (C99) and generation of Abeta1-42 as well as activation of astrocytes in vivo. 3 weeks of pretreatment of sulindac sulfide (3.75 and 7.5 mg/kg, orally), an anti-inflammatory agent, suppressed the LPS-induced amyloidogenesis, memory dysfunction as well as neuronal cell death in vivo. Sulindac sulfide (12.5-50 microM) also suppressed LPS (1 microg/ml)-induced amyloidogenesis in cultured neurons and astrocytes in vitro.

Conclusion: This study suggests that neuro-inflammatory reaction could contribute to AD pathology, and anti-inflammatory agent could be useful for the prevention of AD.

Figure 1

Effect of LPS on step-through type passive avoidance test (A) and water maze test (B, C, D). (A), Each value is mean ± S.E. from 7–9 mice. *Significantly different from control (p < 0.05). Memory function was determined by the escape latencies (B, sec), distance (C, cm) and speed (D, cm/sec) for 3 days at 4 hr (designated 1 day) after administration of LPS. Each value is mean ± S.E. from 7–9 mice. *Significantly different from control (p < 0.05).

Figure 2

Effect of LPS on Aβ accumulation in the cortex and hippocampus. The levels of Aβ_1–42 and Aβ_1–40 (A) were assessed by using a specific Aβ ELISA as described in the Materials and methods section. Values measured from each group of mice were calibrated by amount of protein and expressed as mean ± S.E. (n = 5) *Significant different from control (p < 0.05). Immunostaining of Aβ_1–42 in the cortex and hippocampus (B). Mice were injected intraperitoneally with either 250 μg/kg LPS or sterile saline (0.9% NaCl) daily for 3 or 7 days before sacrifice. Forty μm-thick sections of brains from mice were incubated with rabbit polyclonal anti-Aβ_1–42 antibody and counterstained with hematoxylin. Arrow indicates Aβ_1–42 accumulation which is clearly higher in the cerebral cortex and hippocampus of LPS-treated mouse and was the highest in the mouse treated with daily injection for 7 days. Figure in box shows the intracellular accumulation of Aβ_1–42 (detected anti-Aβ_1–42 immunofluroscene staining after DAPI staining the cells) in the pyramidal neurons of the hippocampus at the high magnification. Arrow bar indicates accumulation of Aβ_1–42.

Figure 3

Effect of LPS on secretase activities and amyloidogenic proteins expression. The activities of α-, β-secretase (A, B) and γ-secretase (C) were assessed by using commercially available assay kits. Data represent mean ± S.E. (n = 5). *Significant different from control group (p < 0.05). The expression of APP, BACE and C99 (D) were detected by Western blotting using specific antibodies. Each blot is representative for five experiments. β-actin protein was used here as an internal control.

Figure 4

Effect of anti-inflammatory agents on expression of amyloidogenic proteins (A, B). The expression of APP, BACE and C99 were detected by Western blotting using specific antibodies in astrocytes (A) and neuronal cells (B). β-Actin protein was used as an internal control. Each blot is representative for five experiments. Sulindac sulfide inhibits expression of amyloidogenic proteins (C) and Aβ_1–42 secretion (D) induced by LPS in cultured neuronal cells. Combined Sulindac sulfide (12.5, 25, 50 μM) and LPS treatment for 24 hr were used. (C), The expression of APP, BACE and C99 in neuronal cells was detected by Western blotting using specific antibodies. β-Actin protein was used as an internal control. (D), Media were collected to determine an Aβ_1–42 secretion by ELISA. Data represent mean ± S.E. of three experiments with duplicated. *Significant different from LPS treated group (p < 0.05).

Figure 5

Effect of sulindac sulfide on the LPS-induced memory impairment (A-C) and elevated Aβ_1–42 level (D). Sulindac sulfide was pretreated for 3 weeks by oral administration. For the passive avoidance performance test, mice were trained one time. At 24 hr later, mice were given LPS (250 μg/kg, i.p.). After 4 hr treatment of LPS, the latency period was measured. Each value is means ± S.E. from 15 mice. *Significantly different from LPS treated control (p < 0.05). (B-C), Mice were pretreated with Sulindac sulfide for 3 weeks, and then trained for 3 days (2 times/day, 6 times training), and then LPS (250 μg/kg, i.p.) was administered into mice. Memory function was determined by the escape latencies (cm, B) and distance (sec, C) at 4 hr (designated day 1). Each value is means ± S.E. from 15 mice. D, The levels of Aβ_1–42 were assessed after finishing the behavioral tests by using a specific Aβ_1–42 ELISA. Values measured from each group of mice were calibrated by amount of protein and expressed as mean ± S.E. (n = 15) *Significant different from LPS treated group (p < 0.05).

Figure 6

Effect of LPS on activation of astrocytes (A) and apoptotic cell death (B). Mice were injected intraperitoneally with either 250 μg/kg LPS or sterile saline (0.9% NaCl) daily for 7 days before sacrifice. Forty μm-thick sections of brains from mice were immunostained with rabbit polyclonal anti-GFAP antibody for evaluation of activation of astrocytes. The broad distribution and deep intensity of GFAP reactive cells increased in the LPS injected mice brain. Each panel is representative of 6 animals. Apoptotic cell death was determined by DAPI staining and TUNEL assay. Apoptosis (%) was defined as the percentage of the number of TUNEL-positive cells per surface of unit. Values are mean ± S.E. (n = 6). *P < 0.05 indicates significantly different from LPS-treated cells.