Involvement of 5-lipoxygenase activating protein in the amyloidotic phenotype of an Alzheimer's disease mouse model

Abstract

Background: The 5-lipoxygenase enzyme is widely distributed within the central nervous system and its activity is regulated by the presence and availability of another protein, called 5-lipoxygenase activating protein. While previous works have shown that 5-lipoxygenase is involved in the pathogenesis of Alzheimer's disease, no data are available on the role that 5-lipoxygenase activating protein plays in Alzheimer's disease.

Methods: In the present paper, we studied the effect of pharmacologic inhibition of 5-lipoxygenase activating protein on the amyloidotic phenotype of Tg2576 mice.

Results: Amyloid β peptide (Aβ) deposition in the brains of mice receiving MK-591, a selective and specific 5-lipoxygenase activating protein inhibitor, was significantly reduced when compared with controls. This reduction was associated with a similar decrease in brain Aβ peptides levels. MK-591 treatment did not induce any change in the steady-state levels of amyloid-β precursor protein, β-site amyloid precursor protein cleaving enzyme 1 or disintegrin and metalloproteinase domain-containing protein 10. By contrast, it resulted in a significant reduction of the γ-secretase complex, at the protein and message level. Furthermore, in vitro studies confirmed that MK-591 prevents Aβ formation by modulating γ-secretase complex levels without affecting Notch signaling.

Conclusions: These data establish a novel functional role for 5-lipoxygenase activating protein in the pathogenesis of Alzheimer's disease-like amyloidosis, and suggest that its pharmacological inhibition could provide a novel therapeutic opportunity for Alzheimer's disease.

Figure 1

Pharmacologic blockade of 5-lipoxygenase activating protein decreases brain Aβ peptides levels and deposition. (A-D) RIPA-soluble (RIPA) and formic acid extractable (FA) Aβ1-40 and Aβ1-42 levels in cortex and hippocampus of Tg2576 receiving MK-591 or placebo for 8 months were measured by sandwich ELISA. (n = 9 for control, and n = 11 for MK-591; *P <0.04). (E) Representative sections of brains from Tg2576 mice receiving MK-591 or placebo (control) for 8 months immunostained with 4 G8 antibody. (F) Quantification of the area occupied by Aβ immunoreactivity in brain of Tg2576 mice receiving MK-591 or placebo for 8 months (*P = 0.03). ELISA: enzyme-linked immunosorbent assay; FA: formic acid; RIPA: radioimmunoprecipitation assay.

Figure 2

Pharmacologic blockade of 5-lipoxygenase activating protein alters amyloid-β precursor protein metabolism via the γ-secretase pathway. (A) Representative western blots of APP, ADAM-10, BACE-1, secreted APPα, secreted APPβ, CTFs, PS1, nicastrin, APH-1 and Pen-2 in brain homogenates of Tg2576 mice receiving MK-591 or placebo for 8 months. (B) Densitometric analysis of some of the immunoreactivities to the antibodies shown in the previous panel (n = 9 control, n = 11 MK-591; *P <0.04). (C) Relative mRNA levels for BACE-1, PS1, nicastrin, APH-1 and Pen-2 in brain homogenates of Tg2576 mice receiving MK-591 or placebo for 8 months, as determined by real-time quantitative RT-PCR amplification (*P <0.02). (D) Representative western blot for total cAMP response element-binding and its phosphorylated form at Ser133, and Sp1 in brain homogenates of Tg2576 mice receiving MK-591 or placebo. (E) Densitometric analysis of the immunoreactivities to the antibodies shown in the previous panel (n = 9 control, n = 11 MK-591; *P <0.03). ADAM-10: disintegrin and metalloproteinase domain-containing protein 10; APH-1: anterior pharynx-defective 1; APP: amyloid-β precursor protein; BACE-1: β-site amyloid precursor protein cleaving enzyme 1; CREB: cAMP response element-binding protein; CTF: C-terminal fragments; Pen-2: presenilin enhancer 2; PS1: presenilin1; RT-PCR: reverse transcriptase polymerase chain reaction.

Figure 3

Pharmacologic blockade of 5-lipoxygenase activating protein reduces neuroinflammation. (A, C) Representative brain sections of the cortex region from Tg2576 receiving MK-591 or vehicle (control) immunostained for CD45 and GFAP (× 20 magnification). (B, D) Quantitative analysis of the immunoreactivity for CD45 and GFAP in the same animals. (E) Representative western blot analysis of GFAP and 5-LO in brain homogenates of Tg2576 receiving MK-591 or control. (F) Densitometric analysis of the immunoreactivities to the antibodies shown in the previous panel. (G) Levels of IL-1β in brain homogenates of Tg2576 mice receiving MK-591 or controls (n = 8 controls, n = 10 MK-591;*P = 0.01). CD45: cluster of differentiation 45; GFAP: glial acidic fibrillary protein; 5-LO: 5-lipoxygenase.

Figure 4

In vitroeffect of MK-591 on Aβ formation and amyloid-β precursor protein metabolism. N2A-APPswe cells were incubated with increasing concentration of MK-591 or vehicle for 24 h, and conditioned media and cell lysates collected. (A) Aβ1-40 levels in the supernatant assayed by sandwich ELISA (n = 4 per each condition; *P <0.01). (B) Representative western blots of APP, ADAM-10, BACE-1, PS1, nicastrin, APH-1, and Pen-2 in the lysates of MK-591 or vehicle-treated cells. (C) Densitometric analyses of the immunoreactivities to the antibodies shown in panel B (*P <0.01). ADAM-10: disintegrin and metalloproteinase domain-containing protein 10; APH-1: anterior pharynx-defective 1; APP: amyloid-β precursor protein; BACE-1: β-site amyloid precursor protein cleaving enzyme 1; ELISA: enzyme-linked immunosorbent assay; N2A-APPswe: neuro-2 A neuroblastoma cells expressing human APP carrying the K670N/M671L Swedish mutation; Pen-2: presenilin enhancer 2; PS1: presenilin1.

Figure 5

In vitroeffect of MK-591 on cAMP response element-binding protein and Notch. N2A-APPswe cells were incubated with increasing concentration of MK-591 or vehicle for 24 hours, and cell lysates collected for immunoassays. (A) Representative western blots of total CREB, its phosphorylated form (p-CREB) at Ser133, and Sp1 levels. (B) Densitometric analyses of the immunoreactivities to the antibodies to CREB, p-CREB and Sp1 (*P <0.01). (C) Representative western blots of NICD in the lysates of cells treated with MK-591, L685,458 or vehicle groups. (D) Densitometric analyses of the immunoreactivities to the antibodies NICD (n = 4; *P <0.01). CREB: cAMP response element-binding protein; NICD: Notch intracellular domain; N2A-APPswe: neuro-2 A neuroblastoma cells expressing human APP carrying the K670N/M671L Swedish mutation.