Pharmacologic blockade of 5-lipoxygenase improves the amyloidotic phenotype of an Alzheimer's disease transgenic mouse model involvement of γ-secretase

Abstract

The 5-lipoxygenase (5-LO) enzyme is widely distributed within the central nervous system. Previous works showed that this protein is up-regulated in Alzheimer's disease (AD) and that its genetic absence results in a reduction of amyloid β (Aβ) levels in Tg2576 mice. In the present study, we examined the effect of 5-LO pharmacological inhibition on the amyloidotic phenotype of these mice. Aβ deposition in the brains of mice receiving zileuton, a selective and specific 5-LO inhibitor, was significantly reduced when compared with control Tg2576 mice receiving vehicle. This reduction was associated with a similar decrease in brain Aβ peptides levels. Zileuton treatment did not induce any change in the steady state levels of amyloid-β precursor protein (APP), BACE1 or ADAM10. By contrast, it resulted in a significant reduction of presenilin 1 (PSEN1, alias PS1), nicastrin (NCSTN) , presenilin enhancer 2 homolog (PSNEN, alias, Pen-2), and anterior pharynx defective 1 (APH-1), the four components of the γ-secretase complex-at the protein and message level. Furthermore, in vitro studies confirmed that zileuton prevents Aβ formation by modulating γ-secretase complex levels without affecting Notch signaling. These data establish a functional role for 5-LO in the pathogenesis of AD-like amyloidosis, whereby it modulates the γ-secretase pathway. They suggest that pharmacological inhibition of 5-LO could provide a novel therapeutic opportunity for AD.

Figure 1

Chronic administration of zileuton decreases brain Aβ peptide levels and deposition in Tg2576 mice. A: Radio-immunoprecipitation assay-soluble and formic acid extractable Aβ1-40 and Aβ1-42 levels in cortex and hippocampus of Tg2576 mice receiving zileuton or placebo (control) for 8 months were measured by sandwich ELISA. n = 8 control, and n = 10 zileuton; *P < 0.05, **P < 0.01). B: Representative sections of brains from Tg2576 mice receiving zileuton or placebo (control) immunostained with 4G8 antibody. Quantification of the area occupied by Aβ immunoreactivity in brain of Tg2576 mice receiving zileuton or placebo (control) (n = 4 control, and n = 4 zileuton; **P < 0.01). Values represent mean ± SEMRIPA, radio-immunoprecipitation assay; FA, formic acid.

Figure 2

Chronic administration of zileuton alters brain APP metabolism via the γ-secretase pathway in Tg2576 mice A: Representative Western blots of APP, ADAM10, BACE1, PS1, nicastrin, Pen2, and APH-1 in cortex homogenates from Tg2576 mice receiving zileuton or placebo (CTL) for 8 months. B: Densitometric analyses of the immunoreactivities to the antibodies shown in the previous panel. C: Relative mRNA levels for BACE1, PS1, nicastrin, APH-1, and Pen2 in the cortex of Tg2576 mice receiving zileuton or placebo (CTL), as determined by real-time quantitative RT-PCR amplification. Values represent mean ± SEM (n = 4 control, and n = 4 zileuton; *P < 0.05, **P < 0.01).

Figure 3

Chronic administration of zileuton alters CREB levels in the brains of Tg2576 mice. Levels of total CREB and its phosphorylated form at Ser133 and Sp1 in the cortex of Tg2576 mice receiving zileuton or placebo (CTL) assayed by Western blot analyses. Densitometric analyses of the immunoreactivities to the antibodies shown in the previous panel. Values represent mean ± SEM (n = 4 control, and n = 4 zileuton; *P < 0.05, **P < 0.01).

Figure 4

In vitro effect of zileuton on Aβ formation and APP metabolism. N2a-APPswe cells were incubated with increasing concentration of zileuton or vehicle (control) for 24 hours, and conditioned media and cell lysates were collected. A: LTB4 and Aβ1-40 levels in the supernatant were assayed by sandwich ELISA (n = 3 control, and n = 3 zileuton; **P < 0.01). B: Representative Western blots of APP, ADAM10, BACE1, PS1, nicastrin, APH-1, and Pen2 in the lysates of zileuton or vehicle-treated (control) cells, and densitometric analyses of the immunoreactivities to the antibodies shown in B (n = 4, *P < 0.05, **P < 0.01). Values represent mean ± SEM. LTB4, leukotriene B4.

Figure 5

In vitro effect of zileuton on CREB and Notch. N2a-APPswe cells were incubated with increasing concentrations of zileuton or vehicle (control) for 48 hours, and cell lysates were collected for immunoassays. A: Representative Western blots of total CREB and its phosphorylated form at Ser133, and Sp1 levels, and densitometric analyses of the immunoreactivities to the antibodies to CREB, p-CREB and Sp1. B: Representative Western blots of NICD in the lysates of cells treated with zileuton, L685485 (1 μmol/L) or vehicle controls (CTL) and densitometric analyses of the immunoreactivities to the antibodies NICD Values represent mean ± SEM (n = 4, *P < 0.05, **P < 0.01).