5-Lipoxygenase gene disruption reduces amyloid-beta pathology in a mouse model of Alzheimer's disease

Abstract

5-Lipoxygenase (5LO), by producing leukotrienes, is a proinflammatory enzyme, and there is evidence suggesting that it is up-regulated with aging and may be involved in Alzheimer's disease (AD). In this paper, we studied the effect of 5LO-targeted gene disruption on the amyloid phenotype of a transgenic mouse model of AD, the Tg2576. Amyloid-beta (Abeta) deposition in the brains of Tg2576 mice lacking 5LO was reduced by 64-80% compared with Tg2576 controls. This reduction was associated with a similar significant decrease in Abeta levels measured by sandwich ELISA. Absence of 5LO did not induce any significant change in amyloid-beta precursor protein (APP) levels and processing, or Abeta catabolic pathways. Furthermore, in vitro studies showed that 5LO activation or 5LO metabolites increase, whereas 5LO inhibition decreases, Abeta formation, secondary to correspondent changes in gamma-secretase activity. These data establish for the first time a novel functional role for 5LO in the pathogenesis of AD-like amyloidosis, thereby modulating gamma-secretase activity. Our work suggests that pharmacological inhibition of 5LO could provide a novel therapeutic tool for AD.

Figure 1

5LO is up-regulated in old Tg2576 mice and in AD brains. A) Relative mRNA levels of 5LO were measured in separate anatomical regions of the brains of 3- (young) and 24-month-old (old) Tg2576 mice by real-time quantitative RT-PCR amplification (n=3 mice per group). *P < 0.05; **P < 0.001 significant, old vs. young. B) Representative Western blot analysis of the hippocampal and cortical homogenates from control and AD brains probed with a specific antibody against 5LO. C) Densitometric analysis of 5LO immunoreactivity in control and AD brains. Values represent mean ± SE. *P < 0.05, significant vs. control.

Figure 2

Genetic ablation of 5LO in Tg2576 mice markedly reduces Aβ deposition in different regions of the brain. A–D) Representative sections of brains of female Tg2576 × 5LO^+/+ (A, C) and Tg2576 × 5LO^−/− mice (B, D) at 12 and 15 months of age, immunostained with 4G8 antibody. E) Quantification of the area occupied by Aβ immunoreactivity in hippocampus, somatosensory cortex, and perihippocampal cortex in Tg2576 × 5LO^−/− (open bars) at 12 (n=8) and 15 months of age (n=10) compared to age-matched Tg2576 × 5LO^+/+ mice (solid bars, n=6 and 12, respectively). Values represent mean ± SE. *P < 0.05; **P < 0.01; ***P < 0.005, significant vs. control.

Figure 3

Aβ1–42, but not Aβ1–40, is significantly decreased in the brains of Tg2576 × 5LO^−/− compared to Tg2576 × 5LO^+/+ mice. RIPA-soluble (RIPA) and formic acid extractable (FA) Aβ1–40 (A, B) and Aβ1–42 (C, D) levels in the cortex and hippocampus from female Tg2576 × 5LO^+/+ (solid bars) and Tg2576 × 5LO^−/− mice (open bars) at 12 and 15 months of age were measured by sandwich ELISA. Values represent mean ± SE (n=5 per group for 12-month-olds; n=7–11 per group for 15-month-olds). *P < 0.05; **P < 0.01, significant vs. control.

Figure 4

Immunohistochemistry and Western blot analyses confirm that Aβ42 is reduced in the brains of Tg2576 mice lacking 5LO. A–F) Representative sections of cortex from 15-month-old female Tg2576 × 5LO^+/+ and Tg2576 × 5LO^−/− mice immunostained for total Aβ (4G8) (A, B), Aβ40 (BA27) (C, D) and Aβ42 (BC05) (E, F). G) Quantification of BA27 and BC05 immunoreactivities in hippocampus, somatosensory cortex, and perihippocampal cortex of Tg2576 mice deficient for 5LO compared to control (n=5–7 per group). Data are expressed as a percentage of control. H) Representative Western blots of cortex homogenates from 15-month-old Tg2576 × 5LO^+/+ and Tg2576 × 5LO^−/− mice probed with 82E1 and anti-β-actin antibodies. I) Densitometric analysis of Aβ1–40 and Aβ1–42 immunoreactivities (n=8 per group). Values represent mean ± SE. *P < 0.05; **P < 0.001, significant vs. control.

Figure 5

The ratio of Aβ1–38:Aβ1–42 is increased in Tg2576 × 5LO^−/− compared to Tg2576 × 5LO^+/+ mice. A, B) Aβ1–38 was measured in the RIPA (A) and FA (B) fractions of the cortex from female Tg2576 × 5LO^+/+ (solid bars) and Tg2576 × 5LO^−/− mice (open bars) at 12 (n=5 per group) and 15 months of age (n=6–8 per group) by sandwich ELISA. C, D) The ratio of Aβ1–38:Aβ1–42 in the same mice. Values represent mean ± SE. *P < 0.05, significant vs. control.

Figure 6

APP metabolism mediated by α- and β-secretase is unaltered in Tg2576 mice deficient for 5LO. A) Representative Western blots of APP, its cleavage products (sAPPβ, sAPPα, and C-terminal fragments), and BACE-1 in the cortex of 15-month-old mice. B) Densitometric analyses of the immunoreactivities to the antibodies shown in panel A. C) γ-secretase activity was significantly decreased in brain homogenates from Tg2576 × 5LO^−/− (n=3) compared to Tg2576 × 5LO^+/+ (n=3). D) Representative Western blots of the cortex homogenates from the 15-month-old mice probed with specific antibodies against Aβ-degrading enzymes, IDE and neprilysin, and against GFAP and apoE. E) Densitometric analyses of the immunore-activities to the antibodies shown in panel C. F) Relative mRNA levels of IDE and neprilysin in the cortex of 15-month-old Tg2576 × 5LO^+/+ (solid bars) and Tg2576 × 5LO^−/− mice (open bars) as determined by real-time quantitative RT-PCR amplification (n=4 per group). Values represent mean ± SE. *P < 0.05, significant vs. control.

Figure 7

Activation of 5LO in MEFs by Ca²⁺ ionophore A23187 results in an increase in the γ-secretase activity and Aβ formation. A) 5LO enzyme was activated in APP × 5LO^+/+ and APP × 5LO^−/− MEFs by adding A23187 (100 nM) in the presence or absence of zileuton, and Aβ peptides secreted in the medium were measured. Data are expressed as percentage of control. Control for each genotype consisted of MEFs with the same genotype not treated with A23187 or zileuton. A23187 is present in all conditions except for control. Values represent mean ± SE of 3 experiments. B) Representative Western blots of total APP measured in cell lysates of APP ×5LO^+/+ and APP × 5LO^−/− MEFs. C) γ-secretase activity was measured in the harvested APP × 5LO^+/+ and APP × 5LO^−/− MEFs. *P < 0.05, significant vs. control.

Figure 8

5LO metabolites increase and 5LO inhibitor decreases Aβ1–40 production in HEK293-C99 cells. A) HEK293-C99 cells were challenged with two different 5LO metabolites, 5-HPETE and LTC4. B) The same cells were treated with a 5LO inhibitor, zileuton (n=3 per condition). *P < 0.05, significant vs. control.