Zileuton improves memory deficits, amyloid and tau pathology in a mouse model of Alzheimer's disease with plaques and tangles

Abstract

The 5-lipoxygenase (5LO) enzyme is widely distributed within the central nervous system. Previous works showed that this protein is up-regulated in Alzheimer's disease (AD), and plays an active role in the development of brain amyloidosis in the APP transgenic mice. In the present paper, we studied the effect of its pharmacological inhibition on the entire AD-like phenotype of a mouse model with plaques and tangles, the 3 × Tg mice. Compared with mice receiving placebo, the group treated with zileuton, a specific 5LO inhibitor, manifested a significant improvement of their memory impairments. The same animals had a significant reduction in Aβ levels and deposition, which was secondary to a down-regulation of the γ-secretase pathway. Additionally, while total tau levels were unchanged for both groups, zileuton-treated mice had a significant reduction in its phosphorylation state and insoluble forms, secondary to a decreased activation of the cdk5 kinase. These data establish a functional role for 5LO in the pathogenesis of the full spectrum of the AD-like phenotype and represent the successful completion of the initial step for the preclinical development of 5LO inhibitors as viable therapeutic agents for AD.

Figure 1. Chronic administration of zileuton ameliorates behavioral deficits of 3×Tg-AD mice.

A. Number of total arm entries for 3×Tg mice receiving zileuton or vehicle. B. Percentage of alternations between 3×Tg mice receiving zileuton or vehicle treatment (*p<0.05). C. Contexual fear memory response in 3×Tg mice receiving zileuton or controls. D. Cued fear memory response in 3×Tg mice receiving zileuton or control (*p<0.05). Values represent mean ± SEM; *p<0.05; n = 9 3×Tg; n = 9 3×Tg zileuton. E. Number of entries to the target platform zone for 3×Tg mice treated with zileuton or vehicle. F. Time in the target platform zone for 3×Tg mice treated with zileuton or vehicle (*p<0.05).

Figure 2. Pharmacologic blockade of 5LO decreases Aβ peptides levels and deposition in the 3×Tg mice via the γ-secretase pathway.

A, B. RIPA-soluble (RIPA) and formic acid extractable (FA) Aβ1-40 and Aβ1-42 levels in cortex of 3×Tg receiving zileuton or vehicle were measured by sandwich ELISA. (n = 9 for control, and n = 9 for zileuton; *p<0.05). C. Representative sections of brains from 3×Tg mice receiving zileuton or placebo (control) immunostained with 4G8 antibody. D. Quantification of the area occupied by Aβ immunoreactivity in brain of 3×Tg mice receiving zileuton or placebo (n = 3 control, and n = 3 zileuton; *p<0.05). E. Representative western blots of APP, ADAM-10, BACE-1, sAPPs, CTFs, PS1, Nicastrin, Pen-2, and APH-1 in the cortex of 3×Tg mice receiving zileuton or vehicle. F. Densitometric analyses of the immunoreactivities to the antibodies shown in the previous panel. (n = 4, *p<0.05). Values represent mean ± SEM.

Figure 3. Pharmacologic blockade of 5LO reduces tau phosphorylation in the 3×Tg mice.

A. Representative western blots of soluble and insoluble total tau (HT7), phosphorylated tau at residues S396 (PHF13), S202/T205 (AT8), T231/S235 (AT180), at T181 (AT270), and S396/S404 (PHF-1) in brain homogenates from 3×Tg mice receiving zileuton or vehicle assayed by western blot analyses. B. Densitometric analyses of the immunoreactivities to the antibodies shown in the previous panel (*p = 0.05). C, D. Representative sections of brains from 3×Tg mice receiving zileuton or vehicle (control) immunostained with HT7, PHF1, PHF13, AT8, AT180, AT270 antibodies.

Figure 4. Pharmacologic blockade of 5LO modulates tau metabolism, ameliorates synaptic integrity and decreases neuroinflammation in the 3×Tg mice.

A. Representative western blots of GSK3α, GSK3β, p-GSK-3α, p-GSK-3β, JNK2, SAPK/JNK, p-JNK2/3, p-JNK1, cdk5, p35, and p25 in brain homogenates of 3×Tg mice treated with zileuton or vehicle. B. Densitometric analyses of the immunoreactivities to the antibodies shown in the previous panel (*p<0.05). C. Representative western blot analyses of synaptophysin (SYP), post-synaptic density protein 95 (PSD-95), and microtubule-associated protein 2 (MAP2) in brain homogenates of 3×Tg mice receiving zileuton or control. D. Densitometric analyses of the immunoreactivities to the antibodies shown in the previous panel (n = 3 per group) (*<0.05). E. Representative sections of brains from 3×Tg mice receiving zileuton or vehicle (control) immunostained with synaptophysin, PDS-95, and MAP2 antibodies. F. Representative western blots of GFAP and CD45 in brain homogenates of 3×Tg mice treated with zileuton or vehicle. G. Densitometric analyses of the immunoreactivities to the antibodies shown in the previous panel (*p<0.05). Values represent mean ± SEM.