Zileuton restores memory impairments and reverses amyloid and tau pathology in aged Alzheimer's disease mice

Abstract

The enzyme 5-lipoxygenase (5LO) is upregulated in Alzheimer's disease (AD), and its pharmacologic blockade with zileuton slows down the development of the AD-like phenotype in young AD mice. However, its efficacy after the AD pathology is established is unknown. To this end, starting at 12 months of age triple transgenic mice (3xTg) received zileuton, a selective 5LO inhibitor, or placebo for 3 months, and then the effect of this treatment on behavior, amyloid, and tau pathology assessed. Although mice on placebo showed worsening of their memory, treated mice performed even better than at baseline. Compared with placebo, treated mice had significantly less Aβ deposits and tau phosphorylation secondary to reduced γ-secretase and CDK-5 activation, respectively. Our data provide novel insights into the disease-modifying action of pharmacologically inhibiting 5LO as a viable AD therapeutic approach. They represent the successful completion of preclinical studies for the development of this class of drug as clinically applicable therapy for the disease.

Keywords: 5-Lipoxygenase; Alzheimer's disease; Amyloid beta; Memory; Tau protein; Transgenic mouse models.

Figure 1. Zileuton inhibits brain 5-lipoxygenase activity

A. Levels of leukotriene B4 (LTB4) in brain homogenates of 15 months old wild type mice (WT), 3xTg mice treated with placebo (CTR) and 3xTg mice treated with zileuton (ZIL) (*p<0.05, ***p<0.001). B. Representative Western blot analyses of 5-lipoxygenase (5-LO) in brain homogenates of 15 months old wild type mice (WT), 3xTg mice placebo treated (CTR) and 3xTg mice zileuton treated (ZIL). C. Densitometric analyses of the immunoreactivities shown in the previous panel (*p<0.05). Results are mean ± sem.

Figure 2. Zileuton restores memory impairments of aged 3xTg mice

A. Total number of entries in the Y-maze for 3xTg 12 months old (12mo) and 3xTg 15 months old receiving placebo (CTR) or zileuton (ZIL) (*p<0.05). B. Percentage of alternation in the Y-maze for the same three groups of mice (*p<0.05). C. Contexual fear conditioning memory responses for the same three groups of mice (*p<0.05). D. Cued fear conditioning memory responses for the same three groups of mice (*p<0.05, ** p<0.01). Results are mean ± sem.

Figure 3. Zileuton reduces Aβ levels and deposition in aged 3xTg mice

A. RIPA-soluble (RIPA) Aβ1-40 and Aβ1-42 levels in brain homogenates of 15 months old 3xTg mice receiving placebo (CTR) or zileuton (ZIL) were measured by sandwich ELISA (** p<0.01). B. Representative sections of brains from the same two groups of mice immunostained with 4G8 antibody. C. Quantification of the area occupied by Aβ immunoreactivity in brains of the same two groups of mice (*p<0.05). D. Representative western blots analyses of APP, ADAM-10, BACE-1, Nicastrin, APH-1, PS1 and Pen-2 levels in brain homogenates from15 months old 3xTg mice receiving placebo (CTR) or zileuton (ZIL). E. Densitometric analyses of the immunoreactivities to the antibodies shown in the previous panel (*p<0.05). Results are mean ± sem.

Figure 4. Zileuton reduces tau phosphorylation in aged 3xTg mice

A. Representative Western blot analyses of soluble total tau (HT-7), tau N-terminal amino acids 7–9 and C-terminal amino acids 312–322 (MC-1), and phosphorylated tau at residues S202/T205 (AT8), T181 (AT270), T231/S235 (AT180), S396/S404 (PHF-1), and S396 (PHF-13) in brain homogenates of 15 months old 3xTg mice receiving placebo (CTR) or zileuton (ZIL). B. Densitometric analyses of the immunoreactivities shown in the previous panel (*p<0.05, **p<0.01). C. Representative images of immunohistochemistry analyses for HT7, MC-1, AT8, AT180, PHF-1 and PHF-13 in brain sections of the same two groups of mice. D. Densitometric analyses of the immunoreactivities shown in the previous panel (*p<0.05). Results are mean ± sem.

Figure 5. The effect zileuton on tau solubility and tau metabolism

A. Representative Western blot analyses of formic acid-soluble total tau (HT-7) in brain homogenates of 15 months old 3xTg mice receiving placebo (CTR) or zileuton (ZIL). B. Densitometric analyses of the immunoreactivities shown in the previous panel (*p<0.05). C. Representative Western blot analyses of CDK-5, P35, P25, GSK-3α and GSK-3β, p-GSK-3α and p-GSK3β, and PP-2A in brain homogenates from the same two groups of mice. D. Densitometric analyses of the immunoreactivities shown in the previous panel. (*p<0.05). Results are mean ± sem.

Figure 6. The effect of Zileuton on synaptic integrity and neuroinflammation in aged 3xTg mice

A. Representative Western blot analyses of synaptophysin (SYP) and post-synaptic protein-95 (PSD-95) in brain homogenates of 15 months old 3xTg mice receiving placebo (CTR) or zileuton (ZIL). B. Densitometric analyses of the immunoreactivities shown in the previous panel. (*p<0.05). C. Representative Western blot analyses of glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule 1 (IBA-1) in brain homogenates of 15 months old 3xTg mice receiving placebo (CTR) or zileuton (ZIL). D. Densitometric analyses of the immunoreactivities shown in the previous panel. (*p<0.05). Results are mean ± sem.