5-Lipoxygenase pharmacological blockade decreases tau phosphorylation in vivo: involvement of the cyclin-dependent kinase-5

Abstract

The 5-lipoxygenase (5LO) enzyme is widely distributed within the central nervous system. Previous works showed that this protein is upregulated in Alzheimer's disease, and that its genetic absence results in a reduction of amyloid beta levels in Tg2576 mice. However, its contribution to tau pathology remains to be investigated. To this end we studied the effect of 5LO chronic pharmacologic inhibition on endogenous tau level and metabolism in the same mice. The phosphorylation of tau at S396 and S396/404 in the brains of mice receiving zileuton, a selective and specific 5LO inhibitor, was significantly reduced when compared with their controls, while there was no significant change of tau phosphorylation at S202/T205, T231/S235, and T181 epitopes. The 5LO-dependent reduction of tau phosphorylation resulted from a significant decrease in the level and activity of the cyclin-dependent kinase-5 but not other kinases. Our findings highlight the novel functional role that neuronal 5LO plays in modulating tau phosphorylation, and suggest that pharmacologic inhibition of 5LO could provide a novel therapeutic opportunity also for Alzheimer's disease-related tau pathology.

Figure 1. Chronic administration of zileuton decreases tau phosphorylation in the brains of Tg2576 mice

A. Representative western blots of total tau (Tau), phosphorylated tau as recognized by PHF13, AT8, AT180, AT270, and PHF-1 antibodies in brain homogenates from mice receiving zileuton or placebo. B. Densitometric analyses of the immunoreactivities to the antibodies shown in the previous panel (*p <0.05) (n=8 placebo, 10 zileuton). C. Representative immunohistochemical staining for tau, PHF13 and PHF-1 positive areas in brain sections of mice receiving zileuton or placebo. D. Densitometric analyses of the immunoreactivities to the antibodies shown in the previous panel (n=4 per group)(*p <0.01). Values represent mean ± SEM.

Figure 2. Chronic administration of zileuton modulates brain tau metabolism via the cdk5 kinase in Tg2576 mice

A. Representative western blot analyses of GSK3α, GSK3β, p-GSK-3α, p-GSK-3β, JNK2, SAPK/JNK, p-JNK2/3, p-JNK1, cdk-5, p35 and p25 in brain homogenates from mice receiving zileuton or placebo. B. Densitometric analyses of the immunoreactivities to the antibodies shown in the previous panel (*p <0.05). C. Cdk5 kinase activity in brain homogenates from mice receiving zileuton or placebo (n=8 placebo, 10 zileuton) (*p <0.01). Values represent mean ± SEM.

Figure 3. Chronic administration of zileuton prevents synaptic dysfunction in Tg2576 mice

A Representative western blot analyses of synaptophysin and protein post synaptic-95 (PDS-95), GFAP and CD45 in brain homogenates from mice receiving zileuton or placebo. B. Densitometric analyses of the immunoreactivities to the antibodies shown in the previous panel (n=4 per group) (*<0.05). Values represent mean ± SEM.

Figure 4. Pharmacological blockade of 5lipoxygenase modulates tau phosphorylation via the cdk5 kinase in neuronal cells

N2A-APPswe cells were incubated with increasing concentration of zileuton or vehicle (control) for 24 hours and cell lysates collected. A. Representative western blots of tau, phosphorylated tau as recognized by the antibody PHF13, AT8, AT180, AT270, PHF-1, cdk-5, p23 and p25 in the lysates of zileuton or vehicle-treated (control) cells. B. Densitometric analyses of the immunoreactivities to the antibodies shown in the previous panel (n = 3 per each condition, **p < 0.05, *p< 0.01). Values represent mean ± SEM