5-Lipoxygenase gene transfer worsens memory, amyloid, and tau brain pathologies in a mouse model of Alzheimer disease

Abstract

Objective: The 5-lipoxygenase (5LO) enzyme is upregulated in Alzheimer disease (AD), and its genetic absence reduces Aβ levels in APP mice. However, its functional role in modulating tau neuropathology remains to be elucidated.

Methods: To this end, we generated triple transgenic mice (3xTg-AD) overexpressing neuronal 5LO and investigated their phenotype.

Results: Compared with controls, 3xTg-AD mice overexpressing 5LO manifested an exacerbation of memory deficits, plaques, and tangle pathologies. The elevation in Aβ was secondary to an upregulation of γ-secretase pathway, whereas tau hyperphosphorylation resulted from an activation of the Cdk5 kinase. In vitro study confirmed the involvement of this kinase in the 5LO-dependent tau phosphorylation, which was independent of the effect on Aβ.

Interpretation: Our findings highlight the novel functional role that neuronal 5LO plays in exacerbating AD-related tau pathologies. They provide critical preclinical evidence to justify testing selective 5LO inhibitors for AD treatment.

Figure 1. 5LO over-expression modulates behavioral deficits of 3xTg-AD mice

A. Number of total arm entries for 3xTg-AD mice and wild type (WT) mice treated with AAV1/2-5LO or AAV-empty vector control (3xTg, WT). B. Percentage of alternations between 3xTg-Ad and WT mice receiving AAV-5LO or AAV-empty vector (*p<0.01). C. Contexual fear memory response in AAV1/2-5LO and empty vector 3xTg-AD and WT mice. D. Cued fear memory response in AAV1/2-5LO and empty vector injected 3xTg-AD and wild type (WT) mice. Values represent mean ± SEM; *p< 0.01; n= 10 AAV1/2-5L0; n=8 empty vector.

Figure 2. 5LO affects brain Aβ peptides levels and deposition in 3xTg-AD mice

A. RIPA-soluble and formic acid (FA) extractable Aβ1–40 and Aβ1–42 levels in cortex of mice receiving empty vector (3xTg) or AAV1/2-5LO gene (3xTg-5LO) were measured by sandwich ELISA. (n= 8 for 3xTg, and n = 10 for 3xTg-5LO; *p=0.01). B. Representative brain sections from mice receiving AAV1/2-5LO (3xTg-5LO) or empty vector (3xTg) immunostained with 4G8 antibody. C. Quantification of the area occupied by Aβ immunoreactivity in the brains of the same mice (*p=0.04). D. Representative western blots of 5LO, APP, ADAM-10, BACE-1, sAPPα, sAPPβ, CTFs, PS1, Nicastrin, APH-1, Pen-2, apolipoprotein E (ApoE), neprilysin, insulin-degrading enzyme (IDE) in cortex homogentates from mice receiving AAV1/2-5LO (3xTg-5LO) or empty vector (3xTg). E. Densitometric analyses of the immunoreactivities to the antibodies shown in the previous panel (* p=0.01). Values represent mean ± SEM.

Figure 3. 5LO regulates tau phosphorylation and metabolism in the brains of 3xTg-AD mice

A. Representative western blots of 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 mice receiving empty vector (3xTg) or AAV1/2-5LO (3xTg-5LO) assayed by western blot analyses. B. Densitometric analyses of the immunoreactivities to the antibodies shown in the previous panel (*p=0.02). C. 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 mice treated with empty vector (3xTg) or AAV1/2-5LO (3xTg-5LO). D. Densitometric analyses of the immunoreactivities for p-25 shown in the previous panel (*p <0.01). E. Representative immunohistochemical staining for HT7, PHF13 and PHF-1 positive areas in brain sections of mice receiving empty vector control (3xTg) or AAV1/2-5LO (3xTg-5LO).

Figure 4. 5LO modulates endogenous tau levels and metabolism in neuronal cells

A. Representative western blots of total tau (Tau-1), phosphorylated tau at residues S396 (PHF13), S396/S404 (PHF-1), S202/T205 (AT8), T231/S235 (AT180), and at T181 (AT270), in lysates from cells transfected with 5LO (5-LO) or vector control (control) assayed by western blot analyses. B. Densitometric analyses of the immunoreactivities to the antibodies shown in the previous panel (*p=0.01). C, D. Representative pictures of immunoflourescence analysis for cellular tau expression (Tau-1) and phopshorylated tau at S396/S404 (PHF-1) in cells transfected with 5LO or vector control.

Figure 5. 5LO activates cdk5 kinase in neuronal cells

A. Representative western blots of GSK3α, GSK3β, p-GSK-3α, p-GSK-3β, JNK2, SAPK/JNK, p-JNK2/3, p-JNK1, p38MAPK, phospho-p38MAPK in lysates from cells transfected with 5LO (5-LO) or vector control (control) assayed by western blot analyses. B. Representative western blots of cdk5, p35, p25 in lysates from cells transfected with 5LO (5-LO) or vector control (control) assayed by western blot analyses. C. Densitometric analyses of the immunoreactivities to the antibodies shown in the previous panel (*p=0.01). D. Representative pictures of immunoflourescence analysis for cdk5 and p35/25 in neuronal cells transfected with 5LO or vector control. E. Cdk5 kinase activity in lysates from neuronal cells transfected with 5LO or vector control (*p=0.03). Values represent mean ± SEM

Figure 6. 5LO modulates tau levels and metabolism via the cdk5 kinase

A. Representative western blots of total tau (Tau-1), phosphorylated tau at residues S396 (PHF13), and S396/S404 (PHF-1), cdk5, p35, p25 and 5-LO in lysates from cells transfected with 5LO (5-LO) or vector control (control) in the presence or absence of zileuton (25μM) and assayed by western blot analyses. B. Densitometric analyses of the immunoreactivities to the antibodies shown in the previous panel (*p<0.01 versus control; #p<0.01 versus 5-LO). C. Representative western blots of total tau (Tau-1), phosphorylated tau at residues S396 (PHF13), and S396/S404 (PHF-1), cdk5, p35 and p25 in lysates from cells transfected with 5LO (5-LO) or vector control (control) in the presence or increasing concentration of roscovitine and assayed by western blot analyses. D. Densitometric analyses of the immunoreactivities to the antibodies shown in the previous panel (#p<0.01 versus control; *p<0.01 versus 5-LO). Values represent mean ± SEM.

Figure 7. 5LO-mediated modulation of tau metabolism is independent from an Aβ effect

A. Neuronal cells were transfected with 5LO then incubated with vehicle or the gamma-secretase inhibitor L685,485 (0.1 and 0.5 μM) and supernatant collected for Aβ 1–40 levels (*p<0.01 versus control; #p<0.01 versus 5-LO). B. Representative western blots of total tau (Tau-1), phosphorylated tau at residues S396 (PHF13), and S396/S404 (PHF-1) in lysates from cells transfected with 5LO (5-LO) or vector control in the presence of vehicle or L685,485 assayed by western blot analyses. C. Densitometric analyses of the immunoreactivities to the antibodies shown in the previous panel (*p<0.01). Values represent mean ± SEM