5-lipoxygenase as an endogenous modulator of amyloid β formation in vivo

Abstract

Objective: The 5-lipoxygenase (5-LO) enzymatic pathway is widely distributed within the central nervous system, and is upregulated in Alzheimer's disease. However, the mechanism whereby it may influence the disease pathogenesis remains elusive.

Methods: We evaluated the molecular mechanism by which 5-LO regulates amyloid β (Aβ) formation in vitro and in vivo by pharmacological and genetic approaches.

Results: Here we show that 5-LO regulates the formation of Aβ by activating the cAMP-response element binding protein (CREB), which in turn increases transcription of the γ-secretase complex. Preventing CREB activation by pharmacologic inhibition or dominant negative mutants blocks the 5-LO-dependent elevation of Aβ formation and the increase of γ-secretase mRNA and protein levels. Moreover, 5-LO targeted gene disruption or its in vivo selective pharmacological inhibition results in a significant reduction of Aβ, CREB and γ-secretase levels.

Interpretation: These data establish a novel functional role for 5-LO in regulating endogenous formation of Aβ levels in the central nervous system. Thus, 5-LO pharmacological inhibition may be beneficial in the treatment and prevention of Alzheimer's disease.

Figure 1

5-LO modulates Aβ formation in a γ-secretase-dependent manner. N2A-APPswe cells were transiently transfected with human 5-LO cDNA and supernatant and cells lysates were collected after 48hr. A. Aβ 1-40 levels in the supernatant. B. Representative immunoblots for 5-LO, APP, BACE-1, ADAM-10, PS1, nicastrin, APH-1 and Pen-2 from cell lysates. C. Densitometric analysis of the immunoblots data after normalization with actin. D. Cells were incubated with increasing concentration of AA-861 overnight and supernatants collected and assayed for Aβ 1-40. E. Representative immunoblots for PS1, nicastrin, APH-1 and Pen-2 in cell lysates harvested from the same experiments. F. Densitometric analysis of the western blots after normalization with actin levels. Values are mean ± s.e.m. of 3 independent experiments repeated in triplicate (*p<0.05; **p<0.01).

Figure 1

5-LO modulates Aβ formation in a γ-secretase-dependent manner. N2A-APPswe cells were transiently transfected with human 5-LO cDNA and supernatant and cells lysates were collected after 48hr. A. Aβ 1-40 levels in the supernatant. B. Representative immunoblots for 5-LO, APP, BACE-1, ADAM-10, PS1, nicastrin, APH-1 and Pen-2 from cell lysates. C. Densitometric analysis of the immunoblots data after normalization with actin. D. Cells were incubated with increasing concentration of AA-861 overnight and supernatants collected and assayed for Aβ 1-40. E. Representative immunoblots for PS1, nicastrin, APH-1 and Pen-2 in cell lysates harvested from the same experiments. F. Densitometric analysis of the western blots after normalization with actin levels. Values are mean ± s.e.m. of 3 independent experiments repeated in triplicate (*p<0.05; **p<0.01).

Figure 2

5-HETE increases Aβ formation, PS1, nicastrin, APH1 and Pen-2 levels, but has no effect on Notch processing. N2A-APPswe cells were incubated with 5-HETE (1 and 10 μM) overnight and supernatants and cell lysates collected. A. Aβ 1-40 levels in supernatant. B. Representative immunoblots for PS1, nicastrin, APH-1 and Pen-2. C. Densitometric analysis of the immunoblots after normalization with actin levels. D. Representative immunoblot and densitometric analysis for NICD. Values are mean ± s.e.m. of 3 independent experiments repeated in triplicate (*p<0.05; **p<0.01).

Figure 3

5-HETE by activating CREB induces PS1, nicastrin, APH-1 and Pen-2 mRNA. N2A-APPswe cells were incubated with 5-HETE overnight and cell lysates collected. A. Representative immunoblots and densitometric analysis for CREB and phosphorylated CREB at Ser 133. B. Representative pictures of immunoflourescence analysis for cellular compartimentalization of p-CREB in cells incubated with vehicle or 5-HETE (a-c) negative control: no primary antibody; (d-f) vehicle; (g-i) 5-HETE. C. Time-dependent activation of CREB and Sp1 after challenge with 5-HETE. D. Time-dependent changes for PS1, nicastrin, APH-1, Pen-2 and BACE-1 mRNAs levels after challenge with 5-HETE. E-G. Cells were pre-incubated with H89 and Rp, two specific PKA inhibitors, then challenged with 5-HETE overnight, supernatant collected for Aβ levels (E), cell lysates for immunoblot analysis of p-CREB (F), PS1, nicastrin, APH-1 and Pen-2 protein levels (G). Values are mean ± s.e.m. of 3 independent experiments repeated in triplicate (*p<0.05; **p<0.01).

Figure 3

5-HETE by activating CREB induces PS1, nicastrin, APH-1 and Pen-2 mRNA. N2A-APPswe cells were incubated with 5-HETE overnight and cell lysates collected. A. Representative immunoblots and densitometric analysis for CREB and phosphorylated CREB at Ser 133. B. Representative pictures of immunoflourescence analysis for cellular compartimentalization of p-CREB in cells incubated with vehicle or 5-HETE (a-c) negative control: no primary antibody; (d-f) vehicle; (g-i) 5-HETE. C. Time-dependent activation of CREB and Sp1 after challenge with 5-HETE. D. Time-dependent changes for PS1, nicastrin, APH-1, Pen-2 and BACE-1 mRNAs levels after challenge with 5-HETE. E-G. Cells were pre-incubated with H89 and Rp, two specific PKA inhibitors, then challenged with 5-HETE overnight, supernatant collected for Aβ levels (E), cell lysates for immunoblot analysis of p-CREB (F), PS1, nicastrin, APH-1 and Pen-2 protein levels (G). Values are mean ± s.e.m. of 3 independent experiments repeated in triplicate (*p<0.05; **p<0.01).

Figure 4

Blockade of CREB activation suppresses 5-LO-dependent Aβ elevation and γ-secretase complex up-regulation. A. Transfection of cells with two mutant CREB negative dominant vectors prevents the 5-HETE-dependent CREB activation, (B) and Aβ formation. C. Densitometric analyses for total CREB and p-CREB after transfection with CREB negative dominant vectors and 5-HETE challenge. D. Representative immunoblot and densitometric analyses for PS1, nicastrin, APH-1 and Pen-2 after transfection with CREB negative dominant vectors and 5-HETE challenge. Values are mean ± s.e.m. of 3 independent experiments repeated in triplicate (*p<0.05; **p<0.01).

Figure 5

In vivo 5-LO modulation of CREB. A. Representative pictures of brain sections from Tg2576 mice and wild type littermates (WT) challenged with a specific anti-5-LO antibody. Panels a, b: entorhinal cortex; panels c,d: hippocampus CA1 region. B. Representative immunoblots and densitometric analyses for p-CREB and total CREB in brains from Tg2576 mice after AA-861 or vehicle (CTL) administration. C. Representative immunoblots and densitometric analyses for p-CREB and total CREB in Tg2576 and Tg2576/5-LO/- mice brains (n=3 per group). Values are mean ± s.e.m. (*p<0.05; **p<0.01).

Figure 6

Genetic absence of 5-LO reduces endogenous Aβ, CREB and γ-secretase complex levels in the central nervous system. A. Groups of 6-month-old 5-LO-/- mice (open symbols) and wild type (WT) littermates (closed symbols) were analyzed for total Aβ 1-40 and 1-42 levels in brain cortex homogenates. B. Representative immunoblot analysis for APP, BACE-1 and ADAM-10 in brain homogenates from these mice. C. Representative immunoblots and densitometric analysis for PS1, nicastrin, APH-1 and Pen 2 protein levels (open bars: WT, closed bars: 5-LO-/- mice). D. Representative immunoblots and densitometric analysis for CREB and p-CREB (open bars: WT, closed bars: 5-LO-/- mice). (*p<0.05; **p<0.01).