Adeno-associated virus-mediated brain delivery of 5-lipoxygenase modulates the AD-like phenotype of APP mice

Abstract

Background: The 5-lipoxygenase (5LO) enzymatic pathway is widely distributed within the central nervous system. Previous works showed that this protein is up-regulated in Alzheimer's disease (AD), and that its genetic absence results in a reduction of Amyloid beta (Aβ) levels in the Tg2576 mice.Here by employing an adeno-associated viral (AAV) vector system to over-express 5LO in the same mouse model, we examined its contribution to their cognitive impairments and brain AD-like amyloid pathology.

Results: Our results showed that compared with controls, 5LO-targeted gene brain over-expression in Tg2576 mice results in significant memory deficits. On the other hand, brain tissues had a significant elevation in the levels of Aβ peptides and deposition, no change in the steady state levels of amyloid-β precursor protein (APP), BACE-1 or ADAM-10, but a significant increase in PS1, nicastrin, and Pen-2, three major components of the γ-secretase complex. Additional data indicate that the transcription factor CREB was elevated and so were the mRNA levels for PS1, nicastrin and Pen-2.

Conclusions: These data demonstrate that neuronal 5LO plays a functional role in the pathogenesis of AD-like amyloidotic phenotype by modulating the γ-secretase pathway. They support the hypothesis that this enzyme is a novel therapeutic target for the treatment and prevention of AD.

Figure 1

AAV1/2-5LO over-expression modulates behavioral deficits of Tg2576 mice. A. Number of total arm entries for AAV1/2-5LO and AAV-empty vector control treated Tg2576 and wild type (WT) mice. B. Contexual fear memory response in AAV1/2-5LO and empty vector treated Tg2576 and wild type (WT) mice. C. Percentage of alternations between AAV-5LO and AAV-empty vector (*p < 0.01). D. Cued fear memory response in AAV1/2-5LO and empty vector injected Tg2576 and wild type (WT) mice. Values represent mean ± SEM; *p < 0.01; n = 10 AAV1/2-5L0; n = 8 empty vector.

Figure 2

AAV1/2-5LO over-expression modulates brain Aβ peptides levels and deposition in Tg2576 mice. A. RIPA-soluble (left panels) and formic acid extractable (right panels) Aβ1-40 and Aβ1-42 levels in cortex of Tg2576 receiving empty vector (CTL) or AAV1/2-5LO gene were measured by sandwich ELISA. (n = 8 for CTL, and n = 10 for AAV-5LO; *p < 0.05). B. Representative sections of brains from Tg2576 mice receiving AAV1/2-5LO or empty vector (CTL) immunostained with 4G8 antibody. C. Quantification of the area occupied by Aβ immunoreactivity in brain of Tg2576 mice receiving AAV1/2-5LO or empty vector (CTL) (*p < 0.05). Values represent mean ± SEM (n-8 CTL; n = 10 AAV-5LO).

Figure 3

AAV1/2-5LO over-expression alters brain APP metabolism via the γ-secretase pathway in Tg2576 mice. A. Representative western blots of 5LO, APP, ADAM-10, BACE-1, sAPPα, sAPPβ, CTFα, CTF-β, PS1, Nicastrin, APH-1, Pen-2, apoE, neprilysin, IDE in brain homogenates from Tg2576 mice receiving AAV1/2-5LO or empty vector (CTL). B. Densitometric analyses of the immunoreactivities to the antibodies shown in the previous panel. Values represent mean ± SEM (* p < 0.01). C. Representative sections of different brain regions from Tg2576 mice receiving AAV1/2-5LO or empty vector (CTL) immunostained for 5-LO (× 20 magnification). D. Correlation between levels of 5LO protein and RIPA soluble Aβ 1-40 peptides (r² = 0.61, p = 0.0003). E. Representative brain sections from Tg2576 receiving AAV1/2-5LO or empty vector (CTL) immunostained for GFAP and CD45 (× 20 magnification). F. Quantitative analysis of the immunoreactivity for GFAP and CD45 in the same animals.(*p < 0.004).

Figure 4

AAV1/2-5LO over-expression modulates CREB levels and transcription of the γ-secretase complex in the brains of Tg2576 mice. A. Levels of total CREB and its phosphorylated form at Ser133 and Sp1 in the cortex of Tg2576 receiving AAV1/2-5LO or empty vector (CTL) assayed by western blot analyses. B. Densitometric analyses of the immunoreactivities to the antibodies shown in the previous panel. C. Relative mRNA levels for PS1, Nicastrin, APH-1 and Pen-2 in the cortex of Tg2576 mice receiving AAV1/2-5LO or empty vector (CTL), as determined by real-time quantitative RT-PCR amplification. Values represent mean ± SEM (*p < 0.01).