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Five lipoxygenase hypomethylation mediates the homocysteine effect on Alzheimer ’ s phenotype

View Article: PubMed Central - PubMed

ABSTRACT

Environmental and genetic risk factors are implicated in the pathogenesis of Alzheimer’s disease (AD). However, how they interact and influence its pathogenesis remains to be investigated. High level of homocysteine (Hcy) is an AD risk factor and associates with an up-regulation of the ALOX5 gene. In the current paper we investigated whether this activation is responsible for the Hcy effect on the AD phenotype and the mechanisms involved. Triple transgenic mice were randomized to receive regular chow diet, a diet deficient in folate and B vitamins (Diet), which results in high Hcy, or the Diet plus zileuton, a specific ALOX5 inhibitor, for 7 months. Compared with controls, Diet-fed mice had a significant increase in Hcy levels, memory and learning deficits, up-regulation of the ALOX5 pathway, increased Aβ levels, tau phosphorylation, and synaptic pathology, which were absent in mice treated with zileuton. In vivo and vitro studies demonstrated that the mechanism responsible was the hypomethylation of the ALOX5 promoter. Our findings demonstrate that the up-regulation of the ALOX5 is responsible for the Hcy-dependent worsening of the AD phenotype in a relevant mouse model of the disease. The discovery of this previously unknown cross-talk between these two pathways could afford novel therapeutic opportunities for treating or halting AD.

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Hcy up-regulates 5LO enzymatic pathway by reducing its DNA methylation. (A) Levels of Aβ 1-40 in conditioned media from N2A APPswe cells incubated with vehicle (Ctrl), 50 μM Hcy, Hcy plus zileuton(100 μM), or zileuton (100 μM) alone for 24 hrs. (B) Representative western blot analyses of 5LO in lysates from the same N2A APPswe cells described in the previous panel. (C) Densitometric analysis of the immune-reactivity shown in panel B. (D) Levels of LTB4 measured by a specific ELISA assay in conditioned media from the same N2A APP cells described in panel A. (E) Quantitative real time Reverse Transcription Polymerase Chain Reaction (q RT-PCR) analysis of 5LO mRNA in N2A APPswe neuronal cells treated with vehicle (Ctrl), Hcy, Hcy + zileuton, or zileuton. (F) 5LO DNA methylation levels in the same cells. Data presented are mean ± s.e.m. (*p < 0.05, n = 6 per condition).
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f6: Hcy up-regulates 5LO enzymatic pathway by reducing its DNA methylation. (A) Levels of Aβ 1-40 in conditioned media from N2A APPswe cells incubated with vehicle (Ctrl), 50 μM Hcy, Hcy plus zileuton(100 μM), or zileuton (100 μM) alone for 24 hrs. (B) Representative western blot analyses of 5LO in lysates from the same N2A APPswe cells described in the previous panel. (C) Densitometric analysis of the immune-reactivity shown in panel B. (D) Levels of LTB4 measured by a specific ELISA assay in conditioned media from the same N2A APP cells described in panel A. (E) Quantitative real time Reverse Transcription Polymerase Chain Reaction (q RT-PCR) analysis of 5LO mRNA in N2A APPswe neuronal cells treated with vehicle (Ctrl), Hcy, Hcy + zileuton, or zileuton. (F) 5LO DNA methylation levels in the same cells. Data presented are mean ± s.e.m. (*p < 0.05, n = 6 per condition).

Mentions: To further corroborate the direct involvement of 5LO activation in the Hcy-dependent effect on Aβ metabolism via the effect that Hcy may have on the 5LO promoter methylation status, we treated the N2A APPswe neuronal cells with 50 μM DL-homocysteine for 24 hr in the presence or absence of zileuton (100 μM), a concentration known to significantly block 5LO activation16. At the end of the incubation time, supernatants collected and assayed for Aβ 1-40 and LTB4, while cell pellets used to investigate 5LO at the protein and message levels. Compared with vehicle controls, conditioned media from cells incubated with Hcy had a significant elevation of Aβ1-40 levels, which was absent in the presence of zileuton (Fig. 6A). Hcy treatment resulted also in a significant increase in the steady state levels of 5LO proteins, which was not influenced by zileuton (Fig. 6B,C). However, while levels of LTB4 were significantly higher in the Hcy-treated cells than in controls, the presence of zileuton completely prevented this increase (Fig. 6D). On the other hand, quantitative RT-PCR analysis showed that beside the protein also the 5LO mRNA levels were significantly elevated in cells treated with Hcy, whereas 5LO DNA methylation status was significantly decreased and that both of these effects were not influenced by the presence of zileuton (Fig. 6E,F).


Five lipoxygenase hypomethylation mediates the homocysteine effect on Alzheimer ’ s phenotype
Hcy up-regulates 5LO enzymatic pathway by reducing its DNA methylation. (A) Levels of Aβ 1-40 in conditioned media from N2A APPswe cells incubated with vehicle (Ctrl), 50 μM Hcy, Hcy plus zileuton(100 μM), or zileuton (100 μM) alone for 24 hrs. (B) Representative western blot analyses of 5LO in lysates from the same N2A APPswe cells described in the previous panel. (C) Densitometric analysis of the immune-reactivity shown in panel B. (D) Levels of LTB4 measured by a specific ELISA assay in conditioned media from the same N2A APP cells described in panel A. (E) Quantitative real time Reverse Transcription Polymerase Chain Reaction (q RT-PCR) analysis of 5LO mRNA in N2A APPswe neuronal cells treated with vehicle (Ctrl), Hcy, Hcy + zileuton, or zileuton. (F) 5LO DNA methylation levels in the same cells. Data presented are mean ± s.e.m. (*p < 0.05, n = 6 per condition).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f6: Hcy up-regulates 5LO enzymatic pathway by reducing its DNA methylation. (A) Levels of Aβ 1-40 in conditioned media from N2A APPswe cells incubated with vehicle (Ctrl), 50 μM Hcy, Hcy plus zileuton(100 μM), or zileuton (100 μM) alone for 24 hrs. (B) Representative western blot analyses of 5LO in lysates from the same N2A APPswe cells described in the previous panel. (C) Densitometric analysis of the immune-reactivity shown in panel B. (D) Levels of LTB4 measured by a specific ELISA assay in conditioned media from the same N2A APP cells described in panel A. (E) Quantitative real time Reverse Transcription Polymerase Chain Reaction (q RT-PCR) analysis of 5LO mRNA in N2A APPswe neuronal cells treated with vehicle (Ctrl), Hcy, Hcy + zileuton, or zileuton. (F) 5LO DNA methylation levels in the same cells. Data presented are mean ± s.e.m. (*p < 0.05, n = 6 per condition).
Mentions: To further corroborate the direct involvement of 5LO activation in the Hcy-dependent effect on Aβ metabolism via the effect that Hcy may have on the 5LO promoter methylation status, we treated the N2A APPswe neuronal cells with 50 μM DL-homocysteine for 24 hr in the presence or absence of zileuton (100 μM), a concentration known to significantly block 5LO activation16. At the end of the incubation time, supernatants collected and assayed for Aβ 1-40 and LTB4, while cell pellets used to investigate 5LO at the protein and message levels. Compared with vehicle controls, conditioned media from cells incubated with Hcy had a significant elevation of Aβ1-40 levels, which was absent in the presence of zileuton (Fig. 6A). Hcy treatment resulted also in a significant increase in the steady state levels of 5LO proteins, which was not influenced by zileuton (Fig. 6B,C). However, while levels of LTB4 were significantly higher in the Hcy-treated cells than in controls, the presence of zileuton completely prevented this increase (Fig. 6D). On the other hand, quantitative RT-PCR analysis showed that beside the protein also the 5LO mRNA levels were significantly elevated in cells treated with Hcy, whereas 5LO DNA methylation status was significantly decreased and that both of these effects were not influenced by the presence of zileuton (Fig. 6E,F).

View Article: PubMed Central - PubMed

ABSTRACT

Environmental and genetic risk factors are implicated in the pathogenesis of Alzheimer&rsquo;s disease (AD). However, how they interact and influence its pathogenesis remains to be investigated. High level of homocysteine (Hcy) is an AD risk factor and associates with an up-regulation of the ALOX5 gene. In the current paper we investigated whether this activation is responsible for the Hcy effect on the AD phenotype and the mechanisms involved. Triple transgenic mice were randomized to receive regular chow diet, a diet deficient in folate and B vitamins (Diet), which results in high Hcy, or the Diet plus zileuton, a specific ALOX5 inhibitor, for 7 months. Compared with controls, Diet-fed mice had a significant increase in Hcy levels, memory and learning deficits, up-regulation of the ALOX5 pathway, increased A&beta; levels, tau phosphorylation, and synaptic pathology, which were absent in mice treated with zileuton. In vivo and vitro studies demonstrated that the mechanism responsible was the hypomethylation of the ALOX5 promoter. Our findings demonstrate that the up-regulation of the ALOX5 is responsible for the Hcy-dependent worsening of the AD phenotype in a relevant mouse model of the disease. The discovery of this previously unknown cross-talk between these two pathways could afford novel therapeutic opportunities for treating or halting AD.

No MeSH data available.


Related in: MedlinePlus