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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.

No MeSH data available.


Diet-induced high Hcy upregulates 5LO enzymatic pathway via epigenetic modification. (A) Representative western blot analysis of 5LO protein in brain cortex homogenates from 3xTg mice receiving vehicle (Ctrl), folate and B vitamins deficient diet (Diet), Diet + zileuton, or zileuton. (B) Densitometric analysis of the immunoreactivity to the antibody shown in panel A. (C) Levels of LTB4 measured by a specific and sensitive ELISA assay in brain cortex homogenates from the same four groups of 3xTg mice. (D) Quantitative real time Reverse Transcription Polymerase Chain Reaction (q RT-PCR) analysis of 5LO mRNA in brain cortices of 3xTg mice receiving vehicle (Ctrl), folate and B vitamins deficient diet (Diet), Diet + zileuton, or zileuton. (E) 5LO DNA methylation levels in brain cortices of the 3xTg mice randomized to the same four treatment groups. (F) Representative western blot analyses for DNMT1, DNMT3α, DNMT3β in brain cortices of the same four groups of mice. (G) Densitometric analyses of the immuno-reactivity shown in the previous panel. Data presented are mean ± s.e.m. (*p < 0.05, n = 6). (n = 5 control, n = 6 Diet, n = 6 Diet + zileuton, n = 5 zileuton).
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f2: Diet-induced high Hcy upregulates 5LO enzymatic pathway via epigenetic modification. (A) Representative western blot analysis of 5LO protein in brain cortex homogenates from 3xTg mice receiving vehicle (Ctrl), folate and B vitamins deficient diet (Diet), Diet + zileuton, or zileuton. (B) Densitometric analysis of the immunoreactivity to the antibody shown in panel A. (C) Levels of LTB4 measured by a specific and sensitive ELISA assay in brain cortex homogenates from the same four groups of 3xTg mice. (D) Quantitative real time Reverse Transcription Polymerase Chain Reaction (q RT-PCR) analysis of 5LO mRNA in brain cortices of 3xTg mice receiving vehicle (Ctrl), folate and B vitamins deficient diet (Diet), Diet + zileuton, or zileuton. (E) 5LO DNA methylation levels in brain cortices of the 3xTg mice randomized to the same four treatment groups. (F) Representative western blot analyses for DNMT1, DNMT3α, DNMT3β in brain cortices of the same four groups of mice. (G) Densitometric analyses of the immuno-reactivity shown in the previous panel. Data presented are mean ± s.e.m. (*p < 0.05, n = 6). (n = 5 control, n = 6 Diet, n = 6 Diet + zileuton, n = 5 zileuton).

Mentions: Compared with controls, brain homogenates from mice receiving the Diet had a significant increase in the steady state levels of 5LO protein, mRNA and activity as shown by the significant elevation of its main metabolic product, the leukotriene (LT)B4 (Fig. 2A–D). However, while the 5LO protein and mRNA levels were unaffected by zileuton, as expected the drug at the concentration used, which was based on our previous work16, prevented the Hcy-dependent LTB4 increase (Fig. 2A–D). Consistent with the elevation in Hcy, the same samples had higher levels of S-adenosyl-homocysteine (SAH), a potent inhibitor of methyl-transferase reactions, and lower S-adenosyl-methionine (SAM) (Table 1). These changes were associated with a significant reduction in all 3 major DNA methyl-transferase enzymes, DNMT1, DNMT3α, DNMT3β, and a significant reduction in 5LO DNA methylation (Fig. 2E–G).


Five lipoxygenase hypomethylation mediates the homocysteine effect on Alzheimer ’ s phenotype
Diet-induced high Hcy upregulates 5LO enzymatic pathway via epigenetic modification. (A) Representative western blot analysis of 5LO protein in brain cortex homogenates from 3xTg mice receiving vehicle (Ctrl), folate and B vitamins deficient diet (Diet), Diet + zileuton, or zileuton. (B) Densitometric analysis of the immunoreactivity to the antibody shown in panel A. (C) Levels of LTB4 measured by a specific and sensitive ELISA assay in brain cortex homogenates from the same four groups of 3xTg mice. (D) Quantitative real time Reverse Transcription Polymerase Chain Reaction (q RT-PCR) analysis of 5LO mRNA in brain cortices of 3xTg mice receiving vehicle (Ctrl), folate and B vitamins deficient diet (Diet), Diet + zileuton, or zileuton. (E) 5LO DNA methylation levels in brain cortices of the 3xTg mice randomized to the same four treatment groups. (F) Representative western blot analyses for DNMT1, DNMT3α, DNMT3β in brain cortices of the same four groups of mice. (G) Densitometric analyses of the immuno-reactivity shown in the previous panel. Data presented are mean ± s.e.m. (*p < 0.05, n = 6). (n = 5 control, n = 6 Diet, n = 6 Diet + zileuton, n = 5 zileuton).
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f2: Diet-induced high Hcy upregulates 5LO enzymatic pathway via epigenetic modification. (A) Representative western blot analysis of 5LO protein in brain cortex homogenates from 3xTg mice receiving vehicle (Ctrl), folate and B vitamins deficient diet (Diet), Diet + zileuton, or zileuton. (B) Densitometric analysis of the immunoreactivity to the antibody shown in panel A. (C) Levels of LTB4 measured by a specific and sensitive ELISA assay in brain cortex homogenates from the same four groups of 3xTg mice. (D) Quantitative real time Reverse Transcription Polymerase Chain Reaction (q RT-PCR) analysis of 5LO mRNA in brain cortices of 3xTg mice receiving vehicle (Ctrl), folate and B vitamins deficient diet (Diet), Diet + zileuton, or zileuton. (E) 5LO DNA methylation levels in brain cortices of the 3xTg mice randomized to the same four treatment groups. (F) Representative western blot analyses for DNMT1, DNMT3α, DNMT3β in brain cortices of the same four groups of mice. (G) Densitometric analyses of the immuno-reactivity shown in the previous panel. Data presented are mean ± s.e.m. (*p < 0.05, n = 6). (n = 5 control, n = 6 Diet, n = 6 Diet + zileuton, n = 5 zileuton).
Mentions: Compared with controls, brain homogenates from mice receiving the Diet had a significant increase in the steady state levels of 5LO protein, mRNA and activity as shown by the significant elevation of its main metabolic product, the leukotriene (LT)B4 (Fig. 2A–D). However, while the 5LO protein and mRNA levels were unaffected by zileuton, as expected the drug at the concentration used, which was based on our previous work16, prevented the Hcy-dependent LTB4 increase (Fig. 2A–D). Consistent with the elevation in Hcy, the same samples had higher levels of S-adenosyl-homocysteine (SAH), a potent inhibitor of methyl-transferase reactions, and lower S-adenosyl-methionine (SAM) (Table 1). These changes were associated with a significant reduction in all 3 major DNA methyl-transferase enzymes, DNMT1, DNMT3α, DNMT3β, and a significant reduction in 5LO DNA methylation (Fig. 2E–G).

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.