Epigenetic regulation of fatty acid amide hydrolase in Alzheimer disease

Abstract

Objective: Alzheimer disease (AD) is a progressive, degenerative and irreversible neurological disorder with few therapies available. In search for new potential targets, increasing evidence suggests a role for the endocannabinoid system (ECS) in the regulation of neurodegenerative processes.

Methods: We have studied the gene expression status and the epigenetic regulation of ECS components in peripheral blood mononuclear cells (PBMCs) of subjects with late-onset AD (LOAD) and age-matched controls (CT).

Results: We found an increase in fatty acid amide hydrolase (faah) gene expression in LOAD subjects (2.30 ± 0.48) when compared to CT (1.00 ± 0.14; *p<0.05) and no changes in the mRNA levels of any other gene of ECS elements. Consistently, we also observed in LOAD subjects an increase in FAAH protein levels (CT: 0.75 ± 0.04; LOAD: 1.11 ± 0.15; *p<0.05) and activity (pmol/min per mg protein CT: 103.80 ± 8.73; LOAD: 125.10 ± 4.00; *p<0.05), as well as a reduction in DNA methylation at faah gene promoter (CT: 55.90 ± 4.60%; LOAD: 41.20 ± 4.90%; *p<0.05).

Conclusions: Present findings suggest the involvement of FAAH in the pathogenesis of AD, highlighting the importance of epigenetic mechanisms in enzyme regulation; they also point to FAAH as a new potential biomarker for AD in easily accessible peripheral cells.

Figure 1. ECS gene expression levels.

ECS genes (panel a: receptors; panel b: metabolic enzymes) expression in PBMCs from LOAD subjects. Bars represent 2^−DDCt values calculated by Delta–Delta Ct (DDCt) method. Expression was normalized to GAPDH, and data are represented as means ± SEM. *p<0.05 vs control.

Figure 2. Genetic and epigenetic regulation of faah gene.

a: Levels of FAAH mRNA in PBMCs from LOAD patients (n = 13) and controls (n = 12); b: Amount of methylated DNA at faah gene in controls (n = 33 ) and LOAD subjects (n = 33); c: Correlation between faah gene expression and % change of DNA methylation in LOAD subjects. Data were compared by Spearman's rank correlation coefficient (p<0.05, r = −0.5326). Scatter dots represent 2^−DDCt values calculated by Delta-Delta Ct (DDCt) method, as described in the Materials and Methods section.

Figure 3. faah DNA methylation levels vs MMSE score.

a: Amount of methylated DNA at faah gene in LOAD subjects subgrouped on the basis of MMSE score; b: Correlation between changes in DNA methylation at faah gene and LOAD subjects with severe AD, based on MMSE score. Data were compared by Pearson's rank correlation coefficient (p<0.05, r = −0.6240).

Figure 4. Levels of FAAH protein and activity.

a: Analysis of FAAH protein levels in PBMCs from LOAD and control (CT) subjects. Values represent means ± SEM, *p<0.05 vs CT. Representative immunoblots of PBMC lysates reacted with specific anti-FAAH or anti-actin antibodies are shown, as well as FAAH immunoreactivity in rat liver extracts and HeLa cell lysates, used as positive and negative controls respectively. Molecular mass markers and the positions of FAAH and actin are indicated to the right. b: FAAH activity in LOAD and CT subjects, expressed as pmol/min per mg of protein (means ± SEM).