One-carbon metabolism modulates miR-29a-DNA methylation crosstalk in Alzheimer's disease

Abstract

Introduction: Alzheimer's disease (AD)'s multifactorial nature stresses the role of epigenetics in affecting different pathological pathways. We demonstrated that one-carbon metabolism epigenetically impacts AD-like phenotype. Here, we investigated the crosstalk between methylation and microRNAs in AD.

Methods: We altered one-carbon metabolism to induce hypo- and hyper-methylation, in SK-N-BE neuroblastoma cells and TgCRND8 mice. miRNAs were profiled through a polymerase chain reaction array, then we focused on miR-29a expression and methylation of its genomic locus. Finally, we assessed miR-29a expression and methylation in the brain of AD subjects.

Results: MiR-29a was repressed in hypomethylating and expressed in hypermethylating conditions. The expression of miR-29a and of its target, BACE1, was inversely correlated.

Discussion: We demonstrated for the first time that miR-29a is modulated by one-carbon metabolism through DNA methylation, disclosing the molecular mechanisms regulating BACE1 expression in AD. These data confirm miR-29a's protective role in AD and support miR-29a as a potential biomarker for AD.

Keywords: Alzheimer's disease; DNA methylation; epigenetics; microRNAs; mir‐29a; non‐CPG methylation; one‐carbon metabolism.

FIGURE 1

miR‐29a expression in AD models. Effects of hypermethylating (SAM‐supplementation) and hypomethylating (B‐deficient) treatments in SK‐N‐BE cell line (A) and TgCNRD8 mice (B). miR‐29a expression in post mortem human brain samples (C). Bar plots show the relative amounts of the miR‐29a cDNA normalized to the mean RNU6‐SNORD68 internal reference obtained using quantitative reverse transcription polymerase chain reaction on the y axis. Data represent the mean ± standard error of the mean. *p < 0.05; **p < 0.01. Cell cultures, N = 3; mice, N = 12; human samples, N = 15. AD, Alzheimer's disease; SAM, S‐adenosylmethionine.

FIGURE 2

miR‐29a transfection in SK‐N‐BE cells. Expression of miR‐29a in SK‐N‐BE cells transfected for 48 hours with 20 µM mimic‐ or inhibitor‐miR29a (A). Effects of miR‐29a transfections on BACE1 mRNA expression (B). Bar plots show the relative amounts of the target genes normalized to the mean β‐ACTIN‐GAPDH internal reference obtained using quantitative reverse transcription polymerase chain reaction on the y axis. Data represent the mean ± standard error of the mean. ***p < 0.001; N = 3.

FIGURE 3

miR‐29a methylation in SK‐N‐BE cells. miR‐29a DNA CpG and non‐CpG methylation patterns in SK‐N‐BE cells. Histograms in (A) Ctrl, (B) Ctrl+SAM, (C) B vitamin deficiency, and (D) B‐def + SAM show methylation % (y axis) of each cytosine; labels on x axis indicate the cytosine position on the reference sequence of the DNA locus hosting the miR‐29a sequence. CpG cytosines are indicated by a dot over the related columns. Data represent the mean ± SEM *p < 0.05; ** p < 0.01; N = 3. Histogram in (E) shows the average methylation % over the total cytosines (y axis) as derived from the respective data and grouped in all the cytosines (TOT), CpG moieties (CpG), and non‐CpG moieties (non‐CpG). Data represent the mean ± SEM versus the related controls (light gray columns). *p < 0.05; ** p < 0.01; N = 3. SAM, S‐adenosylmethionine; SEM, standard error of the mean.

FIGURE 4

miR‐29a methylation in TgCRND8 mice. miR‐29a DNA CpG and non‐CpG methylation patterns in brain (prefrontal cortex) from TgCRND8 mice. Histograms in (A) Ctrl, (B) Ctrl+SAM, (C) B vitamin deficiency, and (D) B‐def + SAM show methylation % (y axis) of each cytosine; labels on x axis indicate the cytosine position on the reference sequence of the DNA locus hosting the miR‐29a sequence. CpG cytosines are indicated by a dot over the related columns. Data represent the mean ± SEM. *p < 0.05; ** p < 0.01; N = 3. Histogram in (E) shows the average methylation % over the total cytosines (y axis) as derived from the respective data and grouped in all the cytosines (TOT), CpG moieties (CpG), and non‐CpG moieties (non‐CpG). Data represent the mean ± SEM vs. the related controls (light gray columns) *p < 0.05; ** p < 0.01; N = 3. SAM, S‐adenosylmethionine; SEM, standard error of the mean.

FIGURE 5

miR‐29a methylation in post mortem human brain. miR‐29a DNA CpG and non‐CpG methylation patterns in post mortem human brain (prefrontal cortex) from healthy and AD subjects. Histograms in (A) Healthy controls, (B) AD Braak stage I‐II, and (C) AD Braak stage V‐VI show methylation % (y axis) of each cytosine; labels on x axis indicate the cytosine position on the reference sequence of the DNA locus hosting the miR‐29a sequence. CpG cytosines are indicated by a dot over the related columns. Data represent the mean ± SEM. *p < 0.05; ** p < 0.01; N = 3. Histogram in (E) shows the average methylation % over the total cytosines (y axis) as derived from the respective data and grouped in all the cytosines (TOT), CpG moieties (CpG), and non‐CpG moieties (non‐CpG). Data represent the mean ± SEM versus the related controls (light gray columns). *p < 0.05; ** p < 0.01; N = 3. AD, Alzheimer's disease; SEM, standard error of the mean.