Genome-wide expression and methylation profiling in the aged rodent brain due to early-life Pb exposure and its relevance to aging

Abstract

In this study, we assessed global gene expression patterns in adolescent mice exposed to lead (Pb) as infants and their aged siblings to identify reprogrammed genes. Global expression on postnatal day 20 and 700 was analyzed and genes that were down- and up-regulated (≥2 fold) were identified, clustered and analyzed for their relationship to DNA methylation. About 150 genes were differentially expressed in old age. In normal aging, we observed an up-regulation of genes related to the immune response, metal-binding, metabolism and transcription/transduction coupling. Prior exposure to Pb revealed a repression in these genes suggesting that disturbances in developmental stages of the brain compromise the ability to defend against age-related stressors, thus promoting the neurodegenerative process. Overexpression and repression of genes corresponded with their DNA methylation profile.

Figure 1. Scatter plots depicting overall gene expression patterns in young and old animals and those developemntally-exposed to Pb

(A) early-life perturbation: PND20 control vs developmentally Pb-exposed PND20 animals; (B) normal aging: control PND20 vs. control PND700; and (c) Pb-reprogrammed aging: control PND20 vs devleopmental exposed PND700. Each group represents data pooled from three animals. Both young and old animals are offspring from the same cohort. The axis numbers represent fluorescent intensity.

Figure 2. Functional annotations of genes from control aging rodents and those with early-life exposure to Pb

The Venn diagram in (A) represents down-regulated genes in the normal aging mouse brain in control animals as well as those exposed to Pb early in life [PND 700 is compared to PND 20 as the baseline (C20)]; (B) shows the functional groups that are down-regulated in normal aging, and (C) displays down-regulated genes due to early-life exposure to Pb. The Venn diagram in (D) represents up-regulated genes in the aging mouse brain in control animals as well as those exposed to Pb early in life [PND 700 is compared to PND 20 as the baseline (C20)]; (E) shows the functional groups that are up-regulated in normal aging, and (F) displays up-regulated genes due to early-life exposure to Pb. Normal aging is shaded in purple to distinguish genes that change solely due to aging. The frontal cortex from three analysis was arrayed separately (n=3) and the data was grouped from the three animals for C20, C700 and E700 groups.

Figure 3. Hierarchical clustering of the most significantly altered genes

Shown through heat maps is the expression patterns of significant two fold differential gene expression (p ≤ 0.01) in the normal aging brain and in aged brains developmentally exposed to Pb. The yellow rows indicate the genes exhibiting the least degree of change; the red rows indicate the highest degree of change as well as the genes relationships to each other. The rows designate the relationship of the genes to each other and the columns indicate the age of the animals. Down-regulated genes (A) and up-regulated genes (B) are shown; different genes are represented horizontally and their relationships to each other in the dendogram are shown vertically.

Figure 4. Gene validation through RT-PCR

Comparison of array and RT-PCR fold change data for selected genes changed from normal aging (C20 vs C700) and Pb-reprogrammed aging (C20 vs E700). Values over 1 (dashed line) are up-regulated and the values under 1 indicate genes that are down-regulated. The values ≥ 2 and values ≤ 0.5 are significantly up-regulated and down-regulated, respectively. “*” denotes significance has been determined by student’s t-test (p≤ 0.01). “#” denotes significance has determined by student’s t-test (p≤ 0.05). The genes that were up-regulated in the array were aldehyde oxidase 1 (Aox1), choline dehydrogenase (Chdh) and transient receptor potential cation channel subfamily C member 6 (TRCP6). The genes that were down-regulated in the array were kelch-like 1 (KLHL1), methyltransferase like 4 (METTL4), protein kinase C-theta (PKC-theta) and 5′ cytidinetriphosphate synthase (CTPS).

Figure 5. Differential Gene Expression and Methylation Correlation

A scatter plot of differential gene expression of control PND20 and developmentally-exposed PND700 (E700 - C20) vs the differential methylation of control PND20 and developmentally-exposed PND700 (E700 - C20). Methylation data were linearly transferred and log₂ was applied to gene expression and methylation signal intensity values.

Figure 6. Gene expression and methylation profiles

A line plot of gene expression intensity values of control PND20 (C20) alongside the methylation intensity values of control PND20 and developmental exposed PND700 (E700) for the selected genes that have ≥2 fold differences in gene expression alterations between samples. The top part of the figure shows all selected genes while the bottom part shows to the first 100 genes. The circled E700 methylation values indicated the genes with noticeable difference between C20 and E700 in methylation values. Gene expression and methylation intensity values were log2-transformed.