Age-related changes in Egr1 transcription and DNA methylation within the hippocampus

Abstract

Aged animals show functional alterations in hippocampal neurons that lead to deficits in synaptic plasticity and changes in cognitive function. Transcription of immediate-early genes (IEGs), including Egr1, is necessary for processes such as long-term potentiation and memory consolidation. Here, we show an age-related reduction in the transcription of Egr1 in the dentate gyrus following spatial behavior, whereas in the area CA1, Egr1 is reduced at rest, but its transcription can be effectively driven by spatial behavior to levels equivalent to those observed in adult animals. One mechanism possibly contributing to these aging-related changes is an age-associated, CpG site-specific change in methylation in DNA associated with the promoter region of the Egr1 gene. Our results add to a growing body of work demonstrating that complex transcriptional and epigenetic changes in the hippocampus significantly contribute to brain and cognitive aging. © 2016 Wiley Periodicals, Inc.

Keywords: aging; epigenetic modulation; memory; transcriptional regulation.

Figure 1

Aged rats are impaired in the spatial version of the Morris swim task. A. Over 4 days of training, adult rats took a significantly shorter path length to reach the hidden platform than did the aged rats. B. On the probe trial, aged rats spent significantly less time in the target quadrant than did adult rats (*p = 0.035). C. During cued trials, in which the escape platform was visible, both adult and aged rats showed improvement in performance over 2 days of training. There was no significant effect of age on either day.

Figure 2

Egr1 in the dentate gyrus. A. Basal levels of Egr1 mRNA are similar in adult rats (n=3) and aged rats (n=3), as measured by RT-PCR (p > 0.05). B. The proportion of dentate granule cells that transcribe Egr1 under resting conditions are similar in adult and aged rats (p > 0.05). C. Representative confocal images from the dentate gyrus in adult and aged rats under rest conditions. Green = Egr1, Blue = cell nuclei stained with TOPRO. Magnification = 40×. D. Behavior-induced relative levels of Egr1 mRNA are significantly lower in aged rats (n=6) compared to adult rats (n=6), as measured by RT-PCR (*p = 0.005). E. The proportion of dentate granule neurons that transcribe Egr1 after exploration is significantly lower in aged rats (*p = 0.030). Representative confocal images from the dentate gyrus of behavior-treated rats in both age groups are shown in F. Green = Egr1, Blue = cell nuclei stained with TOPRO. Magnification = 40×. Scale bar = 20 µm.

Figure 3

Egr1 in area CA1. A. Basal levels of Egr1 mRNA are higher in adult rats (n=3) relative to aged rats (n=3), as measured by RT-PCR (*p = 0.023). B. The proportion of pyramidal neurons that transcribe Egr1 under resting conditions, are similar in adult and aged rats (p > 0.05). C. Representative confocal images from control rats in both age groups for area CA1. D. Behavior-induced levels of Egr1 mRNA are not different between adult (n=6) and aged (n=6) rats, as measured by RT-PCR (p > 0.05). E. The proportion of CA1 pyramidal neurons that transcribe Egr1 after exploration are similar in adult and aged rats (p > 0.05). F. Representative confocal images from CA1 of behavior-treated rats in both age groups. Green = Egr1, Blue = cell nuclei stained with TOPRO. Magnification = 40×. Scale bar = 20 µm.

Figure 4

Methylation analysis of Egr1 dinucleotides from the hippocampus of adult and aged animals. A. Schematic of examined CpG sites in the promoter region of the Egr1 gene. Sequencing primer pair positions are indicated by the left and right arrows. B. In the DG, resting levels of methylation of the Egr1 promoter are significantly lower in adult rats (n=6) compared to aged rats (n=5; #p < 0.05). Following behavior, aged rats (n=4) show a significant decrease in methylation when compared to resting levels. Adult rats (n=6) do not show this dynamic change in methylation of the Egr1 promoter (**p < 0.01). C. Within CA1, there was no overall effect of age or behavior on methylation of Egr1.

Figure 5

Methylation analysis of individual CpG sites within age groups. A. In the dentate gyrus of adult rats, significant differences were not observed at individual CpG sites. B. In the dentate gyrus of aged rats, CpG6 (*p = 0.0001), CpG7 (*p = 0.0001), CpG8 (*p = 0.0001), and CpG12 (**p = 0.05) were significantly different between resting and behavioral conditions. C. In area CA1 of adult rats, there is no difference in methylation between individual CpG sites. D. In area CA1 of aged rats, there is no difference in methylation between individual CpG sites.

Figure 6

Methylation analysis of individual CpG sites between age groups. A. In the dentate gyrus under resting conditions, a significant difference between adult and aged rats was observed at CpG6 (**p < 0.0001), CpG7 (**p < 0.0001), and Cpg8 (*p < 0.001). B. Following behavior, no significant differences in methylation were observed at individual CpG sites. C. In CA1, under resting conditions significant age differences were not observed at individual CpG sites. D. Following behavior, there was a significant difference in methylation between adult and aged rats at CpG6 (*p < 0.05), CpG7 (**p < 0.01) and CpG8 (**p < 0.01).