The role of Igf2 methylation and histone ubiquitination in age-related memory decline

Abstract

Cognitive decline affects nearly 33% of adults over the age of 70 and is a major risk factor for developing dementia and Alzheimer's disease (AD). The hippocampus is one of the major brain regions associated with memory formation, and is one of the first regions impacted in dementia and AD. Although research indicates that molecular mechanisms within the hippocampus become dysregulated with age, the underlying reasons are still not well understood. Epigenetic mechanisms are important regulators of gene transcription, which is a fundamental contributor to memory formation and consolidation and work to regulate the dynamic accessibility of DNA to transcriptional machinery, thereby controlling the expression levels of genes through both DNA methylation and histone post translational modifications (PTM). DNA methylation occurs when a methyl group is attached to the DNA strands generally at a cystine residue followed by a guanine. This phenomenon is generally associated with gene silencing, as genes with high 5-methylcytosine levels in the promoter region have little ability to be expressed. It also plays an important role in genetic imprinting through heavily methylating specific alleles leading to their inactivation. DNA methylation dynamics have been shown to be important in memory formation and become dysregulated with age, possibly contributing to age related memory decline. Insulin-like growth factor 2 (Igf2) is a genomic imprinted gene that is important in development, and remains highly expressed within the brain, specifically in neurons. Administration of IGF2 to the hippocampus of aged animals has been shown to improve memory, and decreased levels of IGF2 have been detected in AD patients. However, whether changes in epigenetic regulation of this gene contribute to age-related memory decline is yet to be explored. There are numerous histone PTMs that play a role in many cellular processes, as well as in memory formation. Specifically, H2B monoubiquitination at lysine 120 (H2Bubi) has recently been shown to be a major regulator of the epigenome and transcriptome during memory formation, which is proposed to occur through the ability to recruit H3 lysine 4 trimethyl (H3K4me3), another which induces gene transcription, as well as act in conjunction with other histone modifications to control gene activation or deactivation. However, to date there has been little research on the role of H2Bubi in memory formation with age. Here we address these gaps in knowledge on how epigenetic mechanisms work in the aged brain and during memory formation late in life. First, we examined the regulation of Igf2 in the aged hippocampus via DNA methylation. Second, we tested the role of H2Bubi in transcriptional regulation during memory in advanced age. We found that Igf2 DNA methylation levels are increased in the promoter region of the hippocampus of 24-month-old rats compared to young (3 months) and middle-aged (12 months), which correlates with a reduction in Igf2 expression compared to young rats. To understand if this impacts memory of aged rats, we used a CRISPR dCas9-TET1 system to oxidate DNA methylation marks at the Igf2 promoter, converting this region to a 5-hydroxymethylation (5-hmC) state that increase gene transcription. Remarkably, this manipulation led to improved memory retention in aged, but not middle-aged, rats. We next showed that baseline levels H2Bubi decrease with age in the hippocampus of male rats. Again, we used CRISPR-dCas9 to upregulate Rnf20, the only E3 ligase for H2Bubi, in the aged hippocampus, which improved memory retention in late life. Using RNA-seq analysis we showed that Rnf20 upregulation not only increased expression of the same 18 genes typically upregulated in the aged hippocampus following contextual fear conditioning but also increased or decreased the expression of an additional 40 and 11 unique genes, respectively, following fear conditioning. This suggests that these 51 genes may be among those critical for memory improvement in advanced age. Together, these projects elaborate upon a significant role that epigenetic mechanisms play in memory formation and identifies contributing factors for memory deficits in the aged hippocampus.