Although normal aging is characterized by a number changes, none are as central to the identity of a person as their memories1. While there has been a large
amount of research into age related diseases of the brain, there is relatively little known about the molecular mechanisms behind how a healthy brain ages. Currently, aging is viewed as risk factor, rather than a cause, for a number of neurodegenerative diseases1. An understanding of the mechanisms behind why some people show relatively little age-related cognitive decline (ARCD), and others show significant and disabling ARCD, will provide great insight for both the basic and clinical sciences.
Initial studies into normal cognitive aging have shown that as we age gene expression patterns in the brain change; particularly genes involved in synaptic plasticity and vesicular transport show decreased expression after age 402. However, the casual link between aging and gene expression patterns has remained unknown until recently. More recent studies have begun to suggest that epigenetic mechanisms are the missing casual link3, 4. Modern epigenetics can be defined as “a mitotically (or meiotically) inheritable change in gene expression, independent of an alteration in DNA sequence5”. Epigenetic mechanisms encompass a number of molecular marks, such as histone modifications, DNA methylation, and microRNAs.
Most molecular research into ARCD has examined mouse models1, 6, 7, which allow for unprecedented observations and interventions, and have focused on the hippocampus, as it is involved in the consolidation of memory from the short-term to the long-term. At this time most studies have chosen to solely examine histone acetylation as the dynamic response to memory consolidation8, 9. These studies have shown that there is an age related failure in establishing histone acetylation in “learning-regulated genes” during memory consolidation. Furthermore, many studies have shown that treatment with histone deacytlase inhibitors (HDACs) can attenuate ARCD and related biomarkers8, 10, 11
While a narrowing of focus into histone acetylation has provided great insight, there is also emerging evidence suggesting that histone modifications act in concert with a number of other epigenetic factors12, 13. Indeed, DNA methyltransferase (DMNT) inhibitors have also been shown to interfere with memory consolidation14. Furthermore, many studies have also narrowed their focus into a relatively small amount candidate genes and/or single pathway being affected as a result of epigenetic modifications8, 15. However, in most cases the epigenome rarely works alone and typically affects gene expression on a much larger scale7, 16. In this research I hypothesize that memory consolidation involves epigenomic changes on a genome-wide scale in the hippocampus and that the dysregulation of these epigenetic marks contributes to ARCD.
2) Specific Objectives:
Specific Objective 1: Evaluate the epigenomic and transcriptomic changes associated with ARCD.
Specific Objective 2: Evaluate the epigenomic and transcriptomic changes associated with interventions that attenuate ARCD.
3) Proposed Research:
Evaluate the epigenomic and transcriptomic changes associated with ARCD.
To evaluate the epigenomic and transcriptomic changes associated with ARCD, I will use a C57BL/6J mouse model6. This model will allow for the collection of a
significant amount (n=10) of hippocampal samples (CA1, CA3, and Dentate Gyrus). These samples will be collected from mice at the ages of 3, 8, and 16 months, which represent young, middle-aged, and old mice respectively6. The use of these 3 age groups will be sufficient for experimentation, as previous research has shown significant memory impairment in 16 month-old mice, with the 8 month-old mice showing an intermediate phenotype8. Samples will be obtained both before and at multiple time points during memory consolidation as initiated by fear conditioning17 (10 min, 30 min, 60 min, and 1d after)8. The different regions of the hippocampus will then be sequenced using next-generation sequencing technologies, including methylated DNA immunoprecipitation paired with sequencing (MeDIP-seq), chromatin immunoprecipitation paired with sequencing (ChIP-Seq), and RNA-Seq methodologies. These technologies will allow for an assessment of repressive epigenetics marks, such as DNA methylation (5-methylcytosine) and H4 lysine-27 trimethylation (H4K27me3). Additional Chip-Seq will also examine the activating marks of H4 lysine-12 acetylation (H4K12ac) and H4 lysine-3 tri-methylation (H4K3me3). Finally, micoRNA and gene expression patterns can also be assessed at a genome wide level by RNA-seq. Bioinformatic analysis will then be done to examine regions of the genome that show overlapping and unique responses of the (epi)genome upon memory consolidation across all age groups (Figure 1). Results from top candidates will be confirmed via sodium bisulfite sequencing, respective qPCR technologies (immunoprecipitation or reverse transcription based), and western blotting to confirm whether the observed alterations in gene expression and/or epigenetic marks translate to the protein level.
It should be noted that the histone modifications chosen only represent a fraction of those that occur, and while the ones chosen are the most extensively studied in this context, it is possible that there are more informative marks. If any of the marks are found to be non-informative in the preliminary experiments, alternative histone modifications will then be examined in a large-scale screening assay based off western blotting.
Evaluate the epigenomic and transcriptomic changes associated with interventions that affect ARCD.
The evaluation of epigenomic and transcriptomic changes associated with interventions that attenuate ARCD will be done using a similar experimental design to
the previous objective, but will focus on time points and regions of interest. The main difference will be the addition of two new groups; mice that serve as a matched-control for the methodology of intervention (i.e: vehicle injection) and those that have received the intervention (Figure 1). The interventions used will include HDAC2 inhibitors, which have received much attention in prior research11. Furthermore, given that DMNT inhibitors interfere with memory consolidation14, methyl-donor rich diets, such as those with choline18, will also be examined. Novel interventions, such as RNAi for transcripts of interest and environmental enrichment19 will also be examined.
It should be noted that there is a large amount of variation in the methodologies of the interventions mentioned. If during preliminary experiments an intervention does not attenuate ARCD, additional experiments will be carried out to optimize the conditions for each intervention (i.e.: dosage and timing of exposure).
4) Expected Findings and Overall Significance of Proposed Research:
The proposed research will provide an unprecedented resolution of the dynamic and transient epigenetic and transcriptomic changes occurring upon memory consolidation. The results are expected to reveal a large number of previously unobserved interactions between the epigenome, genome, and transcriptome due to the relatively unbiased nature of sequencing technologies. The use of mice at different ages will allow for the identification of novel regions that become deregulated with age and contribute to ARCD. The integration of multiple data sets by bioinformatic analysis will also provide novel insight into how epigenetic mechanisms co-regulate changes in gene expression and allow for cross-communication between relatively distinct regions of the genome and/or pathways during memory consolidation. Furthermore, the examination of interventions that attenuate ARCD will also provide a number of novel insights into clinical treatment for ARCD. The use of HDAC inhibitors will allow for an examination of the most commonly used intervention11, and has the potential to reveal the mechanisms behind many of the potential negative side effects associated with a drug of such global nature. The use of RNAi will allow for assessment of the effect in changes the dosage of individual candidate genes. Finally dietary choline supplementation and environmental enrichment serve as noninvasive interventions, with the potential for positive global epigenetic changes18, 19. These interventions, both established and novel, will confirm and reveal much about the inner workings of the aging brain and also further our understanding of the effectiveness of treatments for ARCD.
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- The Newest Cupid on the Block: Epigenetics (knowingneurons.com)
- Mapping the embryonic epigenome (sciencedaily.com)
- Epigenetics Review (biokamikazi.wordpress.com)
- Why do identical twins end up having such different lives? (guardian.co.uk)