H3K4Me3 level is decreased at gene promoters in APP/PS1 mice

As we have noticed the expression levels of many important genes have been up or down-regulated in early AD model of 6-month APP/PS1 mice, we further investigated the epigenetic changes which might contribute to this result. Histone modifications have been proposed as one of the key mechanisms regulating gene expression in eukaryotic organisms (Kouzarides, 2007; B. Li, Carey, & Workman, 2007). Especially trimethylated H3K4 (H3K4Me3) has been reported to be very relevant to contextual memory consolidation, where H3K4Me3 level was elevated after contextual learning, and decreased in mouse models with memory deficits (Gupta et al., 2010; Kerimoglu et al., 2013).

We hypothesized that there might be a reduction in H3K4Me3 levels at promoters in 6-month-old APP/PS1 mice, leading to the down-regulation of various genes.

Hippocampal CA3 regions were dissected from 6-month-old APP/PS1 mice and WT littermates, and nuclei of only neurons were sorted out using FACS (Figure 3.14).

Figure 3.14. Example result of sorted neuronal nuclei by FACS.

Scatter plot of one sample displaying separated neuronal nuclei from non-neuronal cells and debris using FACS. Area P4 (shown in magenta) represents collected neurons, and area P3 (shown in blue) represents non-neuronal components. Note that the yield of neurons per tissue sample is just around 10%.

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In line with the findings from the RNA-seq, ChIP analysis revealed a downregulation of H3K4me3 level at the gene promoters, while very few genes promoter areas showed increased H3K4Me3 level (Figure 3.15 A). To name just a few of the down-regulated genes detected: Lats1 (large tumor suppressor kinase 1), Dop1R1 (Dopamine 1-like receptor 1), Mlf (Myeloid leukemia factor), Nxn (nucleoredoxin).

For a more complete list please refer to Annexes 6.7.

Again, the pathway analysis was performed, and the down-regulated genes with decreased H3K4Me3 at their promoters are listed in categories (Figure 3.15 B).

Figure 3.15. H3K4Me3 level is decreased at the promoter areas of many genes in hippocampal CA3 PCs of 6-month APP/PS1 mice.

A. Volcano plot of ChIP-Seq results displaying H3K4Me3 levels at promoters of various genes in APP/PS1 mice (n=4) compared to WT littermates (n=4). Red dots represent differential binding. To the left side are the numerous gene promoters with low H3K4Me3 levels, and to the right side are few gene promoters with high H3K4Me3 levels. B. Pathway analysis of genes with decreased H3K4Me3 at their promoters.

It is worth noticing that the most affected pathways are mainly involved in neuronal proliferation and function/development of olfactory bulb. This indirectly confirmed that adult neurogenesis could be impaired in 6-month APP/PS1 mice, which have been shown in many AD models. Especially, one study showed that hippocampal neurogenesis is dramatically impaired in 6-month APPswe/PS1dE9 mice. To be more

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specific, the late survival of newborn cells 4-6 weeks later is significantly diminished (Verret et al., 2007). Additional evidence is still needed to confirm whether newborn granule neurons are actively killed by Aβ, or are unable to survive because of changes in the brain associated with Aβ overexpression.

However, a closer look at the down-regulated genes with decreased H3K4Me3 levels showed that they were not overlapping much with the down-regulated genes from RNA-seq result. For this we even chose a more tolerant cut-off for the RNA-seq data to include more genes. Still, we detected only one gene which showed both low expression in RNA-seq and low H3K4Me3 levels at the promoter area, which is Synpr (synaptoporin) (Figure 3.16). Synaptoporin is a synaptic vesicle protein that at first was found to be exclusively enriched in the granule cell axons, the mossy fibers in rat hippocampus (Grabs et al., 1994). Later studies showed that synaptoporin also colocalized well with GAD 65 (a marker for GABAergic terminals) in the mouse hippocampus, and further in situ hybridization combined with immunocytochemistry revealed that various subtypes of interneurons expressed synaptoporin, e.g. in 44.2% of PV+ interneurons, 59.9% of VIP+ interneurons and 38.6% of CCK+ interneurons (Singec et al., 2002). Therefore, a reduction in synaptoporin expression might implicate that the activities of inhibitory synapses in the hippocampal CA3 area in APP/PS1 mice could be impaired, which coincides with our electrophysiological findings.

Figure 3.16. Down-regulated genes from RNA-seq and ChIP-seq in APP/PS1 mice.

Venn diagram depicting down-regulated genes from RNA-seq (shown in yellow) and gene promoters with decreased H3K4me3 from ChIP-seq (shown in purple) and their overlap, in CA3 region of 6-month-old APP/PS1 mice. Note that RNA-seq was performed from whole CA3 tissue and ChIP-seq was performed from sorted neuronal nuclei.

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A fact needs to be taken into consideration is that the RNA-seq was done from whole CA3 tissue whereas the ChIP-seq was performed from sorted CA3 neuronal nuclei. This indicate that the RNA-seq result might include a considerable portion from glia cells in CA3 region, thus can be quite different from the expression profile obtained from ChIP-seq. However, the more plausible explanation is that H3K4Me3 is only one of many epigenetic markers, and other epigenetic mechanisms may play more important role in the down-regulation of genes in early AD mice.

Nevertheless, these results confirmed that H3K4Me3 plays a key role in regulating gene expressions in neurons of hippocampal CA3 region, and H3K4Me3 level was significantly decreased at the promoter areas of many genes in 6-month male APP/PS1 mice.

Chapter 4

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4 Discussion