A popular antiepileptic drug, valproic acid, is a histone-deacetylase inhibitor [thirteen] that induces DNA demethylation inTaprenepag cultured cells [14,15] and in the mind [16]. Additionally, current examination of hippocampi from mice acutely addressed with the chemo-convulsant, kainic acid (KA) demonstrated common adjustments in DNA methylation [seventeen]. To exam whether or not DNA methylation performs a causal role in epileptogenesis, however, it is significant to determine regardless of whether genes essential for epileptogenesis are regulated by DNA methylation in reaction to a transient initial insult and regardless of whether these DNA methylation adjustments are essential for epileptogenesis. In this study we analyzed this hypothesis by examining the modifications in DNA methylation in the upstream regulatory regions of gria2, the gene coding the GluA2 subunit of the AMPA receptor. Proof implies that the existence of GluA2 subunit (encoded by gria2) in the heteromeric AMPA receptors impermeabilizes it to calcium [eighteen,19], preventing achievable calcium-mediated toxicity. Early and long lasting downregulation of gria2 expression noticed in epilepsy designs propose that it performs a important purpose in initiating the epileptogenic cascade, retaining neuronal hyperexcitability [19,twenty] and is crucial for the pathophysiology of mesial temporal lobe epilepsy (MTLE), the most widespread kind of epilepsy acquired in adulthood [21]. On top of that, knockdown of gria2 in youthful rats resulted in seizure-like behavior and neurodegeneration [22]. The molecular system mediating this phenomenon continues to be unclear, but epigenetic changes such as Relaxation targeted regulation of gria2 expression by histone hypoacetylation in response to KA remedy [23], have been implicated. In this study we used a gene-focused tactic by monitoring methylation at precise CpG web sites in gria2, a gene implicated by many lines of knowledge in epileptogenesis, in an in vitro mouse and an in vivo rat product of epileptogenesis brought on by KA. We hypothesize that this alter in methylation is persistent and that inter-particular person variation in gria2 methylation is related with differences in epileptic bursting activity in vitro and the severity of epilepsy created in an in vivo model. We then examined no matter if these methylation events functionally down-regulated the gria2 promoter activity and regardless of whether the non-nucleoside DNA methyltransferase inhibitor N-Phthalyl-L-tryptophan (RG108) [24] blocked methylation of gria2 and epileptogenic bursting.Epileptiform bursts brought on by KA in the hippocampus are affiliated with inter-person variability of quick and persistent changes in a DNA methylation of a 5′ regulatory region of the gria2 gene.KA remedy of experienced organotypic cultured hippocampus slices is a effectively-established in vitro product for inducing epileptiform activity [twenty five]. Using this product we examined the point out of DNA methylation of the proximal promoter of the gria2 gene (Figure 1A) as nicely as a next location upstream to the proximal promoter (boxed -766–804) in hippocampal slices after two hours of cure with KA in contrast to drug-absolutely free cultured slices making use of pyrosequencing (Figure 1B).The proximal promoter was generally unmethylated in all CpG web-sites (<5%) in both KA treated slices and controls with minute changes in DNA methylation between KA and controls (Figure 1B). However, a 5' region positioned at -595 to -804 upstream of the transcription start site showed measurable levels of methylation and small but nevertheless significant differences in methylation between KA and control (CpG sites 35-39). The 5' region (-766-804) that exhibited consistent changes in DNA methylation with KA treatment in slices from individual mice (boxed in Figure 1A and 1B, CpG sites 37-39) was analyzed in silico using TRANSFAC [26]. The analysis identified several transcription factor binding sites, including CCAAT/enhancer-binding protein beta (C/EBP beta) and the Glucocorticoid Receptor (GR), which was previously shown to localize in Gria2 positive cells in the hippocampus and this colocalization was affected by epilepsy [27,28]. These results represent DNA methylation levels in a pool of 5 slices from different mice at the end of two hours KA treatment. However, it is well known that there are interindividual differences in both animals and humans in the liability to developing epilepsy in response to a single brain insult [29]. A plausible hypothesis is that variations in DNA methylation of critical genes in the brain are associated with these differences. Genetically homogenous inbred strains are an ideal system to test this hypothesis. We first tested whether there are differences in the DNA methylation state of the KA responsive 5' region in the gria2 promoter between five individual mice. The results depicted in Figure 1C (see mice I, II, III) demonstrate variability in the basal state of methylation of the three CpG sites in this region between slices derived from littermate mice (the technical variability in measurement of the same mouse is indicated by the standard error), in addition to the differences observed between slices derived from mice of different litters (Figure 1C mice IV and V). In certain cases where an individual CpG site showed high methylation levels in the basal state (e.g CpG 39 in mice I and IV), we observed decreased methylation after the 2 hour KA treatment. This observation further highlights the intriguing finding that the DNA methylation response 2 hours after exposure to KA is different between slices from littermates. We then determined whether the gria2 gene would remain hypermethylated after KA was removed, serving as a "memory" of a transient exposure to the epileptiform inducer. We examined the DNA methylation state of slices that were treated with KA for 2 hours and were then maintained in standard culture media for one week in absence of the agonist and compared it to untreated controls. The results presented in Figure 1D show that transiently treated slices exhibited enduring increases in methylation of all three CpGs in the 5' region (Ctrl 6.8.7, 15.1.36, 20.78 KA 35.5.5, 36.6.2, 36.2.6 respectively) and a 2.4 fold in the average DNA methylation in the whole 5' region of the gria2 promoter (Figure 1A boxed) over control cultures (n=5, p=0.002). The difference in DNA methylation between treated and control slices after 1 week of incubation in drug free medium was higher than immediately after exposure to KA for 2 hours (Figure 1C). Importantly, each of the 3 CpGs sites in this region was more methylation changes in gria2 5' region in response to KA induced epileptiform activity. (A) Physical map of the gria2 5' regulatory region and promoter region. CpG sites are marked by balloons and transcription factor predictions in the analyzed 5' region are indicated above the physical map. (B) State of methylation of CpG sites in the proximal promoter of the gria2 and the 5' region in control and KA treated hippocampal slices (n=3 technical replicates) CpG 32 and 34 were not analyzed due to sequence restrictions. (C) Methylation differences between control and KA treated slices derived from the same mouse (n=4 technical replicates for each individual mouse) immediately after KA treatment for two hours, and (D) 1 week after removal of the drug (n=4 technical replicates, SD indicate technical errors). Inter-individual differences are apparent between littermate mice (Mouse I, II, III) and between mice from different litters (mouse IV, V) (E) Gria2 mRNA expression levels in control and KA treated slices as measured by qPCR immediately after KA treatment (2 hours, n=5) and 1 week after removal of the drug (1 week, n=9). Gria2 mRNA levels were normalized to TBP/GAPDH expression based on NormFinder. p<0.05 p<0.01 p<0.005 as determined by Mann-Whitney U-testmethylated in the treatment cultures than in control cultures (Figure 1D). Inter-individual differences between slices derived from the same littermate mice in the basal DNA methylation state and their response to 2 hours transient KA exposure were amplified following 1 week incubation in absence of the drug (Figure 1D). We therefore tested whether the increase in DNA methylation in response to KA is associated with reduced expression of gria2 mRNA. In order to accurately evaluate the levels of gria2 mRNA levels we used NormFinder analysis to test possible reference genes which have previously been reported to be stable under seizure conditions (Glyceraldehyde-3-phosphate-dehydrogenase - gapdh, TATA binding protein -tbp, Hypoxanthine phosphoribosyl-transferase - hprt1, Neuron specific enolase nse1) [30,31]. We found that all four genes displayed a variability level well below the standard cutoff value previously established of 0.15 (Figure S1), indicating the value of any one of these genes as an accurate single reference gene. In order to increase the accuracy of our measurement, we used the combination of gapdh and tbp which was indicated by NormFinder to be the most stable. We found high variability in the gria2 mRNA expression levels (n=5 cultures per condition) immediately after the 2 hour treatment in the KA-treated slices compared to controls, similar to the methylation response. The average expression of gria2 mRNA in the transiently treated slices that were incubated in drug free medium for 1 week was 21.5% lower than in control slices (p=0.006) (Figure 1E), and showed variable levels of reduction. For each allele, the methylation profile of a specific site is either methylated or not methylated, with each cell contributing an extreme of either zero or one hundred percent methylation to the cumulative measurement (i.e. percent of methylation measured gives an indication of the percentage of cells that are methylated in the slice). We considered an important set of potential confounders namely that the DNA methylation changes observed could have been caused by either DNA synthesis or cell death. These are especially important considering that the hippocampus is a heterogenic tissue and that changes in gria2 levels have been previously associated with neuronal cell death associated with epileptic injuries [32]. Here we used several methods to evaluate the physiological condition of our slices. First, we applied Nissl staining (Figure 2A) to examine the structural integrity of the hippocampal slices. No swelling or vacuolization of cells, typically seen during cell death or apoptosis, was observed suggesting that the integrity of the hippocampus was not affected by 2 hours treatment with KA. Secondly, propidium iodine (PI) staining was performed to test for cell death by necrosis. We evaluated cell death induced by exposure to KA for 12h and found severe cell death throughout the slice similar to previous reports [33,34]. A positive control for the PI staining was also conducted by incubating a slice in high KCl solution prior to the staining (Figure 2B). A small amount of PI positive cells was observed in both the control and treated slices (Ctrl 52.02.78 KA 147.53.40 p=0.025) immediately after the treatment and an even smaller number 1 week post-treatment (Ctrl 43.83.72 KA 35.25.02 p=0.619 Figure 2C). We found a significant increase in PI positive cells after the 2 hour KA treatment. This difference correlates to less than 2% of the overall number of cells (9266.7430.4 as evaluated by manual counting of dissociated neurons in a NeubauerImproved cell counting chamber). 19175605Thirdly, we used BrdU incorporation to evaluate cell proliferation in slices of both treated and untreated samples (Figure 3A). There was no significant difference in the total number of proliferating cells between the treatment and control samples either immediately after the KA treatment (Ctrl 672.51.79, KA 663.70.78, p=0.799) or after 1 week of recovery (Ctrl: 230.5.23, KA 260.02.39, p=0.290 Figure 3B). These minor differences between the number of control and KA treated dying and proliferating cells suggest that the persistent DNA methylation changes in response to KA treatment must represent a much larger percentage of cells that changed DNA methylation than the small number of dying or proliferating cells. In summary, inter-individual DNA methylation differences in this region of the gria2 promoter exist in otherwise genetically identical mice. Most importantly, there are differences in the responsivity of the DNA methylation state of these CpG sites to KA insult and these differences are enhanced during the drugfree incubation period creating large differences in the longterm DNA methylation state between different individuals with a “history” of transient exposure to KA insult. The response of the DNA methylation state does not seem to reflect a variation in cell death or proliferation.The inter-individual differences in DNA methylation as observed in the gria2 promoter region beg the question of whether variations in DNA methylation in response to KA are associated with differences in long-term electrophysiological activity of the hippocampal slices. We therefore conducted membrane potential recording in single-cell current-clamp mode of control and KA-treated slices after one-week recovery (Figure 4A,B). Spikes were detected offline and 3 or more spikes were grouped into bursts if the inter-spike interval was smaller than 600 ms. We found that 10 out of 19 slices exhibited spontaneous bursting activity of the measured pyramidal neurons after the KA treatment, with an average of 6.21.39 bursts per slice (64.790.26 spikes per slice). Two control slices out of 16 did have some bursting activity, with an average of 0.25.14 bursts (3.56.11 spikes per slice, p=0.029). We then compared the DNA methylation level at the gria2 5′ promoter region in slices that exhibited bursting (n=4) versus non-bursting slices (n=4) (Figure 4C). DNA methylation levels were determined in four technical replicates for each slice from the same mouse. Significant hypermethylation was observed in all CpGs in the examined region of gria2 in the bursting slices relative to the non-bursting slices (p<0.001, p<0.001, p=0.017 respectively). The difference in average DNA methylation of the 3 CpGs in this region was highly significant as well (p<0.001). We then correlated bursting and DNA methylation in this gria2 5' region across all individual hippocampal slices. This analysis provided compelling evidence for a correlation cell death in hippocampus organotypic culture slices after KA treatment. (A) Nissl staining of mature hippocampus slices displaying normal neuronal organization in both control and KA treated slices 1 week after removal of the drug. (B) Propidium Iodine (PI) staining of the cultures at 2 hours treatment with KA (KA 2h), control (Ctrl 2h) and 1 week after removal of KA (Ctrl 1 week, KA 1 week) to evaluate cell death. 12h KA (KA 12 hours) was used as a positive control for levels of cell death reported in other KA models and 15.7M KCl was used as PI staining positive control (KCl control). PI positive cells are labeled in red. White arrows point at sample PI positive cells (C) Quantification of total number of PI positive cells in the different conditions show a small but significant increase in the number of positive cells immediately after 2h KA treatment compared to control, and no significant change after 1 week recovery (n=4). p<0.05.
