The ligated patch methylated or unmethylated constructs were directly transfected into SH-Sy5y (human neuronal cell line) cells without passing the plasmid through bacterial cloning to keep the state of methylation of the gria2 5′ region and unmethylated promoter region

BrdU staining for visualization of proliferating Pluripotincells soon after KA therapy. (A) BrdU incorporation [30] in the different regions of hippocampus slice cultures (CA1, CA3 and dentate gyrus DG) after 2 hour cure (Ctrl 2h, KA 2h) and one 7 days restoration (Ctrl -one 7 days, KA -1 week). Neuronal cells (NeuN good) are stained green (B) Quantification of the whole range of proliferating cells (BrdU constructive) display no significant difference among KA treated slices and control instantly right after two hour therapy, or immediately after one week of restoration (n=five).Correlation in between epileptiform burst exercise and gria2 5′ region CpG methylation degrees one week immediately after removal of drug. (A) Sample neurons’ membrane potentials recording in current-clamp method of Manage hippocampal slices and (B) two hours KA dealt with slices one week soon after removal of the drug and incubation in drug totally free medium. A few or a lot more spikes ended up grouped into bursts if the inter-spike interval was smaller than 600 ms. (C) Average methylation stages of gria2 5′ area CpGs in either slices that show spontaneous bursting and people that never show bursting 1 week right after 2 hrs exposure to KA (n=4). (D) Correlation in between bursting frequency and normal probe methylation levels (p=.017, Pearson’s r=.7995). p<0.05 p<0.005.We then examined whether gria2 DNA methylation behaves in-vivo similarly to what was observed ex-vivo. In order to assess this point, we induced Status Epilepticus (SE) in Wistar rats by intraperitoneal injection of KA. SE was terminated after four hours with diazepam. The animals were left to recover for ten weeks, at which point we performed a two-week period of video-EEG recording (Figure 5A) to record the frequency and severity of spontaneous seizures. Our results revealed high variability in the physiological response of the individual rats to the KA treatment (Table 1) ranging from a total of 2 class 0 seizures to 111 seizures (out of which 33 were class IV and V). Collectively, these animals were obtained from 3 litters, but no association existed between severity of the epilepsy and the between burst frequency and DNA methylation levels at the examined gria2 promoter region (Pearson correlation p=0.017 r=0.7995 Figure 4D).DNA Methylation changes in rat gria2 gene promoter 5. ` region in epileptic and control rats and their correlation with seizures. (A) Representative electrographic recording of a seizure with synchronous video using Compumedics software. (B) Physical map of the rat gria2 promoter (+320 - -664). CpG sites are marked by balloons and predicted transcription factors common to the mouse and rat 5' region (-562--664) are indicated above the physical map. (C) Correlation between bursting frequency and average methylation levels of the gria2 5' region (p=0.006, Pearson's r=0.7190, n=13). (D) mRNA gria2 expression levels in control and KA treated rats measured by qPCR 10 weeks after initial SE. p<0.005 litter. This observation demonstrates that the severity of the epilepsy which develops from this insult (all animals become epileptic following KA-induced SE) varies between different individuals from the same litter similarly to what was observed with the mouse hippocampal slices in culture (Figures 1C and D and 4D). We used this model to address the following questions: First, would the gria2 promoter in rat hippocampus become hypermethylated in response to KA-induced SE and second, whether there are inter-individual differences in DNA methylation that correlate with the frequency of seizures We focused on a 5' upstream region (Figure 5B) that was previously shown to regulate gria2 in the rat [35] and contains binding sites for transcription factors found in the differentially methylated gria2 5' region in the mouse (Figure 1A). DNA was isolated from whole hippocampi from the rats after completion of the video-EEG recording. Similar to the mouse in vitro model, we found very low methylation levels (<10%) and no significant differences throughout the promoter, in both the control and high seizing animals (data not shown), and high inter-individual differences in the methylation state of the gria2 5' promoter region in different rats. The high convulsive seizure rats exhibited higher DNA methylation in all CpG sites in the gria2 5' promoter region (Figure 5C). We determined whether inter-individual differences in seizure behaviour (recorded number of convulsive seizures class IV and V) correlated with these inter-individual differences in DNA methylation of the tested gria2 region (n=13). Our results plotted in Figure 5C show that, similar to the in vitro results, there is a highly significant correlation between the state of DNA methylation of gria2 in the hippocampus of individual epileptic rats and their seizure behaviour (p=0.006, Pearson's r=0.72). Furthermore, we found a significant reduction of 26.3% (p<0.001 n=13) in mRNA expression levels of gria2 in the same treated rats relative to the controls, 10 weeks after induction of SE with KA (Figure 5D), as have been reported previously in the literature, and as we found in the in vitro model. The low seizure rats expressed on average 11.5% higher levels of mRNA than the high seizure rats but the difference between the high and low seizure groups did not reach statistical significance due to high variability in expression in the group and reduced numbers once the treated group was subdivided to low and high seizures. It is important to note that, on review of the videoEEG, no animals experienced Class IV or V in the 24 hours immediately preceding cull, which suggests that the changes in methylation are not due to the acute effects of convulsive seizures.The gria2 promoter was previously found to contain several regulatory regions that interact with different transcription factors such as REST and NRF-1 [36]. We utilized transient transfection luciferase reporter assays to determine whether the gria2 5' region (-528--719) that exhibited differential methylation in rats functionally regulated transcriptional activity and whether that activity was silenced by DNA methylation. We first generated a plasmid that contained the gria2 5' region (-528--719) (Figure 6A) upstream to a Luciferase reporter gene in a pCpGL-Basic plasmid in either sense or antisense (reverse) direction (scheme in Figure 6A). The vector sequences were previously engineered to have no CpGs and are therefore not methylated by CpG methyltransferases [37], thus any effect of DNA methylation on expression of the reporter gene would be caused by the methylated CpG sites in the inserted test regions. We then subjected the plasmid to either in vitro methylation with the bacterial CpG methyltransferase M.SssI or to mock methylation. Following in vitro methylation of the gria2 5' region we inserted by ligation the proximal unmethylated gria2 promoter (+320 -528) in the sense orientation (Figure 6A) downstream to the methylated 5' region. The state of methylation of this construct recapitulates the state of methylation of the gria2 promoter in vivo in high convulsive rats. The ligated patch methylated or unmethylated constructs were directly transfected into SH-Sy5y (human neuronal cell line) cells without passing the plasmid through bacterial cloning to keep the state of methylation of the gria2 5' region and unmethylated promoter region. Our results show that DNA methylation of the gria2 5' region silenced luciferase activity when it was inserted in the sense orientation to the promoter but not in the antisense orientation (Figure 6B). Enhancer regions could direct activity in both orientations but promoters act only in the 5`orientation to transcription start site. We therefore tested whether this 5' region has independent promoter activity in absence of the proximal gria2 promoter. Our results show that the 5` region of gria2 could direct luciferase transcriptional activity independently and that this activity is silenced by methylation of its CpG sites. We further validated the presence of a transcript upstream to the known TSS and downstream to the gria2 5' region by RT- PCR using forward 5' primers residing on the 3' edge of gria2 5'-region and reverse 3' primers around the known TSS. The amplified fragment was sequenced to verify that the fragment indeed represents mRNA upstream to the known TSS (see Figure S2 for physical map and sequence). Together, these results suggest that the gria2 5' region (-528--719) that we found to be differentially methylated in response to KA treatment in vivo has promoter activity in vitro that is silenced by DNA methylation and that methylation of this region silences also the downstream unmethylated proximal gria2 promoter.Our results established that new DNA methylation events occur in response to KA treatment in vitro, that they could persist and amplify following removal of KA and they correlate with bursting electrical activity. The remaining crucial question is that of cause and effect are the DNA methylation events a result of epileptogenesis and the changing cellular landscape in the brain [17], or do the DNA methylation changes triggered by the initial insult play a causal role in persistent bursting in vitro DNA methylation of the 5' region silences the activity of the gria2 promoter as determined by a transient transfection Luciferase reporter assay. (A) Physical map of the gria2-Luciferase reporter construct. The 5' region CpGs in the probe were in-vitro methylated (black lollypop), or mock methylated (empty lollypop). The CpGs in the promoter region were left unmethylated (empty lollypop). Additional constructs were designed as controls One containing the full promoter but with the 5' region in a reverse direction (Antisense) second, containing only the 5' region (5' region) third containing only the unmethylated promoter (Promoter) forth containing plasmid without any promoter sequence (Empty vector). (B) The indicated constructs were transfected into SH-Sy5y (human neuronal cell line). 48h after transfection the cells were harvested, extracts were prepared and assayed for Luciferase activity and the values were normalized to total protein concentration. Results are average of (n=3) transfections +/SEM. p<0.05 p<0.01 p<0.005 determined by a Student t test with Holm-Bonferroni correction and seizures in vivo We used a catalytic inhibitor of DNA methyltransferase RG108 to test whether DNA methylation activity is required for the development of bursting activity in hippocampal slices in response to KA. Mature organotypic hippocampal slices were treated for 2 hours with either the DNA methylation inhibitor RG108 (100), KA (6), a combination of KA and RG108 or left untreated as controls. The slices were left to recover for one week without the drugs. The results presented in Figure 7A show that while KA treatment induced methylation changes as displayed by the hypermethylation of all the CpG sites in the gria2 5' region, the DNA methylation inhibitor RG108 blocked this persistent increase in DNA methylation in response to KA. RG108 by itself had no effect on the state of DNA methylation. This suggests that there is no active demethylation or new DNA synthesis of this region in untreated slices that will necessitate the presence of DNA methylation activity to maintain the DNA methylation state. However, DNA methylation activity is required for KA induced DNA hypermethylation. We then determined whether blocking KA induced DNA methylation would also block bursting activity. As seen by the bursting frequency in Figure 7B,C, while RG108 had no significant effect on cultures that were not exposed to KA with an average of 0.58.34 bursts (6.25.69 spikes, n=12), it completely blocked bursting activity that is normally induced by KA in all of the slices (n=14) showing bursting activity (3.86.70 spikes per slice). The percentage of bursting slices were Ctrl 12.5%, KA 53%, RG108 25%, RG108 + KA 0%. When bursting activities and DNA methylation of the gria2 5' region were correlated across samples of all conditions, there was a significant correlation between gria2 DNA methylation and bursting activity (Figure 7D, p=0.003 Pearson's r=0.7763). This data supports the conclusion that DNA methylation activity is required for the development of epileptogenesis in response to KA and is consistent with the hypothesis that increased DNA methylation of gria2 5' region as well as other putative genes that were not measured in this study is involved in epileptogenesis.Epilepsy can be triggered by either physical or neurochemical insults to the brain in humans and animals. The critical question is what are the mechanisms that mediate longterm consequences of these transient insults, such as chronic seizures Another unresolved question is what are the mechanisms responsible for the inter-individual variation in the chronic response to a transient epileptoform insult Human and animal epilepsy studies show changes in gene expression profiles [38-40], suggesting that mechanisms involved in epileptogenesis are registered in the genome and that there must be genomic mechanisms that mediate the long-lasting changes in gene expression in response to transient neural insult. We used in vitro and in vivo rodent models of epileptogenesis triggered by transient KA exposure to test the plausibility that DNA methylation mechanisms are involved in long-term genomic memory of earlier neural insults. 19176528We effect of DNA methylation inhibitor RG108 on KA induced DNA methylation of the gria2 5′ region and epileptiform bursting 1 week following a transient 2 hour exposure. (A) Methylation differences between control (Ctrl, n=4 technical replicates on 3 different mice of origin), KA treated (KA, n=4 technical replicates on 3 slices from different individual mice), RG108 (RG108 n=4 technical replicates on slices from 2 different mice) and RG108 and KA treated slices (RG108+KA n=4 technical replicates on 2 different mice of origin) 1 week after removal of the drug and incubation in drug free medium. (B) Sample neurons’ membrane potentials recording in current-clamp mode of Ctrl, KA, RG108 and RG108 + KA treated samples. (C) Average spontaneous bursting activity of hippocampus slices after treatment with the different drugs. Significant increase in bursting activity can be observed in KA treated slices (n=19) vs. control (n=16). RG108 combined with KA treatment (RG108+KA, n=14) blocks the spontaneous bursting induces by KA treatment (n=19). Treatment with RG108 alone (n=12) does not have any significant effect on bursting compared to control slices (n=16). Significance between the different conditions in multiplecomparisons was calculated using Student t-test with HolmBonferroni correction for multiple comparisons.