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  • Similar to Bdnf acute administration of RG


    Similar to Bdnf4, acute administration of RG108 did not alter gene expression of Gria1 and Hdac2, despite their relevance for the task. Gria1 belongs to the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic syk inhibitor receptor (AMPAR) family that is known to be crucial for LTP and the strengthening of synapses that is important for learning and memory (Lisman, Yasuda, & Raghavachari, 2012). For that, rapid trafficking of GluA1- containing AMPARs at the postsynaptic membrane is required. The trafficking may involve delivery of already existing AMPARs or synthesis of new AMPARs (Ju et al., 2004, Nayak et al., 1998, Oh et al., 2006, Penn et al., 2017). Nevertheless, the timing of these dynamic changes is difficult to be predicted. Therefore, it is possible that 1 h after treatment represents a time-point that does not involve transcription of GluA1-AMPARs, implying it to be too early or too late in this respect. Regarding Hdac2, its expression levels were determined because it works in close concert with DNMTs. Opposed to methylation, acetylation normally leads to an increase in gene-transcription, by opening the chromatin and making it more accessible to transcription factors. The strong interplay between DNMT and HDAC2 makes it interesting to assess whether DNMT inhibition also induces Hdac2 expression changes (Feng et al., 2007, Tsankova et al., 2007). In contrast to Hdac1 that is mainly expressed in astrocytes, Hdac2 is mainly found in neurons and HDAC2 has a crucial role in normal brain development and cell survival (Hagelkruys et al., 2014). HDAC2 however is only linked to one of the two mechanism of methylation-induced gene silencing via binding in the MBDs and could therefore remain unchanged even if methylation changes (Feng et al., 2007). In order to extend our Bdnf1 findings, we performed DNA methylation analysis in 14 CpG sites in the promoter I of Bdnf and observed an increase in the methylation status of 3 of them. Specifically, for the treatment that had an intermediate effect (0.1 mg/kg) in OPS performance, there was an increase at the CpG 2, 3 and 6 in promoter I, while for the most effective treatment (0.3 mg/kg) the increased methylation was restricted in CpG2. The methylation status of the remaining CpG islands tested was unaltered between the different experimental groups. Considering that the animals were treated with a DNMT inhibitor, the observed increased methylation was not anticipated. A paradoxical effect in the action of DNMTs was also observed in a study showing that stress induced an increase in DNA methylation levels in the hippocampus and a decrease in methylation levels in the prefrontal cortex. Administration of RG108 was able to compensate for these changes, suggesting that both increase and decrease in stress-induced DNA methylation could be regulated by DNMT activity (Sales & Joca, 2016). In contrast to the methylation that is catalyzed exclusively by DNMTs, there have been suggested several pathways that could induce demethylation (Watt & Molloy, 1988). However, the existence and influencing of enzymes that promote active demethylation remains elusive. Studies aiming to gain more insight into the demethylation mechanism in vertebrates suggested that, under specific conditions, mammalian DNMTs could act as active DNA demethylases by removing the methyl or hydroxymethyl group from 5-methylcytosine (5-mC) or 5-hydroxy-mC (5-hmC), respectively (Chen et al., 2012, Chen et al., 2013). Importantly, the demethylase activity of DNMTs is Ca2+-dependent (Chen, Wang, & Shen, 2013). Considering that increased Ca2+ mediates signal transduction in neuronal populations, it could be speculated that in activated neurons DNMTs promote active demethylation. In our study, the animals were sacrificed thirty minutes after the mnemonic test raising the possibility that the action of DNMTs was shifted towards the demethylation pathway. In that case, administration of RG108 could prevent demethylation rather than methylation explaining the initially counterintuitive finding of increased Bdnf1 methylation after treatment. Additional studies are required in order to shed light into the complex epigenetic changes occurring after DNMT inhibition as well as to determine the exact location of DNMT binding in the promoter region of Bdnf1.