Investigation On The Mechanism Of DNA Methyltransferase Inhibitor RG108 In HT22 Hippocampal Neuron Cell Line And Dentate Gyrus

Objective:To study the effect of epigenetic changes on not only cancer prevention and treatment,but also mental illness caused by fear memory.Our group has previously found that DNMTs play an important role in fear memory,and the DNA methyltransferase inhibitor RG108 is a non-nucleoside analogue small molecule drug that directly binds to inhibit methyltransferase sites and transiently blocks DNMTs activity.We therefore continued to investigate the role of RG108 in fear memory-related disorders through in vitro and in vivo experiments.In vitro experiments:To investigate the effects of RG108 on the growth and synaptic plasticity related genes of HT22 hippocampal neuronal cell line;In vivo experiments: To investigate the effects of RG108 on neuronal activity,early reporter gene c-fos expression,and DNMT3 A expression in the dentate gyrus(DG)of the hippocampus,and to explore the effects of RG108 on the extinction pattern of fear memory that is not updated after extinction(DG neurons are activated during extinction training)in combination with behavioral and optogenetic techniques.To provide a basis for further development of DNA methylation-related regulators in fear memory-related diseases and neurological diseases.Methods:The experiment was carried out in vitro and in vivo.In vitro experiments: Hippocampal neuronal cell line HT22 was cultured,1.the optimal drug concentration and the viability of RG108 on cells were screened using CCK8 assay;2.the expression of synaptic plasticity-related genes and protein amounts were screened after RG108 intervention.In vivo experiments: 1.The calcium indicator r AAV-syn-j GCa MP7s-WPRE was stereotaxically injected into the dentate gyrus of the hippocampus,and the neuronal activity in the DG was measured by fiberoptic photometry while conditioned fear was performed in behavioral experiments after 14 days of expression or greater.2.Photosensitive channel proteins p AAV-Ca MKIIa-EGFP-P2A-Cre-WPRE and p AAV-Ef1a-DIO-h Ch R2(H34R)m Cherry were injected into the dentate gyrus of the hippocampus by stereotaxic localization to specifically express excitatory neurons,which were expressed for more than 14 days,and hippocampal dentate gyrus neurons were activated while fear memory subsided on the second day of behavior to detect whether RG108 functioned in the presence of neuronal activation and whether DNMTs were involved.3.After RG108 intervention,fear conditioning regression test was performed,and the brains were removed within 90 minutes after the end and cryopreserved at low temperature for subsequent experiments.Immunofluorescence was used to detect the expression of DNMT3a;immunohistochemistry was used to detect the expression of c-fos,an early gene;q PCR was used to detect the expression of synaptic plasticity-related genes;WB was used to detect the expression of synaptic plasticity-related proteins;and Golgi staining was used to detect the density of dendritic spines.Results:1.Hippocampal neuronal cell line HT22 was cultured,and CCK8 was used to detect cell viability.It was demonstrated that 50 μmol/L RG108 could promote the proliferation of HT22 hippocampal neuronal cell line at 48 h and 72 h.2.Cultured hippocampal neuronal cell line HT22,RT-q PCR detected gene expression,nr1,reln significantly decreased,the difference was statistically significant(p <0.05).3.Hippocampal neuronal cell line HT22 was cultured and protein expression was detected by WB.The expression of nr1 showed a decreasing trend but was not significant,and the expression of reln was not significant.4.Conditional fear conditioning was performed in C57/BL6 mice by injecting calcium ion indicators(j GCa MP7s)into the dentate gyrus of the hippocampus and intraperitoneal injection of RG108,and the results demonstrated that RG108 inhibited the extinction of fear memory and promoted the renewal of fear memory.At the same time of fear conditioning experiment,the activity of neurons in DG region of hippocampus was detected by fiberoptic photometry.The results showed that RG108 inhibited the extinction of fear memory and inhibited the activity of neurons.5.Photosensitive channel protein(Ch R2)was injected into the dentate gyrus of the hippocampus of C57/BL6 mice and RG108 was injected intraperitoneally to activate neurons in the DG of the hippocampus using optogenetic activation while performing fear conditioning experiments,and previous results demonstrated that RG108 could inhibit fear memory extinction,but did not promote its renewal,after activating neurons in the dentate gyrus of the hippocampus.Ninety minutes after injection of RG108 into the dentate gyrus of the hippocampus of C57/BL6 mice and behavioral fear conditioning experiment Extinction(regression),the brains were removed and immunofluorescence assay demonstrated that RG108 inhibited the expression of DNMT3 a in the DG of the hippocampus,inhibited the expression of c-Fos in the DG of the hippocampus,and reduced the density of dendritic spines in the DG of the hippocampus.Conclusions:1.DNA methyltransferase inhibitor RG108 promotes the growth of hippocampal neuronal cell line HT22 and affects the expression of synaptic plasticity-related genes.2.DNA methyltransferase inhibitor RG108 inhibits fear memory extinction and inhibits neuronal activity.3.In optogenetic experiments,RG108 inhibited fear memory extinction,but did not promote its renewal.After stimulation,the recovery experiment(renistatement)further verified that RG108 had a significant effect in the context fear experiment,but not in the renistatement experiment.Therefore,DNMTs may play a stronger role in fear memory extinction.4.Regimens that increase methylation combined with behavioral intervention may promote extinction of fear memory,and we hypothesize that regimens that increase methylation may assist in the treatment of fear memory-related disorders such as PTSD.