Studying The Effect Of Selective Deletion Of Dnmt1 And Dnmt3a In Excitatory Neurons Of Hippocampal CA1 On Memory And Underlying Mechanisms

In recent years,a large amount of researchs indicate that epigenetic modification plays an important role in regulating normal brain function?such as learning and memory?.Moreover,the pathogenesis of various neuropsychiatric diseases associated with cognitive dysfunction is related to abnormal epigenetic modification.Currently DNA methylation is the most studied form of epigenetic modification.During the learning process,external environment stimulation can dynamically regulate DNA methylation,and the methylation regulates memory formation and storage by regulating the transcription of specific genes in the brain.A large number of studys have shown that DNA methylation participates in the processes of memory consolidation by regulating the expression of memory-related genes.DNA methylation refers to the transfer of a methyl group from the methyl donor S-adenosyl-methionine?SAM?to the 5?position on the cytosine-pyrimidine ring in the CpG dinucleotide via the catalysis of DNA methyltransferase?DNMT?.In mammals,DNA methylation is primarily regulated by three DNMTs.DNMT1 is a maintenance methyltransferase,which is mainly responsible for the addition of a methyl group for hemimethylated DNA to maintaining methylation in cells;DNMT3A and DNMT3A are de novo methyltransferases since they can methylate the cytosine of CpG dinucleotides previously unmethylated on both strands,which can establish new methylation patterns in cells.Those three methyltransferase are coordinated so that methylation can maintain continuity and stability in cell division.DNMT1 and DNMT3A are two major DNA methyltransferases expressed in mature neurons.Many recent studies suggested that DNA methylation mediated by DNMT1 and DNMT3A plays an important role in regulating adult brain functions such as learning and memory.There is also evidence showing that DNMT1and DNMT3A are both involved in the de novo synthesis of DNA methylation,but the mechanism is unclear yet.To further study the effect of DNA methylation on synaptic plasticity and memory,and to explore the underlying molecular and cellular mechanisms,we first applied viral vector-mediated Cre expression?AAV-Cre-GFP?in dorsal CA1 region of the hippocampus,in order to specifically knock out Dnmt1 and Dnmt3a expression in mature neurons of Dnmt1,3a²flox/2floxflox/2flox mice?work done during master's degree?.Meanwhile,we established a conditional Dnmt1 and Dnmt3a double knockout mouse strain,?CaMKII-Cre;Dnmt1,3a²flox/2floxflox/2flox mice,by crossing B6.Cg-Tg?CaMK2?-cre?T29-1Stl/J mice with Dnmt1,3a²flox/2floxflox/2flox mice to genetically knockdown Dnmt1 and Dnmt3a expression in?CaMKII-expression neurons in CA1 region of the hippocampus.First,a series of behavioral paradigms,such as elevated plus maze,open field,novel position recognition,social preference,Morris water maze and rotarod were used to evaluate behavioral performance of those two conditional knockout mice.At cellular levels,whole-cell patch clamp recording and field excitatory postsynaptic potential?fEPSP?recording was used to investigate the synaptic function and intrinsic excitability of CA1 pyramidal neurons.At molecular levels,we combined gene broad-spectrum chips,transcriptome sequencing,whole genome methylation sequencing,Western Blotting and quantitative reverse transcription polymerase chain reaction?qRT-PCR?techniques to explore the molecular mechanism underlying synaptic dysfunction and memory deficits observed in Dnmt1 and 3a double knock-out mice.Our results are as follow:1.the effect of selective deletion of Dnmt1 and Dnmt3a in excitatory neurons of hippocampal CA1 on memory.1.1?CaMKII-Cre mice were crossed with Gt?ROSA?26Sor^tm1?EYFP?Cos reporter mice to show CRE recombinase expression mainly in the CA1 region of the hippocampus.And the knockout rates of Dnmt1 and Dnmt3a were about 40%and 30%,respectively,in?CaMKII-Cre;Dnmt1,3a²flox/2floxflox/2flox mice?shown in Fig.3?.1.2?CaMKII-Cre;Dnmt1,3a²flox/2floxflox/2flox mice displayed normal baseline anxiety and locomotor activity compared to the Dnmt1,3a²flox/2floxflox/2flox control mice in elevated plus maze and open field?shown in Fig.4?.These results are consistent with our previous results in the Dnmt1,3a²flox/2floxflox/2flox mice,whose Dnmt1,3a in hippocampal CA1 region were knock-down via localized brain region injection of AAV-Cre-GFP virus.1.3?CaMKII-Cre;Dnmt1,3a²flox/2floxflox/2flox mice showed memory impairment,including object place recognition impairment,social recognition memory impairment and spatial memory impairments respectively in novel position recognition,social preference and Morris water maze?shown in Fig.5?.These results are also consistent with our previous results in the Dnmt1,3a²flox/2floxflox/2flox mice,whose Dnmt1,3a in hippocampal CA1 region were knock-down via localized brain region injection of AAV-Cre-GFP virus.1.4?CaMKII-Cre;Dnmt1,3a²flox/2floxflox/2flox mice showed enhanced motor learning compared to the Dnmt1,3a²flox/2floxflox/2flox control mice in rotarod?shown in Fig.6?.2.Cellular mechanism underlying the effect of deletion of Dnmt1 and Dnmt3a on memory2.1?CaMKII-Cre;Dnmt1,3a²flox/2floxflox/2flox mice exhibited abnormal synaptic plasticity in hippocampal neuronal circuit compared to the Dnmt1,3a²flox/2floxflox/2flox control mice,specifically enhanced long-term depression?LTD?,but normal long-term potentiation?LTP?,paired-pulse ratio?PPR?and basic excitatory synaptic transmission in field excitatory postsynaptic potentiation?fEPSP?recording?shown in Fig.7?.2.2?CaMKII-Cre;Dnmt1,3a^2flox/2floxmice exhibited reduced frequency?but normal amplitude?of spontaneous and miniature inhibitory postsynaptic current?sIPSC and mIPSC?,and unchanged excitatory postsynaptic current?sEPSC and mEPSC?in CA1pyramidal neurons,revealed by whole-cell patch-clamp recording?shown in Fig.8?.2.3 The intrinsic excitability of the CA1 pyramidal neurons in?CMKII-Cre;Dnmt1,3a²flox/2floxflox/2flox mice was significantly enhanced in comparison to control Dnmt1,3a²flox/2floxflox/2flox mice,specifically enhanced actin potential?AP?in whole-cell patch clamp recording.Moreover the memory impairment of?CaMKII-Cre;Dnmt1,3a²flox/2floxflox/2flox mice was ameliorated by calcium-activated potassium channel agonist CyPPA?shown in Fig.9?.3.Molecular mechanism underlying the effect of deletion of Dnmt1 and Dnmt3a on memory3.1 The hippocampal CA1 gene expression chips analysis showed that there were 166up-regulated genes and 138 down-regulated genes with a fold change more than 1.5 times;transcriptome sequencing analysis showed that there were 2490 up-regulated genes and 3466down-regulated genes with a fold change of expression more than 2 times.The whole genome methylation sequencing analysis showed that the overall DNA methylation levels were up-regulated in 2,980 genes and were down-regulated in 4986 genes?changes more than 10%?in aCaMKII-Cre;Dnmt1,3a²flox/2floxflox/2flox mice.We found 4 genes with consistent expression trends in mRNA levels,meanwhile they were along with changed CG methylation levels:beta-galactoside alpha-2,6 sialyltransferase 2?St6gal2?,activating transcription factor 7 interacting protein 2?Atf7ip2?,insulin receptor-related receptor?Insrr?,2-5 oligoadenylate synthetase-like 1?Oasl1,shown in Fig.10?.3.2 Differential expression of candidate genes in hippocampal CA1 region of?CaMKII-Cre;Dnmt1,3a²flox/2floxflox/2flox mice were confirmed by qRT-PCR and Western blot analysis.We found that both mRNA and protein levels of St6gal2 were increased and viral vector-mediated St6gal2 shRNA expression in the CA1 region of dorsal hippocampus,rescues hippocampal-dependent memory deficits in?CaMKII-Cre;Dnmt1,3a²flox/2floxflox/2flox mice?shown in Fig.11?.3.3 The mRNA expression of kcnn4,which encoding the intermediate-conductance Ca²⁺-activated K⁺channels?KCa3.1?,was decreased and virus-mediated overexpression of Kcnn4 in the CA1 region improved memory impairment observed in?CaMKII-Cre;Dnmt1,3a²flox/2floxflox/2flox mice?shown in Fig.12?.3.4 Western blotting analysis showed the phosphorylation of Akt is significantly reduced in?CaMKII-Cre;Dnmt1,3a²flox/2floxflox/2flox mice,compared to the Dnmt1,3a²flox/2floxflox/2flox control mice?shown in Fig.13?.In summary,on the basis of our previous finding showing that virus-mediated Dnmt1and Dnmt3a deletion in hippocampal CA1 neurons impaired hippocampus-dependent learning and memory,we further demonstrated that selective knock-out of Dnmt1 and Dnmt3a in excitatory neurons of hippocampal CA1 region caused consistent,hippocampal-dependent memory impairment,but enhanced motor learning.At cellular levels,we found abnormal synaptic transmission and neuronal intrinsic excitability in CA1pyramidal neurons,and abnormal synaptic plasticity in hippocampal SC-CA1 path as well,which may contribute to memory deficits observed in double knock-out mice.At molecular levels,we found that the DNA methylation and mRNA expression of St6gal2 were both up-regulated,meanwhile kcnn4 expression was down-regulated at the transcriptional level.Silencing St6gal2 expression or increasing kcnn4 expression in CA1 region of the dorsal hippocampus ameliorated memory deficits in Dnmt1 and Dnmt3a double knock-out mice.We thus concluded that St6gal2 and kcnn4 genes can interact to affect neuronal morphology,intrinsic membrane excitability,synaptic transmission and plasticity,and ultimately produce long-term impact on learning and memory processes.Further studies are still needed to disclose the precise mechanism.Our study provides new targeting genes,St6gal2 and Kcnn4,for the study of methylation-related mental disorders,including mental disorders and learning disabilities.In the long run,these studies will not only help to deepen our understanding of learning and memory and its molecular and cellular mechanisms,but also have a profound impact on the etiology and treatment of neuropsychiatric disorders with cognitive impairment.