St6gal2 Gene Is Involved In The Cognition Deficits Caused By Dnmt3b Deletion In Mice

Recent years,there are mounting evidences indicating that DNA methylation,as one of the most important branches of epigenetics,involves in the process of memory acquisition and consolidation by regulating the expression of associated genes.DNA methylation is catalyzed primarily by three methytransferases:DNMT3a and DNMT3b belong to de novo methyltransferase,which are responsible for the novel methylation marker;while DNMT1 is classified as maintenance methyltransferase that maintain the methylation of both DNA strands via recognizing the existed methylation markers.Cooperation among the three methyltransferases leads to lifelong modification during the cell division.Most studies focused on dnmt3a and dnmt1,while little was known on the role of dnmt3b in learning memory.Here we carried out a series of experiment to explore the effect and underlying mechanism of dnmt3b in learning memory.We found by RNA-chips and RNA-seq analysis,the abnormal upregulation of st6gal2 in hippocampus CA1 of dnmt3b knockout mice.It was known that st6gal2 belongs to sialytransferase family,locating mainly in the adult brain（prefrontal cortex and hippocampus）and expressing in specific-tissue stage（embryonic stage）.Some studies suggested that the expression of st6gal2 was high in the embryonic development phase while was decreased to a lower level in adult.st6gal2 functions by transferring the sialic acid from CMP-Sia to the surface of glycoprotein or glycolipid viaα2,6-glycosidic link.The hypophilicity and cidification mediated by st6gal2 and associated proteins play an essential role in ultiple biological processes,such as cell migration,celladhesion.Some previous studies clarified that a few members of sialytransferase family participate in the synaptic plasticity due to the acidification in the glycoprotein and glycolipid of neurons.Nevertheless,much attention about st6gal2 has been paid on cancer and tumor cells instead of neurons.In this study,we aim to demonstrate the effect and mechanism of st6gal2 in learning memory.In previous study,we discovered that dnmt3b knockout mice exhibited impaired recognition memory accompanied with st6gal2.We then proposed that deletion of dnmt3b affects synaptic function in certain neural network by modulating st6gal2expression.In order to test this hypothesis,we first applied real-time qPCR and Western blot analysis to confirm the expression of st6gal2 in the hippocampal CA1 of conditionalαCaMKII-Cre;dnmt3b^flox/floxlox/flox mice.We also analyzed the alteration in DNA methylation by Whole Genome Bisulfite Sequencing（WGBS）.As for functional study,we compared hippocampal synaptic function ofαCaMKIICre;dnmt3b^flox/flox mice with control dnmt3b^flox/flox mice.Finally we investigated whether reverse st6gal2 upregulation via either antibody or shRNA virus injection into hippocampal CA1 could ameliorate abnormal synaptic function and recognition memory deficit observed inαCaMKII-Cre;dnmt3b^flox/flox mice.The results are listed as follows:1.Real-time qPCR and Western blot demonstrated st6gal2 expressed upregulation in αCaMKII-Cre;dnmt3b^flox/flox mice in basal condition and object-in-position NPR 1h（P<0.0001）.2.Overexpressiom of st6gal2 was accompanied with deregulation of ncam（P<0.05）,st8sia2（P<0.05）while st8sia4 wasn’t affected.3.Differentiated methylation in st6gal1,st8sia2,st8sia4,ncam affected the expression of associated genes or proteins.4.PPR showed normal transmitter release betweenαCaMKII-Cre;dnmt3b^flox/floxlox/flox and dnmt3b^flox/floxlox/flox mice（P>0.05）.5.Short-term potentiation E-LTP（P<0.05）and long-term potentiation L-LTP suggested synaptic plasticity was impaired inαCaMKII-Cre;dnmt3b^flox/flox mice.6.Downregulation of st6gal2 expression could rescue the impaired LTP caused by dnmt3b deletion inαCaMKII-Cre;dnmt3b^flox/flox mice（P>0.05）.7.Antagonism of ST6Gal2 protein by microinfusing anti-ST6Gal2（P>0.05）and interference of st6gal2 gene by injecting AAV-st6gal2-shRNA（P<0.01）could rescue the impaired NPR memory performance inαCaMKII-Cre;dnmt3b^flox/flox mice.In sum,the differentiated methylation in st6gal1,st8sia2,st8sia4,ncam caused by dnmt3b knockout in mice led to the abnormal overexpression of st6gal2 and resulted in the impaired LTP which is associated with synaptic plasticity.Consensuses have reached into that synaptic plasticity plays a key role in memory formation.Thus,the synaptic plasticity deficits in our transgenic mice might fail to promote the synthesis of memory-associated genes or proteins leading to the disorder of recognition memory.