Decipher Molecular Mechanism Underlying The Regulating Effect Of Conditional Dnmt1,3a Knockout On Learning And Memory Of Aging Mice

Aging is the greatest risk factor for the development of Alzheimer’s disease（AD）.Aging and AD are clinically characterized by memory loss and common neuropathological manifestations including amyloid-beta（Aβ）plaques accumulation.However,how aging contributes to the development of neurodegenerative diseases is largely unknown.DNA methylation,one of the important epigenetic mechanisms,has been reported to regulate hippocampal synaptic plasticity and long-term memory formation.The DNA methylation process is mediated by DNA methyltransferase（DNMT）,including DNMT1,DNMT3A,and DNMT3B.DNMTs are required to maintain cognitive function in old age.Dysregulation of DNA methylation has been reported in the aged brain,which is related to memory loss or even contributes to memory impairment,suggesting that dysregulation of DNA methylation is a driver of aging-related cognitive dysfunction.Our previous studies showed that conditional deletion of Dnmt1 and Dnmt3a resulted in a significant up-regulation of ST6Gal2 expression in the hippocampus,meanwhile,the mice exhibited hippocampal-dependent memory impairment.ST6Gal2 belongs to the sialyltransferase family,which binds sialic acid to cell surface glyco-proteins and glyco-lipids.Sialylation has a variety of biological functions such as regulating the immune system and modulating synaptic plasticity.ST6Gal family contains two enzymes ST6Gal1and ST6Gal2.The expression of ST6Gal2 in human tissues specifically differs from that of ST6Gal1.ST6Gal1 is ubiquitously expressed in human tissues,while ST6Gal2 is dominantly expressed in the adult central nervous system and fetal tissues,especially in the hippocampus and prefrontal cortex.ST6Gal1 facilitates the sialylation of amyloid precursor protein（APP）and promotes the aggregation of Aβpeptides.However,so far there are few reports on the possible effect of ST6Gal2 on brain functions,especially the memory processes.The purpose of our study is to investigate the effects of Dnmt1 and Dnmt3a deficiency on learning and memory of the aging mice,as well as to explore the role of ST6Gal2 in mediating aging-related memory impairment and the possible mechanisms.In this study,we used mice with genetic knockdown of Dnmt1 and Dnmt3a in forebrainαCa MKII-expressing neurons(αCa MKII-Cre;Dnmt1,3a^2flox/2floxor CKO)and control mice(Dnmt1,3a^2flox/2flox).First,we compared the expression of St6gal2 in the hippocampus of aging CKO mice（over 18 months of age）and adult CKO mice（6 months of age）by quantitative reverse transcription PCR（RT-q PCR）.We used the elevated plus maze test and the open field test to evaluate the spontaneous activity and anxiety state,and used object-place recognition and Morris water maze to evaluate the learning and memory of these conditional knockout mice.Meanwhile,we overexpressed St6gal2 in the hippocampus d CA1 region of C57BL/6J mice by stereotaxic viral injection to investigate whether virus-mediated St6gal2 expression in the hippocampus mimics the effect of Dnmt1 and Dnmt3a deficiency on memory.Finally,we investigated whether memory impairment in the agingαCa MKII-Cre;Dnmt1,3a^2flox/2floxmice could be alleviated by interfering St6gal2 expression in the hippocampus.Enzyme-linked immunosorbent assay（ELISA）was used to quantify Aβ1-40,Aβ1-42,and IL-6 levels in the hippocampus.The aggregation of Aβplaques in the hippocampus was observed using fluorescence immunohistochemistry.Our main findings are as follows:1.In comparison to the same-age control mice(Dnmt1,3a^2flox/2flox),both young and aging Dnmt1,3a CKO mice(αCa MKII-Cre;Dnmt1,3a^2flox/2flox)showed elevated St6gal2expression in the hippocampus;however,the St6gal2 up-regulation was more dramatic in the aging mice than in young mice with Dnmt1,3a deficiency.2.In comparison to the same-age control mice,the aging Dnmt1,3a-deficient mice exhibited more severe hippocampus-dependent memory deficits.3.Virus-mediated St6gal2 overexpression in the hippocampus of aging C57BL/6J mice caused memory deficits;St6gal2 overexpression did not affect spontaneous activity and basal anxiety.4.Aging mice with Dnmt1,3a deficiency exhibited increased Aβ1-40,Aβ1-42,IL-6 levels,and elevated Aβaccumulation in the hippocampus when compared to control mice at the same age.5.Interfering St6gal2 expression in the hippocampus reduced abnormal Aβaccumulation,meanwhile alleviated memory deficits in aging mice with Dnmt1,3a deficiency.In conclusion,our study demonstrates that hippocampal ST6GAl2 regulates memory.The abnormal upregulation of ST6GAl2 may promote the transition of physiological aging into pathological neurodegeneration,leading to memory impairment.Specifically,aging exacerbates DNA hypomethylation of St6gal2 associated with Dnmt1 and Dnmt3a deficiency,leading to abnormal increases in St6gal2 transcription and resultant ST6Gal2expression.Upregulated ST6Gal2 expression participates in the pathological process of Aβplaque accumulation and promotes the transition from aging to neurodegenerative changes,which ultimately causes AD-associated memory impairment in aging mice.Therefore,dynamic monitoring of ST6Gal2 levels may be helpful for early screening and diagnosis of neurodegenerative diseases.