The Effect Of Conditional Knockout Of Dnmt3b Gene In Mature Hippocampal Neurons On Learning And Memory And Its Mechanism

Recent studies have shown that epigenetic modulation is involved in neuronal development and synaptic functions,and its abnormality is associated with many neuropsychiatric disorders with cognitive deficits.Epigenetic modifications,including DNA methylation and demethylation,histone acetylation,micro RNA interference,are reversible processes that regulate gene expression and function without change of DNA sequence.DNA methylation is a major form of epigenetic modification by covalent binding of methyl groups to cytosine C5 of Cp G dinucleotides on DNA.DNA methylation is catalyzed by DNA methyltransferases(Dnmts).Dnmt1,Dnmt3 a and Dnmt3 b are three major Dnmts expressed in the central nervous system of eukaryotes.Although the expression level of the Dnmt3 b is significantly decreased in postnatal neurons,studies have shown that it regulates synaptic plasticity and memory formation in adult brains;however the underlying molecular biological mechanisms have not been elucidated yet.To further study the role of Dnmt3 b in learning and memory and explore itsunderlying mechanisms,we injected AAV-Cre-GFP virus into CA1 region of the dorsal hippocampus to delete Dnmt3 b expression in adult Dnmt3b2lox/2lox transgenic mice.Meanwhile,we established a conditional Dnmt3 b knockout mouse strain(αCa MKII-Cre;Dnmt3b2lox/2lox)by crossing αCa MKII-Cre transgenic mice with Dnmt3b2lox/2lox mice to genetically knockdown Dnmt3 b expression in postnatal forbrain excitatory neurons,including CA1 neurons in the hippocampus.A serial of behavioral paradigms,including elevated plus maze,open field experiment,new object recognition,object-in-place recognition and Morris water maze etc.,were performed to observe learning and memory ability in Dnmt3b2lox/2lox mice received AAV-Cre-GFP virus infusion into CA1 region,αCa MKII-Cre;Dnmt3b2lox/2lox mice and the controls.In combination of primary neuron culture,calcium imaging,m RNA expression array genechip technology,we further explored the possilbe mechanisms.Our results are as follows:1.AAV-Cre-GFP-virus-mediated deletion of Dnmt3 b gene in CA1 region of dorsal hippocampus did not change locomotor activity,anxiety and exploration activity in mice.2.AAV-Cre-GFP-virus-mediated deletion of Dnmt3 b in hippocampal CA1 region impaired NPR memory performance in adult mice.3.αCaMKII-Cre;Dnmt3b2lox/2lox mice showed normal anxiety-and depression-like behavior.4.αCa MKII-Cre;Dnmt3b2lox/2lox mice also showed significant NPR memory deficits,which is consistent with the behavioral phenotype observed after virus-mediated deletion of Dnmt3 b in CA1 of dorsal hippocampus.5.Dnmt3 b deletion in cultured hippocampal neurons did not affect dendrites morphology and excitatory synapse formation.6.Calcium imaging study showed that Dnmt3 b deletion enhanced glutamate-evoked cytoplasmic [Ca2+] elevation in primary cultured hippocampal cells(DIV 21).7.The differential genes expression after Dnmt3 b deletion in the hippocampus were first screened by m RNA microarrays analysis and confirmed with real-time quantitative RT-PCR techniques.Our results showed that,the expression of β-galactosamide alpha-2,6-sialyltranferase 2(St6gal2)in the hippocampus were significantly up-regulated(2.5 folders)after Dnmt3 b deletion in both CA1 of hippocampus and forebrain excitatory neurons.8.In addition,we found that object-place recognition(NPR)training downregulates Dnmt1 m RNA expression in the hippocampus as tested 1h after the training session,indicating the involvement of Dnmt1 plasticity in the formation of NPR memory.Noticeably,dnmt3 b deletion induced compensatory upregulation of Dnmt1 and Dnmt3 a expression in the hippocampus.In conclusion,with both virus-mediated gene deletion and genetic,conditional gene knockout methods,we consistently found that conditional knockout Dnmt3 b expression in the hippocampus leads to object-place recognition deficit in adult mice.Dnmt3 b gene deletion does not change dendrites morphology and excitatory synapse formation in primary cultured hippocampal neurons;however it significantly facilitates glutamateevoked cytoplasmic [Ca2+] elevation measured by calcium images,suggesting enhanced neuronal activity.RNA expression chips and followed real-time q RT-PCR further confirmed that dnmt3 b deletion induces up-regulation of beta-galactosamide alpha-2,6-sialyltranferase 2(St6gal2)m RNA expression in the hippocampus.We thus suspect that St6gal2 might be a direct target of dnmt3 b.The reduced activity of dnmt3 b could increase St6gal2 expression and change the sialylation pattern of glycoproteins and glycolipids in hippocampal neurons,which may cause neuronal hyperactivity and Ca2+ overload leading to abnormal synaptic transmission and plasticity,and recognitive memory deficits as well.To test this hypothesis,we are doing western blot analysis to check the protein expression of dnmts,St6gal2 and its reaction substrate PSA-NCAM;meanwhile we are measuring synaptic transmission and plasticity in acute hippocampal slices.Finally we will test whether suppression of St6gal2 by antibody antagonist or sh RNA in hippocampus can rescue recognitive memory deficits caused by dnmt3 b deletion.Noticeably,we found that object-place recognition(NPR)training downregulates Dnmt1 m RNA expression in the hippocampus as tested 1h after the training session,indicating the involvement of Dnmt1 plasticity in the formation of NPR memory.In the other hand,Dnmt3 b deletion in our study induces compensatory increase of Dnmt1 and Dnmt3 a expression in the hippocampus.The compensatory increase in Dnmt1 after dnmt3 b deletion may dampen activity-dependent down-regulation of Dnmt1 and contributes to object-place recognition deficits in αCa MKII-Cre;Dnmt3b2lox/2lox mice.Therefore,we concluded that Dnmt3 b deletion in the hippocampus causes recognitive memory deficits,both direct(St6gal2)and indirect(Dnmt1 and gene target)molecular mechanisms may be involved in this process.