Conditional Knock-out Of Dnmt3a And Dnmt1in The Hippcampus CA1Impairs Memories In Adult Mice

Dnmt1and Dnmt3a are two main DNA Methyltransferase. Recently a lot of reseach indicate that the methylation mediated by Dnmtl and Dnmt3a plays an important role in the functions of adult nervous system, such as learning and memory. To further study the effect of DNA methylation on hippocampal synaptic plasticity and memory, and explore the underlying mechanisms, we first injected adeno-associated virus expressing both Cre recombinase and Green Fluorescent Protein (GFP) into the CA1of Dnmt12flox2flox Dnmt3a2flox2flox mice hippocampus. By Cre-I.oxP recombinase systerm, the Cre virus successfully induced Dnmt1and Dnmt3a knock-down in the CA1of hippocampus with a spatial and temporal manner. In combination with a series of behavior experiments, we checked the effect of conditional Dnmt1and Dnmt3a double knock-down in the CA1of hippocampus on learning and memory formation in adult mice. Meanwhile, we applied the AAV-Cre-2A-GFP virus to primary?cultured hippocampal neurons isolated from Dnmtl2flox2floxDnmt3a2f mice to delete Dnmtl and Dnmt3a in vitro. With the technology of Immunofluorescence. western blot and RT-PCR, we observed the effect of double knock-down of Dnml1and Dnml3a on the morphology of hippocampal neurons and its network formation. We found that:1. Double knock-down of Dnml3a and Dnmtl (～50%) in CA1of hippocampus did not affect anxiety in Dnmt12flox2flox Dnmt3a2flox2flox mice.2. Double knock-down of Dnmt3a and Dnmtl (～50%) in CA1interfered new objective recognition in Dnmtl2flox2flox Dnmt3a2flox2flox mice.3. Double knock-down of Dnmt3a and Dnmtl (～50%) in CA1interfered social novelty preference in in Dnml12flox2flox Dnml3a2flox2flox mice mice.4. Double knock-down of Dnmt3a and Dnmtl (～50%) in CA1had no effect on spatial memory and contextual fear memory in mice.5. Double knock-down of Dnmt3a and Dnmtl in primary cultured Dnmtl2flox2flox Dnml3a2flox2flox neurons interfered its late development. Virus infected neurons showed smaller cell bodies and simplified networks of axons and dendrites compared to the controls, however the cell numbers were intact indicating that knock-down of Dnmt3a and Dnmtl did not induce apoptosis. Only knock-down of Dnmt3a or Dnmtl had no such effect. 6. Double knock-down of Dnmt3a and Dnmtl in primary cultured Dnmt12flox2flox Dnml3a2flox2flox neurons significantly reduced the protein levels of type Ⅲ β-tubulin. actin and synapsin I. Only knock-down of Dnmt3a or Dnmt1had no such effect.7. Double knock-down of Dnmt3a and Dnmtl in primary cultured Dnmt12flox2flox Drmt3c2flox2flox neurons significantly reduced the protein levels of Akt, GSK3β rpS6and ERK1/2. Only knock-down of Dnmt3a or Dnmt1had no such effect.In summary, our present data showed that Dnmt3a and Dnml1double knock-out in CA1interfered with certain forms of hippocampus-dependent recognition memory in mice. Double knock-down of Dnmt3a and Dnmt1in primary cell culture interfered late development of the hippocampal neurons. Our study confirmed that Dnmtl and Dnmt3a have overlapping roles in maitaining DNA methylation. which are required for synaptic plasticity, learning and memory. Further studies are required to illustrate the underlying molecular and cellular mechanisms which mediating the effect of Dnmtl and Dnmt3a on neuronal morphology, plasticy and memory.