Dissecting The Role Of DNA Methyltransferases (DNMTs) In Motor Learning In Normal And PD Model Mice

Epigenetic modification refers to reversible and heritable changes in gene expression without changing the primary DNA genetic code sequence.DNA methylation is an important epigenetic modification.DNA methylation refers to the covalent modification with the methyl group on the 5’carbon position of DNA Cp G dinucleotide cytosine,which is mediated by the DNA methyltransferase（DNMTs）.DNMT1,DNMT3a and DNMT3b are three major DNMTs expressed in the central nervous system of the eukaryotes.The former is mainly involved in the maintenance of the DNA methylation state,and the other two are the de novo methyltransferases that participate in the elongation of DNA methylation.Abnormal epigenetic modification is close associated with the pathogenesis of motor disorders,such as Parkinson’s disease,Huntington’s disease,etc.Meanwhile,studies have demonstrated that exercise can promote human health,reduce the incidence of disease and delay aging by epigenetic mechanisms including DNA methylation.Motor learning is a learning process to acquire muscle response or motor skills,as well as to promote body coordination.Studies have shown that the basal ganglia is the main structure regulating motor learning,which mainly includes the striatum,red nucleus,substantia nigra and the subthalamic nucleus.The basal ganglia plays an important role in motor regulation.It is closely related to the stability of voluntary movement,the control of muscle tension,and the processing of impulse information from proprioception,and participates in the formation and regulation of delicate movement.The basal ganglia regulates motor learning through two classical pathways,the direct cortex-striatum-medial pallidus and nigra reticular-thalamus pathway,and the indirect cortex-striatum-subthalamic nucleus-medial pallidus and nigra reticular-thalamus pathway.Many studies have reported the role of DNA methylation in regulation of learning and memory processes,however,its effect on motor learning is less explored.Previously,we found that conditional knockdown of mice Dnmt3a and Dnmt1 in specific population of neurons affected motor learning,suggesting that Dnmts and mediated DNA methylation participate in motor learning control.In this study,we established the conditional Dnmt3b knockout mice and MPTP-induced PD model mouse.We then combined different techniques,such as stereotactic virus injection into targeted brain region,behavioral assays,immunofluorescent staining and RT-q PCR,to further investigate the role of Dnmts in motor learning.Specifically,we aimed 1)Toinvestigate the effect of motor learning on the expression of Dnmts and Fos in different brain regions involved in motor learning.2)To investigate alteration of Dnmts expression in different brain regions of PD model mice,both in basal state and after motor learning.3)To investigate the effect of conditional Dnmt3b knockout in excitatory or inhibitory neurons on motor learning.4)To investigate the effect of conditional Dnmt（3a,1）double knockout selectively in dorsolateral striatum excitatory or inhibitory neurons on motor learning.We found that:1.Motor learning promoted the expression of Fos in multiple brain regions,including motor cortex,hypothalamus,and dorsolateral striatum.2.Motor learning led to downregulation of Dnmt3b expression in the motor cortex,hypothalamus,and striatum of WT mice.In contrast,Dnmt3a and Dnmt1 transcripts were not changed significantly.3.The PD model mice exhibited motor learning impairment.Differently from WT mice,none of Dnmt1,Dnmt3a and Dnmt3b transcripts was altered after motor learning,in either motor cortex,dorsolateral striatum,or hypothalamus of PD model mice.4.Dlx5/6-Cre;Dnmt3b^flox/floxmice showed improved motor learning.5.αCa MKII-Cre;Dnmt3b^flox/floxmice also exhibited enhanced motor learning.6.Conditional knockdown of Dnmt1 and Dnmt3a in dorsolateral striatalαCa MKII⁺neurons(AAV-Camk2a-Cre-GFP::Dnmt（3a,1）^2flox/2floxmice)did not affect motor learning.7.Conditional knockdown of Dnmt3a and Dnmt1 in dorsolateral striatal Vgat⁺neuronal(AAV-Vgat-Cre-GFP::Dnmt（3a,1）^2flox/2floxmice)suppressed motor learning.In summary,we found in this study that motor learning induces downregulation of Dnmt3b expression in related brain regions,without alterations in Dnmt1 or Dnmt3a expression..Supportively,we demonstrated that conditional knockout of Dnmt3b either inαCa MKII⁺neurons or Dlx5/6⁺neuron improved motor learning,while conditional knockdown of Dnmt3a and Dnmt1 in dorsolateral striatalαCa MKII⁺neurons did not affect motor learning.PD model mice showed motor learning impairment and the abnormal expression of Dnmts in related brain regions.Interestingly,the plasticity of Dnmt3b expression induced by motor learning disappeared in PD model mice.We thus conclude that the plasticity of Dnmt3b expression is required for motor learning,and the abnormal plasticity of Dnmt3b may contribute to motor learning deficit of PD model mice.Our findings therefore suggest that suppressing Dnmt3b expression may help to improve motor impairment associated with PD.