GluN2B Tyrosine 1070 Phosphorylation Regulates Depression-related Behaviors By Affecting The Function Of Extrasynaptic NMDA Receptors

Depression is a serious threat to human life and health.This stress-related emotional disorder has caused a very high rate of disability and suicide,making society and families bear a huge economic burden and mental loss.Both clinical and basic studies have shown that glutamate system the main excitatory neurotransmission system in the brain plays an important role in the regulation of depression.Especially in recent years,many studies have reported that Ketamine has a rapid and long-lasting effect on alleviating the symptoms of depression patients and depressed animals,when it be used at a subanaesthetic dose.This provides a strong research support for the subsequent treatment of depression and the development of antidepressants.The previous studies have shown that Ketamine plays an antidepressant role by blocking ionic glutamate NMDARs,although previous studies believe that Ketamine is a non-selective NMDARs channel blocker,but a growing body of research suggests that the antidepressant effect of Ketamine is the specificity of blocking the Glu N2B-containing NMDARs.There are currently two hypotheses regarding the antidepressant effect of inhibiting Glu N2 B.One is the direct inhibition hypothesis.The theoretical basis of this hypothesis is that the glutamate level is increased in some brain regions of depression patients and animals,such as m PFC,which indicates that the activity of extrasynaptic NMDA receptors is enhanced,and Ketamine inhibits the extrasynaptic receptors to play an antidepressant role.Another disinhibition hypothesis suggests that low-dosed Ketamine inhibits the Glu N2 Bcontainig NMDARs on interneurons,so that pyramidal neurons are de-inhibited and the relative excitatory synaptic transmission is enhanced,thereby playing an antidepressant effect.This suggests that Glu N2B-containing NMDARs play a very important role in the regulation of depressive disorders,but the specific molecular mechanism remains to be explored.It is known that the membrane trafficking and synaptic localization of Glu N2 B subunits are regulated by phosphorylation modification,endoplasmic reticulum retention signals,and microtubule motor protein interactions,among which tyrosine phosphorylation modification regulation is particularly important.Many studies have shown that the tyrosine phosphorylation of the Glu N2 B subunit C-terminus can affect the channel activity or receptor trafficking.Among them,the tyrosine 1472 site is located in an internalization motif(YEKL motif)of Glu N2 B,regulating the surface expression of NMDARs,and then participating in the pathophysiological processes,such as synaptic plasticity,excitotoxicity and pain.Previous studies in our laboratory have reported that the phosphorylation of tyrosine 1070 of Glu N2 B can coordinately regulate the phosphorylation of tyrosine 1472 and facilitate surface expression of the receptors in vitro,but the regulation and function of the tyrosine 1070 in vivo are still unclear.Using CRISPR-Cas9 technology,we obtained and bred Glu N2 B Y1070F KI mice.Through behavioral testing,we found that KI mice exhibited obvious antidepressant behaviors at the basic level and after chronic restraint stress(CRS)exposure.However,the behaviours related to the hippocampus and amygdala were not affected in the KI mice,showing a completely different behavioral phenotype from that of the 1472 KI mice.The biochemical results showed that the phosphorylation level of tyrosine 1070 site in the m PFC of WT mice were increased significantly after 6-min FST and CRS exposure,indicating that the phosphorylation of tyrosine 1070 participates in the regulation of depression-related processes.Interestingly,we found that the basic level of tyrosine 1472 phosphorylation was decreased significantly in the m PFC of the KI mice,but not in other brain regions we detected.Further studies showed that the synaptic receptor function of pyramidal neurons in the layer 5 of m PFC was not affected by the Y1070 F mutation,but the function of extrasynaptic Glu N2B-containing NMDARs was down regulated.Furthermore,we found that the m TORC1 activity was increased in the m PFC ofthe KI mice,the number of excitatory synapses of layer 5 pyramidal neurons was increased,suggesting that the antidepressant-like behaviors the KI mice may be mediated by down regulating the extrasynaptic NMDARs.Taken above results together,we reveal a new mechanism for regulating NMDARs functions via Glu N2 B subunit tyrosine phosphorylation in m PFC and mediating depressive behaviours,which provides a new idea for the treatment of depression.