Role Of NME2,A Histidine Phosphorylation Kinase,in Anxiety—Like Behaviors In Mice

Phosphorylation is a common post-translational modification of proteins.Protein kinases catalyze the covalent attachment of a phosphate group to the serine,threonine or tyrosine residues of the substrate.Phosphorylation is the most common mechanism of regulating protein function and transmitting signals throughout the cell,playing critical roles in regulating many cellular processes,including cell cycle,growth and apoptosis.In addition to the formation of stable phosphoester bonds（P-O bonds）on the hydroxyl group of serine,threonine or tyrosine,phosphorylation can also form high-energy but unstable phosphoramidate bonds（P-N bonds）on histidine’s imidazole group,which is called histidine phosphorylation.Histidine phosphorylation plays a key role in signal transduction in lower prokaryotes,but its roles in mammalians are still unclear.NME1（non-metastatic）and NME2 are two histidine kinases in mammalian cells.So far,it has been found that NME2 can phosphorylate the ion channels,including KCa3.1 and TRPV5,and G proteinβsubunit GNB1 in non-neural cells to regulate their functions.However,little is known about the roles of NME2 in the nervous system.To this end,we first detected the expression of NME2 in the brain by Western blotting and found NME2 was expressed abundantly in different brain regions,as well as at different developmental stages.Moreover,by PSD（postsynaptic density）fractionation assay,NME2 was found to be distributed only to the presynaptic area,but not PSD.Next,we obtained NME2 knockout mice(Nme2^-/-)to explore the roles of NME2in the brain.Nissl staining and immunostaining with anti-Neu N antibody showed that gross brain morphology,structure and density of neurons were not changed in Nme2^-/-mice.In the open-field test（OFT）,Nme2^-/-mice traveled similar distances but spent less time in the central area than wild type（WT）mice.And in the elevated plus maze（EPM）,Nme2^-/-mice spent less time in opened arms than WT.These results suggest NME2 deficiency leads to anxiety-like behaviors.The medial prefrontal cortex（mPFC）is a main component of the limbic system,and abnormal function or structure in mPFC may cause anxiety.Therefore,we first recorded the action potentials of pyramidal neurons in the prelimbic cortex（Pr L）of mPFC,and found no change between Nme2^-/-and WT mice.Then the spontaneous excitatory postsynaptic currents（s EPSCs）and miniature excitatory postsynaptic currents（m EPSCs）were recorded,and the frequencies of s EPSCs and m EPSCs were decreased in Nme2^-/-mice,while the amplitudes were not altered.These results indicate NME2 deficiency results in decreased glutamatergic transmission.NME2 was systemically knocked out in Nme2^-/-mice.We wonder if the anxiety-like behaviors in Nme2^-/-mice were resulted from NME2 deficiency in mPFC.We packaged adeno-associated viruses（AAV）expressing small hairpin RNA against NME2（AAV-sh-NME2）and injected them stereotactically into WT mPFC to knockdown NME2 expression.Compared with the control virus（AAV-sh-ctrl）,the mice injected with AAV-sh-NME2 exhibited anxiety-like behaviors in OFT and EPM,as well as decreased frequencies of s EPSCs and m EPSCs.These data demonstrate NME2 deficiency in mPFC induces anxiety-like behaviors and abnormal glutamatergic transmission.So,if these deficits in Nme2^-/-mice could be rescued by overexpressing NME2 in mPFC?Finally,we packaged AAV overexpressing NME2（AAV-o/e-NME2）and injected them stereotactically into the mPFC of Nme2^-/-mice.Compared with the control virus（AAV-o/e-ctrl）,injection of AAV-o/e-NME2 in Nme2^-/-mice could alleviate the anxiety-like behaviors in OFT and EPM.And the decreased frequencies of s EPSCs and m EPSCs in Nme2^-/-mice were also be restored by overexpressing NME2.These results furtherly suggest the anxiety-like behaviors and decreased glutamatergic transmission in Nme2^-/-mice are caused by the NME2 deficiency in mPFC.Taken together,our study found histidine kinase NME2 regulates glutamatergic transmission of pyramidal neurons in mice mPFC,and its deficiency in mPFC induces anxiety-like behaviors.These findings provide new clues and evidence for the roles of NME2 and histidine phosphorylation in the brain.