A Study Of The Tyrosine Phosphotase Shp2 Regulating The Tyrosine Phosphorylation Of NMDAR And Synaptic Plasticity

Src homology 2 domain containing phosphotyrosine phosphatase 2(Shp2),is a highly evolutionary conserved non-receptor tyrosine phosphatase.About 50 percent of the Noonan syndrome(NS)is caused by gain-of-function mutation in Shp2,while about 80 percent of LEOPARD syndrome(LS)is caused by loss-of-function mutation.These two diseases are both developmental disorders,and a certain percentage of the patients showed mental retardation.Although there are reports that Shp2 regulates downstream Ras/ERK MAP kinase signaling in memory processes,however,the underlying mechanism is still poorly understood.It is reported that Shp2 is in N-methyl-D-aspartate receptor(NMDAR)complex,and there is evidence that Shp2 directly bind to GluN2B subunit of NMDAR.NMDAR,which mainly mediates Ca2+,is one of the excitatory ionotropic glutamate receptors.NMDAR is a tetramer that usually composed of two obligatory G1uN1 and two modulatory G1uN2 or G1uN3 subunits.It opens only when receiving glutamate released from presynapse and sensing postsynaptic depolarization,and it plays an important role in synaptic plasticity and memory.Based on the information mentioned above,we crossed Shp2 FloxP/FloxP mice with CaMKⅡα-Cre mice to generate Shp2 forebrain knockout mice(Shp2 FloxP/FloxP:CaMKIIa-Cre +/-,CaSKO),and took advantage of these mice to study whether Shp2 regulated the phosphorylation of NMDAR subunits and further synaptic plasticity and the learning and memory.Immunoblot analysis showed that,the phosphorylation level of tyrosine at 416 of Src(Src Y416),which represented its activity,was increased in the hippocampus of CaSKO mice.Accordingly,the phospohrylation level of tyrosine site at 1472 in GluN2B(GluN2B Y1472)and tyrosine site at 1325 in GluN2A(GluN2A Y1325)were also markedly enhaced.Electrophysiological analysis indicated that excitatory synaptic transmission in hippocampal CA1 pyramidal neuron of CaSKO mice was also increased along with NMDAR mediated currents.Our analysis further revealed that the increased synaptic NMDA receptors in CaSKO mice were triheteromeric GluN1/GluN2A/GluN2B.We also found impairment of field recording of long-term potentiation(LTP)in hippocampal CA1 of CaSKO mice,and this was due to the increased sensitivity of NMDAR to zinc inhibition.In addition,we found that long-term contextual fear memory of CaSKO mice was decreased without any change in the formation and short-term memory.In hippocampus of WT mice,the phosphorylation level of Src Y416 and GluN2B Y1472 after contextual fear memory was elevated.However,in CaSKO mice the phosphorylation level of them showed no further increase after fear memory test.Using Src kinase inhibitor PP2 treatment in CaSKO mice,we found that all the phenomenones seen above in CaSKO mice were reversed when the activity of Src was restored.Taken together,we conclude that Shp2 deficiency causes increased Src kinase activity and results in increased levels of phosphorylation of NMDA receptor subunits that promotes NMDARs expression at synapses with increased functional synapses.The saturated synatic function occludes LTP induction,and further represses the long-term contextual fear memory.Our finding provides a new insight to understand the role of Shp2 in synaptic plasticity and mental retardation in NS and LS.