Preliminary Functional Study Of Neuroligin(NL)/APPL1 Complex

Background Neuroligin(NL)is a postsynaptic adhesion protein which play a key role in the regulation of excitatory and inhibitory synapse.Consistent with its localization,Neuroligin 1 and Neuroligin 3 induce excitatory synapse formation while neuroligin 2 exclusively induce inhibitory synapse formation.Dysfunctions of the NL proteins such as deletion or point mutation have been found involved in autism spectrum disorders(ASD).Mice with the reported deletions or mutations of NLs also show autism-like behaviors.Adaptor protein containing PH domain,PTB domain and Leucine zipper motif(APPL)is an adaptor protein,including APPL1 and APPL2.APPL1 can regulate dendritic spine and synapse formation in hippocampal neurons through APPLl/Akt signaling complex.Co-Immunoprecipitation/MS analysis results indicate that NLs have an interaction with APPL1.Objectives Verify the interaction between NLs and APPL1 in vitro and in vivo and further explore the molecular mechanism of NLs and APPL1 in synapse formation.Methods Using cell transfection,Co-Immunoprecipitation,viral-mediated shRNA knockdown,western blot,immunocytochemistry staining and fluorescence imaging analysis to verify the interaction between NLs and APPL1 and further explore the molecular mechanism of NLs and APPL1 in synapse formation.Using western blot,immunocytochemistry staining,immunohistochemical staining and fluorescence imaging analysis to test the infection efficiency of NL-AAV.Results NLs can interact APPL1 in vitro and in vivo.The transfected HA-NL1 can colocalize with endogenous APPL1 in cultured hippocampal neurons.NL1/APPL1/PSD95/GluN2A form a complex in cultured neurons.In cultured hippocampal neurons,there is a decrease(p<0.05)or an increase(p<0.05)in pAkt473 phosphorylation level after overexpression or knockdown of NL1 and there is a decrease(p<0.05)in pAkt473 phosphorylation level after overexpression of APPL1 while there is no change in Akt expression level.There is also a decreased pAkt473 phosphorylation level in total brain,cortex and cerebellum of NL1 KO mouse.NL-AAV can infect cultured neurons effectively while no effective for in vivo infection.Conclusion These results suggest that NL1 may regulate the synapse formation through APPL1/Akt signaling complex.