VGLUT1 Novel Binding Partner Screening And APP Regulates The Expression Of VGLUT1

Glutamate is the most important excitatory neurotransmitter in the central nervous system(CNS)of mammals,and it involves in long-term potentiation(LTP)and longterm depression(LTD),which is the molecular basis of learning and memory.Therefore,strict regulation of glutamate concentration in the brain is inevitably necessary to the balance of the important role of glutamate in life support and its harmful effects.While type I vesicular glutamate transporter 1(VGLUT1)is responsible for 80% of the total vesicular glutamate uptake in the adult central nervous system,so the regulation of VGLUT1 is very important.The regulation of VGLUT1 activity,transcription and translation depends on the molecules interacting with VGLUT1.In this study,we studied the isolation and preparation of rat synaptic vesicles(SV),recombinant expression of full-length twelve-transmembrance VGLUT1 in vitro,screening and verification of VGLUT1 interactome in synaptic vesicles,and regulation of VGLUT1 expression by the candidate VGLUT1 interacting proteins.Specifically,we improved the current method at first to optimize the yield and purity of synaptic vesicles(SVs)and got higher yield than that reported in the literature in a shorter time,and the size and purity of the of SVs obtained could meet the requirements of our follow-up research.In the meantime,we aslo optimized the recombinant expression conditions of full-length VGLUT1 in vitro,and finally determined to use the HIS tag,while the induction conditions were determined as 23 ℃and 0.4m M isopropyl-β-D-thiogalactoside(IPTG)induction for 4 hours(hr).Western Blot confirmed that the recombinant VGLUT1 protein extract needed for the subsequent study of VGLUT1 interactome in synaptic vesicles was obtained.In order to obtain VGLUT1 interactome in synaptic vesicles as comprehensively as possible,we used Pull-Down technique and co-immunoprecipitation(Co-IP)technique to screen VGLUT1 interacting proteins in situ against recombinant expressed VGLUT1 and synaptic vesicle protein extract in vitro,and 255 and 225 VGLUT1 interacting proteins were obtained respectively.Then,the Membrane Yeast-TwoHybrid(MYTH)technique was used to screen VGLUT1 interacting proteins against rat brain c DNA library,and 215 VGLUT1 interacting proteins were obtained.Among those interacting proteins obtained,4 of them were detected by Pull-Down and MYTH,and there were 6 proteins identified by Co-IP and MYTH.While only 2 of them were detected by Pull-Down,Co-IP and MYTH.And,surprisingly,only MYTH also detected the interaction between amyloid precursor protein(APP)and VGLUT1,while none of these methods detect the reported VGLUT1 binding partner Endophilin-A1.After comprehensive analysis,we finally used Co-IP to verify the interaction of 9 proteins including Lynx1,Stx12,SV2 A,Gja1,Atp6ap2,EMD,Cox4i1,APP and EndophilinA1 with VGLUT1 in situ against the SV protein extract,and confirmed the interaction of 5 proteins including Stx12,SV2 A,Gja1,Atp6ap2 and APP with VGLUT1.Subsequently,the effect of the above 5 proteins on VGLUT1 expression were verified by gene knockout experiments in PC12 cells.The results showed that only APP could negatively regulate the transcription and translation of VGLUT1.In order to further confirm the regulation of APP on VGLUT1 expression,we further verified this negative regulation by APP gene knockout and overexpression in PC12 cells.And results of animal experiments also confirmed the negative regulatory effect of APP on VGLUT1,the expression levels of VGLUT1 m RNA and protein were significantly upregulated in the cerebral cortex and hippocampus of APP knockout mice,while significantly down-regulated in the cerebral cortex and hippocampus of 2-month-old and 8-month-old APP overexpression mice.In conclusion,this study found and confirmed the interaction between APP and VGLUT1,and APP can negatively regulate the transcription and translation of VGLUT1.In conclusion,this study is the first to use synaptic vesicles to study the VGLUT1 interactome,and also for the first time we found and confirmed the interaction between APP and VGLUT1,which nagetively regulated the VGLUT1 transcription and translation.