USP47 Regulates Excitatory Synaptic Transmission In Hippocampal Neurons And Modifies Seizure Susceptibility

Part one: Localization and expression of USP47 in the central nervous system Objective: To explore the expression and localization of USP47 in the central nervous system.Methods:1.To study the developmental expression characteristics of USP47 in the central nervous system by immunoblotting.2.To invest the expression and distribution of USP47 in the central nervous system through immunofluorescence.Results:1.USP47 is abundantly expressed in mouse hippocampus and cortex at birth,and its expression level gradually decreases with maturity.2.Immunofluorescence showed that USP47 was co-expressed with neuron marker NeuN in the human cortex and adult mouse hippocampus and cortex,but not co-expressed with astrocyte marker GFAP.Besides,USP47 was mainly localized in the cytoplasm of the neuron,but no obvious localization was found in the nucleus.Conclusion: The expression level of USP47 decreased gradually with the development of the central nervous system,and it was mainly located in neurons,instead of astrocytes.Part two: Function study of USP47 in the central nervous system Objective: To investigate the possible function of USP47 in the central nervous system.Methods:1.Lentivirus carrying USP47-shRNA sequence was constructed.To knock down the expression of USP47 and evaluate the knockdown efficiency,the lentivirus was transfected in primary hippocampal neurons.2.Label-free quantitative proteomics and tandem mass spectrometry were used to identify downstream effector proteins and interacting proteins of USP47,and the acquired data were analyzed by bioinformatics software.3.Cell electrophysiology was performed to analyze the effect of USP47 on synaptic transmission in neurons.4.Immunofluorescence was used to confirm the synaptic localization of USP47.Results:1.Through bioinformatics analysis,we found that USP47 mainly exists in neuronal cytoplasm and cytomembrane,and may be involved in earlyendosome-mediated endocytosis.Meanwhile,it is mainly localized in the postsynaptic membrane and related to glutamate synaptic transmission.Furthermore,it may be involved in the regulation of AMPA receptors.2.The results of cell electrophysiology indicate that USP47 mainly affects the amplitude of miniature excitatory postsynaptic currents(mEPSCs)other than the frequency.Meanwhile,USP47 does not affect the amplitude and frequency of miniature inhibitory postsynaptic currents(mIPSCs).3.The results of Immunofluorescence suggested that USP47 was mainly co-located with the excitatory postsynaptic marker PSD95,but not with the presynaptic marker Synaptophysin.Conclusion: USP47 is mainly located at the postsynaptic membrane of neuron and is involved in glutamatergic excitatory synaptic transmission.Part three: Study of the mechanism of USP47 modulates AMPA receptor Objective: To invest the interaction of USP47 and AMPA receptor and to detect the mechanism of USP47 regulating AMPA receptor.Methods:1.The hippocampal tissue of mice was extracted and the binding of USP47 with glutamate receptor was verified by co-immunoprecipitation.2.The pcDNA3.1-mUSP47-Flag plasmid was co-transfected respectively with pCI-SEP plasmid vectors containing GluR1、GluR2(R)、NR1、NR2Aor NR2 B into HEK293 cells,and the binding of USP47 with glutamate receptor was verified by co-immunoprecipitation.3.Immunoblotting was conducted to verify the effect of USP47 on AMPAR protein expression.4.Immunofluorescence was carried out to detect the co-localization of USP47 with the protein markers of organelle involved in receptor trafficking.Results:1.In vivo co-immunoprecipitation confirmed that USP47 can bind with AMPAR subunits(GluR1 and GluR2)and NMDAR subunits(NR1,NR2 A and NR2B),that is,USP47 is a member of the complex receptors.2.In vitro co-immunoprecipitation was used to confirm the direct binding of USP47 with AMPAR subunits(GluR1 and GluR2)and NMDAR subunits(NR1,NR2 A and NR2B).3.Reducing the expression of USP47 can reduce the total protein expression levels of AMPAR subunits(GluR1 and GluR2)in primary neurons.4.USP47 was mainly co-located with the marker EEA1 of early endosome(involved in the endocytosis of membrane receptor)and the marker Lamp2 of lysosomal(involved in protein degradation,and membrane AMPAR is mostly degraded by lysosomal pathway),but not with the late endosome marker Rab7 and the recycling endosome marker Rab11.Conclusion: USP47 may directly interact with the AMAPR subunits GluR1 and GluR2 and regulate the total protein expression level of AMPAR.Also,it was found that USP47 may mediate the endocytosis of AMPA receptor and affect its degradation by the lysosomal pathway.Part four: Expression of USP47 in the epileptic brain tissue and its effect on seizure susceptibility Objective: To investigate the expression of USP47 in epilepsy and its effect on seizure susceptibility.Methods:1.Samples of temporal lobe cortex were collected after surgical excision of refractory epilepsy as the epilepsy group and samples of the temporal lobe cortex after craniocerebral decompression after brain injury as the control group.Human brain tissue samples were collected to verify the differential expression of USP47 in human cortex.2.The pentylenetetrazol-kindled mouse model was established to verify the differential expression of USP47 in the cortex and hippocampus of the mice.3.Lentivirus was stereotactically injected into the hippocampus of mice to evaluate the effect of USP47 knockdown on seizure susceptibility of the mice.Results:1.Expression of UPS47 was increased in the human cortex as well in hippocampus and cortex of kindled mice.2.After the knockdown of USP47,the latency of seizure was prolonged,meanwhile,the duration of grade 4-5 seizures was shortened and the number of seizures of grade 4-5 seizures was reduced on days 14-28.Conclusion: The expression of USP47 was increased in patients with epilepsy and PTZ-kindled mice,and knockdown of USP47 can reduce susceptibility to seizures.