Neuroplastin Inhibits The Internalization Of GABA Type A Receptors And Exerts Antiepileptic Effects By Binding To The α1 Subunit Of GABA Type A Receptors

Objective Status epilepticus（SE）is one of the most common neurological emergencies,with a mortality rate of up to 20%.Studies have shown that SE is related to the imbalance of excitatory/inhibitory function（E/I）of the nervous system,and the reduction of GABAa Rs on the membrane surface caused by the internalization ofγaminobutyric acid Type A receptor（GABAa Rs）is the key factor causing E/I imbalance.The amount of GABAa Rs on cell membrane surface may be related to postsynaptic protein interactions such as Rho guanine nucleotide Exchange factor 9（ARHGEF9）and Gephyrin（GPN）.These two proteins mainly bind to theαsubunit to inhibit receptor internalization,but they bind more strongly to theα2-containing receptor（Gabra2）,conversely,they bind less to the largest number ofα1-containing receptor（Gabra1）,so Gabra1 has a weaker anti-epileptic ability.However,it is not clear how Gabra1 participates in the pathogenesis of SE.Studies have shown that Neuroplastin（Nptn）is involved in the formation of GABAa Rs synapses by regulating theαsubunit,but whether Nptnis involved in the pathogenesis of SE remains unclear.This project intends to study the expression changes of Nptnin SE and the regulation mechanism of Gabra1.Methods（1）twelve SD male rats were randomly divided into control group（Ctl）and SE group（6 rats in each group）.The control group was intraperitoneally injected with normal saline,and the SE group was intraperitoneally injected with PTZ to induce SE.Animal behavior was observed and EEG was recorded.Western blot was used to detect the expression of Nptn and Gabra1 in hippocampal total protein and membrane protein.（3）Establishment of adeno-associated virus（AAV）animal model:12 rats were randomly divided into negative control group（AAV-NC）and Nptnoverexpression group（AAV-OE-Nptn）.AAV was injected into hippocampus to construct vector transfection model.Four weeks later,the transfection situation of the two groups of rats was detected.The expression of Nptnwas detected by Western blot.（4）The SE model of AAV was established.The RATS in the two groups were injected intraperitoneally with AAV,respectively,and the EEG data,SE latency time and duration of epileptic seizures of grade 4 and above were recorded.（5）Western blot was used to detect the expression of Nptnand Gabra1 in total protein and membrane protein.（6）The interaction between Nptnand Gabra1 was detected by immunoprecipitation（COIP）.Pearson correlation coefficient（PCC）between Nptnand Gabra1 was detected by immunofluorescence co-location.（7）Construction of vitro SE model:Construction of epileptic cell model:Neurons were divided into Ctl group The neurons were divided into Ctl group and magnesium free group（Mg2+free）.The neurons were exposed to normal extracellular fluid or magnesium free extracellular fluid for 3 h,respectively.The spontaneous action potentials（SAPs）,action potentials（APs）and miniature inhibitory postsynaptic currents（m IPSCs）of the neurons were detected by patch clamp.The internalization rate of Gabra1 in each group was detected and compared.（8）Lentivirus（LV）neuron model was constructed and divided into negative control group（LV-NC）and Nptn overexpression group（LV-OE-Nptn）.After lentivirus transfection,neurons were internalized and their internalization rates were compared.（9）The transfected neurons were exposed to magnesium-free extracellular fluid for 3h,and the APs and m IPSCs of the two groups of neurons were detected and compared.Results（1）PTZ can induce SE:in SE group,all 6 rats showed recurrent epileptic seizures and met SE criteria.EEG showed repeated epileptic discharges,multispinous slow wave discharge during seizures,and low flat wave after each seizure.（2）SE reduced the expression of Nptnand Gabra1 on cell surface:the relative gray value of Nptnof total protein was 0.57±0.12 in Ctl group and0.04±0.01 in SE group（P=0.009）.The relative gray value of Nptnof membrane protein was:Ctl group was 0.71±0.51,SE group was 0.02±0.01（P=0.023）.The expression of Gabra1 of total protein in SE group was not significantly changed,and the relative gray value of cell surface was 6.02±3.56 in Ctl group and2.35±1.53 in SE group.（3）Nptn had antagonistic effect on PTZ-induced SE:Incubation period,AAV-NC was 76.15±19.31s,AAV-OE-Nptn was 115.50±21.01s,P=0.007)（Figure.2E）.Duration of epileptic seizures at stage 4 and above:It was 1025±601.03s in AAV-NC group and 264.83±220.33s in AAV-OE-Nptngroup,P=0.016).（5）Overexpression of Nptn could increase the expression of Gabra1 on cell surface:the relative gray value of total protein Nptnwas 0.11±0.02 in AAV-NC group and 0.41±0.10 in AAV-OE-Nptn group（P=0.002）.The relative gray value of surface protein Nptn was 0.57±0.01 in AAV-NC group and 1.38±0.34 in AAV-OE-Nptn group（P=0.00）.（6）SE weakened the correlation between Gabra1 and Nptn,and the relative gray value of Nptn dropped by Gabra1 decreased（Ctl group was 0.76±0.39,SE group was0.35±0.21,P=0.04）.PCC decreased（Ctl group 0.75±0.04,SE group 0.63±0.04,P=0.000）.（7）Overexpression of Nptn can promote the interaction between Gabra1 and Nptn,and the relative gray value of Nptn decreased by Gabra1increased（AAV-NC was 0.26±0.06,AAV-OE-Nptn was 0.83±0.42,P=0.03）.（8）（8）Neurons exposed to magnesium-free extracellular fluid showed increased excitability and decreased inhibitory function:The frequency of SAPs in the Ctl group was 0.50±0.50Hz and the amplitude was 23.76±22.18m V,while in the Mg²⁺-free group,multiple SAPs were released with the frequency of42.10±21.83Hz（vs.Ctl,P=0.013）and the amplitude was 124.23±9.95m V（vs.Ctl,P=0.013）.P=0.00);The APs frequency was 3.00±1.58Hz and the amplitude was 54.40±6.30m V in Ctl group,and the amplitude was 172.80±19.70m V（vs.Ctl,P=0.036）in Mg²⁺-free group.Neuron m IPSCs:The amplitude of m IPSCs in Ctl group was 23.36±9.24p A and the frequency was 0.78±1.04Hz,while the amplitude of m IPSCs in Mg²⁺-free group was 7.54±1.12p A（vs.Ctl,P=0.018）and the frequency was 0.37±0.11Hz（vs.Ctl,P=0.40）.Conclusion The interaction between Nptn and Gabra1 is the key factor to inhibit the internalization of Gabra1 and increase the stability of Gabra1 in cell membrane,which promote the nerve inhibition function and play an antiepileptic role.