Research On The Regulatory Effect Of SNX32 On Epileptic Seizures And Epidemiological Investigation Of Autoimmune Encephalitis

PART ONE RESEARCH ON THE REGULATORY EFFECT OF SNX32 ON EPILEPTIC SEIZURESEpilepsy is one of the most common neurological disorders.According to statistics,there are about 70 million epilepsy patients in the world,of which about 90% are from developing countries,and there are about 10 million epilepsy patients in China.The pathogenesis of epilepsy is complex and still unclear.Although more and more antiepileptic drugs(AEDs)are being developed for clinical use,there are still a large number of patients who do not respond to multiple drug treatments.Therefore,finding new drug targets by studying the mechanism of epilepsy formation has become an important task in the prevention and treatment of epilepsy.SNX32 is a member of the sorting nexins(SNXs)family of proteins involved in the regulation of membrane trafficking in the endocytic pathway.Based on the amino acid sequence analysis and functional classification of the SNXs family,it was suggested that SNX32 is likely to be a component of the Retromer complex.Whereas the Retromer complex may be involved in the transport of receptors from the postsynaptic membrane as well as in presynaptic membrane vesicle recycling,thereby affecting synaptic transmission and regulating seizures.Objective: To explore the regulatory effect of SNX32 on epilepsy and its possible mechanism,and provide an experimental basis for the development of new antiepileptic drugs.Methods: Immunofluorescence was used to examine SNX32 localization in the human cortex as well as in the cortex and hippocampus of C57BL/6 mice;Expression changes of SNX32 in patients with refractory temporal lobe epilepsy(TLE)and two epilepsy mouse models were studied by western blotting;Adeno-associated virus was used to change the expression of endogenous SNX32,and the behavioral changes of epilepsy mouse animal models in each group were observed;Changes of EEG of mice in each group were recorded by local field potential(LFP)recording;Changes of action potential(AP)of pyramidal neurons in each group were detected by whole-cell patch clamp recordings.Using liquid chromatography-tandem mass spectrometry(LC-MS/MS)qualitative proteomics to identify the interacting proteins of SNX32 in the central nervous system,and further using high-throughput tandem mass tags(TMT)quantitative proteomics to screen differential proteins to provide a theoretical basis for the downstream mechanism of SNX32 in epilepsy.Results: Immunofluorescence indicated that SNX32 was widely expressed in the brain,mainly located in neurons rather than glial cells,and its localization did not change in epilepsy.Western blot analysis showed that the expression of SNX32 was significantly increased in the temporal lobe of TLE patients and the brain tissue of two mouse epilepsy models(P<0.01).The behavioral results of the kainic acid(KA)epilepsy model showed that overexpression of SNX32 prolonged the seizure latency(P<0.01)and reduced the number of spontaneous recurrent seizure(SRS)(P<0.05),while inhibition of SNX32 shortened the seizure latency(P<0.05)and increased the number of SRS(P<0.01).In the PTZ kindling model,overexpression of SNX32 decreased the average seizure grade(P<0.05)and improved the survival rate(P<0.05),while SNX32 inhibition increased the average seizure grade(P<0.05)and decreased the survival rate(P<0.05).The LFPs recording showed,overexpression of SNX32 reduced the seizure-like events(SLEs)frequency and total time spent in SLEs during 30 minutes(P<0.01),and inhibition of SNX32 showed the opposite effect(P<0.01).Whole-cell patch-clamp results suggested that overexpression of SNX32 decreased the frequency of APs(P<0.01)and inhibition of SNX32 increased the frequency of Aps(P<0.01)compared with the respective controls.LC-MS/MS identified 141 proteins interacting with SNX32,and GO(Gene Ontology)enrichment analysis indicated that its biological process(BP)was mainly concentrated in synapse formation;cellular component(CC)were also associated with interneuron synapses.TMT quantitative proteomics screened 726 proteins with significant differences,of which 462 were up-regulated and 264 were down-regulated.Go enrichment analysis was mainly concentrated in synapse organization,asymmetric synapse,neuron to neuron synapse,postsynaptic density,postsynaptic specialization.According to statistical power analysis,SNX5,SNX6 and VPS26 were found to be significantly different,suggesting that SNX32 may be related to Retromer complex.Conclusions: Intervention of endogenous SNX32 can modulate epileptic seizures.Overexpression of SNX32 can reduce seizure activity,and inhibition of SNX32 can increase seizure activity,which may be involved in the seizure process by affecting synaptic transmission through the Retromer complex.PART TWO EPIDEMIOLOGY OF AUTOIMMUNE ENCEPHALITISIn recent years,as more and more neuronal autoantibodies are detected and used for clinical diagnosis,and clinicians’ understanding of autoimmune encephalitis continues to spread and deepen,the incidence of autoimmune encephalitis is increasing year by year.However,there is no large-scale epidemiological survey of autoimmune encephalitis in adults and children so far,and its epidemiological characteristics are unclear.Understanding the epidemiological characteristics of autoimmune encephalitis can help us better understand its regularity,make a faster diagnosis,and improve patient outcomes.Objective: To understand the epidemiological characteristics of 189 patients who were clearly diagnosed with autoimmune encephalitis and were positive for neuronal autoantibodies in Chongqing,Southwest China,and thus to better understand the development of the disease and provide a basis for clinical decision-making and prognosis analysis.Methods: Clinical data of 189 patients who were clearly diagnosed with autoimmune encephalitis and antibody positive between January 2012 and February 2018 from 6 large general hospitals in Chongqing were reviewed.Baseline demographic characteristics were analyzed.The χ2 test,Fisher’s exact test,independent sample t test or paired sample rank sum test were used to analyze the differences in disease severity among different age and gender populations,and Spearman correlation analysis was used to clarify the correlation between antibody titers and disease severity.Results: Among the patients with antibody-positive autoimmune encephalitis,there are more female patients than male patients,and children and young adults account for the vast majority.In terms of antibody composition,anti-NMDA receptor encephalitis accounts for the vast majority.In the detection of antibodies against NMDA receptor encephalitis,the sensitivity of cerebrospinal fluid antibodies is higher than that of serum,and the titer of cerebrospinal fluid antibodies is higher than that of serum.Men and children are more likely to have seizures.Women have a better prognosis than men,and adults are more likely to develop non-NMDA receptor encephalitis than children,and adults are more likely to have tumors.In addition,cerebrospinal fluid antibody titers were positively correlated with ICU admission,ventilator use,and comorbid tumors,which reflected the severity of the disease.Conclusions: Autoimmune encephalitis in Chongqing,Southwest China,has its own epidemic characteristics.Knowing its laws and characteristics will help us better understand the disease,make a relatively quicker diagnosis,and improve the prognosis of patients.