The Roles And Mechanisms Of GPR30 In The Epileptogenesis Of Female Patients With Focal Cortical Dysplasia Ⅱb And Tuberous Sclerosis Complex

Malformations of cortical development（MCD）are the major cause of refractory epilepsy.Focal cortical dysplasia IIb（FCDⅡb）and tuberous sclerosis complex（TSC）are typical MCD with similar pathological characteristics,such as dysmorphic neurons（DN）,bright eosinophilic giant cells（GC）,and balloon cells（BC）.Resected epileptogenic focus of FCDⅡb and TSC patients exhibit increased expression of glutamate transporters,NMDA receptors,and proinflammatory factors.Increased neuronal excitability and activated inflammation ultimately lead to epileptogenesis in patients with FCDⅡb and TSC.However,the detailed molecular mechanisms underlying the epilepsy of patients with FCDⅡb and TSC remain unclear.Estrogen is a crucial regulator of neurodevelopment,neuronal excitability,and neuroinflammation in the central nervous system（CNS）.Clinical evidence and animal experiments have shown that estrogen has a proconvulsant effect.Estrogen acts through nuclear receptors（estrogen receptor ERαand ERβ）and G protein-coupled receptor 30（GPR30）.ERαand ERβmediate the genomic effects of estrogen,and ERαhas been reported to increase seizure susceptibility.GPR30 is a membrane receptor that regulates the non-genomic effects of estrogen,including kinase activation,calcium mobilization,and nitric oxide production.It is reported to be two to four times more abundant than ERαor ERβin the rat prefrontal cortex and binds estradiol with a higher affinity than ERαand ERβ.Moreover,GPR30 is selectively activated by the 17αand 17βisomers of estradiol,but not bind other steroids,including testosterone,progesterone,or cortisol.GPR30 has been reported to regulate neuronal excitability in myelinated vagal afferent neurons and reduce the release of TNF-α,IL-1β,and IL-6 in microglia,but the role of GPR30 in the epileptogenesis of FCDⅡb and TSC patients is still unknown.To explore the effect of GPR30 in the epilepsy of FCDⅡb and TSC patients,we detected the expression and distribution of GPR30,analyzed the correlation between GPR30expression and clinical variables,and explored the expression of the downstream PKA signaling pathway.In addition,we also explored GPR30-mediated NF-κB inflammatory signaling and cortical neuronal excitability.Furthermore,the correlation between GPR30 and¹⁸F-FDG PET-CT standardized uptake values（SUVs）were analyzed in FCDⅡb and TSC patients,and the potential predictive value of SUV on epileptogenic tubers was assessed in female patients with TSC.Main results were listed as follows:1.Expression and distribution of GPR30 in female patients with FCDⅡb and TSC1.1 The RNA and protein levels of GPR30 were examined in surgical specimens from the FCDⅡb,TSC,and control groups.For specimens from male patients,there were no significant differences in the RNA and protein levels of GPR30 compared with controls.However,there were significantly decreased GPR30 RNA and protein levels in the specimens from female FCDⅡb and TSC patients compared with controls.Next,the correlations between GPR30 expression and different clinical variables of female patients were assessed.There were significant inverse correlations between GPR30 expression and seizure frequency in female patients with FCDⅡb and TSC.1.2 The distribution of GPR30 in specimens from female patients and controls was evaluated by immunohistochemistry（IHC）and immunofluorescence（IF）.GPR30 expression were found in both DN and BC.In addition,the mean optical density of GPR30 was decreased in female patients compared with controls.IF revealed that GPR30 was widely distributed in the neurons,astrocytes,and microglia of female patients and controls,and GPR30 expression was significantly decreased in the microglia.2.Expression of PKA signaling pathway in Female Patients with FCDⅡb and TSCIHC was used to evaluate the immunostaining of GPR30 downstream PKA signaling pathways in the specimens of female patients with FCDⅡb and TSC.PKA and p-PKA staining were observed in DN and BC.Furthermore,quantitative analysis of PKA signaling pathways was performed using western Blot.The expression of PKA and p-PKA was decreased in FCDⅡb and TSC compared with controls.In addition,GPR30 expression was positively correlated with the expression of PKA and p-PKA in FCDⅡb and TSC.We found that seizure frequency was negatively correlated with PKA and p-PKA expression in FCDⅡb and TSC.3.Inflammation in Female Patients with FCDⅡb and TSCNext,we explored the expression of microglia and the activation of the NF-κB-mediated inflammatory pathway in female patients with FCDⅡb and TSC.IF revealed there were more Iba1-positive microglia in FCDⅡb and TSC than controls.IHC showed both DN and BC were immunoreactive for NF-κB.Besides,NF-κB signaling pathway was activated in female patients compared with controls.RNA levels of the NF-κB mediated inflammatory factors（IL-1b,IL-6,and TNF-α）were also increased in female patients.The correlation between NF-κB and GPR30 expression and seizure frequency was evaluated,and we found that NF-κB expression was negatively correlated with GPR30 expression and positively correlated with seizure frequency.4.Effects of G-1 and G-15 on the GPR30 Signaling PathwaysGPR30 regulation of the PKA and NF-κB signaling pathways was detected in cultured cortical neurons after treatment with DMSO,GPR30 agonist（G-1）,or GPR30 antagonist（G-15）.G-1 treatment increased the expression of PKA and p-PKA,whereas G-15 treatment decreased the expression of PKA and p-PKA.In addition,NF-κB expression was decreased in cortical neurons treated with G-1,and increased in cortical neurons treated with G-15.These results suggested that GPR30 regulates the PKA and NF-κB signaling pathways.5.Role of GPR30 in Cortical Neuronal ExcitabilityTo explore the role of GPR30 in cortical neuronal excitability,we evaluated the s EPSCs and s IPSCs of cortical neurons treated with G-1 or G-15 in our in vitro epileptic model.The s EPSC frequency was decreased after G-1 treatment and increased after G-15 treatment in the in vitro epileptic model,but the s EPSC amplitude was not affected by either G-1 or G-15treatment.Both the frequency and amplitude of s IPSC were not affected by G-1 or G-15treatment in the in vitro epileptic model.These results indicate that GPR30 regulates excitatory synaptic transmission.Therefore,we detected the expression of NR2A and NR2B in cultured cortical neurons treated with G-1 or G-15.G-1 treatment decreased the expression of both NR₂A and NR₂B,whereas G-15 treatment increased the expression of both NR₂A and NR₂B.6.Correlation between GPR30 and the SUV in Patients with FCDⅡb and TSC¹⁸F-FDG PET-CT can reflect glycometabolism by the maximum and mean standardized uptake values（SUVmax and SUVmean）,and GPR30 has been reported to regulate glycometabolism.We found that the SUV were positively correlated with GPR30 expression in female patients with FCDⅡb and TSC.Female patients who had significantly increased premenstrual seizure frequency were further selected,and we found a strong positive correlation between GPR30 expression and SUV in those with FCDⅡb and TSC.In contrast,we found no significant correlation between the SUV and GPR30 in all patients and in male patients with FCDⅡb and TSC.7.Expression and Correlation of GPR30 and the SUV in the Epileptogenic Tubers of the Female Patients with TSCWe assayed the GPR30 protein levels in epileptogenic tubers（n=11）and non-epileptogenic tubers（n=13）obtained from female patients with TSC by western Blotting and found that GPR30 expression was significantly decreased in epileptogenic tubers.In addition,GPR30 expression was positively correlated with SUV in the epileptogenic tubers,but not in the non-epileptogenic tubers.Notably,epileptogenic tubers had decreased SUV（n=11）compared with non-epileptogenic tubers（n=30）.The ROC curve demonstrated that both SUVmax and SUVmean could predict the location of epileptogenic tubers,and that SUVmax was a better predictor than SUVmean.Therefore,SUV could be used as a marker to predict epileptogenic tubers.Conclusions:In summary,we found that GPR30 potentially affected epileptogenesis by modulating neuroinflammation and neuronal excitability in female patients with FCDIIb and TSC.In addition,we found that decreased GPR30 expression was positively correlated with hypo-glycometabolism in the epileptogenic foci of female patients with FCDIIb and TSC by analyzing the correlation between GPR30 and PET-CT SUV.Intriguingly,both GPR30expression and SUV were decreased in the epileptogenic tubers of female TSC patients,and SUV could be used to predict the location of epileptogenic tubers.Therefore,our results offered a potential therapeutic strategy for female patients with FCDIIb and TSC,and showed that PET-CT SUV might be a noninvasive marker to reflect GPR30 expression and the localization of epileptogenic tubers in female patients.However,there were some limitations in this study.The number of human participants in each group was barely acceptable,limiting the universality of our results.Moreover,the specific molecular indicator of PET should be synthesized to image GPR30 and confirm our results in this study.