Studies Of Molecular Pathology And Biomarker In Drug-refractory Epilepsy

PART ONE: UP-REGULATION OF SERUM- AND GLUCOCORTICOID- INDUCED PROTEIN KINASE 1 IN THE BRAIN TISSUE OF HUMAN AND EXPERIMENTAL EPILEPSYPurpose: Several studies have shown that serum- and glucocorticoid-induced protein kinase 1(SGK1) can regulate both glutamate receptors and glutamate transporters and may participate in the regulation of neuroexcitability in neuronal diseases. In our previous study, we analyzed differential gene expression in the anterior temporal neocortex of drug-refractory epilepsy patients relative to control patients by using a complementary DNA microarray and found that the SGK1 gene was up-regulated more than two-fold in the brain tissues of epileptic patients. In the current study, we measured SGK1 expression in the brain tissues of humans and in an experimental model of rat epilepsy in order to explore the relationship between SGK1 and epilepsy.Methods: The SGK1 expression was detected in thirty human brain tissues derived from patients undergoing operation for drug-refractory epilepsy and was also detected in eight samples from autopsies using RT-PCR, immunostaining and western blot analysis. Meanwhile, we investigated SGK1 and EAAT3 expression during the epileptic process in Lithium- pilocarpine induced rats and in rats after knockdown SGK1 via lentivirus vector.Results: SGK1 expression was detected in neurons and located in cytoplasm. SGK1 expression was enhanced in the temporal neocortex of patients with drug-refractory epilepsy. In rat model, SGK1 mRNA and protein was up-regulated after the onset of seizure, reached peak expression at the 24 h time point and last higher levels during different phases of the epileptic process. SGK1 expression was also related with the elevation of EAAT3, which expression reduced after knockdown SGK1.Conclusions: These results provide new insight into the potential role of SGK1 in the pathophysiology of epilepsy. The up-regulation of SGK1 may serve to stimulate EAAT3 and thus to reduce neuroexcitotoxicity after seizure.PART TWO: THE CSF AND SERUM CONCENTRATION OF APOE AND TETRANECTIN IN EPILEPTIC PATIENTSPurpose: The previous proteomics study showed identified ApoE and tetranectin (TN) in cerebrospinal fluid (CSF) of epileptic patients, but not in healthy controls. Here, we detect the CSF-ApoE to determine whether it is changed after seizures and measured the concentrations of TN in CSF and serum of epileptic patients to study the changes of TN levels after seizures.Methods: We detected CSF-ApoE in 60 epileptic patients and 28 subjects with no evidence of any neurological diseases, and TN concentration in CSF and serum of 64 epileptic patients and 26 healthy subjects by using sandwich enzyme-linked immunosorbent assay (ELISA).Results:?The CSF-ApoE levels in epilepsy and control groups were 5.78±2.15 mg/l and 13.60±12.11 mg/l (p<0.05). In epilepsy group, the CSF-ApoE concentration was 6.53±2.55 mg/l in male patients, and 4.98±1.21 mg/l in female (p<0.05); In secondary epilepsy group was 5.06±1.31 mg/l, and in idiopathic epilepsy was 6.04±2.34 mg/l (p<0.05); In different seizure types groups, including complex partial seizure (CPS), secondarily generalized tonic-clonic seizure (SGTC), generalized tonic-clonic seizure (GTCS), and absence seizure (AS), the mean concentrations of CSF-ApoE were 6.62±3.13 mg/l, 5.21±1.22 mg/l, 5.00±1.09 mg/l and 7.25±1.88 mg/l respectively (p<0.05);?The CSF-TN levels in epileptic patients and control group were 0.368±0.105 mg/l and 0.281±0.056 mg/l (p<0.05). Serum-TN concentration in epileptic patients and control group were 6.709±1.265 mg/l and 8.883±1.335 mg/l (p<0.05). The decrease of serum-TN in the patients with drug-refractory epilepsy was 6.369±1.134 mg/l and decreased obviously. Conclusions:?CSF-ApoE concentration decreases after seizures, correlated with the gender, etiological factor and seizure types. The decrease of CSF-ApoE after seizure might be related with the brain injury severity;?CSF-TN levels increased in epileptic patients while serum-TN levels decreased. The serum-TN in epileptic patients could reflect the effect of anti-epileptic drugs and might be a candidate biomarker for drug-refractory epilepsy.PART THREE: DNA METHYLATION PROFILING REVEALS DIFFERENTIAL METHYLATION PATTERNS CORRELATED WITH GENE EXPRESSION IN HUMAN EPILEPSYPurpose: DNA methylation is an epigenetic modification that plays important roles in regulating gene expression and is one of the key processes underlying neuronal functions. However, few studies to date have addressed whether abnormal DNA methylation contributes to differential gene expression in epilepsy. This study aimed to define differential genomic DNA methylation patterns in the anterior temporal neocortex of drug-refractory epilepsy patients relative to control patients and to investigate the role of DNA methylation in epileptogenesis.Methods: We first evaluated DNA methyltransferase 1 (Dnmt1) and Dnmt3a expression in brain tissues of epileptic patients by immunostaining and immunoblotting. We then performed global DNA methylation profiling in brain tissues from epileptic and control patients (n = 3 of each) via a methylated-cytosine DNA immunoprecipitation microarray chip. Differentially methylated loci were validated by bisulfite sequencing-PCR, and the mRNA levels of candidate genes were evaluated by RT-PCR.Results: We found that protein expression of Dnmt1 and Dnmt3a was up-regulated in the brain tissues of epileptic patients. Through the microarray analysis of 385k CpG islands and promoters, we found 224 genes that showed differential DNA methylation between epileptic patients and controls. These genes included those involved in the regulation of Rho, microtubule-based processes and mitogen-activated protein kinase scaffold activity. Among the seven candidate genes evaluated, three showed clear transcriptional regulation by DNA methylation. TUBB2B and ATPGD1 exhibited relatively hypermethylated promoters and decreased mRNA levels, whereas HTR6 displayed a relatively hypomethylated promoter and higher mRNA levels in the epileptic samples.Conclusions: Our findings suggest that DNA methylation changes may play a causative role in the progression of epileptogenesis and that certain genes become differentially regulated by DNA methylation after the onset of epileptic seizures.