| Epilepsy is characterized by a long term risk of recurrent seizures.Epilepsy isusually controlled, but not cured, with medication. However, more than30%ofpeople with epilepsy do not have seizure control even with the best availablemedications. Intractable epilepsy is a seizure disorder in which a patient’s seizuresfail to come under control with treatment. For several decades, both in vitro and invivo models of seizures and epilepsy have been employed to unravel the molecularand cellular mechanisms underlying the occurrence of spontaneous recurrentseizures (SRS) the defining hallmark of the epileptic brain. However, despitegreat advances in our understanding of seizure genesis, investigators have yet todevelop reliable biomarkers and surrogate markers of the epileptogenic process.Sadly, the pathogenic mechanisms that produce the epileptic condition, especiallyafter precipitating events such as head trauma, inflammation, or prolonged febrile convulsions, are poorly understood. A major challenge has been the inherentcomplexity and heterogeneity of known epileptic syndromes and the differentialgenetic susceptibilities exhibited by patients at risk.Therefore, it is unlikely thatthere is only one fundamental pathophysiologic mechanism shared by all theepilepsies.Identification of antiepileptogenesis targets has been an overarching goalover the last decade, as current anticonvulsant medications appear to influence onlythe acute process of ictogenesis. Clearly, there is an urgent need to develop noveltherapeutic interventions that are disease modifying therapies that eithercompletely or partially prevent the emergence of SRS.An important secondary goalis to develop new treatments that can also lessen the burden of epilepsycomorbidities (e.g., cognitive impairment, mood disorders) by preventing orreducing the deleterious changes during the epileptogenic process. So far, themolecular and cellular changes reported during the presumed epileptogenesisprocess in animal models include neuronal injury and cell death, axonal anddendritic plasticity, presynaptic and postsynaptic modifications, neurogenesis,neuroinflammation, glial cell activation, vascular damage and angiogenesis,disruption of extracellular matrix integrity, as well as structural and functionalchanges in ion channels properties. In recent years, a variety of studies have foundthat the incidence of epilepsy and energy metabolism imbalances are closely related,the the energy metabolites imbalance can induce epileptic occurs.Glutamate is the primary excitatory neurotransmitter in the central nervous system(CNS), mechanisms that promote glutamatergic neurotransmission could playimportant roles in the development of epilepsy. Increased levels of extracellularglutamate in fact have been observed in human epileptic serum. In vivomicrodialysis studies also show elevated extracellular concentration of glutamate inthe epileptogenic cortex as compared to the nonepileptogenic cortex in patientswith temporal lobe epilepsy (TLE). These reports suggest that dysfunctionalextracellular glutamate cycling and reuptake may play a vital role in the genesisand maintenance of focal epileptic activity.Glutamate transporters, also referred toas excitatory amino acid transporters (EAAT), represent the sole mechanism of active reuptake of glutamate into the astrocytes and neurons and are essential todampen neuronal excitation following glutamate release at synapses.Monocarboxylate transporters (MCT) facilitates the transport of monocarboxylatefuels (lactate and pyruvate) and acidic drugs, such as valproic acid, across cellmembranes. A resent research shows that MCT1is deficient on microvessels in theepileptogenic hippocampal formation in patients with medicationrefractoryepilepsy. To further define the role of MCT in the pathophysiology ofmedicationrefractory epilepsy, we used immunohistochemistry, western blot andwhole cell clamp analysis to localize and quantify the transporters in thehippocampal formation in the novel and highly relevant rat model ofedicationrefractory epilepsy and in nonepileptic control animals. We were also todetermine the expression and distribution of MCT in tissue samples from therefractory cortex of patients who had been surgically treated for refractory epilepsy.We compared these tissues with histologically normal samples from controls. Inour present study, we found MCT4was lost on hippocampus and piriform corticesin the lithium-pilocarpine model. In addition, we were able tocoimmunoprecipitate MCT4and EAAT1in primary rat astrocytes. We also foundRNAi-mediated inhibited of MCT4can decrease the expression of EAAT1inprimary rat astrocytes. It is possible that EAAT1is a substrate for MCT4, implyingMCT4may directly modulate EAAT1in primary rat astrocytes. Therefore, wehypothesize that the loss of MCT in brain is mechanistically involved in thepathophysiology of intractable epilepsy, and propose that re-expression of MCTmay represent a novel therapeutic approach for this disease.This study is supposedto confirm the above hypothesis. The results from this study will shed light on newstrategic treatment for epilepsy. The following research has been conducted on thisbasis.Part1The expression of monocarboxylate transporter4in epilepsy animalmodels and clinical epilepsy sampleObjective： The purpose of our research is to explore the expression of monocarboxylate transporter4in epilepsy animal models and clinical epilepsysample. Methods：The cases included in the study were obtained from the files ofthe Department of Neurosurgery of Tangdu Hospital of the Fourth Military MedicalUniversity. We examined30specimens obtained from patients undergoing surgeryfor medically intractable epilepsy. The control samples obtained from12patientswho underwent neurosurgical intervention for increased intracranial pressure due tohead trauma. The li-pilo model has been established. The rats were randomlydivided into a normal control and eight Li-pilo model (SE4h、SE12h、SE1d、 SE3d、SE7d、SE14d、SE28d、SE60d) groups. After the success of the model, Rattemporal lobe and hippocampal tissue collected at different time points. The epilepsyrat cortex and hippocampus expression of MCT4was studied by Western blot. Theexpression of MCT4in the cortex of patients with intractable epilepsy was studiedby western blot and immnohistochemistry. Meanwhie, these clinical samples wasstudied by methylation-specific PCR. Results: In western blot test, the temporalcortex MCT4protein level was shown to be low in the acute phase of the epilepsymodel, the expression of MCT4protein decreased dramatically in the latent phase ofthe epilepsy model. The expression of MCT4in the hippocampus is consistent withthe expression in temporal cortex. In brain tissue, decreased MCT4proteinexpression has been found in the cortex of patients with intractable epilepsy. We alsofound hypermethylation of MCT4Promoter Contributes to the down-regulation ofMCT4in human epileptic brian tissue. Conclusion: Both in the epilepsy model orclinical epilepsy sample found MCT4expression reduced dramatically. The studyalso found that hypermethylation of MCT4Promoter Contributes to the decreasedexpression of MCT4in epileptic tissue.Based on the phenomenon of decreasedMCT4expression in the cortex of patients with intractable epilepsy, we suggest thatMCT4may play a role in intractableepilepsy.Part2The role Of MCT4in epileptogenesisObjective: To explore the role of MCT4in epileptogenesis. Methods: The model of primary culture rat cortical astrocytes has been established. Primary culturecells were transduced with the lentiviral particles containing MCT4shRNAsequences, these cells were identified by immunofluorescence. Measurement ofthese cells growth by Methyl Thiazolyl Tetrazolium Assay (MTT). Apoptosis andcell cycle of these cells were analyzed by flow cytometry. Moreover, the MCT4blockers are used to observe the effects of the blockers on the electrical activity ofneurons. Results: The model of MCT4shRNA primary culture rat corticalastrocytes was successfully established. In our study, the cell growth curvesshowed that the growth of MCT4shRNA cells was notably inhibited in atime-dependent manner. We also found the decreased cell number was attributableto apoptosis induced by MCT4-targeting shRNA. To explore the potentialcontribution of MCT4shRNA to cell cycle progression, we used flow cytometry toevaluate the cell cycle distribution. The results showed that MCT4shRNA cellsaccumulated in G0/G1phase, but the cell numbers in G2/M phase were reducedsharply. The patch clamp results show that the MCT4signal blockers CHCincreased neuronal excitability. Conclusion: In the current study, we demonstratedthat suppression of MCT4inhibited primary culture rat cortical astrocytes growth.In the model of primary culture rat cortical astrocytes, we found suppression ofMCT4inhibited proliferation and induced apoptosis. We also demonstrated thatsuppression of MCT4increased neuronal excitability. Because CHC increasedneuronal excitability and that depletion of MCT4in primary culture rat corticalastrocytes suppressed proliferation and induced apoptosis, MCT4may play animportant role in epileptogenesis.Part3The potential molecular mechanisms of MCT4in epileptogenesisObjective: To explore the potential molecular mechanisms of MCT4inepileptogenesis. Methods: The model of primary culture rat cortical astrocytes hasbeen established. Primary culture astrocytes were transduced with the lentiviralparticles containing MCT4shRNA sequences, Western blot analyses wereperformed as described above. Whole primary culture rat cortical astrocytes lysates were obtained by resuspending cell pellets in RIPA buffer. Lysates were incubatedovernight with EAAT1antibody before being absorbed with proteinA/G PLUS agarose beads. Precipitated immunocomplexes were released by boilingwith2×SDS electrophoresis sample buffer and were prepared for Western blotanalysis. For immunohistochemical analysis, clinical sample sections were stainedand evaluated. Primary culture rat cortical astrocytes stably transfected with thelentiviral particles containing MCT4shRNA sequences were treated with20mg/mlcycloheximide (CHX) for0,1,3,6, and12h. After treatments, Western blotanalyses were performed. Results: Western blot results demonstrated decreasedexpression levels of EAAT1in MCT4silence astrocytes, compared with controlcells. We found MCT4was coimmunoprecipitated with EAAT1when anti-EAAT1was used to pull down EAAT1protein and its associated proteins. Wedemonstrated that the expression of MCT4and EAAT1has clinical relevance inclinical epilepsy sample. We also found the decrease in the EAAT1protein levelfor MCT4silence astrocytes correlated with a decrease in its stability. Conclusion:In the current study, we demonstrated that suppression of MCT4contributes to thedecreased expression of EAAT1in primary culture rat cortical astrocytes. Inaddition, we were able to coimmunoprecipitate MCT4and EAAT1in imary culturerat cortical astrocytes. A clinical relevance comparison of the expressions of MCT4and EAAT1demonstrated that EAAT1expression in clinical epilepsy sample wassimilarly associated with MCT4expression.CHX treatment revealed that MCT4bindingEAAT1could stabilize EAAT1expression in primary culture rat corticalastrocytes. Taken together, these results provide the biological basis for MCT4as acandidate therapeutic target for antiepileptic drugs. |
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