The Potential Mechanism Of ZDHHC8 On Seizure Susceptibility In Epilepsy

Part one: Localization and expression of ZDHHC8 in patients with TLE and chronic epileptic mice Objective: To investigat whether the activity of brain tissue pharmaco-resistant TLE patients and pilocarpine-induced epileptic mice with SRSs in vivo affects ZDHHC8 levels.Method:1.Sixteen patients who had undergone surgical resection of the temporal lobe were randomly chosen from 400 specimens in our epileptic brain tissue bank.For comparison,we randomly selected 16 controls who were not associated with any known neurological disease or exposure to AEDs.Controls were patients treated for increased intracranial pressure due to head trauma and corresponded to histologically normal temporal neocortical samples.2.Male adult C57Bl/6 mice(20-30 g,10-14 weeks of age)were used in the test.Hippocampus and cortex between pilocarpine-induced epileptic mice with SRSs group and without SRSs group(n=6).3.Western-blot was used to examine the ZDHHC8 protein levels in epileptic mice and control mice.Localization of ZDHHC8 was investigated by immunofluorescent staining.Result:1.Western blot showing ZDHHC8 levels were increased in temporal lobe samples from patients with TLE compared to controls(n=16,**P<0.01 compared to control).ZDHHC8 levels were also increased in hippocampal and cortex lysates of epileptic mice compared to controls(non-epileptic mice)(**P<0.01,n=5).2.ZDHHC8 and MAP2 but not GFAP are colocalized(merged)in the cortex of temporal lobe samples from patients with TLE and epileptic mice as well as in the hippocampus of epileptic mice.Conclusion:ZDHHC8 was showed increased in TLE patient and pilocarpine-induced epileptic mice with SRSs.ZDHHC8 was localized in neurons of sections in these brain tissues.These indicated that ZDHHC8 may play an important role in the development of epilepsy.Part two: The effect of ZDHHC8 on seizure susceptibility in animal models in vivo and neuronal hyperexcitability and hypersynchrony zero-Mg2+ seizure model in vitro.Objective:To determine if changes in the levels of ZDHHC8 had any gross effects on seizure susceptibility,we constructed r AAVs to knockdown or overexpress ZDHHC8.Two mouse models: pilocarpine-and kainic acid(KA)-induced chronic epilepsy models in vivo and zero-Mg2+ seizure model in vitro were utilized in this test.Method:1.Adult male C57BL/6 mice were randomly divided into five groups: control group,r AAV-ZDHHC8-sh group,r AAV-Empty(sh)-GFP group,r AAV-ZDHHC8 group and r AAV-Empty(over)-GFP group.r AAV-ZDHHC8-sh,r AAV-Empty(sh)-GFP,r AAV-Empty(over)-GFP and r AAV-ZDHHC8 were intracerebroventricularly(i.c.v.)injected into mice.The mice of control group were just peformed sham operration.2.To confirm that the r AAV vectors were efficient,Western-blot and immunofluorescent staining were performed to detect hippocampal r AAV-ZDHHC8-sh and r AAV-ZDHHC8 in mice at 1,3 and 5 weeks after injection.To verify that AAV-induced knockdown or overexpression was specific to only ZDHHC8,we detected the expression of ZDHHC5.HE staining and TUNEL staining of sections from mice injected with ZDHHC8 was used to detect the neuronal loss and apoptotic positive cells.3.Behavioral assays.Mice were injected injectd with pilocarpine(320mg/kg)to establish pilocarpine-induced chronic epileptic mouse model.Another model was induced by unilateral intrahippocampal injection of KA(0.3 ?g /mouse).We observed the latency to the first SRS and accumulated the SRS times for 1-30 days.The latency to fully-kindled and the seizure class scores were decided by Racine score for 1 hour after each injection.Western-blot was used to examine the ZDHHC8 protein levels of mice with SRSs.To examine whether ZDHHC8 regulates epileptiform activity,we recorded LFPs(analogous to EEG)in C57BL/6 KA model mice with stable baseline SRSs.The frequency and duration of epileptiform-like discharge events were recorded.4.In vitro zero-Mg2+ seizure model,the parameters of neuronal hyperexcitability and hypersynchrony were recorded by combining whole-cell patch-clamp and extracellular field recordings in acute hippocampal slices.Result:1.rAAV with GFP were localized in the CA1 and dentate gyrus of the hippocampus 3 weeks after r AAV i.c.v.injection.Under the same conditions,the expression of ZDHHC8 was significantly increased 3 and 5 weeks after overexpression r AAV-ZDHHC8 injection.The expression of ZDHHC5,no significant change in ZDHHC5 was observed.HE staining indicated that brains of mice injected with r AAV had normal gross anatomy.No obviously evidence of hippocampal neuronal death was observed upon Td T-mediated deoxy-uridine triphosphate nick end labeling(TUNEL)staining of sections from mice injected with ZDHHC8(P>0.05).2.In the pilocarpine-and kainic acid(KA)-induced chronic seizure models,we observed that the latency to SRS was delayed and the number of SRS was decreased in r AAV-ZDHHC8-sh RNA group compared to control group and r AAV-Empty(sh)-GFP group(*P<0.05,**P<0.01).In contrast,we up-regulated ZDHHC8 and found that the latent period was shortened and the number of SRSs increased significantly(**P<0.01,***P<0.001).In C57BL/6 KA model,the frequency and duration of epileptiform-like discharge events were reduced by ZDHHC8 knockdownn,whereas ZDHHC8 overexpression increased event frequency but not duration(*P<0.05,**P<0.01,***P<0.001).The protein levels in the hippocampus was found down-regulated expression in ZDHHC8 knockdown mice and up-regulated expression in ZDHHC8-overexpressing mice,which was consistent with the behavioral results(*P<0.05,**P<0.01,***P<0.001).3.The APs was markedly decreased compared with control and r AAV-Empty(sh)-GFP slices,and the mean frequency of epileptiform events(PDS or bursts)and the average number of APs in PDSs markedly decreased in ZDHHC8 knockdown neurons but were markedly increased in ZDHHC8-overexpressing neurons(*P<0.05,**P<0.01,***P<0.001).The frequency(number of events/min),event durations of ictal-like events and interictal-like events were markedly decreased in r AAV-ZDHHC8-sh RNA group but were markedly increased in ZDHHC8-overexpressing slices compared with control and r AAV-Empty(over)-GFP slices(*P<0.05,**P<0.01,***P<0.001).Conclusion:1.The expression of ZDHHC8 in hippocampus was significantly inhibited after r AAV-ZDHHC8-sh RNA intracerebroventricularly injected;r AAV-ZDHHC8 intrahippocampal injection specifically increased the expression of ZDHHC8 in hippocampus.2.The ZDHHC8 in vivo intervention could change the latency and severity of seizure and LFPs in pilocarpine-induced and kainic acid(KA)-induced chronic seizure models.3.ZDHHC8 knockdown reduces neuronal hyperexcitability and hypersynchrony,and in contrast,ZDHHC8 overexpression results in hyperexcitability and hypersynchrony the zero-Mg2+ seizure model in vitro.Part three: The potential mechanism of ZDHHC8 involved in seizure susceptibility Objective:To investigat the potential mechanism of ZDHHC8 involving in seizure susceptibility,we investigated the synaptic function of epileptic mice performed by r AAV mediated knockdown and overexpression of ZDHHC8 in the hippocampus.Method:1.Male C57BL/6 mice with intracerebroventricularly injected of r AAV-ZDHHC8-sh RNA,r AAV-Empty(sh)-GFP,r AAV-ZDHHC8,and r AAV-ZDHHC8 respectivly.Three weeks after injected with r AAV,the mice were prepared to acute hippocampal slice.2.In zero-Mg2+ seizure model,whole cell patch-clamp recordings of CA1 pyramidal neurons in hippocampal slices were performed,and the m IPSCs and m EPSCs were recorded.Localization of ZDHHC8 was investigated by immunofluorescent staining in TLE patients and epileptic mice.3.In zero-Mg2+ seizure model,whole cell patch-clamp recordings of CA1 pyramidal neurons in hippocampal slices were performed,the ratio of evoked AMPAR-mediated current to NMDA receptor-mediated response(ratio of e AMPA/NMDA),PPR,RI of Glu A2-lacking calcium-permeable AMPARs and ratio of e AMPA/NMDA before and after IEM-1460(50 ?M)application.4.Immunoprecipitation was used for analysis the relationship between ZDHHC8 with Glu A1?Glu A2 ?Glu A3?Glu A4 receptors in the epileptic mice.5.Western blot analysis was used for investigate of the surface/total ratio of Glu A1,the intracellular/ total ratio and the cell surface expression of GABA-A receptors ?2/3 recepters expression in the pilocarpine-induced chronically epileptic model and kainic acid-induced epilepsy model hippocampus.Result:1.We observed a significantly decreased mean amplitude of m EPSCs but not m IPSCs in the slices of ZDHHC8 knockdown mice compared with those of control and r AAV-Empty(sh)-GFP mice(***P<0.001).In ZDHHC8-overexpressing mice,we found a significant increase in m EPSC amplitude without a change in m IPSC amplitude(*P<0.05).The frequencies of the m EPSCs and m IPSCs were not changed in all groups(P>0.05).Immunofluorescent labeling for ZDHHC8 in the hippocampus of chronic epileptic mice and in the neocortex of patients with TLE indicated ZDHHC8 expression in the cell membrane but not cytoplasm;Meanwhile,ZDHHC8 and PSD95 are colocalized but VFLUT1,GAD67 and Gephyrin.2.The ratio of AMPA-mediated to NMDA-mediated currents was decreased in slices from ZDHHC8 knockdown mice compared with those from control and AAV-Empty(sh)-GFP mice,and increaced in ZDHHC8 overexpression slices(*P<0.05,**P<0.001,***P<0.0001).ZDHHC8 did not alter the probability of release at AMPAR-expressing synapses(P>0.05).AMPAR rectification index(RI)was decreased in slices from ZDHHC8 knockdown mice and increased in ZDHHC8-overexpressing mouse slices(**P<0.001).The AMPA/NMDA ratio before perfusing slices with IEM-1460 and found that the ratio of AMPA/NMDA markedly deceased in all groups(*P<0.05).3.In immunoprecipitation of mouse hippocampus lysate,ZDHHC8 coimmunoprecipitates with Glu A1 but Glu A2,Glu A3 and Glu A4.4.In ZDHHC8 knockdown neurons of pilocarpine-induced chronic seizure model mice and KA-induced seizure model mice,cell surface expression of Glu A1 was significantly reduced(*P< 0.05,**P <0.01),while GABAAR ?2/3 was unchanged(P>0.05).In ZDHHC8 knockdown neurons,the more Glu A1 protein was retained in the cytoplasm of ZDHHC8 knockdown neurons(*P<0.05).In addition,immunoblotting showed that the total expression levels of Glu A1 did not significantly change among groups.The cell surface expression of Glu A1 was increased but cytoplasm of ZDHHC8 decsreaced in neurons overexpressing ZDHHC8 via r AAV of ZDHHC8 overexpression experiments(*P< 0.05,**P < 0.01,***P < 0.001).Conclusion:1.ZDHHC8 localizes at excitatory postsynaptic membranes and may alter postsynaptic AMPA receptor responses and the number of postsynaptic receptor excitatory synapses.2.ZDHHC8 regulates postsynaptic neurotransmission via Glu A2-lacking calcium-permeable AMPARs in the hippocampus.3.The results suggested that ZDHHC8 only interacts with Glu A1 protein and may involve in Glu A1 trafficking.