The Effect And Protential Mechanism Of Neuroligin-1Involved In Epilepsy

Part one: The expression of neuroligin-1and neurexin-1β in temporallobe epileptic foci in patients and experimental animalsObjective: Abnormally synchronized synaptic transmission in the brainleads to epilepsy. Neuroligin-1(NL1) is an synaptic cell adhesion moleculelocated at excitatory synapse, which modulates synaptic transmission anddetermines the properties of neuronal networks in the mammalian centralnervous system. Here we investigated the expression of NL1and its bindingpartner neurexin-1β (NRX1β) in temporal lobe epileptic foci in patients andexperimental animals in order to explore the probable relationship betweentheir expression and temporal lobe epilepsy (TLE).Method:1. The NL1and NRX1β expression were assessed in twenty two humanbrain tissues derived from patients undergoing operation for intractable TLEand was also detected in ten temporal lobes from controls.2. Adult male Sprague–Dawley (SD) rats were used in this study,including lithium–pilocarpine-treated rats (n=70) on days1,2,3,7,14,30,and60post-seizure, and control rats (n=10). 3. Expression of NL1and NRX1β were assessed byimmunohistochemistry, immunofluorescence, and Western blot analysis.Result:1. NL1protein was mainly expressed in the membrane and cytoplasmof neurons in the temporal neocortex from both the control and intractableTLE groups by immumohistochemical staining. the mean optical density(OD) value of NL1was significantly higher in the temporal neocortex of theTLE group compared with the control group (p<0.05) Double-labelimmunofluorescent staining showed that NL1and GFAP were seldomcoexpressed in astrocytes, but NL1-positive cells coexpressed withNMDAR1. Western blot analysis showed the expression of NL1and NRX1βwas strong in the subjects with intractable TLE, whereas it was relativelyweak in the control subjects.2. The NL1immumohistochemical staining was extensively observedin neocortex and all regions of the hippocampus, and the intense staining inhippocampus was in the dentate gyrus, CA1and CA3regions. Comparedwith the controls, the mean OD values of NL1expression were elevateddynamically. Double-labeling immunofluoresence showed NL1-positivecells did not co-express with GFAP in astrocytes but co-expressed withNMDAR1in neurons. Western blot analysis showed compared with thecontrols, both NL1and NRX1β expression elevated in the acute period andthe chronic period, and their expression profile presented nearly the same. The difference of the mean OD ratio in the NL1expression level inhippocampus between the control and every treated group was significant(p<0.05), except the7-d and14-d groups (p>0.05). There was a significantdifference of the mean OD ratio in the NRX1β expression between thecontrol and every treated group (p<0.05), except the7-d group (p>0.05).Conclusion: The expression of NL1and its binding partner NRX1βwere increased in temporal lobe epileptic foci in patients andlithium–pilocarpine-treated epileptic rats. Our results suggest that NL1andNRX1β may play an important role in the development of TLE.Part two: The effect of knockdown of neuroligin1on epileptic seizuresObjective: To further study whether supression of NL1could preventseizures, we investigated the behavioral changes of epileptic rats performedby lentivirally mediated knockdown of NL1in the hippocampus.Method:1. The following three groups with hippocampal injection wereincluded, namely sham operation group with hippocampal injection of saline(EP), group with hippocampal injection of vehicle virus (EP+sh-con.), andgroup with hippocampal injection of NL1shRNA (EP+sh-NL1).2. Western blot analysis of NL1expression in hippocampus was used toverify that in vivo RNA interference for NL1was effective and selective. Onthe other hand, EGFP antibody was used to identify and amplify the virus-infected neurons in the hippocampus by immunofluoresence.3. We perform rats behavioral investigation7days after virus infusion.We observed the behavioral changes of the rats within60-min afterpilocarpine injection. Scoring in rats for each20-min interval, the maximumRacine score during the60-min trial,the percentage of the rats reached above5score, and the seizure latency in the three groups were recorded.Result:1. Western blot analysis showed that the mean OD ratio of NL1significantly decreased in the epileptic group at days3,7respectively afterinfusion (p<0.05). There was no significantly difference between the3-d and7-d groups (p>0.05). The results revealed that at days3and7after infusion,the amount of NL1was respectively decreased by36.3±4.3%, and44.2±5.3%of control levels. The immunfluresene showed that EGFP waslocalized in the hippocampus, especially in CA1area.2. With time, all the rats developed more severe seizures. The seizuresin sh-NL1group are less severe than that in the other groups in each20-minintervals (P<0.05). There was a significant main effect of time (P<0.05) butno significant of time×group interaction (P>0.05). The maximum racinescore in sh-NL1group was less severe than that in the other groups (P<0.05), but there was no significant difference between the two control groups(P>0.05). Finally, only33.3%rats in sh-NL1group showed generalizedclonic or tonic seizures (seizure score4or5) compared with83.3%rats in both other control groups. NL1knockdown significantly reduced theincidence of generalized clonic or tonic seizures (P<0.05). NL1knockdownsignificantly delayed seizure latency compared with the control goups (P<0.05), but there was no significant difference between the two control groups(P>0.05).Conclusion:1. The expression of endogenous NL1in the hippocampus wassuccessfully suppressed in vivo by sh-NL1, and the efficiency of RNAinterference in vivo for NL1got to a stable level at day7.2. Knockdown of NL1in epileptic rats could reduce seizure severityand prolonged seizure latency.Part three: The protential mechanism of neuroligin1involved inepileptigenesisObjective: To further explore the probable underlying mechanisms ofof NL1involved in spontaneous recurrent seizure(SRS), we investigated theelectrophysiological changes of epileptic rats performed by lentivirallymediated knockdown of NL1in the hippocampus.Method:1. Three epileptic groups with hippocampal injection of saline (EP),vehicle virus (EP+sh-con.), and NL1shRNA (EP+sh-NL1) respectivly, aswell as one normal control group with hippocampal injection of saline (control) were included.2. Western blot analysis and immunfluresene were used for analysis ofthe efficiency of RNA interference in the epileptic rats at day30.3. Whole-cell recordings of CA1pyramidal neurons in brain slices wereperformed, and the action potential (AP), miniature excitatory postsynapticcurrents (mEPSCs) and evoked excitatory postsynaptic currents (mEPSCs)were recorded.4. Western blot analysis was used for analysis of the total or the surface/total ratio of NMDAR1expression in the epileptic rats at day30.Result:1. Western blot analysis showed the difference in the NL1expressionlevel in hippocampus between the sh-NL1epileptic group and sh-con.epileptic group or epileptic group was significant (p<0.05), but there was nosignificant difference between the sh-con. epileptic group and epilepticgroup as well as the the sh-NL1epileptic group and normal control group(p>0.05). The immunfluresene of EGFP indicated that the lentivirus wassuccessfully transfected into hippocampal neurons.2. the frequency of spontaneous AP in sh-NL1epileptic group waslower than that in the sh-con. epileptic group (P <0.05). Compared with thenormal control group, the spontaneous AP was increased in both sh-con.epileptic and sh-NL1epileptic groups (P <0.05). There was no significantlydifference of the mean mEPSC amplitude between the sh-NL1epileptic group and the sh-con. epileptic group (P>0.05). The mean mEPSCamplitude averaged from the sh-con. epileptic group and the sh-NL1epileptic group were significantly increased respectively compared with thenormal control group (P <0.05). the mEPSC frequency averaged from thesh-NL1epileptic group was lower than that in the sh-con. epileptic group(P <0.05). Compared with the normal control group, the mean mEPSCfrequency in both sh-con. epileptic group and sh-NL1epileptic weredramaticlly increased (P <0.05). There was a significant difference betweenthe normal control group and the sh-NL1epileptic group (P <0.05).3. A clear reduction in NMDAR/AMPAR ratio was observed in thesh-NL1epileptic group comparing with the normal control group or sh-con.epileptic group(P <0.05). Compared with the sh-con. epileptic group, thesh-NL1epileptic group showed a significant decrease in the averageamplitude of NMDAR-dependent EPSCs (P<0.05), but not AMPAR-dependent EPSCs (P>0.05).4. The total or the surface/total ratio of NMDAR1expression in bothepileptic group and sh-con. epileptic group were significantly higher thanthat in the control group (P <0.05). The total or the surface/total ratio ofNMDAR1protein expression in the sh-NL1epileptic group wassignificantly lower than that in the epileptic group and sh-con. epilepticgroup (P <0.05).Conclusion: 1. The expression of endogenous NL1in the hippocampus remainssuppressed in vivo by shNL1in the rats at day30.2. Knockdown of NL1in epileptic rats inhibits hyperexcitability inepileptic hippocampal slices.3. NL1knockdown in epileptic rats could reduces NMDAR-mediatedsynaptic currents in hippocampal slices via postsynaptic NMDAR1.