LIM Kinase1 Modulates Actin Dynamics In Hippocampal Cells And Modifies Seizures In Vivo And Vitro

PART ONE:The expression of LIMK1/P-LIMK1 in Mg²⁺-free treated hippocampal neurons and epileptic rat modelsObjective:The important pathological changes in the epileptogensis prosess are the synaptic remodeling and formation of the pathological discharge loops.The LIMK1 plays a very important role in the process of synaptic remodeling.In this study,we analyze the expression of LIMK1and P-LIMK1 in brain tissues of hippocampal neurons induced by Mg²⁺-free and epileptic rat models,so that explore whether LIMK1 and P-LIMK1 are related to epilepsy activity.Methods:1.hippocampal neurons of Neonatal Sprague Dawley（SD）rats were selected to culture.After the primary cultured hippocampal neurons were treated for 3 hours at DIV10 with Mg²⁺-free medium.The epileptiform discharge group was successfully selected as the Mg²⁺-free epilepsy group.2.The animal experiment,we divided about 250g male Sprague Dawley（SD）rats into two groups:the pentylenetetrazol epilepsy experimental group and the normal saline control group.The two groups of rats were given intraperitoneal injection of pentylenetetrazol or saline.According to the Racines classification,rats of grades 4 to 5 were selected into the experimental group.3.immunofluorescence and Westernblotting were used to detect the expression of LIMK1 and P-LIMK1 in the brain tissues of pentylenetetrazol kindled chronic epilepsy rats and the magnesium-free induced hippocampal neuronsResults:1.LIMK1 and P-LIMK1 are abundantly expressed in the cytoplasm and membrane of primary hippocampal neurons which induced by Mg²⁺-free epilepsy model.2.in primary cultured neurons,the levels of LIMK1 and p-LIMK1were significantly higher in the Mg²⁺-free group than in the control group.（P<0.05）.3.The expression level of LIMK1 was significantly higher in the hippocampus tissue of pentylenetetrazol epileptic rats’brain compared with the control group（P<0.05）.Conclusion:LIMK1 and P-LIMK1 are mainly expressed in the cytoplasm and membrane of neurons.In a Mg²⁺-free treated hippocampal neurons and a pentylenetetrazol-induced chronic epilepsy model,the expression levels of LIMK1 and P-LIMK1 were significantly up-regulated compared with the control group,showing that the LIMK1/P-LIMK1 might be closely related to the epileptogensis process and played an important role.PART TWO:Effect of LIMK1 on chronic spontaneous seizures in epileptic rats and on the synaptic remodeling of primary hippocampal neurons treated with Mg²⁺-freeObjective:To analyze the the change of chronic spontaneous seizures in PTZ kindled rats by intracranial transfection of lentiviral vector.Silencing the expression of LIMK1 in a vivo model of primary cultured hippocampal neurons,detecting the expression changes of Actin,and observing its effect on the morphology and density of spines.Methods:1.The lentiviral vector（LV）containing the LIMK1-RNAi sequence was constructed.the male SD adult rats（140g-160g）,were randomly divide them into an control group（Con-RNAi）and experimental group（LIMK1-RNAi）,and the corresponding lentivirus（LV）were injected into the hippocampal DG（Dentate Gyrus）area.The westernblotting was used to verify whether LIMK1 was successfully transfected.2.After injection of the lentiviral virus for two weeks,a regular daily intraperitoneal injection of pentylenetetrazol（35mg/kg）was used to construct a rat model of chronic epilepsy for 28 days.The rats were observed for 30 minutes during the injection of pentylenetetrazol,and the rats were ranked according to Racines classification.The time of first to level four or five seizure,the duration of level to four or five seizures,and the number rats of level to four or five seizures were recorded.3.The lentiviral vector（LV）was transfected into primary neurons and verified the rate of LIMK1 transfection by immunoblotting and immunofluorescence techniques;we also verify whether down-regulating LIMK1 has an impact on activities of protein in this pathway.4.Filament actin（F-actin）and globular actin（G-actin）in primary hippocampal neurons were extracted,and the gray intensity values were measured by Western blot experiment,and the ratio of F-actin/G-actin was obtained.5.The morphology and the density of spinous processes of the neurons of silencing the expression of LIMK1 in a vivo model were analyzed by Immunofluorescence that was to determine the effect of LIMK1 on synapse remodeling in primary cultured neurons.Results:1.After transfection with the lentiviral vector in rats,immunoblot experiments showed that the expression level of LIMK1 was significantly lower than that of the Con-RNAi control group.（P<0.05）.2.In the epileptic rat model,knockdown the expression level of LIMK1 significantly prolonged the latency of the first grade 4 or 5 seizures,and the total duration of grade 4 and 5 seizures was significantly reduced.The number of grade 4 and 5 seizures was significantly reduced（P<0.05）.3.After the transfection test,the optimal multiplicity of infection（MOI）=6 was obtained.After transfecting the neuron with this multiplicity of infection,the expression level of LIMK1 was significantly down-regulated by Westernblotting.After the neurons were transfected with this multiple of infection,the growth of neurons was not affected.There was no obvious effects in Rock-2、PAKs、LIMK2、和cofilin activities after LIMK1 expression was knockdown.4.In the primary neurons of the Mg²⁺-free epilepsy model,the ratio of F-actin/G-actin was significantly higher than that of the control group,indicating that the aggregation of actin was significantly increased in the vitro model.Compared with the control group（Con-RNAi）in the primary neuron experimental group that knocked down LIMK1,and both groups treated with Mg²⁺-free,we found that the ratio of F-actin/G-actin was more Group significantly reduced.5.In the primary neurons treated with Mg²⁺-free,the proportion of normal functional dendritic spines and the density of dendritic spines were significantly reduced.Whereas the density of dendritic spines and the rate of mature spines significantly increased in silencing LIMK1 in a vivo model of primary cultured hippocampal neurons.Conclusion:1.The LIMK1-RNAi can successfully knock down the expression level of LIMK1 in primary hippocampal neurons and neurons in the hippocampal DG region of SD rats;the silencing of LIMK1 in hippocampal neurons did not affect its upstream activity of Racl and Cdc42.2.Knocking down LIMK1 can reduce the susceptibility to seizures in rats.3.LIMK1 may affect seizures by regulating actin polymerization4.In the hippocampal neurons treated with Mg²⁺-free,the density of dendritic spines and the proportion of mature spines were significantly increased when silencing LIMK1,proving that LIMK1 participates in the pathological process of synaptic remodeling during seizure...