| Background Abnormal neural network reconstruction to form neural circuits with superexcitation capabilities of hippocampal neurons may be one of the important pathological mechanisms of medial temporal lobe epilepsy,such as remodeling of dendritic spine morphology/number of hippocampal neurons and the formation of abnormal synaptic connections.Cofilin mainly affects cytoskeletal protein dynamics by cutting actin F-actin to regulate actin fiber dispolymerization and polymerization,resulting in dynamic changes in dendritic spines which is involved in regulating synaptic plasticity.Phosphorylation and dephosphorylation balance of cofilin regulates cytoskeleton recombination and is essential for dendritic spine remodeling of epilepsy neurons.When Ca2+ in nerve cells is elevated,CaMK? can be activated to affect synaptic reconstruction,neurotransmitter release and other processes,which plays an important role in the occurrence and development of epilepsy.Therefore,it has a profound impact on searching for potential drug research targets for temporal lobe epilepsy by to explore whether the CaMK?/cofilin/F-actin pathway has regulating neuronal cytoskeletal recombination to mediate dendritic spine remodeling.Objective 1.Observe the abnormal neural network of medial temporal lobe and hippocampal brain tissue that are surgically removed in patients with medial temporal lobe epilepsy;2.Explore the differential expression profile of epileptic-causing brain tissue proteins in temporal lobe epilepsy patients and verify the expression of CaMK?,and look for suspicious pathogenic proteins for the occurrence and development of temporal lobe epilepsy;3.Explore whether the CaMK?/cofilin/F-actin pathway regulates the recombination of cytoskeleton in the hippocampus to mediate dendritic spine remodeling,and to find effective clinical treatment strategies and potential drug development targets for epilepsy.Methods 1.Collect surgically removed temporal lobe and hippocampal brain tissue from epilepsy patients(n=6)and glioma tumor patients without seizure(n=5),and prepare paraffin sections of human brain tissue.HE staining and Nissl staining were performed to observe histopathological changes under microscopy;Neurofilament-L immunofluorescence staining was used to observe nerve fiber morphology and arrangement under confocal microscopy;Neu N/Doublecortin immunofluorescence staining wea used to observe neuronal cell maturity,Tau/MAP2/Synaptophysin immunofluorescence staining was used to observe synaptic recombination and neurite morphology;2.Perform 4D label free quantitative proteomics sequencing of brain tissue to find differential expression protein profiles,and then perform protein annotation,GO classification,KEGG pathway enrichment analysis,protein function cluster analysis,protein interaction analysis;further,human brain tissue paraffin sections MAP2/CaMK? immunofluorescence double-staining confocal microscopy imaging and CaMK? immunofluorescence staining STED ultra-high resolution imaging were performed to verify the expression and localization of epileptogenic focal brain tissue;3.Prepare rats epilepsy model and explore the correlation between the dynamic changes of hippocampal brain histopathological damage and the dynamic expression trend of CaMK? protein in epilepsy rats.Sham(Sham)group,KA epilepsy model(KA),KA epilepsy model+CaMK? selective inhibitor KN-93(KA+KN-93)group and KA epilepsy model + KN-93 inactive analogue KN-92(KA+KN-92)group were set up to evaluate the behavioral score of seizures in rat groups;the level of malonaldehyde(MDA)and superoxide dismutase(SOD)activity in the hippocampal brain tissue of rats was detected to assess oxidative stress damage;Western Blot was used to detecte the expression levels of CaMK?,cofilin,p-cofilin,F-actin protein;Golgi staining was used to observe the number and morphology of hippocampal neuronal dendrites,and Image J software was used to calculate dendritic spine density and proportion of mature dendritic spines;rat brain tissue paraffin sections for Tau/MAP2/Synaptophysin immunofluorescence were used to observe synaptic recombination and synaptic morphology of hippocampal neurons in epileptic rats.Results 1.HE and Nissl staining results showed selective loss of hippocampal neuron cells,neuronal damage to varying degrees,glial hyperplasia and hippocampal sclerosis in the brain tissue of patients with medial temporal lobe epilepsy;Neurofilament-L immunofluorescence staining results showed that the neural fibers in the brain tissue of patients with medial temporal lobe epilepsy were disordered,budding and tangled;Neu N/Doublecortin immunofluorescence double staining results suggested an increase in the proportion of immature neuronal cells;Tau/MAP2/Synaptophysin immunofluorescence results showed abnormal tangles,increased synapses and abnormal accumulation of neuronal dendrites and axonal arrangements in the brain tissue of patients with medial temporal lobe epilepsy;2.Proteomics results showed that compared with the control group,there were 569 upregulated protein and 841 down-regulated protein in the brain tissue of patients with medial temporal lobe epilepsy,among which the upregulated proteins involved in axon-guided,glutamatergic neuron synapses,dopaminergic neuron synapses,GABA neuronal synapses,calcium ion signaling and other pathways may play important roles in the occurrence and development of epilepsy;MAP2/CaMK? immunofluorescence staining results showed significant increase in CaMK? expression and abnormal accumulation along dendritic processes in the brain tissue of patients with medial temporal lobe epilepsy;3.The pathological damage of hippocampal brain tissue in epileptic rats was most serious at 24 hours of seizures,and the expression of CaMK? protein in hippocampal brain tissue of epileptic rats increased significantly at 24 hours of seizures(p<0.001);Rats in the KA group had significant increases in MDA levels in hippocampal brain tissue(p<0.001),SOD activity decreased significantly(p<0.001),CaMK? protein expression was significantly upregulated(p<0.001),cofilin expression was increased(p<0.05),p-cofilin expression was significantly increased(p<0.01),p-cofilin/cofilin ratio was significantly increased(p<0.01),and F-actin expression was significantly reduced(p<0.001);Golgi staining results showed that the dendrite arrangement of hippocampal neurons was significantly disordered,curved,germinated,tangled,the dendritic spine density decreased(p<0.05),and the proportion of mature dendritic spines decreased significantly(p<0.01);Tau/MAP2/Synaptophysin immunofluorescence trichroma results showed that epileptic rats with hippocampal neuron dendrites,axons bent,sprouted,tangled,synaptic anomalies recombined and accumulated synaptic plaques.Compared with the KA group,the behavioral score of seizures in rats in the KA+KN-93 group was significantly improved.Significantly reduced MDA levels were(p<0.01)and increased SOD activity(p<0.05)were assesed in hippocampal tissue homogenates;decreased p-cofilin expression(p<0.01),significantly reduced p-cofilin/cofilin ratio(p<0.001)and increased F-actin expression(p<0.001)in hippocampal brain tissue,but no significant effect on the total level of cofilin protein;Golgi staining results showed that the dendrite arrangement of hippocampal neurons was significantly regular after selective inhibition of CaMK? activity by KN-93,with significant reductions in curvature,germination,and tangles,increased dendritic spine density(p<0.05),and significantly increased the ratio of mature dendritic spines(p<0.0 1);Tau/MAP2/Synaptophysin immunofluorescence trichromation results showed that the dendrites and axonal morphologies of hippocampal neurons in epileptic rats were significantly improved and the synaptic plaques formed by abnormal synaptic recombination and accumulation were significantly reduced after KN-93 selective inhibition of CaMK? activity.Conclusion 1.Disordered neural fiber arrangement,synaptic recombination,abnormal remodeling of neural networks,and increased proportion of immature neurons were found in the anterior temporal lobe and hippocampal brain tissuse of patients with medial temporal lobe epilepsy;2.The brain tissuse proteomics sequencing results of the anterior temporal lobe and hippocampus of medial temporal lobe epilepsy patients showed abnormal protein expression,among which axon guidance,glutamatergic neuron synapses,dopaminergic neuron synapses,GABA neuron synapses,calcium ion signals and other pathways may play an important role in the occurrence and development of epilepsy; 3.CaMK? expression were significantly upregulated in hippocampal brain tissue of epileptic rats and patients with medial temporal lobe epilepsy;4.CaMK?/cofilin/F-actin pathway mediates dendritic spine remodeling and synaptic recombination by regulating epilepsy rat hippocampal neuron actin cell cytoskeletal recombination,resulting in abnormal neural networks in epileptic rats. |
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