| Epilepsy is a common neurologic disorder with complex pathogenesis and various onset forms. The children morbidity is significant higher than any other age group, about3.5%o-6.6%o and the harm to the patients is serious. Mesial temporal lobe epilepsy (MTLE) is one more common intractable epilepsy character with hippocampal sclerosis. Nowadays, the treatment for MTLE is still a difficult problem and researches on the mechanism will be a hot issue in recent decades.Recently, more and more researches have demonstrated that the synapses in epileptics were distinct dysfunction. The morbid synapses could speed up the nervous impulse from several or ten times per second in normal to dozens or even hundreds times per second, through the rapid cycle of synaptic vesicle, and subsequently spread the epileptic discharge fastly. The synaptic vesicle is the foundation to maintain the transition of neurotransmitter, completed by exocytosis and endocytosis. Further studies on details of morbid synaptic vesicle recycling and the regulatory mode and site would be great theoretic and practical significance for searching the therapeutic target in clinic.Dynamin-1, a member of GTPase family with a molecular weight of96kD, is highly and selectively expressed in neurons and represents by far the major dynamin isoform expressed in the brain. Researches in recent 20years had proved dynamin-1was crucial for the fission stage of synaptic vesicle recycling and vesicle trafficking mediated by clathrin, involved gene regulation, cell signaling pathway and protein function. Non-dynamin-1dependent regulatory mechanism could maintain the endocytosis of synaptic vesicle in physiology, while dynamin-1played a key role in keeping the discharge process of high frequency and long-time interval. So intervention to dynamin-1as a target could block the rapid cycle in the high frequent and long-time interval discharge process, with little effect of synaptic transmission in physiology. Dynamin-1might be a very promising target to design antiepileptic drugs (AED), totally different from the mechanism of conditional AED.In this study, we investigated the expression change of dynamin-1in the development of MTLE integrally and continuously by using a MTLE model in immature rat, and verified in MTLE patients. Then we established cell model of epilepsy in primary hippocampal neurons and explored the mechanism of dynamin-1in epilepsy development.Subsequently, we constructed prokaryotic expression plasmids for the four functional domains of dynamin-1. Glutathione S-transferase pull-down, co-immunoprecipitation (co-IP), and liquid chromatography/mass spectrometry were used to screen and identify dynamin-1interacting proteins in rat brain synaptosomes. We identified a set of63candidate protein interactions, including36proteins interacting with dynamin-1C-terminal proline-rich domain (PRD),14with pleckstrin-homology domain (PH),7with GTPase effector domain (GED) and6with GTPase domain, consisting of synaptic vesicle-associated proteins, cytoskeletal proteins, metabolic enzymes and other proteins. We selected three previously unreported dynamin-1interacting proteins (Rab GDI, CIC-3and TUC-4b) to verify their interaction with dynamin-1under native conditions.Methods:1. Three weeks old sprague-dawley (SD) rats were induced to epileptic seizure by lithium-pilocarpin and were divided into control group and model group. According to the development of MTLE, rats in both groups were monitored in the acute, latent and chronic stages of disease (1.5hr,3weeks and8weeks after induced status eiplepticus by lithium-pilocarpine, respectively). We performed western blot and immunohistochemistry to detect the expression of phosphor-dynamin-1and dynamin-1on the hippocampus of the rats. Similar expression methods were applied to hippocampus obtained from children with MTLE and controls from autopsy cases.2. Epileptic cell models were established by magnesium-free infusion induction in cultured hippocampal neuron of newly born SD rat. Dynasore, an inhibitor of dynamin-1, was used to inhibit the activity of dynamin-1. Western blot and immunofluorescence techniques were performed to observe the expression change of dynamin-1and phosphor-dynamin-1in epilepsy cell models, and then transferrin was used to detect the change of endocytosis in epileptic cell models after the activity of dynamin-1was inhibited.3. We constructed prokaryotic expression plasmids for the four functional domains of dynamin-1to interact with rat brain synaptosomes. Glutathione S-transferase pull-down and liquid chromatography/mass spectrometry were used to screen and identify dynamin-1interacting proteins in rat brain synaptosomes. Then we validated two defined proteins of endophilins and amphiphysins both interacted with dynamin-1by Western blot. Three previously unreported dynamin-1interacting proteins (Rab GDI, CIC-3and TUC-4b) were selected to verify their interaction with dynamin-1under native conditions by co-IP.Results:1. Western blot and immunohistochemistry showed the expression of phosphor-dynamin-1decreased significantly in acute stage and chronic stage in MTLE rats and children (p<0.05), while there was no difference at the latent stage. The expression of total dynamin-1protein of MTLE rats at each stage and MTLE children had no significant difference between the control group and model group.2. Compared to the control group, western blot and immunohistochemistry showed feeble phosphor-dynamin-1expression in SE hippocampal neurons compared with the control group (p<0.05), whereas the expression of dynamin-1had no difference comparing to control group. SE had a positive effect on transferrin uptake in hippocampal neurons, whereas after pretreated by dynasore, the endocytosis of transferrin was decreased significantly and meanwhile,for control group, pretreating with dynasore didn’t affect the endocytosis.3. We identified a set of63candidate interacting proteins, including36proteins interacting with C-terminal proline-rich domain (PRD) of dynamin-1,14with pleckstrin-homology domain (PH),7with GTPase effector domain (GED) and6with GTPase domain. The known interacting proteins, endophilins and amphiphysins had been demonstrated the interaction with dynamin-1. We also found that Rab GDI and C1C-3did interact with dynamin-1for the first time.Conclusion:1. Phosphor-dynamin-1but not dynamin-1down-regulation on the hippocampus of MTLE children and MTLE rats in acute and chronic stages which were related to seizure activities, which indicated that the phosphorylation/dephosphorylation of dynamin-1might be related to the epileptogensis of MTLE.2. Less phosphor-dynamin-1expression and a positive effect on transferrin uptake in SE hippocampal neurons compared with the control group. Inhibiting the activity of dynamin-1had an effect on SE neurons but not on normal neurons, which suggested the activity of dynamin-1might play the role in epilepsy only but not in normal physiological status through regulating the synaptic vesicle endocytosis.3. Sixty-three candidate dynamin-1interacting proteins were identified. Two known dynamin-1interacting proteins, endophilins and amphiphysins had been demonstrated the interaction with dynamin-1. Rab GDI and CIC-3were reported to have interactions with dynamin-1for the first time. |
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