Dynamin-1与内侧颞叶癫痫发生的关系及机制研究
发布时间：2018-07-27 13:03
【摘要】：研究背景与目的： 癫痫(epilepsy)是一种常见的神经系统疾病,在儿童期发病率明显高于其他年龄阶段,发病率约为3.5‰-6.6‰,其发病形式多样,病因复杂,对病人的危害巨大。其中难治性癫痫以内侧颞叶癫痫(mesial temporal lobe epilepsy, MTLE)较为常见,海马硬化为其突出的临床病理特征。迄今为止,MTLE的治疗一直是癫痫治疗的难点,MTLE的发病机制仍是近几十年来癫痫研究的热点与难点。 近几年来,关于突触囊泡循环相关调控蛋白的研究已成为一个新的热点。突触囊泡循环是维持神经递质传递的基础,突触囊泡通过胞吐和胞吞作用完成一次突触囊泡循环。大量研究证实,癫痫患者神经元突触有明显的功能异常,这种病态突触通过突触囊泡的快速循环再生使正常情况下每秒仅能传播数次或数十次神经冲动的突触传递功能增加到每秒数十次到数百次,使痫性放电得以迅速扩布。深入研究囊泡的再循环细节以及生理调控的方式和位点,为临床寻找有效治疗靶点,具有重要的理论和实际意义。 Dynamin-1,又称发动蛋白-1,是一个96kD的大分子量三磷酸鸟苷酶(GTPase),为GTPase家族成员,近20年的研究从基因、细胞信号调控、蛋白功能等多方面已证实,dynamin-1在网格蛋白介导的突触囊泡胞吞及囊泡循环利用中发挥至关重要的作用。非dynamin-1依赖的其他调控机制能够维持生理状态下基本正常的突触囊泡胞吞功能,而dynamin-1在维持神经元高频、长时程放电过程中发挥了关键作用。因此,以dynamin-1作为靶点进行干预,可以阻断囊泡在高频、长时程电兴奋事件中的快速循环而发挥作用,而对生理状态下的突触传递活动影响甚少。由此可见,dynamin-1有希望成为一个非常有前景、完全不同于传统抗癫痫药物作用机制的抗癫痫新药研发靶点。 本实验通过建立幼年大鼠MTLE模型,从一个整体、连续发展的角度探讨dynamin-1在MTLE发生发展过程中的表达变化,同时在人MTLE患儿海马组织中进行验证。并进一步建立离体原代海马神经元癫痫细胞模型,探讨dynamin-1在癫痫发生过程中的机制。从而为难治性MTLE寻找新的药物靶点提供了一个全新的视角。 随后,本实验通过构建dynamin-14个结构域的融合蛋白,在大鼠脑神经突触小体中筛选出63个可能与dynamin-1存在相互作用的蛋白,并选取了3个以前从未报道过与dynamin-1有相互作用的蛋白：Rab GDⅠ、C1C-3以TUC-4b,利用免疫共沉淀对其进行自然条件下与dynamin-1相互作用的研究。 本研究分为三部分： 研究方法： 1.利用本实验室已建立的方法,用氯化锂-匹罗卡品诱导3周龄Sprague-Dawley (SD)幼鼠出现癫痫发作,根据MTLE发生发展过程取三个时间点(急性期、潜伏期、慢性期)一共分为6个组：急性期对照组、急性期模型组、潜伏期对照组、潜伏期模型组、慢性期自发发作组、慢性期对照组。运用western blot及免疫组化技术动态观察MTLE大鼠海马组织中dynamin-1及磷酸化dynamin-1蛋白的表达变化。并收集人难治性MTLE术后海马标本,western blot及免疫组化方法检测其表达变化。 2.利用新生SD大鼠培养海马神经元,应用无镁细胞外液灌流方法诱导痫性放电,建立癫痫细胞模型。应用dynamin-1抑制剂dynasore抑制dynamin-1的活性,western blot及免疫荧光技术观察癫痫细胞模型中dynamin-1及磷酸化dynamin-1蛋白的表达变化,并利用转铁蛋白进一步研究抑制dynamin-1蛋白活性后癫痫细胞模型胞吞量的改变。 3.构建dynamin-14个功能结构域的融合蛋白,与大鼠脑突触小体相互作用,利用GST-pull down联合质谱技术筛选出dynamin-1在大鼠脑神经突触小体中的相互作用蛋白。验证2个已有报道中确切与dynamin-1存在相互作用的蛋白,endophilins以及amphiphysins。并选取3个以前从未报道过的dynamin-1相互作用的蛋白,Rab GDⅠ、C1C-3以及TUC-4b,应用免疫共沉淀对其进行自然条件下与dynamin-1相互作用的研究。 研究结果： 1. Western blot及免疫组化结果显示磷酸化dynamin-1蛋白在MTLE大鼠急性期及慢性期以及MTLE患儿海马组织中表达较同时期对照组显著降低(P0.05),在MTLE大鼠潜伏期的表达与同时期对照组相比无差异。而dynamin-1总蛋白在MTLE大鼠各期、MTLE患儿及对照组海马之间表达无明显差异。 2. Western blot及免疫组化结果显示磷酸化dynamin-1蛋白在癫痫细胞模型组中表达较对照组显著降低(P0.05),经dynamin-1抑制剂dynasore预处理的癫痫细胞模型组中磷酸化dynamin-1蛋白表达则与正常对照组基本一致,同时,dynasore对正常对照组磷酸化dynamin-1蛋白表达无明显影响。Dynamin-1总蛋白在海马神经元痫性放电模型及对照组中表达无明显差异。激光显微共聚焦显示癫痫细胞模型组海马神经元胞吞量较正常对照组明显增多(P0.05)。应用dynasore抑制dynamin-1蛋白活性表达后,痫性放电组海马神经元胞吞量显著减少。同时,dynasore对正常对照组海马神经元胞吞量无明显影响。 3.本实验共筛选出63个可能与dynamin-1存在相互作用的蛋白,其中PRD域鉴定出36个蛋白,PH域14个蛋白,GED域7个蛋白,GTPase域6个蛋白。已知的dynamin-1相互作用蛋白endophilins和amphiphysins被证实与dynamin-1存在相互作用关系。未报道蛋白RabGDI以及C1C-3与dynamin-1存在相互作用关系,然而,TUC-4b与dynamin-1之间未检测出相互作用。 研究结论： 1.磷酸化dynamin-1蛋白在MTLE大鼠急性期、慢性期以及MTLE患儿海马组织中表达较同时期对照组显著降低,提示dynamin-1可能通过磷酸化／去磷酸化过程在癫痫发生发展过程中发挥重要作用。 2.癫痫细胞模型组磷酸化dynamin-1蛋白表达下降,转铁蛋白胞吞量增多,但抑制dynamin-1活性后,其表达与正常对照组无明显变化,同时正常对照组dynamin-1活性抑制前后磷酸化dynamin-1蛋白表达无明显差异。提示dynamin-1可能通过影响其磷酸化／去磷酸化及调节突触囊泡胞吞在癫痫发病机制中起重要作用,但对生理状态下正常细胞表达无影响。 3.筛选了63个可能与dynamin-1存在相互作用的蛋白。证实endophilins和amphiphysins蛋白与dynamin-1存在相互作用关系。首次报道蛋白Rab GDI以及C1C-3与dynamin-1作用相关。
[Abstract]:Research background and purpose:
Epilepsy (epilepsy) is a common nervous system disease. The incidence of epilepsy in childhood is obviously higher than that of other ages. The incidence of epilepsy is about 3.5 per thousand -6.6 per thousand. The incidence of epilepsy is varied, the cause is complex and the disease is very harmful to the patients. Among them, the intractable epilepsy (mesial temporal lobe epilepsy, MTLE) is more common, and the hippocampus is sclerosis. So far, the treatment of MTLE has been a difficult point for the treatment of epilepsy, and the pathogenesis of MTLE is still a hot and difficult point in recent decades.
In recent years, the study of synaptic vesicle cycle related regulatory proteins has become a new hot spot. Synaptic vesicle circulation is the basis for maintaining neurotransmitter transmission. Synaptic vesicles complete a synaptic vesicle cycle through exocytosis and endocytosis. A large number of studies have proved that the neuronal synapses in epileptic patients have obvious functional abnormalities. The rapid circulatory regeneration of synapses through synaptic vesicles makes synaptic transmission functions that can only transmit several or dozens of nerve impulses per second to hundreds of times per second to hundreds of times per second in normal conditions, making the epileptic discharge rapidly spread. Effective therapeutic targets have important theoretical and practical significance.
Dynamin-1, also known as the starting protein -1, is a 96kD macromolecular weight three guanosine enzyme (GTPase), a member of the GTPase family. In the past 20 years, the research has been confirmed in many aspects, such as gene, cell signal regulation, protein function, and so on. Dynamin-1 plays a vital role in the use of grid protein mediated synaptic vesicle endocytosis and vesicle recycling. Other regulatory mechanisms of IN-1 dependence can maintain the basic normal synaptic vesicle function under physiological state, and dynamin-1 plays a key role in maintaining the high frequency and long term discharge process of neurons. Therefore, the intervention of dynamin-1 as a target can block the rapid circulation of vesicles in high frequency and long term electrical excitatory events. It can be seen that dynamin-1 is expected to be a very promising, completely different from the traditional antiepileptic drug mechanism of antiepileptic drugs research and development target.
In this experiment, the MTLE model of young rats was established to explore the expression changes of dynamin-1 during the development of MTLE from a whole and continuous development point of view. At the same time, it was verified in the hippocampal tissue of children with MTLE, and the epileptic cell model of the isolated primary hippocampal neurons was further established to explore the mechanism of dynamin-1 in the process of epilepsy. This provides a new perspective for the search of new drug targets for refractory MTLE.
Then, we screened 63 proteins that might interact with dynamin-1 in the synaptic body of the rat brain by constructing the fusion protein of dynamin-14 domain, and selected 3 proteins that had never been interacted with dynamin-1 previously reported: Rab GD I, C1C-3 with TUC-4b, and use immunoprecipitation for its nature. The study of the interaction with dynamin-1 under conditions.
This study is divided into three parts:
Research methods:
1. using the method established in this laboratory, lithium chloride pilocarpine was used to induce epileptic seizures in young rats of 3 weeks old Sprague-Dawley (SD). According to the development process of MTLE, three time points (acute, latent, chronic) were divided into 6 groups: acute phase control group, acute stage model group, latent period control group, latent period model group, slow The changes in the expression of dynamin-1 and phosphorylated dynamin-1 protein in the hippocampus of MTLE rats were dynamically observed by Western blot and immunohistochemical technique. The expressions of the hippocampal dynamin-1 after intractable MTLE were collected, and the expression changes were detected by Western blot and immunohistochemistry.
2. the hippocampal neurons were cultured in the newborn SD rats. The epileptic discharge was induced by the method of MGC external fluid perfusion. The epileptic cell model was established. The activity of dynamin-1 was inhibited by the dynamin-1 inhibitor dynasore. The expression of dynamin-1 and phosphorylated dynamin-1 protein in the epileptic cell model was observed by Western blot and immunofluorescence. Further studies on the changes of endocytosis in the epileptic cell model after inhibiting the activity of dynamin-1 protein were carried out with transferrin.
3. a fusion protein of dynamin-14 functional domains was constructed and interacted with the synaptosomes in the rat brain. The interaction proteins of dynamin-1 in the synaptic body of the rat brain were screened by GST-pull down combined mass spectrometry. The protein, endophilins and amphiphysins that had been confirmed to be exactly interacting with dynamin-1 were reported. 3 proteins interacting with dynamin-1, Rab GD I, C1C-3 and TUC-4b, which have never been reported before, were selected to study the interaction with dynamin-1 in natural conditions by immunoprecipitation.
The results of the study:
1. Western blot and immunohistochemical results showed that the expression of phosphorylated dynamin-1 protein in the acute and chronic phase of MTLE rats and in the hippocampus of MTLE children was significantly lower than that of the same period control group (P0.05). The expression of the latent period in the MTLE rats was no difference compared with the same period control group, but the total dynamin-1 protein was in the MTLE rat and MTLE children. There was no significant difference in the expression of the hippocampus between the control group and the control group.
2. Western blot and immunohistochemical results showed that the expression of phosphorylated dynamin-1 protein in the epileptic cell model group was significantly lower than that in the control group (P0.05). The expression of phosphorylated dynamin-1 protein in the epileptic cell model group pretreated by dynamin-1 inhibitor dynasore was basically consistent with that of the normal control group, and dynasore against the normal control group. The expression of dynamin-1 protein had no obvious effect on the expression of.Dynamin-1 total protein in the epileptic discharge model of hippocampal neurons and in the control group. Laser microconfocal microscopy showed that the amount of hippocampal neurons in the epileptic cell model group increased significantly (P0.05). After the application of dynasore to inhibit the expression of dynamin-1 protein, the epileptogenic activity was observed. The amount of cytosolic carbon in hippocampal neurons of the discharge group was significantly reduced. Meanwhile, dynasore had no significant effect on the amount of neurons in the hippocampus of the normal control group.
3. we screened 63 proteins that might interact with dynamin-1, in which 36 proteins were identified in the PRD domain, 14 in PH domain, 7 in the GED domain and 6 in the GTPase domain. The known dynamin-1 interacting protein endophilins and amphiphysins were confirmed to interact with dynamin-1. No protein RabGDI and C1C were reported. There is interaction between -3 and dynamin-1. However, no interaction was found between TUC-4b and dynamin-1.
The conclusions are as follows:
The expression of 1. phosphorylated dynamin-1 protein in the acute, chronic, and hippocampal tissues of MTLE rats was significantly lower than that of the same period control group, suggesting that dynamin-1 may play an important role in the development of epilepsy through the process of phosphorylation / dephosphorylation.
2. the expression of phosphorylated dynamin-1 protein in the epileptic cell model group decreased and the amount of transferrin swallowed increased, but the expression of the dynamin-1 activity was not significantly changed after the inhibition of the activity of the normal control group, and there was no significant difference in the expression of phosphorylated dynamin-1 protein before and after the inhibition of dynamin-1 activity in the normal control group. It suggested that dynamin-1 may affect its phosphorylation. Dephosphorylation and regulation of synaptic vesicle endocytosis play an important role in the pathogenesis of epilepsy, but have no effect on normal cell expression in physiological condition.
3. 63 proteins that might interact with dynamin-1 were screened. It was confirmed that endophilins and amphiphysins proteins interact with dynamin-1. The first reports of protein Rab GDI and C1C-3 are related to the action of dynamin-1.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R742.1
[Abstract]:Research background and purpose:
Epilepsy (epilepsy) is a common nervous system disease. The incidence of epilepsy in childhood is obviously higher than that of other ages. The incidence of epilepsy is about 3.5 per thousand -6.6 per thousand. The incidence of epilepsy is varied, the cause is complex and the disease is very harmful to the patients. Among them, the intractable epilepsy (mesial temporal lobe epilepsy, MTLE) is more common, and the hippocampus is sclerosis. So far, the treatment of MTLE has been a difficult point for the treatment of epilepsy, and the pathogenesis of MTLE is still a hot and difficult point in recent decades.
In recent years, the study of synaptic vesicle cycle related regulatory proteins has become a new hot spot. Synaptic vesicle circulation is the basis for maintaining neurotransmitter transmission. Synaptic vesicles complete a synaptic vesicle cycle through exocytosis and endocytosis. A large number of studies have proved that the neuronal synapses in epileptic patients have obvious functional abnormalities. The rapid circulatory regeneration of synapses through synaptic vesicles makes synaptic transmission functions that can only transmit several or dozens of nerve impulses per second to hundreds of times per second to hundreds of times per second in normal conditions, making the epileptic discharge rapidly spread. Effective therapeutic targets have important theoretical and practical significance.
Dynamin-1, also known as the starting protein -1, is a 96kD macromolecular weight three guanosine enzyme (GTPase), a member of the GTPase family. In the past 20 years, the research has been confirmed in many aspects, such as gene, cell signal regulation, protein function, and so on. Dynamin-1 plays a vital role in the use of grid protein mediated synaptic vesicle endocytosis and vesicle recycling. Other regulatory mechanisms of IN-1 dependence can maintain the basic normal synaptic vesicle function under physiological state, and dynamin-1 plays a key role in maintaining the high frequency and long term discharge process of neurons. Therefore, the intervention of dynamin-1 as a target can block the rapid circulation of vesicles in high frequency and long term electrical excitatory events. It can be seen that dynamin-1 is expected to be a very promising, completely different from the traditional antiepileptic drug mechanism of antiepileptic drugs research and development target.
In this experiment, the MTLE model of young rats was established to explore the expression changes of dynamin-1 during the development of MTLE from a whole and continuous development point of view. At the same time, it was verified in the hippocampal tissue of children with MTLE, and the epileptic cell model of the isolated primary hippocampal neurons was further established to explore the mechanism of dynamin-1 in the process of epilepsy. This provides a new perspective for the search of new drug targets for refractory MTLE.
Then, we screened 63 proteins that might interact with dynamin-1 in the synaptic body of the rat brain by constructing the fusion protein of dynamin-14 domain, and selected 3 proteins that had never been interacted with dynamin-1 previously reported: Rab GD I, C1C-3 with TUC-4b, and use immunoprecipitation for its nature. The study of the interaction with dynamin-1 under conditions.
This study is divided into three parts:
Research methods:
1. using the method established in this laboratory, lithium chloride pilocarpine was used to induce epileptic seizures in young rats of 3 weeks old Sprague-Dawley (SD). According to the development process of MTLE, three time points (acute, latent, chronic) were divided into 6 groups: acute phase control group, acute stage model group, latent period control group, latent period model group, slow The changes in the expression of dynamin-1 and phosphorylated dynamin-1 protein in the hippocampus of MTLE rats were dynamically observed by Western blot and immunohistochemical technique. The expressions of the hippocampal dynamin-1 after intractable MTLE were collected, and the expression changes were detected by Western blot and immunohistochemistry.
2. the hippocampal neurons were cultured in the newborn SD rats. The epileptic discharge was induced by the method of MGC external fluid perfusion. The epileptic cell model was established. The activity of dynamin-1 was inhibited by the dynamin-1 inhibitor dynasore. The expression of dynamin-1 and phosphorylated dynamin-1 protein in the epileptic cell model was observed by Western blot and immunofluorescence. Further studies on the changes of endocytosis in the epileptic cell model after inhibiting the activity of dynamin-1 protein were carried out with transferrin.
3. a fusion protein of dynamin-14 functional domains was constructed and interacted with the synaptosomes in the rat brain. The interaction proteins of dynamin-1 in the synaptic body of the rat brain were screened by GST-pull down combined mass spectrometry. The protein, endophilins and amphiphysins that had been confirmed to be exactly interacting with dynamin-1 were reported. 3 proteins interacting with dynamin-1, Rab GD I, C1C-3 and TUC-4b, which have never been reported before, were selected to study the interaction with dynamin-1 in natural conditions by immunoprecipitation.
The results of the study:
1. Western blot and immunohistochemical results showed that the expression of phosphorylated dynamin-1 protein in the acute and chronic phase of MTLE rats and in the hippocampus of MTLE children was significantly lower than that of the same period control group (P0.05). The expression of the latent period in the MTLE rats was no difference compared with the same period control group, but the total dynamin-1 protein was in the MTLE rat and MTLE children. There was no significant difference in the expression of the hippocampus between the control group and the control group.
2. Western blot and immunohistochemical results showed that the expression of phosphorylated dynamin-1 protein in the epileptic cell model group was significantly lower than that in the control group (P0.05). The expression of phosphorylated dynamin-1 protein in the epileptic cell model group pretreated by dynamin-1 inhibitor dynasore was basically consistent with that of the normal control group, and dynasore against the normal control group. The expression of dynamin-1 protein had no obvious effect on the expression of.Dynamin-1 total protein in the epileptic discharge model of hippocampal neurons and in the control group. Laser microconfocal microscopy showed that the amount of hippocampal neurons in the epileptic cell model group increased significantly (P0.05). After the application of dynasore to inhibit the expression of dynamin-1 protein, the epileptogenic activity was observed. The amount of cytosolic carbon in hippocampal neurons of the discharge group was significantly reduced. Meanwhile, dynasore had no significant effect on the amount of neurons in the hippocampus of the normal control group.
3. we screened 63 proteins that might interact with dynamin-1, in which 36 proteins were identified in the PRD domain, 14 in PH domain, 7 in the GED domain and 6 in the GTPase domain. The known dynamin-1 interacting protein endophilins and amphiphysins were confirmed to interact with dynamin-1. No protein RabGDI and C1C were reported. There is interaction between -3 and dynamin-1. However, no interaction was found between TUC-4b and dynamin-1.
The conclusions are as follows:
The expression of 1. phosphorylated dynamin-1 protein in the acute, chronic, and hippocampal tissues of MTLE rats was significantly lower than that of the same period control group, suggesting that dynamin-1 may play an important role in the development of epilepsy through the process of phosphorylation / dephosphorylation.
2. the expression of phosphorylated dynamin-1 protein in the epileptic cell model group decreased and the amount of transferrin swallowed increased, but the expression of the dynamin-1 activity was not significantly changed after the inhibition of the activity of the normal control group, and there was no significant difference in the expression of phosphorylated dynamin-1 protein before and after the inhibition of dynamin-1 activity in the normal control group. It suggested that dynamin-1 may affect its phosphorylation. Dephosphorylation and regulation of synaptic vesicle endocytosis play an important role in the pathogenesis of epilepsy, but have no effect on normal cell expression in physiological condition.
3. 63 proteins that might interact with dynamin-1 were screened. It was confirmed that endophilins and amphiphysins proteins interact with dynamin-1. The first reports of protein Rab GDI and C1C-3 are related to the action of dynamin-1.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R742.1
【共引文献】
相关期刊论文 前10条
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7 李听松;叶园珍;蒋莉;陈恒胜;熊佳佳;孔敏;胡君;;幼鼠癫痫发生过程中胼胝体内髓鞘变化的研究[J];第三军医大学学报;2013年20期
8 武晨;江文;;突触囊泡蛋白2A:抗癫痫药物的新靶点[J];国际神经病学神经外科学杂志;2014年03期
9 邢晶晶;刘海娇;范路生;宋凯;陈彤;林金星;;植物细胞胞吞途径及其研究方法[J];电子显微学报;2014年05期
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