神经调节素1-ErbB4信号对GABA能中间神经元功能的调节及其在癫痫等疾病中的作用

发布时间：2018-05-28 15:37

  本文选题：ErbB4受体 + 神经调节素1　； 参考：《浙江大学》2014年博士论文

【摘要】：神经调节素(NRG1)是神经生长因子家族的一员,其通过作用于ErbB受体家族发挥作用,包括ErbB4受体。近年来,来自世界不同研究组的遗传学研究发现Nrg1和Erbb4是两个独立的精神分裂症易感基因。在成年脑内,Erbb4 mRNA在中间神经元上比较丰富。最近的研究结果显示,ErbB4受体特异性表达在GABA能中间神经元上,特别是PV中间经元上。这些研究提示PV神经元可能是NRG1-ErbB4信号在成年大脑中的主要细胞靶点。在大脑皮层中,约有20-30%的GABA能中间神经元,尽管数量不多,但是GABA能中间神经元在兴奋抑制平衡、感觉信息的处理、神经环路的同步化以及情绪,动机的传递和调节中发挥了重要作用。GABA能抑制性神经元主要在局部环路中起作用,比如PV中间神经元,主要投射到锥体细胞近胞体端,可以非常有效地调控锥体细胞的输出和同步化活动。大量研究指出,PV神经元参与调控大脑的高级认知功能。多项研究提示GABA能神经元介导的微环路异常是癫痫、精神分裂症、自闭症等神经精神疾病的致病基础。所以,我们想以NRG1-ErbB4为切入点,研究PV介导的微环路在神经精神疾病中的作用及机制。癫痫(Epilepsy)是大脑神经元突发性异常放电,进而导致短暂的大脑功能障碍的一种慢性疾病。目前,经过现有的抗癫痫药物治疗,仍有大约30%的患者不能控制。药物不能控制的癫痫,即难治性癫痫的死亡率可达50%。因此,寻找有效和安全的治疗药物是生物医学重要的目标之一。尽管癫痫的起始和发生的机制仍没有完全明白,但是大脑中兴奋与抑制的失平衡被认为是一个关键因素。PV神经元主要以近胞体端投射的方式投射到锥体神经元,即投射到锥体细胞胞体、基树突以及轴突起始端,这种投射可以控制锥体细胞的发放模式和发放时机,进而以gamma和theta震荡的方式同步化锥体细胞。来自不同实验室David Lau, Ikuo Ogiwara等人用在体脑电记录的方式显示PV中间神经元的低功能可以导致癫痫发生。PV神经元抑制能力的下降与癫痫的这种联系让我们猜测NRG1-ErbB4信号的失常在癫痫发生中发挥怎样的作用。此项研究综合运用转基因小鼠、生化、药理学以及电生理学的方法证明NRG1通过其受体ErbB4,增加了parvalbumin阳性神经元的兴奋性。这一作用是通过调节Kvl.1型钾离子通道来降低神经元动作电位发放的阈值来实现的。敲除ErbB4的parvalbumin阳性神经元也表现为兴奋性的降低。所以说NRG1-ErbB4信号通路的异常可能参与到由parvalbumin日性神经元介导的抑制性环路的异常。我们发现,在parvalbumin阳性神经元上特异性敲除ErbB4受体的小鼠,对匹鲁卡品,戊四唑等癫痫模型有很高的易感性。这种敲除小鼠发作的癫痫等级更高,小鼠发作癫痫的几率更高。另外,脑室内注射重组NRG1可以降低致痫药诱导的癫痫的发生和发展。我们想进一步探讨NRG1-ErbB4信号在人类癫痫中潜在生物学价值。我们比较了继发性癫痫与对照组织中NRG1-ErbB4信号及其下游蛋白的表达。第一,我们比较了正常组织的颞叶,额叶皮层与癫痫患者相应区域的蛋白表达量,发现癫痫组织中ErbB4的含量显著增高；第二,癫痫组织中,颞叶皮层具有升高的Src家族蛋白表达量,但是Src-pY416/Src比例下降。那是否有可能是NRG-ErbB4信号参与了src家族激酶的活性调控呢?有意思的是,NRG1孵育脑片可以逐渐降低Src-pY416/Src比例水平。第三,我们知道GluN2B是一个重要的Src酪氨酸激酶调节靶点,而GluN2B-pY1472位点磷酸化水平可以影响GluN2B在细胞膜的滞留和功能。所以我们试图观察癫痫组织中GluN2B-pY1472的水平是否发生变化。我们发现在癫痫皮层组织中GluN2B表达显著增多,但其1472位点的磷酸化水平并没有相应程度的增加。所以,我们推测这可能是由于癫痫组织中升高的ErbB4引起的Src激酶活性下降而受到的调节。第四,我们对NRG-ErbB4是否能通过Src调节GluN2B的磷酸化水平感兴趣。我们观察到NRG1孵育可以降低src-pY416水平,同时降低了GluN2B-pY1472的水平。第五,癫痫组织中抑制性神经元更兴奋,NRGl可以增加癫痫或对照组织抑制性神经元兴奋性。这些实验为NRG1-ErbB4信号参与PV神经元兴奋性的调节及GABA能神经元保护及抑制性突触传递对癫痫的影响提供了有力的证据,也为解析癫痫发展中GABA能抑制系统的作用及其接受的调节提供了人脑水平的研究。综上,系统利用电生理、行为学、药理学和生化的方法,借助精神分裂症的易感基因Nrgl和Erbb4,我们研究了PV神经元介导的抑制性微环路在癫痫疾病中的作用及机制。同时,我们还找到Neuregulinl-ErbB4信号的作用靶点,为后续的癫痫等疾病治疗提供了细胞水平的候选分子。
[Abstract]:Neuroregulator (NRG1) is a member of the nerve growth factor family, which plays a role in the ErbB receptor family, including the ErbB4 receptor. In recent years, genetic studies from different research groups in the world have found that Nrg1 and Erbb4 are two independent susceptibility genes for schizophrenia. In the adult brain, Erbb4 mRNA is more abundant in the middle neurons. Rich. Recent results show that the ErbB4 receptor is specifically expressed on the GABA energy intermediate neurons, especially the PV intermediate. These studies suggest that the PV neurons may be the major target of the NRG1-ErbB4 signal in the adult brain. In the cerebral cortex, there are about 20-30% GABA in the intermediate neurons, although the number is not much, but GABA can Intermediate neurons play an important role in stimulating inhibition of balance, processing of sensory information, synchronization of neural circuits, emotion, transmission and regulation of moods,.GABA suppressor neurons play an important role in local loops, such as PV intermediate neurons, which are mainly projected into the near cell end of the pyramidal cells, and can be very effective in regulating cones. The output and synchronization of somatic cells. A large number of studies have pointed out that PV neurons are involved in the control of the brain's advanced cognitive functions. A number of studies suggest that the microloop abnormalities mediated by GABA neurons are the pathogeny basis of epilepsy, schizophrenia, autism and other neuropsychiatric disorders. Therefore, we want to use NRG1-ErbB4 as the breakthrough point to study the microsphere mediated by PV. The role and mechanism of the loop in neuropsychiatric disorders. Epilepsy (Epilepsy) is a chronic disease of the brain neuron sudden abnormal discharge, which leads to transient brain dysfunction. At present, about 30% of the patients are still unable to control it after the current antiepileptic drug treatment. The drug can not control epilepsy, that is, the death of intractable epilepsy. The death rate is up to 50%., so finding effective and safe therapeutic drugs is one of the important biomedical targets. Although the mechanism of onset and occurrence of epilepsy is still not fully understood, the imbalance of excitation and inhibition in the brain is considered to be a key factor in the projection of.PV neurons to the pyramidal nerve mainly in the way of the near cell end projection. The element, projecting into the pyramidal cell body, the base dendrites and the beginning of the axon protuberance, can control the distribution patterns and timing of the pyramidal cells, and then synchronize the pyramidal cells in the manner of gamma and theta oscillations. From different laboratories David Lau, Ikuo Ogiwara and others display the PV intermediate neurons in the manner of the body brain electrical recording. Low function can lead to the decline of.PV neuron inhibition in epileptic seizures and the association of epilepsy, which let us guess what the role of the NRG1-ErbB4 signal is in the occurrence of epilepsy. This study combined transgenic mice, biochemical, pharmacological, and electrophysiological methods to prove that NRG1 increased parval through its receptor ErbB4. The excitability of the bumin positive neurons. This effect is achieved by reducing the threshold of the action potential distribution by regulating the Kvl.1 type potassium channel. The parvalbumin positive neurons that knock off the ErbB4 also show a decrease in the excitability. Therefore, the abnormalities of the NRG1-ErbB4 signaling pathway may be involved in the parvalbumin diurnal neurons. We found that the mice that specifically knocked out the ErbB4 receptor on the parvalbumin positive neurons have high susceptibility to the pilocarpine, penttrazol and other epileptic models. This type of knockout mice has a higher level of epileptic seizures and a higher incidence of epileptic seizures in mice. In addition, intraventricular injection of recombinant NRG1 can be reduced. The occurrence and development of epilepsy induced by epileptic drugs. We want to further explore the potential biological value of NRG1-ErbB4 signal in human epilepsy. We compared the expression of NRG1-ErbB4 signal and its downstream protein in secondary epilepsy and control tissue. First, we compare the temporal lobe of normal tissue, the frontal cortex and the corresponding area of epileptic patients. It was found that the content of ErbB4 in the epileptic tissues increased significantly. Second, in the epileptic tissues, the temporal lobe cortex had an elevated Src family protein expression, but the proportion of Src-pY416/Src decreased. Is it possible that the NRG-ErbB4 signal is involved in the activity regulation of the Src family kinase? It is interesting that the NRG1 incubating brain slices can gradually be developed. Reduce the Src-pY416/Src level. Third, we know that GluN2B is an important regulatory target of Src tyrosine kinase, and the level of phosphorylation of GluN2B-pY1472 loci can affect the retention and function of GluN2B in the cell membrane. So we try to observe whether the level of GluN2B-pY1472 in the epileptic tissue changes. The expression of GluN2B in the tissue was significantly increased, but the level of phosphorylation at the 1472 site did not increase correspondingly. Therefore, we speculate that this may be due to the regulation of the decrease in the activity of Src kinase caused by the elevated ErbB4 in the epileptic tissues. Fourth, we are interested in whether NRG-ErbB4 can regulate the phosphorylation level of GluN2B through Src. We observed that NRG1 incubation can reduce the level of src-pY416 and reduce the level of GluN2B-pY1472. Fifth, the inhibitory neurons in the epileptic tissues are more excited, and NRGl can increase epilepsy or control tissue inhibitory neuronal excitability. These experiments are involved in the involvement of NRG1-ErbB4 signals in the regulation of the excitability of the PV deity and the protection of GABA neurons. And the effect of inhibitory synaptic transmission on epilepsy has provided strong evidence, and also provides a human brain level study for the analysis of the role of GABA in the development of epilepsy and the regulation of its acceptance. To sum up, the system uses electrophysiological, behavioral, pharmacological and biochemical methods to help the susceptibility genes of schizophrenia Nrgl and Erbb4. The role and mechanism of the inhibitory microloop mediated by PV neurons in epileptic disease are investigated. At the same time, we also find the target of the Neuregulinl-ErbB4 signal to provide a cell level candidate for the subsequent treatment of epilepsy and other diseases.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R742.5
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本文编号：1947265