本文選題：ErbB4受體 + 神經(jīng)調(diào)節(jié)素1　； 參考：《浙江大學(xué)》2014年博士論文

【摘要】：神經(jīng)調(diào)節(jié)素(NRG1)是神經(jīng)生長(zhǎng)因子家族的一員,其通過(guò)作用于ErbB受體家族發(fā)揮作用,包括ErbB4受體。近年來(lái),來(lái)自世界不同研究組的遺傳學(xué)研究發(fā)現(xiàn)Nrg1和Erbb4是兩個(gè)獨(dú)立的精神分裂癥易感基因。在成年腦內(nèi),Erbb4 mRNA在中間神經(jīng)元上比較豐富。最近的研究結(jié)果顯示,ErbB4受體特異性表達(dá)在GABA能中間神經(jīng)元上,特別是PV中間經(jīng)元上。這些研究提示PV神經(jīng)元可能是NRG1-ErbB4信號(hào)在成年大腦中的主要細(xì)胞靶點(diǎn)。在大腦皮層中,約有20-30%的GABA能中間神經(jīng)元,盡管數(shù)量不多,但是GABA能中間神經(jīng)元在興奮抑制平衡、感覺(jué)信息的處理、神經(jīng)環(huán)路的同步化以及情緒,動(dòng)機(jī)的傳遞和調(diào)節(jié)中發(fā)揮了重要作用。GABA能抑制性神經(jīng)元主要在局部環(huán)路中起作用,比如PV中間神經(jīng)元,主要投射到錐體細(xì)胞近胞體端,可以非常有效地調(diào)控錐體細(xì)胞的輸出和同步化活動(dòng)。大量研究指出,PV神經(jīng)元參與調(diào)控大腦的高級(jí)認(rèn)知功能。多項(xiàng)研究提示GABA能神經(jīng)元介導(dǎo)的微環(huán)路異常是癲癇、精神分裂癥、自閉癥等神經(jīng)精神疾病的致病基礎(chǔ)。所以,我們想以NRG1-ErbB4為切入點(diǎn),研究PV介導(dǎo)的微環(huán)路在神經(jīng)精神疾病中的作用及機(jī)制。癲癇(Epilepsy)是大腦神經(jīng)元突發(fā)性異常放電,進(jìn)而導(dǎo)致短暫的大腦功能障礙的一種慢性疾病。目前,經(jīng)過(guò)現(xiàn)有的抗癲癇藥物治療,仍有大約30%的患者不能控制。藥物不能控制的癲癇,即難治性癲癇的死亡率可達(dá)50%。因此,尋找有效和安全的治療藥物是生物醫(yī)學(xué)重要的目標(biāo)之一。盡管癲癇的起始和發(fā)生的機(jī)制仍沒(méi)有完全明白,但是大腦中興奮與抑制的失平衡被認(rèn)為是一個(gè)關(guān)鍵因素。PV神經(jīng)元主要以近胞體端投射的方式投射到錐體神經(jīng)元,即投射到錐體細(xì)胞胞體、基樹(shù)突以及軸突起始端,這種投射可以控制錐體細(xì)胞的發(fā)放模式和發(fā)放時(shí)機(jī),進(jìn)而以gamma和theta震蕩的方式同步化錐體細(xì)胞。來(lái)自不同實(shí)驗(yàn)室David Lau, Ikuo Ogiwara等人用在體腦電記錄的方式顯示PV中間神經(jīng)元的低功能可以導(dǎo)致癲癇發(fā)生。PV神經(jīng)元抑制能力的下降與癲癇的這種聯(lián)系讓我們猜測(cè)NRG1-ErbB4信號(hào)的失常在癲癇發(fā)生中發(fā)揮怎樣的作用。此項(xiàng)研究綜合運(yùn)用轉(zhuǎn)基因小鼠、生化、藥理學(xué)以及電生理學(xué)的方法證明NRG1通過(guò)其受體ErbB4,增加了parvalbumin陽(yáng)性神經(jīng)元的興奮性。這一作用是通過(guò)調(diào)節(jié)Kvl.1型鉀離子通道來(lái)降低神經(jīng)元?jiǎng)幼麟娢话l(fā)放的閾值來(lái)實(shí)現(xiàn)的。敲除ErbB4的parvalbumin陽(yáng)性神經(jīng)元也表現(xiàn)為興奮性的降低。所以說(shuō)NRG1-ErbB4信號(hào)通路的異�？赡軈⑴c到由parvalbumin日性神經(jīng)元介導(dǎo)的抑制性環(huán)路的異常。我們發(fā)現(xiàn),在parvalbumin陽(yáng)性神經(jīng)元上特異性敲除ErbB4受體的小鼠,對(duì)匹魯卡品,戊四唑等癲癇模型有很高的易感性。這種敲除小鼠發(fā)作的癲癇等級(jí)更高,小鼠發(fā)作癲癇的幾率更高。另外,腦室內(nèi)注射重組NRG1可以降低致癇藥誘導(dǎo)的癲癇的發(fā)生和發(fā)展。我們想進(jìn)一步探討NRG1-ErbB4信號(hào)在人類癲癇中潛在生物學(xué)價(jià)值。我們比較了繼發(fā)性癲癇與對(duì)照組織中NRG1-ErbB4信號(hào)及其下游蛋白的表達(dá)。第一,我們比較了正常組織的顳葉,額葉皮層與癲癇患者相應(yīng)區(qū)域的蛋白表達(dá)量,發(fā)現(xiàn)癲癇組織中ErbB4的含量顯著增高；第二,癲癇組織中,顳葉皮層具有升高的Src家族蛋白表達(dá)量,但是Src-pY416/Src比例下降。那是否有可能是NRG-ErbB4信號(hào)參與了src家族激酶的活性調(diào)控呢?有意思的是,NRG1孵育腦片可以逐漸降低Src-pY416/Src比例水平。第三,我們知道GluN2B是一個(gè)重要的Src酪氨酸激酶調(diào)節(jié)靶點(diǎn),而GluN2B-pY1472位點(diǎn)磷酸化水平可以影響GluN2B在細(xì)胞膜的滯留和功能。所以我們?cè)噲D觀察癲癇組織中GluN2B-pY1472的水平是否發(fā)生變化。我們發(fā)現(xiàn)在癲癇皮層組織中GluN2B表達(dá)顯著增多,但其1472位點(diǎn)的磷酸化水平并沒(méi)有相應(yīng)程度的增加。所以,我們推測(cè)這可能是由于癲癇組織中升高的ErbB4引起的Src激酶活性下降而受到的調(diào)節(jié)。第四,我們對(duì)NRG-ErbB4是否能通過(guò)Src調(diào)節(jié)GluN2B的磷酸化水平感興趣。我們觀察到NRG1孵育可以降低src-pY416水平,同時(shí)降低了GluN2B-pY1472的水平。第五,癲癇組織中抑制性神經(jīng)元更興奮,NRGl可以增加癲癇或?qū)φ战M織抑制性神經(jīng)元興奮性。這些實(shí)驗(yàn)為NRG1-ErbB4信號(hào)參與PV神經(jīng)元興奮性的調(diào)節(jié)及GABA能神經(jīng)元保護(hù)及抑制性突觸傳遞對(duì)癲癇的影響提供了有力的證據(jù),也為解析癲癇發(fā)展中GABA能抑制系統(tǒng)的作用及其接受的調(diào)節(jié)提供了人腦水平的研究。綜上,系統(tǒng)利用電生理、行為學(xué)、藥理學(xué)和生化的方法,借助精神分裂癥的易感基因Nrgl和Erbb4,我們研究了PV神經(jīng)元介導(dǎo)的抑制性微環(huán)路在癲癇疾病中的作用及機(jī)制。同時(shí),我們還找到Neuregulinl-ErbB4信號(hào)的作用靶點(diǎn),為后續(xù)的癲癇等疾病治療提供了細(xì)胞水平的候選分子。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R742.5
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本文編號(hào)：1947265