本文選題：脆性X綜合癥　切入點(diǎn)：突觸可塑性　出處：《第四軍醫(yī)大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：【目的】 脆性X綜合癥（Fragile X syndrome, FXS）是臨床最常見(jiàn)的遺傳性智力低下癥，探索其發(fā)病機(jī)制及治療途徑是社會(huì)和臨床都急需解決的一個(gè)重要課題。脆性X綜合癥是由Fmr1基因的突變而導(dǎo)致體內(nèi)缺乏其所編碼的蛋白質(zhì)產(chǎn)物FMRP引起。FMRP是一種與核糖體相關(guān)的mRNA結(jié)合蛋白，參與mRNA的聚集并調(diào)控靶基因的轉(zhuǎn)錄效率，主要抑制蛋白表達(dá)，從而影響神經(jīng)元的發(fā)育成熟和突觸可塑性。目前廣泛接受的觀點(diǎn)認(rèn)為，脆性X綜合癥中FMRP表達(dá)缺失，引起代謝型谷氨酸受體（mGluR）依賴性翻譯過(guò)表達(dá)，導(dǎo)致神經(jīng)元成熟早期形態(tài)異常和突觸傳遞紊亂，最終表現(xiàn)出發(fā)育遲滯、癲癇發(fā)作和智力障礙等典型臨床癥狀�？梢�(jiàn)，，F(xiàn)MRP調(diào)控蛋白翻譯異常是脆性X綜合癥發(fā)病的關(guān)鍵。 脆性X綜合癥的突觸可塑性異常，主要表現(xiàn)為結(jié)構(gòu)上樹(shù)突發(fā)育障礙和功能上突觸傳遞紊亂。對(duì)Fmr1基因敲除（knock-out, KO）小鼠的形態(tài)學(xué)研究發(fā)現(xiàn)，突觸部位谷氨酸AMPA受體密度降低，棘突變得細(xì)長(zhǎng)；腦片電生理記錄發(fā)現(xiàn)，前扣帶回（anteriorcingulated cortex, ACC）皮層錐體神經(jīng)元長(zhǎng)時(shí)程增強(qiáng)（long-term plasticity, LTP）明顯減弱；行為學(xué)發(fā)現(xiàn)，額葉皮層參與的恐懼記憶能力降低。 星形膠質(zhì)細(xì)胞是神經(jīng)系統(tǒng)中膠質(zhì)細(xì)胞的主要組成部分。近年來(lái)研究發(fā)現(xiàn)，星形膠質(zhì)細(xì)胞除了對(duì)神經(jīng)元有營(yíng)養(yǎng)和保護(hù)作用，還具有一些免疫學(xué)特性，所以對(duì)神經(jīng)性疾病的防治有重要的研究?jī)r(jià)值。星型膠質(zhì)細(xì)胞可表達(dá)多種神經(jīng)遞質(zhì)受體，并能釋放許多已知或者未知的神經(jīng)活性成分，從而促進(jìn)發(fā)育中神經(jīng)元的存活、分化及正確遷移。研究表明，脆性X綜合癥患者體內(nèi)神經(jīng)元樹(shù)突長(zhǎng)度降低并伴隨分支增多。當(dāng)神經(jīng)元與Fmr1KO星形膠質(zhì)細(xì)胞共培養(yǎng)時(shí)，樹(shù)突生長(zhǎng)發(fā)育障礙；而與野生型（wild-type,WT）星形膠質(zhì)細(xì)胞共培養(yǎng)時(shí)，神經(jīng)元形態(tài)明顯得到改善。這提示星形膠質(zhì)細(xì)胞參與了脆性X綜合癥中神經(jīng)元的發(fā)育障礙，但其作用機(jī)制仍不清楚。 調(diào)節(jié)突觸可塑性的因素有多種，雌激素作為類固醇激素的代表，在生殖系統(tǒng)、骨骼和心血管中發(fā)揮重要作用，近年來(lái)其在神經(jīng)系統(tǒng)中的作用成為關(guān)注的焦點(diǎn)。17β-雌二醇能易化LTP的誘導(dǎo)，促進(jìn)樹(shù)突棘形成和突觸發(fā)生，提高認(rèn)知能力。但是，雌激素在FXS的作用尚不清楚。由于FXS中FMRP缺失導(dǎo)致臨床最重要的特征是智力低下，因此我們研究FMRP在學(xué)習(xí)記憶中的作用，以及雌激素調(diào)節(jié)異常突觸可塑性的信號(hào)通路。 本研究擬分別從星形膠質(zhì)細(xì)胞調(diào)節(jié)突觸結(jié)構(gòu)可塑性，以及雌激素調(diào)節(jié)突觸功能可塑性的角度，闡明脆性X綜合癥中突觸可塑性異常的機(jī)制，為發(fā)掘潛在的藥物治療靶點(diǎn)和臨床治療策略提供新的思路和理論依據(jù)。 【方法】 1.先將野生型（wild-type, WT）和Fmr1基因敲除（knock-out, KO）星型膠質(zhì)細(xì)胞分別與神經(jīng)元共培養(yǎng)，觀察星型膠質(zhì)細(xì)胞對(duì)神經(jīng)元樹(shù)突發(fā)育的影響；再分別收集WT與KO星型膠質(zhì)細(xì)胞的條件培養(yǎng)液（astrocytic conditioned medium, ACM）用以培養(yǎng)神經(jīng)元，從形態(tài)學(xué)比較7天后神經(jīng)元樹(shù)突發(fā)育的差異。 2.采用分子克隆手段構(gòu)建FMRP蛋白的表達(dá)載體，對(duì)Fmr1KO星型膠質(zhì)細(xì)胞進(jìn)行瞬時(shí)轉(zhuǎn)染后，再收集其ACM用以培養(yǎng)神經(jīng)元，觀察星型膠質(zhì)細(xì)胞中FMRP蛋白對(duì)神經(jīng)元樹(shù)突發(fā)育的作用。 3.利用HPLC法檢測(cè)WT和KO ACM中神經(jīng)遞質(zhì)谷氨酸的濃度，通過(guò)活性氧和丙二醛兩個(gè)指標(biāo)評(píng)價(jià)過(guò)量谷氨酸對(duì)氧化應(yīng)激的影響。 4.利用ELISA法分別檢測(cè)體外ACM和體內(nèi)腦勻漿液中神經(jīng)營(yíng)養(yǎng)因子NGF，BDNF，NT-3，GDNF和CNTF的濃度。在WT ACM中外源性給予過(guò)量神經(jīng)營(yíng)養(yǎng)因子，在KO ACM中用抗體中和過(guò)量神經(jīng)營(yíng)養(yǎng)因子，觀察神經(jīng)元形態(tài)和突觸蛋白表達(dá)的變化，進(jìn)一步證實(shí)異常含量的神經(jīng)營(yíng)養(yǎng)因子可以影響神經(jīng)元樹(shù)突發(fā)育。 5.根據(jù)FMRP為RNA結(jié)合蛋白的特性，采用RNA結(jié)合蛋白共沉淀（RNA-BindingProtein Immunoprecipitation, RIP）技術(shù)，觀察FMRP是否結(jié)合某些RNA，抑制其在正常水平中的過(guò)量表達(dá)。 6.通過(guò)shRNA降低Fmr1KO星形膠質(zhì)細(xì)胞中過(guò)量表達(dá)的神經(jīng)營(yíng)養(yǎng)因子，觀察其ACM對(duì)神經(jīng)元生長(zhǎng)狀態(tài)的改善作用，再將干擾后的細(xì)胞定位注射到Fmr1KO小鼠的前扣帶回區(qū)，建立條件性恐懼記憶模型，觀察其學(xué)習(xí)記憶能力的變化。 7.采用免疫印跡技術(shù)，觀察E2或者協(xié)同使用mGluR5拮抗劑DL-AP3，對(duì)WT和KO神經(jīng)元中AMPA受體GluR1亞基膜表達(dá)和磷酸化水平的調(diào)節(jié)作用。 8.應(yīng)用膜片鉗和Med64電生理技術(shù)，觀察E2或者協(xié)同使用mGluR5拮抗劑DL-AP3，對(duì)WT和KO腦片ACC區(qū)中LTP的誘導(dǎo)作用。 9.采用免疫共沉淀技術(shù)，觀察E2對(duì)WT和KO神經(jīng)元中ER-CAV1-mGluR1/5復(fù)合物形成的作用。 10.通過(guò)shRNA降低Fmr1KO神經(jīng)元中CAV1表達(dá)，觀察體外培養(yǎng)神經(jīng)元GluR1的胞膜分布，體內(nèi)腦片LTP的誘導(dǎo)、樹(shù)突棘形態(tài)的變化和動(dòng)物行為的變化。 【結(jié)果】 1.不論是WT還是KO神經(jīng)元，只要與Fmr1KO星形膠質(zhì)細(xì)胞共培養(yǎng)，都會(huì)發(fā)生形態(tài)異常。神經(jīng)元在KO ACM中出現(xiàn)生長(zhǎng)障礙，對(duì)Fmr1KO星形膠質(zhì)細(xì)胞轉(zhuǎn)染FMRP真核表達(dá)載體后，其ACM可顯著改善神經(jīng)元樹(shù)突的生長(zhǎng)。 2. Fmr1KO星形膠質(zhì)細(xì)胞通過(guò)合成和轉(zhuǎn)運(yùn)機(jī)制，釋放過(guò)量神經(jīng)遞質(zhì)谷氨酸，引起神經(jīng)元過(guò)度氧化應(yīng)激。 3. Fmr1KO小鼠大腦皮層和ACM中NT-3的含量均顯著高于正常組，而其余四種星形膠質(zhì)細(xì)胞來(lái)源的神經(jīng)營(yíng)養(yǎng)因子沒(méi)有明顯差異。這是由于FMRP結(jié)合NT-3基因，導(dǎo)致Fmr1KO星型膠質(zhì)細(xì)胞中的NT-3基因水平?jīng)]有改變，但蛋白水平升高。 4.當(dāng)在WT ACM中外源性給予過(guò)量NT-3時(shí)，樹(shù)突發(fā)育異常程度隨NT-3濃度的升高而增加。相反地，在KO ACM中用抗體中和NT-3時(shí)，神經(jīng)元發(fā)育狀態(tài)與中和抗體的使用劑量呈非線性相關(guān)。只有合適的濃度才可以恢復(fù)神經(jīng)元形態(tài)，而過(guò)高和過(guò)低濃度的抗體均不能有效恢復(fù)神經(jīng)元形態(tài)異常。 5.慢病毒介導(dǎo)小干擾RNA從基因水平抑制NT-3在Fmr1KO星形膠質(zhì)細(xì)胞中的過(guò)量表達(dá)后，通過(guò)收集其ACM可顯著改善神經(jīng)元生長(zhǎng)狀態(tài)，將干擾后的細(xì)胞定位注射到幼年Fmr1KO小鼠的前扣帶回區(qū)，其腦內(nèi)NT-3水平降低，缺失的恐懼記憶能力顯著恢復(fù)。 6. E2通過(guò)作用于膜上受體介導(dǎo)的非基因組效應(yīng)，可快速調(diào)節(jié)AMPA受體GluR1亞基向膜轉(zhuǎn)移，以及增強(qiáng)GluR1磷酸化水平。但在Fmr1KO神經(jīng)元中E2介導(dǎo)的上述效應(yīng)消失；并且，在Fmr1KO小鼠的ACC腦片中E2易化LTP誘導(dǎo)作用也消失。 7.協(xié)同使用E2和mGluR1/5拮抗劑DL-AP3可以在Fmr1KO小鼠腦片上誘導(dǎo)出明顯的LTP，并且可以促進(jìn)Fmr1KO神經(jīng)元中GluR1向膜轉(zhuǎn)移和Ser831位點(diǎn)的磷酸化。 8. Fmr1KO小鼠中雌二醇濃度、ERs表達(dá)與定位均正常，但是雌激素受體ER偶聯(lián)過(guò)度激活的I型mGluR，致使Gq蛋白-PLC-PKC信號(hào)異常。 9.微囊蛋白CAV1通過(guò)形成細(xì)胞內(nèi)凹陷使ER與mGluR1/5偶聯(lián)，E2促進(jìn)WT神經(jīng)元中ER和CAV1的結(jié)合；而在Fmr1KO神經(jīng)元中，由于缺失FMRP蛋白結(jié)合CAV1基因，導(dǎo)致CAV1過(guò)表達(dá)并與ER過(guò)度結(jié)合，對(duì)E2的信號(hào)傳遞無(wú)響應(yīng)。 10.體外干擾Fmr1KO神經(jīng)元中CAV1的表達(dá)后，E2可促進(jìn)GluR1Ser831位點(diǎn)磷酸化，GluR1向胞膜分布以及ER-CAV1-mGluR1/5復(fù)合物的形成。Fmr1KO小鼠ACC區(qū)注射CAV1shRNA后，E2可以誘導(dǎo)出LTP，并增加錐體神經(jīng)元中蘑菇狀樹(shù)突棘比例，不論是成年雄性還是卵巢切除后的雌性小鼠，E2均可提高其恐懼記憶能力。 【結(jié)論】 1.證實(shí)星形膠質(zhì)細(xì)胞在脆性X綜合癥中的重要作用，F(xiàn)mr1KO星形膠質(zhì)細(xì)胞釋放過(guò)量神經(jīng)遞質(zhì)谷氨酸和神經(jīng)營(yíng)養(yǎng)因子NT-3，引起神經(jīng)元樹(shù)突生長(zhǎng)發(fā)育障礙。 2.闡明FMRP缺失導(dǎo)致NT-3分泌過(guò)多的分子機(jī)制，干擾星形膠質(zhì)細(xì)胞來(lái)源NT-3，可顯著改善脆性X綜合癥中神經(jīng)元樹(shù)突形態(tài)和動(dòng)物發(fā)育早期的恐懼記憶行為。 3.發(fā)現(xiàn)Fmr1KO小鼠中雌二醇濃度、ERs表達(dá)與定位均正常，但是雌激素不能通過(guò)作用于膜上受體介導(dǎo)的非基因組效應(yīng)調(diào)節(jié)其突觸可塑性。 4. Fmr1KO神經(jīng)元中ER-CAV1-mGluR1/5復(fù)合物過(guò)度偶聯(lián)，抑制CAV1過(guò)表達(dá)可恢復(fù)E2對(duì)GluR1分子的調(diào)節(jié)、樹(shù)突棘形態(tài)的改善、LTP誘導(dǎo)的易化和動(dòng)物行為的糾正。
[Abstract]:[Objective]
Fragile X syndrome (Fragile X, syndrome, FXS) is the most common inherited mental retardation disease in clinic, and explore its pathogenesis and treatment methods is an important subject in social and clinical are urgently needed to solve. Fragile X syndrome is caused by mutations in the Fmr1 gene encoding the lack of protein products induced by FMRP.FMRP a ribosome associated mRNA binding protein, transcription efficiency and aggregation of target genes regulated in mRNA, inhibition of protein expression, thus affecting neuronal maturation and synaptic plasticity. The widely accepted view is that the lack of FMRP expression of fragile X syndrome, caused by metabotropic glutamate receptor (mGluR) dependence the Chinese expression, lead to neuronal maturation early abnormal morphology and synaptic transmission disorder, showing the retardation, epilepsy and mental retardation are typical clinical symptoms. Visible, FMRP Abnormal translation of protein control is the key to the pathogenesis of fragile X syndrome.
Synaptic plasticity of fragile X syndrome, mainly manifested as disordered structure on dendritic development and function disorder of synaptic transmission. Fmr1 gene knockout (knock-out, KO) morphological study on mice found that glutamate AMPA receptor density of synapses decreased, the spinous process becomes slender; electrophysiology, anterior cingulate (anteriorcingulated cortex, ACC) cortex neurons of long term potentiation (long-term plasticity, LTP) significantly decreased; behavior that reduce the fear memory in the frontal cortex involved.
Astrocytes are the main component of glial cells in the nervous system. In recent years, the study found that astrocytes in addition to nutritional and protective effects on neurons, but also has some immunological characteristics, prevention and treatment of neurological diseases has important research value. Astrocytes express many neurotransmitter receptors, nerve active components and to release many known or unknown, so as to promote the survival of neuron development, differentiation and migration of the right. The study shows that the fragile X syndrome in patients with decreased length of dendrite branches increased. When co cultured neurons and astrocytes, Fmr1KO, dendritic growth and developmental disorders; and with the wild type (wild-type, WT) Co cultured astrocytes, neurons were obviously improved. This suggests that astrocytes are involved in nerve element of fragile X syndrome. However, the mechanism of its action is still not clear.
There are many factors in regulating synaptic plasticity, estrogen as a representative of steroid hormones in the reproductive system, play an important role in bone and cardiovascular, in recent years, its role in the nervous system become the focus of the.17 beta estradiol induced facilitation of LTP, promote the formation of dendritic spines and synapses, improve cognitive ability however, the role of estrogen in FXS is not clear. Due to the FMRP deletion in FXS clinic is the most important feature of low intelligence, so we study FMRP in learning and memory, and estrogen signaling pathway dysregulation of synaptic plasticity.
This study intends to respectively regulate synaptic structural plasticity from astrocytes, and estrogen regulate synaptic functional plasticity of the angle, to clarify the mechanism of fragile X syndrome in aberrant synaptic plasticity, provide new ideas and theoretical basis for exploring potential drug targets and treatment strategies.
[method]
1. of the first wild type (wild-type, WT) and Fmr1 knockout (knock-out, KO) astrocytes were co cultured with neurons, observe the effect of astrocytes on neuronal dendritic development; then WT and KO were collected from astrocyte conditioned medium (astrocytic conditioned, medium, ACM) with in cultured neurons from the comparison of morphological differences after 7 days of dendritic development of neurons.
2., molecular cloning was used to construct FMRP protein expression vector. After transiently transfected Fmr1KO astrocytes, ACM was collected to culture neurons. The role of FMRP protein in astrocytes in neuronal dendrite development was observed.
3., the concentration of neurotransmitter glutamate in WT and KO ACM was detected by HPLC. The effects of excessive glutamate on oxidative stress were evaluated by two indicators of reactive oxygen species and malondialdehyde.
4. the ELISA method was used to detect the neurotrophic factor NGF and ACM in vitro and in vivo brain homogenate in BDNF, NT-3, GDNF and CNTF. The concentration of WT ACM in excess of exogenous neurotrophic factor, using antibodies to neutralize the excess neurotrophic factor in KO ACM, and observe the expression of neuronal morphology and synaptic proteins. Neurotrophic factors can affect the content of further confirmed abnormal neuronal dendritic development.
5., according to the characteristics of FMRP as RNA binding protein, RNA binding protein RNA-BindingProtein Immunoprecipitation (RIP) technology was used to observe whether FMRP combined with some RNA and inhibit its over expression in normal level.
By 6. shRNA decreased neurotrophic factor overexpression of Fmr1KO in astrocytes, observe the effect of its ACM on the growth of neurons, then the front button after the interference localization in cells injected into Fmr1KO mice to establish the model of conditioned fear memory area, and observe the changes of learning ability and memory.
7., Western blotting was used to observe the regulatory effect of E2 or mGluR5 antagonist DL-AP3 on AMPA receptor GluR1 submembrane expression and phosphorylation level in WT and KO neurons.
8. the application of patch clamp and Med64 electrophysiological techniques to observe the induction of E2 or the synergistic use of mGluR5 antagonist DL-AP3 to LTP in WT and KO brain slices ACC region.
9. the effect of E2 on the formation of ER-CAV1-mGluR1/5 complex in WT and KO neurons was observed by immunoprecipitation.
10., the expression of CAV1 in Fmr1KO neurons was reduced by shRNA, the distribution of GluR1 membrane, the induction of LTP in vivo, the morphological changes of spines and the behavior of animals in vitro were observed.
[results]
1., no matter WT or KO neurons, as long as co cultured with Fmr1KO astrocytes, there will be morphological abnormalities. Neurons grow in KO ACM. After ACM transfection of Fmr1KO eukaryotic expression vector, ACM can significantly improve the growth of dendritic cells.
2. Fmr1KO astrocytes, through the mechanism of synthesis and transport, release excessive neurotransmitter glutamic acid, causing excessive oxidative stress in neurons.
The content of NT-3 3. Fmr1KO and ACM in the mouse cerebral cortex were significantly higher than the normal group, and other four kinds of neurotrophic factors from astrocytes had no obvious difference. This is because the FMRP combined with NT-3 gene, NT-3 gene in Fmr1KO level astrocytes did not change, but the protein level increased.
4. when WT ACM in exogenous excessive NT-3, abnormal dendritic development degree increased with the increase of NT-3 concentration. On the contrary, in the KO ACM with NT-3 antibody, is related to the use of nonlinear neurons and dose of neutralizing antibody. Only the appropriate concentration can restore neuronal morphology, and high antibody low concentration and can not effectively restore neuron morphological abnormalities.
5. overexpression of lentivirus mediated RNA interference suppression in Fmr1KO NT-3 from the gene level in astrocytes, by collecting the ACM can significantly improve the growth of neurons, the cell injection after the interference to the anterior cingulate in young Fmr1KO mice back to the area, the level of NT-3 in the brain decreased, fear memory loss the recovery was significant.
Non genomic effects of 6. E2 by acting on the membrane receptor mediated, fast regulation of AMPA receptor GluR1 subunit to the membrane transfer, and enhanced the phosphorylation of GluR1. But the effect in Fmr1KO neurons mediated by E2 and ACC in disappear; brain slices in Fmr1KO mice E2 facilitated LTP induction also disappeared.
7., the synergistic use of E2 and mGluR1/5 antagonist DL-AP3 can induce obvious LTP in the slices of Fmr1KO mice, and promote GluR1 to membrane transfer and Ser831 site phosphorylation in Fmr1KO neurons.
In 8. Fmr1KO mice, estradiol concentration, ERs expression and localization were normal, but estrogen receptor ER coupling overactivated I mGluR resulted in abnormal -PLC-PKC signal of Gq protein.
The 9. microcapsule protein CAV1 formed the cell sag to make ER and mGluR1/5 coupled. E2 promoted the binding of ER and CAV1 in WT neurons. In Fmr1KO neurons, the deletion of FMRP protein combined with CAV1 gene led to over expression of the CAV1 and the over binding with the FMRP, which did not respond to the signal transduction of C.
The expression of CAV1 in Fmr1KO neurons in vitro after interference of 10., E2 can promote GluR1Ser831 phosphorylation, GluR1 to cell membrane distribution and ER-CAV1-mGluR1/5 complex formation in.Fmr1KO mice ACC after injection of CAV1shRNA, E2 can induce LTP, and increased the mushroom shaped spines in tree pyramidal neuron proportion, whether adult males or ovariectomized female mice, E2 could improve their fear memory ability.
[Conclusion]
1., we confirmed the important role of astrocytes in fragile X syndrome. Fmr1KO astrocytes released excessive neurotransmitter glutamate and neurotrophic factor NT-3, resulting in neuronal dendrite growth and development disorders.
2., elucidate the molecular mechanism of FMRP deficiency leading to excessive secretion of NT-3, and interfere with astrocyte NT-3. It can significantly improve the dendritic morphology of neurons in fragile X syndrome and the fear memory behavior at early stage of animal development.
3., it was found that estradiol concentration, ERs expression and localization were normal in Fmr1KO mice, but estrogen could not regulate synaptic plasticity by acting on the receptor mediated non genomic effect on the membrane.
4., the over coupling of ER-CAV1-mGluR1/5 complexes in Fmr1KO neurons and inhibition of over expression of CAV1 can restore the regulation of E2 on GluR1 molecules, improve the morphology of dendritic spines, induce LTP facilitation and correct animal behavior.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R964

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