脆性X综合症神经元发育与突触可塑性异常机制研究

发布时间：2018-03-17 23:35

本文选题：脆性X综合症　切入点：突触可塑性　出处：《第四军医大学》2014年博士论文　论文类型：学位论文

【摘要】：【目的】脆性X综合症（Fragile X syndrome, FXS）是临床最常见的遗传性智力低下症，探索其发病机制及治疗途径是社会和临床都急需解决的一个重要课题。脆性X综合症是由Fmr1基因的突变而导致体内缺乏其所编码的蛋白质产物FMRP引起。FMRP是一种与核糖体相关的mRNA结合蛋白，参与mRNA的聚集并调控靶基因的转录效率，主要抑制蛋白表达，从而影响神经元的发育成熟和突触可塑性。目前广泛接受的观点认为，脆性X综合症中FMRP表达缺失，引起代谢型谷氨酸受体（mGluR）依赖性翻译过表达，导致神经元成熟早期形态异常和突触传递紊乱，最终表现出发育迟滞、癫痫发作和智力障碍等典型临床症状。可见，，FMRP调控蛋白翻译异常是脆性X综合症发病的关键。脆性X综合症的突触可塑性异常，主要表现为结构上树突发育障碍和功能上突触传递紊乱。对Fmr1基因敲除（knock-out, KO）小鼠的形态学研究发现，突触部位谷氨酸AMPA受体密度降低，棘突变得细长；脑片电生理记录发现，前扣带回（anteriorcingulated cortex, ACC）皮层锥体神经元长时程增强（long-term plasticity, LTP）明显减弱；行为学发现，额叶皮层参与的恐惧记忆能力降低。星形胶质细胞是神经系统中胶质细胞的主要组成部分。近年来研究发现，星形胶质细胞除了对神经元有营养和保护作用，还具有一些免疫学特性，所以对神经性疾病的防治有重要的研究价值。星型胶质细胞可表达多种神经递质受体，并能释放许多已知或者未知的神经活性成分，从而促进发育中神经元的存活、分化及正确迁移。研究表明，脆性X综合症患者体内神经元树突长度降低并伴随分支增多。当神经元与Fmr1KO星形胶质细胞共培养时，树突生长发育障碍；而与野生型（wild-type,WT）星形胶质细胞共培养时，神经元形态明显得到改善。这提示星形胶质细胞参与了脆性X综合症中神经元的发育障碍，但其作用机制仍不清楚。调节突触可塑性的因素有多种，雌激素作为类固醇激素的代表，在生殖系统、骨骼和心血管中发挥重要作用，近年来其在神经系统中的作用成为关注的焦点。17β-雌二醇能易化LTP的诱导，促进树突棘形成和突触发生，提高认知能力。但是，雌激素在FXS的作用尚不清楚。由于FXS中FMRP缺失导致临床最重要的特征是智力低下，因此我们研究FMRP在学习记忆中的作用，以及雌激素调节异常突触可塑性的信号通路。本研究拟分别从星形胶质细胞调节突触结构可塑性，以及雌激素调节突触功能可塑性的角度，阐明脆性X综合症中突触可塑性异常的机制，为发掘潜在的药物治疗靶点和临床治疗策略提供新的思路和理论依据。【方法】 1.先将野生型（wild-type, WT）和Fmr1基因敲除（knock-out, KO）星型胶质细胞分别与神经元共培养，观察星型胶质细胞对神经元树突发育的影响；再分别收集WT与KO星型胶质细胞的条件培养液（astrocytic conditioned medium, ACM）用以培养神经元，从形态学比较7天后神经元树突发育的差异。 2.采用分子克隆手段构建FMRP蛋白的表达载体，对Fmr1KO星型胶质细胞进行瞬时转染后，再收集其ACM用以培养神经元，观察星型胶质细胞中FMRP蛋白对神经元树突发育的作用。 3.利用HPLC法检测WT和KO ACM中神经递质谷氨酸的浓度，通过活性氧和丙二醛两个指标评价过量谷氨酸对氧化应激的影响。 4.利用ELISA法分别检测体外ACM和体内脑匀浆液中神经营养因子NGF，BDNF，NT-3，GDNF和CNTF的浓度。在WT ACM中外源性给予过量神经营养因子，在KO ACM中用抗体中和过量神经营养因子，观察神经元形态和突触蛋白表达的变化，进一步证实异常含量的神经营养因子可以影响神经元树突发育。 5.根据FMRP为RNA结合蛋白的特性，采用RNA结合蛋白共沉淀（RNA-BindingProtein Immunoprecipitation, RIP）技术，观察FMRP是否结合某些RNA，抑制其在正常水平中的过量表达。 6.通过shRNA降低Fmr1KO星形胶质细胞中过量表达的神经营养因子，观察其ACM对神经元生长状态的改善作用，再将干扰后的细胞定位注射到Fmr1KO小鼠的前扣带回区，建立条件性恐惧记忆模型，观察其学习记忆能力的变化。 7.采用免疫印迹技术，观察E2或者协同使用mGluR5拮抗剂DL-AP3，对WT和KO神经元中AMPA受体GluR1亚基膜表达和磷酸化水平的调节作用。 8.应用膜片钳和Med64电生理技术，观察E2或者协同使用mGluR5拮抗剂DL-AP3，对WT和KO脑片ACC区中LTP的诱导作用。 9.采用免疫共沉淀技术，观察E2对WT和KO神经元中ER-CAV1-mGluR1/5复合物形成的作用。 10.通过shRNA降低Fmr1KO神经元中CAV1表达，观察体外培养神经元GluR1的胞膜分布，体内脑片LTP的诱导、树突棘形态的变化和动物行为的变化。【结果】 1.不论是WT还是KO神经元，只要与Fmr1KO星形胶质细胞共培养，都会发生形态异常。神经元在KO ACM中出现生长障碍，对Fmr1KO星形胶质细胞转染FMRP真核表达载体后，其ACM可显著改善神经元树突的生长。 2. Fmr1KO星形胶质细胞通过合成和转运机制，释放过量神经递质谷氨酸，引起神经元过度氧化应激。 3. Fmr1KO小鼠大脑皮层和ACM中NT-3的含量均显著高于正常组，而其余四种星形胶质细胞来源的神经营养因子没有明显差异。这是由于FMRP结合NT-3基因，导致Fmr1KO星型胶质细胞中的NT-3基因水平没有改变，但蛋白水平升高。 4.当在WT ACM中外源性给予过量NT-3时，树突发育异常程度随NT-3浓度的升高而增加。相反地，在KO ACM中用抗体中和NT-3时，神经元发育状态与中和抗体的使用剂量呈非线性相关。只有合适的浓度才可以恢复神经元形态，而过高和过低浓度的抗体均不能有效恢复神经元形态异常。 5.慢病毒介导小干扰RNA从基因水平抑制NT-3在Fmr1KO星形胶质细胞中的过量表达后，通过收集其ACM可显著改善神经元生长状态，将干扰后的细胞定位注射到幼年Fmr1KO小鼠的前扣带回区，其脑内NT-3水平降低，缺失的恐惧记忆能力显著恢复。 6. E2通过作用于膜上受体介导的非基因组效应，可快速调节AMPA受体GluR1亚基向膜转移，以及增强GluR1磷酸化水平。但在Fmr1KO神经元中E2介导的上述效应消失；并且，在Fmr1KO小鼠的ACC脑片中E2易化LTP诱导作用也消失。 7.协同使用E2和mGluR1/5拮抗剂DL-AP3可以在Fmr1KO小鼠脑片上诱导出明显的LTP，并且可以促进Fmr1KO神经元中GluR1向膜转移和Ser831位点的磷酸化。 8. Fmr1KO小鼠中雌二醇浓度、ERs表达与定位均正常，但是雌激素受体ER偶联过度激活的I型mGluR，致使Gq蛋白-PLC-PKC信号异常。 9.微囊蛋白CAV1通过形成细胞内凹陷使ER与mGluR1/5偶联，E2促进WT神经元中ER和CAV1的结合；而在Fmr1KO神经元中，由于缺失FMRP蛋白结合CAV1基因，导致CAV1过表达并与ER过度结合，对E2的信号传递无响应。 10.体外干扰Fmr1KO神经元中CAV1的表达后，E2可促进GluR1Ser831位点磷酸化，GluR1向胞膜分布以及ER-CAV1-mGluR1/5复合物的形成。Fmr1KO小鼠ACC区注射CAV1shRNA后，E2可以诱导出LTP，并增加锥体神经元中蘑菇状树突棘比例，不论是成年雄性还是卵巢切除后的雌性小鼠，E2均可提高其恐惧记忆能力。【结论】 1.证实星形胶质细胞在脆性X综合症中的重要作用，Fmr1KO星形胶质细胞释放过量神经递质谷氨酸和神经营养因子NT-3，引起神经元树突生长发育障碍。 2.阐明FMRP缺失导致NT-3分泌过多的分子机制，干扰星形胶质细胞来源NT-3，可显著改善脆性X综合症中神经元树突形态和动物发育早期的恐惧记忆行为。 3.发现Fmr1KO小鼠中雌二醇浓度、ERs表达与定位均正常，但是雌激素不能通过作用于膜上受体介导的非基因组效应调节其突触可塑性。 4. Fmr1KO神经元中ER-CAV1-mGluR1/5复合物过度偶联，抑制CAV1过表达可恢复E2对GluR1分子的调节、树突棘形态的改善、LTP诱导的易化和动物行为的纠正。
[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.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R964

【参考文献】