CRTC1-BDNF相关信号通路在阿尔茨海默病早期突触可塑性及认知功能障碍中的机制研究

发布时间：2018-06-09 03:04

本文选题：阿尔茨海默病 + β-淀粉样蛋白　；参考：《宁波大学》2017年硕士论文

【摘要】：研究背景:阿尔茨海默病(Alzheimer's disease,AD)是一种进行性神经退行性疾病,主要表现为进行性的认知功能障碍、记忆力减退及性格行为改变。众多研究表明,可溶性β-淀粉样蛋白(Amyloid-βprotein,Aβ)在AD早期认知功能障碍中发挥重要作用。神经可塑性是学习记忆的神经基础。长时程增强(long-term potentiation,LTP)是指兴奋性突触传递通路在强直刺激的作用下诱发的突触传递效率长时间增强的现象,海马LTP与学习记忆功能密切相关。晚期时相长时程增强(late-phase LTP,L-LTP)是编码长时记忆的突触可塑性形式,需要cAMP反应元件结合蛋白(cAMP-responsive element binding protein,CREB)调控的cAMP反应元件(cAMP-responsive element,CRE)基因转录和新蛋白质合成。CREB转录共激活因子1(CREB-regulated transcription coactivators,CRTC1),通过感受胞浆内Ca~(2+)和cAMP信号分子,以去磷酸化方式入核与CREB的bZIP区域结合,加强CREB与CBP/P300的结合,以非依赖CREB Ser133磷酸化的方式介导CRE靶基因转录和表达,如脑源性神经营养因子(Brain derived neurotrophic factor,BDNF)等。研究表明,CRTC1活动依赖性的细胞核迁移和聚集是海马L-LTP维持的必需过程。BDNF受体是原肌球蛋白相关酶受体B(tropomyosin-related kinase receptors,TrkB)。BDNF对神经元发育和生存、突触可塑性及学习和记忆功能均有重要作用。课题组前期研究表明:Aβ_(42)寡聚体可以引起L-LTP损伤,注射RNA干扰CRTC1慢病毒(LV-Sh-CRTC1)也可以引起L-LTP的损伤,但是LV-Sh-CRTC1和Aβ_(42)寡聚体联合应用不能够进一步引起L-LTP损伤;而注射CRTC1过表达慢病毒(LV-OE-CRTC1)可以保护由Aβ_(42)寡聚体引起的L-LTP损伤。提示CRTC1可能是Aβ_(42)寡聚体引起海马L-LTP损伤的靶点之一。本研究进一步从CRTC1-BDNF信号通路入手,探寻CRTC1参与AD发病过程中突触可塑性损害及认知功能下降的分子机制。研究方法:我们通过蛋白免疫印迹(Western blotting,WB)技术对12月龄双转基因APP/PS1小鼠海马和皮层组织C末端淀粉样前体蛋白(Amyloid Precursor Protein,CTerminal,APP)表达水平进行检测;通过WB技术对APP/PS1小鼠海马和皮层组织CRTC1及CRE靶基因表达水平进行检测;通过免疫荧光化学技术对APP/PS1小鼠海马齿状回(dentate gyrus,DG)区域CRTC1表达水平进行检测;通过行为学实验检测海马脑立体定位注射Aβ_(42)寡聚体及LV-Sh-CRTC1对大鼠学习和记忆功能的影响,运用WB技术对行为学实验后大鼠海马组织CRTC1、CREB及CRE靶基因进行检测;通过海马DG区场电位记录技术检测Aβ_(42)寡聚体是否通过CRTC1-BDNF通路引起L-LTP损伤。研究结果:1)12月龄APP/PS1小鼠海马和皮层组织APP表达量显著增高,表明转基因小鼠模型构建成功;2)12月龄APP/PS1小鼠海马和皮层组织CRTC1、BDNF、c-Fos表达水平显著降低,pCRTC1表达水平显著增高;3)12月龄APP/PS1小鼠海马DG区CRTC1低表达;4)Aβ_(42)寡聚体及LV-Sh-CRTC1处理组在新物体识别实验中表现出认知水平显著降低,在水迷宫实验中表现出空间学习和记忆能力显著降低;5)外源性孵育BDNF可以逆转由Aβ_(42)寡聚体和LV-Sh-CRTC1单独作用引起的海马DG区L-LTP损伤;6)外源性孵育BDNF可以逆转由LV-Sh-CRTC1和Aβ_(42)寡聚体共同作用引起的海马DG区LLTP损伤;7)BDNF受体TrkB被其抑制剂TrkB-FC或K252a抑制后,预先过表达CRTC1不能够保护由Aβ_(42)寡聚体引起的海马DG区L-LTP损伤。研究结论:在AD发病过程中,海马及皮层组织CRTC1、BDNF表达显著下调,CRTC1-BDNF通路可能是Aβ_(42)寡聚体引起AD突触可塑性损伤及认知功能障碍的靶点之一。
[Abstract]:Background: Alzheimer's disease (AD) is an progressive neurodegenerative disease characterized by progressive cognitive impairment, memory impairment and personality behavior changes. Numerous studies have shown that soluble beta amyloid (Amyloid- beta, A beta) plays an important role in the early cognitive dysfunction of AD. Neural plasticity is the neural basis for learning and memory. Long-term potentiation (LTP) refers to the long time enhancement of synaptic transmission efficiency induced by the excitatory synaptic transmission pathway under the action of tetanic stimulation. The hippocampus LTP is closely related to the learning and memory function. At the late stage, the long period enhancement (late-phase LTP, L-LTP) is a compilation. The synaptic plasticity of memory at code length requires the cAMP reactive element binding protein (cAMP-responsive element binding protein, CREB) for the cAMP reaction element (cAMP-responsive element, CRE) gene transcription and the synthesis of a new protein,.CREB transcriptional activator 1 (CREB-regulated), through the receptor cell. The Ca~ (2+) and cAMP signal molecules in the pulp are combined with the bZIP region of CREB by dephosphorylation, and the binding of CREB to CBP/P300 is strengthened, and the transcription and expression of CRE target genes are mediated by non dependent CREB Ser133 phosphorylation, such as the brain derived neurotrophic factor (Brain derived). Nuclear migration and aggregation are essential for the maintenance of hippocampal L-LTP. The.BDNF receptor, B (tropomyosin-related kinase receptors, TrkB).BDNF, plays an important role in the development and survival of neurons, synaptic plasticity, and learning and memory functions. L-LTP injury, RNA interfering CRTC1 lentivirus (LV-Sh-CRTC1) can also cause L-LTP damage, but the combination of LV-Sh-CRTC1 and A beta oligomers can not further cause L-LTP damage, while CRTC1 overexpressed lentivirus (LV-OE-CRTC1) can protect the L-LTP damage caused by A beta (42) oligomers. Oligomer is one of the targets of hippocampal L-LTP damage. This study further starts with the CRTC1-BDNF signaling pathway to explore the molecular mechanism of CRTC1 participating in the synaptic plasticity damage and cognitive decline during the pathogenesis of AD. Research methods: we use protein immunoblotting (Western blotting, WB) technology for 12 month old pairs of genetically modified APP/PS1 mice. The expression level of C terminal amyloid precursor protein (Amyloid Precursor Protein, CTerminal, APP) in the horse and cortex tissues was detected, and the expression level of CRTC1 and CRE target genes in hippocampal and cortical tissues of APP/PS1 mice was detected by WB technology. The expression level was detected, and the effects of A beta oligomer (42) oligomer and LV-Sh-CRTC1 on the learning and memory function of rats were detected by behavioral experiment. WB technique was used to detect the target genes of CRTC1, CREB and CRE in hippocampus of rats after behavioral experiment. The oligomerization of A beta (42) was detected by the field potential recording technique in the hippocampal DG region. Whether the body could cause L-LTP damage through the CRTC1-BDNF pathway. 1) the expression of APP in the hippocampus and cortex of 12 month old APP/PS1 mice increased significantly, indicating that the transgenic mice model was constructed successfully; 2) the hippocampus and cortical tissues of 12 month old APP/PS1 mice were CRTC1, BDNF, c-Fos expression was significantly reduced, the expression level of pCRTC1 significantly increased; 3) 12 month old AP. The low expression of CRTC1 in the DG region of P/PS1 mice; 4) A beta (42) oligomer and LV-Sh-CRTC1 treatment group showed a significant reduction in the cognitive level in the new object recognition experiment. In the water maze experiment, the spatial learning and memory ability decreased significantly; 5) exogenous incubating BDNF could reverse the effect of A beta (42) oligomer and LV-Sh-CRTC1 alone. L-LTP damage in hippocampal DG region; 6) exogenous incubating BDNF can reverse the LLTP damage in hippocampus DG region caused by LV-Sh-CRTC1 and A beta (42) oligomers; 7) BDNF receptor TrkB is inhibited by its inhibitor TrkB-FC or K252a. In the course of the disease, the expression of CRTC1 and BDNF in the hippocampus and cortex is down significantly. The CRTC1-BDNF pathway may be one of the targets of the A beta (42) oligomer to cause the plasticity damage and cognitive impairment of AD synapses.
【学位授予单位】：宁波大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.16

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