【作者】 王鑫；

【导师】 王建枝；

【作者基本信息】 华中科技大学 ， 病理学与病理生理学， 2021， 博士

【摘要】 研究背景:阿尔茨海默病(Alzheimer disease,AD)是神经退行性疾病中最为流行的一种,最主要的病理特征之一是细胞内病理性tau的异常聚集。而tau最先出现病理性改变的脑区为内嗅皮层(Entorhinal cortex,EC)。近期有研究表明,tau的异常聚集不仅能在细胞内沉积形成神经纤维原缠结,而且能够通过多种形式,以内嗅皮层为起始脑区向外传播,导致临近脑区或者有突触联系的其他脑区成扩散样病理改变。但是传播的具体机制尚不清楚。Tau的乙酰化是tau重要的翻译后修饰,在阿尔茨海默病患者或转基因小鼠的大脑中,我们观察到tau的K174,K274,K280和K281位点乙酰化增加,有研究表明,tau的乙酰化升高会导致病理性tau聚集增多,降解出现障碍。那么异常聚集的tau的传播是否受tau乙酰化的影响,尚不清楚。目的:研究内嗅皮层和海马区tau蛋白乙酰化对其传播及学习记忆功能的影响。方法:使用体重没有明显差异、健康成熟的野生型雄性小鼠作为实验对象。构建模拟tau乙酰化的病毒AAV-Tau-4Q和模拟非乙酰化tau的病毒AAV-Tau-4R,以及未突变的野生型tau病毒AAV-Tau-WT和空载病毒AAV-Vec作为对照。病毒带有P2A水解元件,AAV-syn-e GFP-2a-tau-3flag,与e GFP蛋白非融合,2a遇水断开,使tau与GFP蛋白共表达后解离,不影响tau的传播。使用脑立体定位注射在小鼠EC区或者DG区过表达tau病毒,对照组注射空载病毒。设置时间梯度来观察过表达病毒对小鼠学习记忆和情绪的影响,以及EC区到投射脑区的传播情况。使用新事物识别(Novel object recognition,NOR),水迷宫(Morris water maze,MWM)和场景恐惧(Fear condition,FC)行为学检测小鼠学习记忆能力;采用开放旷场(Open field,OF)和高架十字迷宫(Elevated plus maze,EPM)检测小鼠情绪变化;采用MED64离体微电极阵列记录系统检测长时程增强效应(LTP);采用免疫组化和免疫荧光检测tau的传播和胶质细胞的激活状态。结果:1.AD模型鼠的EC区tau蛋白的乙酰化水平升高与WT小鼠相比,AD模型鼠的EC区tau蛋白赖氨酸位点174和280乙酰化水平明显升高。2.EC区过表达Tau-4Q 3个月或6个月不引起tau蛋白向海马区传播、不导致学习记忆损伤在C57小鼠EC区过表达tau蛋白3个月后,除了在EC区可见小胶质细胞明显激活外,在海马的DG区未见明显胶质细胞激活、未见明显tau蛋白向海马区传播、小鼠未见明显学习记忆损伤。过表达tau蛋白6个月后,在EC区可见小胶质细胞和星形胶质细胞明显激活,但未见明显tau蛋白向海马区传播、小鼠未见明显学习记忆损伤。3.EC区过表达Tau-4Q 13.5个月促进tau蛋白向海马DG区传播并导致小鼠出现学习记忆损伤在C57小鼠EC区过表达tau蛋白13.5个月后,表达tau的三个组在EC区小胶质细胞和星形胶质细胞明显激活,且Tau-4Q组激活更加明显;表达Tau-WT诱导tau传播到齿状回(Dentate gyrus,DG)的颗粒细胞层,而表达Tau-4Q促进tau传播到DG的内层;表达tau蛋白的三个组在DG区小胶质细胞和星形胶质细胞都有不同程度的激活,且Tau-4Q组激活更加明显,小鼠出现记忆功能损伤。4.海马DG区过表达Tau-4Q促进tau蛋白向对侧传播并加重Tau-WT-引起的突触和记忆损伤在C57小鼠单侧DG区过表达tau蛋白8周后,在Tau-4Q小鼠对侧海马中HT7阳性细胞数比Tau-WT小鼠多,过表达Tau-4R消除了tau向对侧海马的传播;过表达Tau-4Q在海马DG区同侧和对侧诱导非常明显的小胶质细胞和星形胶质细胞激活,在Tau-WT组只有同侧海马中有较少的胶质细胞激活;过表达tau的三组EPSP斜率均下降,而在Tau-4Q组下降最为明显,Tau-WT组和Tau-4R组之间没有差异;表达Tau-WT和Tau-4Q损伤小鼠学习记忆能力,Tau-WT,Tau-4Q和Tau-4R导致情绪障碍,Tau-WT和Tau-4Q之间没有差异,Tau-4R恢复小鼠学习记忆能力。结论:Tau蛋白乙酰化促进其从EC向DG区的时间依赖性传播;单纯EC区tau蛋白聚集不损伤学习记忆功能;海马区tau蛋白聚集可导致认知功能缺陷。研究背景:阿尔茨海默病(Alzheimer disease,AD)是神经退行性疾病中最为流行的一种,最主要的病理特征之一是细胞内病理性tau的异常聚集。最近的一项研究发现,与脑脊液(cerebral spinal fluid,CSF)磷酸化tau T181相比,早期轻度认知障碍(mild cognitive impairment,MCI)和AD患者血浆中tau T217的磷酸化作为AD的早期预测,比已建立的基于血浆和MRI的生物标记物具有更高的准确性,可以更好地与其他神经退行性疾病区分开。然而,tau T217的早期磷酸化对AD病理过程的影响尚不清楚。目的:研究tau T217-磷酸化的毒性作用。方法:在本研究中,使用体重没有明显差异的、健康成熟的野生型雄鼠作为实验对象。构建模拟tau磷酸化的病毒AAV-tau-T217E和模拟非磷酸化tau的病毒AAVtau-T217A,以及未突变的tau病毒AAV-tau-WT和空载Vec作为对照。病毒带有P2A水解元件,AAV-syn-e GFP-2a-tau-3flag,与e GFP蛋白非融合,2a遇水断开,使tau与GFP蛋白共表达后解离,不影响tau的传播。使用脑立体定位注射在小鼠CA3区过表达tau病毒,对照组注射空载病毒。使用新事物识别(Novel object recognition,NOR),水迷宫(Morris water maze,MWM)和场景恐惧(Fear condition,FC)行为学检测小鼠学习记忆能力;采用开放旷场(Open field,OF)和高架十字迷宫(Elevated plus maze,EPM)检测小鼠情绪变化;采用免疫组化和免疫荧光检测tau的传播和胶质细胞的激活状态。使用蛋白免疫印迹法检测蛋白质表达。使用高尔基染色法检测树突棘数目变化。使用体外重组tau检测蛋白聚集。结果:1.3x Tg小鼠海马中tau-T217磷酸化水平升高通过蛋白质印迹技术,我们观察到在3月龄和6月龄的海马中tau-T217磷酸化的水平显著增加。通过免疫组织化学技术,我们观察到tau在小鼠CA3区早期就出现tau-T217磷酸化水平升高。2.模拟tau-T217磷酸化可增强tau其他位点的磷酸化,同时抑制tau降解并增加其聚集HEK293细胞转染质粒48小时后,与野生型tau和tau-T217A组相比,过表达tau-T217E显著增加了tau其他位点的磷酸化水平,抑制了tau降解。通过体外纯化蛋白实验,用硫黄素T染色,与野生型tau和tau-T217A组相比,tau-T217E的聚集显著增强。3.模拟tau-T217磷酸化加重Tau-WT引起的小鼠认知功能损伤在小鼠海马CA3中过表达tau病毒一个月后,不同类型的tau会引起认知功能障碍,tau-T217E组的变化比野生型tau和tau-T217A组更显著,T217A的突变恢复了小鼠的行为表现。4.模拟tau-T217磷酸化加重tau病理和树突棘损伤在小鼠海马CA3中过表达tau病毒一个月后,与野生型tau相比,过表达tau-T217E显著增加了tau在T181,T205,S214,T231,S262和S422的磷酸化水平,并显著提高了截短的tau-N368(经AEP剪切的tau)和tau-C3的水平(半胱天冬酶在Asp421上剪切的tau)。通过高尔基染色,所有表达tau的组在海马CA3中神经元树突棘的数量明显减少。以上,在过表达tau-T217A组恢复到野生型tau组的水平。5.模拟tau-T217磷酸化加重Tau-WT引起的神经胶质细胞激活,但不影响tau蛋白传播在小鼠海马CA3中过表达tau病毒一个月后,所有tau表达组在同侧和对侧海马CA3中均观察到小胶质细胞激活,并且在tau-T217E组中激活最为明显,并且T217A使IBA1恢复至正常对照水平。对于星形胶质细胞,过表达tau只能诱导同侧的星形胶质细胞激活,其程度与IBA1类似,但不会影响对侧。6.在tau-P301L基础上模拟T217磷酸化没有进一步增加tau其他位点的磷酸化HEK293细胞转染质粒48小时后,过表达的tau-L,tau-LE,tau-LA不会在tau的S202,T205,S214,T231,S262,S396位点诱导磷酸化进一步升高。7.在tau-P301L基础上模拟T217磷酸化虽然会加重tau剪切,但是不会恶化认知障碍在2月龄小鼠CA3过表达病毒一个月后,过表达tau-P301L-T217E显著增加了被剪切的tau-N368的水平,但不影响其在T181和S214处的磷酸化,并且通过开放旷场,新事物识别和水迷宫实验测得,所有三种类型的tau诱导的行为异常水平相似。8.在tau-P301L基础上模拟T217磷酸化促进tau传播和神经胶质细胞激活在2月龄小鼠CA3过表达病毒一个月后,在tau-LE组的对侧海马CA1上有明显的HT7阳性信号。通过IBA1和GFAP免疫荧光染色,我们还观察到T217磷酸化加剧了tau-P301L诱导的神经胶质细胞激活。结论:基于野生型tau的T217模拟磷酸化加剧了tau诱导的其他位点磷酸化,加重了tau的裂解,纤维化和小鼠的认知障碍,而基于P301L的T217的模拟磷酸化虽然促进了tau的传播和裂解,但是不会加重其他位点的磷酸化,也不会加剧认知能力缺陷。更多还原

【Abstract】 Background: Alzheimer’s disease(AD)is the most prevalent neurodegenerative disease among aging population.One of pathological hallmarks of AD is the abnormal aggregation of intracellular pathological tau.The brain area where tau first showed pathological changes is the entorhinal cortex(EC).Recent studies have shown that the intracellular abnormal aggregation of tau can not only be deposited to form neurofibrillary tangles,but also propagate through various forms with the entorhinal cortex as the starting brain area to spread outward,and lead to adjacent brain areas or other brain areas with synaptic connections into pathological changes,the specific mechanism of transmission is elusive.Tau acetylation is an important modification after tau translation.Increased tau acetylation at K174,K274,K280,and K281 has been observed in the brains of Alzheimer’s disease(AD)patients or the transgenic mice.Many studies have shown that increased tau acetylation will lead to increased tau aggregation and obstruction of degradation.The specific mechanism is still unclear.It is unknown whether the spread of abnormally aggregated tau is affected by tau acetylation.Objective: To study the effect of tau acetylation in the entorhinal cortex and hippocampus on tau transmission and learning and memory.Methods: In this study,healthy and mature wild-type male mice with no significant difference in body weight were used as experimental subjects.AAV-Tau-4Q,which mimics tau acetylation,and AAV-Tau-4R,which mimics non-acetylated tau,as well as unmutated tau virus AAV-Tau-WT and AAV-Vector was used as controls.all virused have GFP tag which is not fused with tau.Brain stereotaxic injection was used to overexpress tau virus in mouse EC,or DG subset,and the control group was injected with the vector virus.Using novel object recognition(NOR),Morris water maze(MWM)and fear condition(FC)behaviors to test the learning and memory.Using open field(OF)and elevated plus maze(EPM)to detect mood changes in mice.Using MED64 isolated microelectrode array recording system to detect a long-term potentiation(LTP).Using immunohistochemistry and immunofluorescence to detect the glial activation and the transmission of tau in brain slices.Results:1.The acetylation level of tau in EC region is increased in AD model mice Compared with WT mice,the acetylation levels of tau K174 and K280 were significantly increased in EC subset of AD model mice.2.Overexpressing Tau-4Q in EC for 3 months and 6 months neither induce tau transmission to hippocampus nor learning and memory deficitOverexpressing different AAV-tau constructs in the EC subset of 2-month-old C57 mice after 3 months,except for microglial activation in the EC subset,there was no significant glial cell activation in the dentate gyrus(DG)subset of hippocampus,no obvious tau protein transmission to the hippocampus and no obvious learning and memory deficit in mice.Overexpressing different AAV-tau constructs in the EC subset after 6 months,microglia and astrocytes were obviously activated in the EC subset,but there was no significant tau protein transmission to the hippocampus and no obvious learning and memory deficit in mice.3.Overexpressing Tau-4Q in EC for 13.5 months promotes tau propagation to DG subset with memory deficitOverexpressing different AAV-tau constructs in the EC subset of 2-month-old C57 mice after 13.5 months,microglia and astrocyte in all three tau expression groups were significantly activated in the EC subset,the more significant effect was shown in Tau-4Q group;expressing Tau-WT induced tau propagation to the granular cell layer of DG,while expressing Tau-4Q promoted tau propagation to the inner hilus layer of DG;microglia and astrocyte in all three tau expression groups were activated in the DG subset,the more significant effect was shown in Tau-4Q group,the mice suffered memory deficit.4.Overexpressing Tau-4Q in hippocampal DG promotes its contralateral transmission and deteriorate Tau-WT induced impairment of synaptic function and cognitive deficitsOverexpressing different AAV-tau constructs in the unilateral hippocampus of 2-month-old C57 mice after 8 weeks,the HT7-positive cell number was much higher in Tau-4Q mice than the Tau-WT mice.Overexpressing Tau-4R abolished tau propagation to the contralateral hippocampus;overexpressing Tau-4Q induced very significant activation of microglial and astrocyte in both ipsilateral and the contralateral sites of the hippocampal DG,though fewer microglial activation was also shown in the ipsilateral hippocampus of Tau-WT group;the EPSP slope was decreased in all three groups with tau proteins overexpression,and the decrease was most significant in Tau-4Q group than the Tau-WT group,and no difference was shown between Tau-WT and the Tau-4R groups;significant cognitive impairments were detected in Tau-4Q and Tau-WT,Tau-4R restores learning and memory,significant anxiety-depression were detected in Tau-WT,Tau-4Q and Tau-4R.Conclusion: Tau protein acetylation promotes a time-dependent propagation from EC to DG,and only hippocampus but not EC tau accumulation induces cognitive deficits.Background: Alzheimer’s disease(AD)is the most prevalent neurodegenerative disease among aging population.One of pathological hallmarks of AD is the abnormal aggregation of intracellular pathological tau.A recent study found that the phosphorylation of tau T217 in the plasma of early mild cognitive impairment(MCI)and AD patients,with higher accuracy than established plasma-and MRI-based biomarkers,can be better distinguished from other neurodegenerative diseases compared with the cerebral spinal fluid(CSF)phosphorylation tau T181,the earlier prediction of AD.However,the effect of early phosphorylation of tau T217 on the pathological process of AD is unclear.Objective: To study the potential toxic role of tau T217-phosphorylation.Methods: In this study,healthy and mature wild-type male mice with no significant difference in body weight were used as experimental subjects.AAV-tau-T217 E,which mimics tau phosphorylation,and AAV-tau-T217 A,which mimics non-phosphorylation tau,and AAV-tau-P301L-T217 E,AAV-tau-P301L-T217 A,which mimics tau phosphorylation and non-phosphorylation on the basis of tau P301 L,as well as unmutated tau virus AAV-tau-WT and vector was used as controls.all virused have GFP tag which is not fused with tau.Brain stereotaxic injection was used to overexpress tau virus in mouse CA3 subset,and the control group was injected with the vector virus.Using novel object recognition(NOR),Morris water maze(MWM)and fear condition(FC)behaviors to test the learning and memory ability of mice.Using open field(OF)and elevated plus maze(EPM)to detect mood changes in mice.Using immunohistochemistry and immunofluorescence to detect the glial activation and the transmission of tau in brain slices.Using Western blotting to detect proteins expression.Using Golgi staining to detect spine.Using in vitro recombinant tau to detect polymerization.Results:1.Level of T217-phosphorylated tau is increased in the hippocampus of 3x Tg AD miceBy Western blotting,we observed that the level of tau-p T217 in the total hippocampal extracts was significantly increased when compared with the age-and sex-matched controls.2.T217-phosphorylation on wild-type tau enhances tau phosphorylation at other sites with inhibited tau degradation and increased fibrillization in vitroHEK293 cells were transfected with plasmids after 48 hours,tau-T217 E significantly increased tau phosphorylation level at other sites,inhibited tau degradation compared with wild-type tau and tau-T217 A groups.By Thioflavin T staining,a significantly enhanced aggregation of tau-T217 E was shown compared with wild-type tau and tauT217 A groups.3.T217-phosphorylation on wild-type tau exacerbates tau-induced cognitive damages in miceOverexpressing different AAV-tau constructs in the CA3 subset of 2-month-old C57 mice after 1 month,different types tau induce cognitive deficit,the changes were more significant in tau-T217 E group than wild-type tau and tau-T217 A group,and the T 217 A restored the behavioral performance of the mice.4.T217-phosphorylation on wild-type tau exacerbates tau pathologies with aggravated spine in miceOverexpressing different AAV-tau constructs in the CA3 subset of 2-month-old C57 mice after 1 month,tau-T217 E significantly increased the phosphorylation level of tau at T181,T205,S214,T231,S262,and S422 compared with wild-type tau and remarkably increased the levels of truncated tau-N368(AEP-cleaved tau)and tau-C3(caspase-cleaved tau at Asp421)compared with tau-T217.By Golgi staining,all tauexpressing groups showed a significantly reduced number of neuronal dendritic spine in hippocampal CA3 subset.Overexpressing tau-T217 A was restored to the level of the wild-type tau group.5.T217-phosphorylation on wild-type tau exacerbates tau-induced glial activation without affecting tau propagationOverexpressing different AAV-tau constructs in the CA3 subset of 2-month-old C57 mice after 1 month,all tau-overexpressing groups showed microglial activation in both ipsilateral and contralateral hippocampal CA3,and the activation was most significant in tau-T217 E group,and T217 A restored IBA1 to normal control level.For astrocytes,overexpressing tau only induced astrocytes activation in ipsilateral side with similar feature as IBA1,but without affecting the contralateral side.6.T217-phosphorylation on tau-P301 L does not enhance tau phosphorylation at other sitesHEK293 cells were transfected with plasmids after 48 hours,all tau-overexpressing groups did not induce significant elevation of tau phosphorylation at S202,T205,S214,T231,S262,S396.7.T217-phosphorylation on tau-P301 L does not exacerbate cognitive impairment though it promotes tau cleavageOverexpressing different AAV-tau constructs in the CA3 subset of 2-month-old C57 mice after 1 month,tau-P301L-T217 E remarkably increased the level of cleaved tauN368 without affecting its phosphorylation at T181 and S214 in mice and all three types of tau induced similar level of behavioral abnormalities measured by open field test,novel object recognition test,and Morris water maze test.8.T217-phosphorylation on tau-P301 L promotes propagation and glial activationOverexpressing different AAV-tau constructs in the CA3 subset of 2-month-old C57 mice after 1 month,significant HT7-positive signal was detected on the contralateral hippocampal CA1 in tau-LE group.By immunofluorescence co-staining with IBA1 and GFAP,we also observed that T217 phosphorylation exacerbated tau-P301L-induced glial activation.Conclusion: T217-phosphorylation exacerbates wild-type tau hyperphosphorylation with aggravated tau cleavage/fibrillization and cognitive impairments,while overexpressing T217 E on the basis P301 L does not exacerbate tau phosphorylation or the P301L-induced cognitive deficits although it aggravates tau cleavage and propagation.更多还原