【作者】 周艳；

【导师】 刘飞； 储丹丹；

【作者基本信息】 南通大学 ， 医学生物化学与分子生物学， 2021， 博士

【摘要】 目的Tau的异常过度磷酸化和聚集在阿尔茨海默病(Alzheimer’s disease,AD)等相关神经退行性疾病中的作用非常关键,最近的研究提示病理性tau蛋白在AD脑中朊样传播可能是其发生发展的分子基础。本论文提出tau磷酸化和截断是否在其朊样聚集传播中发挥作用以及能否通过自噬激活剂干预病理性tau朊样传播等两个尚待回答的问题,从下列五个方面展开了一系列研究。第一部分:过度磷酸化与截断是AD脑内tau聚集体的生化特征方法通过Western blots对AD患者和对照脑组织中tau及SDS和还原剂具有抗性的高分子量tau的磷酸化水平和截断特点进行了系统的分析。结果我们发现(1)AD脑组织中从低分子量(LMW)到高分子量(HMW)tau蛋白均表现为弥散性条带,AD组和对照组的LMW-tau水平相似,而HMW-tau仅在AD脑内表达。(2)AD患者脑中tau的磷酸化水平明显增加,HMW-tau在多个位点过度磷酸化,且不含有Ser46和Ser198/199/202未磷酸化的tau。(3)识别tau蛋白的N端抗体43D(a.a.6-18)和HT7(a.a.159-163)只能在HMW-tau中检测到微弱的信号,而C端的抗体Tau46和Tau46.1可以呈现较强免疫反应性。(4)从N端到C端tau抗体依次检测到的HMW-tau与LMW-tau的比值增加。(5)在AD脑中tau在Asp421处截断明显升高,但与HMW-tau相关性较差。结论AD患者脑内聚集的高分子量tau在多个位点发生过度磷酸化并且发生截断,且该截断主要发生在tau的N端。第二部分诱导tau朊样聚集过程中其磷酸化的时空演化方法从阿尔茨海默病(Alzheimer’s disease,AD)患者(Braak stage VI)脑组织中提取tau的寡聚体(AD oligomeric tau,AD O-tau);采用培养细胞模型和小鼠海马注射模型,分别通过Western blots和免疫荧光染色的方法,分析AD O-tau诱导tau朊样聚集过程中磷酸化和tau聚集的时空演变特点。结果(1)AD O-tau在Ser199、Thr205、Thr214位点磷酸化水平相对较低,但在Ser262、Ser396/404(PHF-1)以及Ser422位点的磷酸化水平较高,其中在Ser422位点尤为突出。(2)AD O-tau可以有效地在培养细胞模型中诱导tau的朊样聚集。(3)细胞模型中AD O-tau诱导的tau过度磷酸化和朊样聚集呈时间依赖性。(4)AD O-tau在C57BL/6野生型小鼠脑内诱导的朊样聚集呈时间依赖性并与动物的年龄相关。结论AD O-tau本身是异常过度磷酸化的,能诱导SH-SY5Y细胞和野生型小鼠脑内tau朊样聚集和过度磷酸化,且该效应具有时间依赖性。第三部分:Tau的截断决定其自聚集和朊样活性方法我们构建一系列tau截断体,采用培养细胞模型研究这些截断体的自聚集活性,进而选取tau151-391和全长tau(tau1-441)作为代表,从真核细胞中表达提取tau151-391聚集体和tau1-441聚集体,通过小鼠海马内注射模型比较截断体tau聚集体和全长tau聚集体对小鼠脑内tau朊样聚集的诱导活性。结果(1)HEK-293FT细胞表达的tau151-391自聚集能力最强。(2)截断体tau151-391不但具有更强的自聚集活性,而且也更容易被诱导发生朊样聚集。(3)截断体tau151-391蛋白聚集体在小鼠脑内有类似AD O-tau的诱导朊样传播作用,该作用明显强于全长tau蛋白聚集体。结论截断体tau151-391比全长tau更容易产生自聚集,也更易被AD O-tau诱导发生朊样聚集;从培养真核细胞中分离的tau151-391不可溶性蛋白聚集体与AD O-tau类似,可以诱导小鼠脑内tau产生tau朊样聚集。第四部分:过度磷酸化助推tau朊样传播方法采用培养细胞模型和小鼠脑内注射模型,通过冈田酸(okadaic acid,OA)处理和敲减或过表达蛋白磷酸酶2A(protein phosphatase 2 A,PP2A)分别上、下调内源性tau的磷酸化水平,分析不同磷酸化水平的tau被AD O-tau诱导朊样聚集的特点。结果(1)过度磷酸化的tau明显更容易被AD O-tau猎获,更易被AD O-tau诱导朊样聚集。(2)表达tau151-391的HEK-293FT细胞,用sh PP2Ac干扰蛋白磷酸酶2A催化亚基(PP2Ac),则经AD O-tau诱导后发生更多的朊样聚集;相反,若共过表达PP2Ac,则被AD O-tau诱导的tau朊样聚集减少。(3)PP2Ac的敲减促进C57BL/6小鼠内源性tau被AD O-tau朊样诱导聚集,而敲减PP2Ac本身并不足以引起内源性tau的自聚集;过表达PP2Ac明显减少AD O-tau诱导的内源性tau的朊样聚集。结论内源性tau的磷酸化水平影响AD O-tau诱导的tau病理性聚集和朊样传播,磷酸化水平越高,越容易被诱导发生朊样聚集。第五部分:自噬激活剂对tau朊样聚集的影响方法采用已知的自噬激活剂雷帕霉素(rapamycin)和海藻糖(trehalose),通过培养细胞模型和小鼠脑内注射模型,研究自噬激活剂对tau朊样聚集的影响。结果(1)在AD O-tau诱导的tau聚集过程中,雷帕霉素能够激活自噬。(2)体外培养细胞模型中,雷帕霉素处理明显抑制AD O-tau诱导的sarkosyl不溶性总tau的聚集。(3)自噬激活剂雷帕霉素和海藻糖能够抑制AD O-tau所致的小鼠脑内tau朊样聚集和过度磷酸化。结论雷帕霉素和海藻糖能够抑制AD O-tau诱导的朊样聚集和位点特异性磷酸化。更多还原

【Abstract】 Objective Abnormal hyperphosphorylation and aggregation of the microtubule-associated protein tau(tau)play a key role in Alzheimer’s disease(AD)and other related neurodegenerative diseases.Recent studies suggest that the prion-like propagation characteristics of pathological tau in the AD brain may be the molecular basis for its development.In this study,we explored whether tau truncation and phosphorylation contribute to its prion-like propagation and whether activation of autophagy could inhibit the progression of tau pathology.The research is divided into five parts.Part I: Hyperphosphorylation and truncation were the biochemical characteristics of tau aggregates in AD brainMethods In the present study,we investigated the phosphorylation level and truncation of total tau and SDS-and reducing agent-resistant high-molecular weight tau(HMW-tau)in control and AD brains by Western blots.Results(1)We found that tau in AD brain appears as a smear from low molecular weight(LMW)to HMW in western blots developed with pan-tau antibodies.Similar levels of LMW-tau were presented in AD and control brain,while HMW-tau only expressed in AD brain.(2)The phosphorylation levels of tau in the brain of AD patients were significantly increased.HMW-tau is hyperphosphorylated at multiple sites and does not contain unphosphorylated Ser46 and Ser198/199/202.(3)The N-terminal antibodies 43D(recognizes 6-18 a.a.of full length tau)and HT7(recognizes 159-163 a.a.)could only detect weak signals in HMW-tau,while the C-terminal antibodies tau46 and tau46.1 showed strong immunoreactivity.(4)The ratio of HMW-tau to LMW-tau detected by tau antibodies was increasing as the epitope of the antibodies ranges from N-terminal to C-terminal.(5)Tau truncation at Asp421 was significantly increased in AD brain,but is poorly correlated with the levels of HMW-tau.Conclusions These findings suggest that HMW-tau in AD brain was hyperphosphorylated at multiple sites and was mostly truncated at the Nterminus.Part II: Spatiotemporal evolution of phosphorylation during induced prion-like aggregation of tauMethods AD oligomeric tau(AD O-tau)was extracted from AD brains(Braak stage VI)and added into cell culutre or injected into the hippocampus of mouse brain.Western blots and immunofluorescence were used to analyze the temporal and spatial evolution of tau phosphorylation and aggregation during AD O-tau-induced prion-like propagation of tau pathology.Results(1)The phosphorylation levels of AD O-tau at Ser199,Thr205 and Thr214 sites were relatively low,but the phosphorylation levels at Ser262,Ser396/404(PHF-1)and Ser422 sites were relatively high,especially at Ser422.(2)AD O-tau effectively induced prion-like aggregation of tau in cultured cell model.(3)In cell model,tau hyperphosphorylation and prion-like aggregation induced by AD O-tau were time-dependent.(4)The prion-like aggregation induced by AD O-tau in the brain of C57BL/6wild-type mouse was time-dependent and age-related.Conclusions AD O-tau is abnormally hyperphosphorylated,which can induce tau prion-like aggregation and hyperphosphorylation in SHSY5 Y cells and wild-type mice brain in a time-dependent manner.Part III: The truncation of tau determines its self-aggregation and prion-like activityMethods We constructed a series of tau truncations and studied their self-aggregation activity in cell model.As a representative,tau151-391 or tau1-441(full-length tau)were expressed in eukaryotic cells.Tau151-391 and tau1-441 aggregates were then extracted from the cells and injected into the hippocampus of mouse brain.The activity of truncated tau(tau151-391)aggregates and full-length tau aggregates in inducing the prion-like propagation of pathological tau in vivo was compared.Results(1)The self-aggregation activity of tau151-391 was the strongest among all the truncations expressed in HEK-293 FT cells.(2)Tau151-391 not only has stronger self-aggregation activity,but also is easier to induce prion-like aggregation.(3)The tau151-391 protein aggregates have a prion-like spreading effect similar to AD O-tau in the mouse brain,which is significantly stronger than the full-length tau aggregates.Conclusions Tau151-391 is more prone to self-aggregate and be seeded to aggregate by AD O-tau than the full-length tau.Tau151-391 insoluble aggregates isolated from cultured eukaryotic cells can induce tau prion-like aggregation in mouse brain,similar to that of AD O-tau.Part IV: Hyperphosphorylation promotes tau prion-like propagationMethods The phosphorylation levels of endogenous tau were up-regulated or down-regulated by okadaic acid treatment or protein phosphatase 2A(PP2A)knockdown or overexpression.Tau ofdifferent phosphorylation levels were analyzed for their characteristics in prionlike aggregation induced by AD O-tau.Results(1)Hyperphosphorylated tau was more easily captured by AD O-tau,and to aggregate when induced by AD O-tau.(2)When HEK-293 FT cells expressing tau151-391 were treated with sh PP2 Ac to interfere the expression of protein phosphatase 2A catalytic subunit(PP2Ac),more prion-like aggregation occurred after being induced by AD O-tau.On the contrary,when overexpressing PP2 Ac,tau prion-like aggregation induced by AD O-tau decreased.(3)Knockdown of PP2 Ac promoted the aggregation of endogenous tau induced by AD O-tau in C57BL/6 mice,but knockdown of PP2 Ac itself was not enough to induce the autoaggregation of endogenous tau.Overexpression of PP2 Ac significantly reduced the aggregation of endogenous tau induced by AD O-tau.Conclusions The phosphorylation level of endogenous tau affects the pathological aggregation of tau and prion-like propagation induced by AD O-tau.The higher the phosphorylation level of tau,the easier it is to form aggregation.Part V: The effect of autophagy activator on tau prion-like aggregationMethods Rapamycin and trehalose,known as autophagy activators,were used to study the effect on tau prion-like aggregation and propagation through cell culture model and mouse hippocampus injection model.Results(1)Rapamycin could activate autophagy in the process of tau aggregation induced by AD O-tau.(2)In the cell culture model,rapamycin treatment significantly inhibited the aggregation of sarkosylinsoluble total tau induced by AD O-tau.(3)The autophagy activators rapamycin and trehalose can inhibit the aggregation and hyperphosphorylation of tau induced by AD O-tau in the brain of C57BL/6 mice.Conclusions Autophagy activators rapamycin and trehalose inhibit the prion-like aggregation and site-specific phosphorylation of tau induced by AD O-tau.更多还原