Caspase-3 对磷酸化 tau 蛋白截断作用的研究

磷酸化 tau 是阿尔茨海默病 (Alzheimer's disease , AD) 的特征性病理改变———神经原纤维缠结 (neurofibrillary tangles , NFTs) 的主要组成部分 . 最近的研究显示： NFT 存在 Glu391 和 Asp421 位点被截断的 tau 片段,然而, tau 蛋白的磷酸化是否会影响 caspase-3 的切割作用尚不清楚 . 首先纯化重组 tau 蛋白,然后利用蛋白激酶 A (PKA) 、钙 / 钙调蛋白依赖性蛋白激酶Ⅱ (CaMK Ⅱ ) 和乳鼠海马组织抽提液对其磷酸化,并用 caspase-3 对不同磷酸化的 tau 蛋白进行切割,比较 caspase-3 对非磷酸化和不同蛋白激酶磷酸化的 tau 蛋白的切割特性 . 结果显示：除切割非磷酸化 tau 蛋白外, caspase-3 在体外可分别切割被 PKA 、 CaMK Ⅱ和乳鼠海马组织抽提液磷酸化的 tau 蛋白 . 这一结果提示：磷酸化修饰的 tau 蛋白仍然是 caspase-3 的底物 .     

急性缺氧和急性低糖对脑片tau蛋白磷酸化的影响

为探讨急性缺氧对tau蛋白磷酸化的影响,将Wistar大鼠脑片进行不同时间的缺氧培养后,对tau蛋白的磷酸化状态及相关磷酸酯酶的活性和表达进行检测.结果显示,急性缺氧使tau蛋白多个丝氨酸位点磷酸化水平下降,蛋白磷酸酯酶～2A(PP-2A)的活性升高,其催化亚单位表达上调,而蛋白磷酸酯酶-1(PP-1)的活性及催化亚单位表达均无明显改变.该研究结果表明:急性缺氧可能通过蛋白磷酸酯酶-2A的上调而使tau蛋白多个丝氨酸位点发生去磷酸化作用.     

肝素对人类神经tau蛋白分子聚集及磷酸化的影响

在老年性痴呆患者的脑中,肝素与超磷酸化的tau蛋白共存[7].采用NCLK(neuronalcdc2-likekinase)及PP2B(phosphoproteinphosphatase2B)在含肝素的体系中对人类神经tau蛋白进行磷酸化和脱磷酸化,结果表明,肝素具有促进tau蛋白被磷酸化的功能,并促进该蛋白磷酸化分子二聚体的形成和单体的减少,其一级动力学常数分别为2.88×10-3s-1和1.74×10-3s-1.PP2B可使磷酸化的tau蛋白脱磷酸化,并且脱磷酸化作用随肝素浓度的增加而增强,提示肝素可能具有调节tau蛋白磷酸化状态的作用     

糖尿病大鼠脑GSK-3与PP-2A失调诱导tau蛋白过度磷酸化

探讨胰岛素缺乏的糖尿病大鼠皮层糖原合酶激酶-3(GSK-3)及蛋白磷酯酶-2A(PP-2A)变化及其对tau蛋白磷酸化的作用.用链脲佐菌素(streptozotocin,STZ)建立胰岛素缺乏的糖尿病大鼠模型,用放射性配体结合实验检测了GSK-3和PP-2A的活性,蛋白质印迹检测了tau蛋白的磷酸化水平及PP-2A的表达.结果提示:在糖尿病大鼠皮层,GSK-3活性升高,PP-2A活性及表达降低,tau蛋白在Ser198/Ser199/Ser202和Ser396/Ser404位点磷酸化.应用GSK-3的选择性抑制剂Li2CO3后,GSK-3活性降低,PP-2A活性及表达恢复,tau蛋白在Ser198/Ser199/Ser202和Ser396/Ser404位点磷酸化水平降低.研究提示:糖尿病大鼠皮层GSK-3升高可能抑制PP-2A的活性,升高的GSK-3和降低的PP-2A协同促进tau蛋白的磷酸化.     

罗格列酮改善胰岛素抵抗大鼠海马Alzheimer病样tau蛋白磷酸化水平

Tau蛋白过度磷酸化是Alzheimer病(AD)发病的关键事件.由于2型糖尿病是AD的风险因子,并且胰岛素抵抗是2型糖尿病的特征,检测了胰岛素抵抗大鼠大脑海马tau蛋白磷酸化水平,以及运用胰岛素增敏剂罗格列酮(TZD)后磷酸化的变化,发现胰岛素抵抗组大鼠海马tau蛋白呈过度磷酸化改变,但运用TZD后,tau蛋白的磷酸化状态有所恢复.由于糖原合成激酶-3β(GSK-3β)位于胰岛素信号转导途径中,并且是tau蛋白的重要磷酸激酶,研究检测罗格列酮干预前后GSK-3β活性,发现均升高.研究结果表明,肥胖时胰岛素抵抗导致细胞内胰岛素信号转导途径中,GSK-3β活性上调可能是引起大鼠海马内tau蛋白过度磷酸化的一个重要原因;虽然TZD可抑制tau蛋白的过度磷酸化,但可能不是通过下调GSK-3β活性的途径.     

褪黑素对异丙肾上腺素诱导大鼠tau蛋白过度磷酸化的预防作用

异常过度磷酸化的微管相关蛋白tau是阿尔茨海默病(Alzheimer's disease,AD)患者大脑中神经原纤维缠结的主要组成部分.迄今为止,尚无有效的措施阻止tau蛋白的过度磷酸化.为探讨褪黑素(melatonin,Mel)对AD样tau蛋白过度磷酸化的预防作用,我们以β受体激动剂异丙肾上腺素(isoproterenol,IP)来复制AD样tau蛋白过度磷酸化的动物模型,在大鼠双侧海马注射IP前,以褪黑素作为保护组药物,于腹腔连续注射5 d.应用磷酸化位点特异性抗体(PHF-1和Tau-1)作免疫印迹和免疫组织化学检测tau蛋白的磷酸化水平,并用非磷酸化依赖的总tau蛋白抗体(111e)进行标准化.免疫印迹结果显示在注射IP 48 h后,tau蛋白在PHF-1表位的免疫反应显著增强,在Tau-1表位显著减弱,表明tau蛋白在Ser396/Ser404(PHF-1)和Ser199/Ser202(Tau-1)位点有过度磷酸化.免疫组织化学染色结果与免疫印迹结果相似,主要检测到在大鼠海马CA3区的神经纤维有tau蛋白过度磷酸化.褪黑素预处理大鼠可有效地阻止IP诱导tau蛋白在Tau-1和PHF-1位点的过度磷酸化.上述结果提示褪黑素可预防大鼠脑组织中由异丙肾上腺素引起的AD样tau蛋白的过度磷酸化.     

Anisomycin过度激活丝裂原活化蛋白激酶使tau发生过度磷酸化

阿尔茨海默病(Alzheimer's disease,AD)的病理特征之一是神经元内存在神经原纤维缠结(neurofibrillary tangles,NFTs),后者是由过度磷酸化的微管相关蛋白tau形成的双股螺旋细丝(paired helical filaments,PHFs)构成.为了探讨丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)在微管相关蛋白tau磷酸化中的作用及机制,本实验用0.1 μg/mL、0.2 μg/mL和0.4μg/mL三种不同浓度的MAPK激动剂anisomycin处理小鼠成神经瘤细胞株(mouse neuroblastoma cells,N2a),检测MAPK活性的变化及其与tau蛋白多个AD相关位点过度磷酸化的关系,并检测糖原合酶激酶-3(glycogen synthase kinase-3,GSK-3)和蛋白激酶A(protein kinase A,PKA)的活性变化.结果显示,anisomycin以剂量依赖的方式激活MAPK活性,但免疫印迹结果显示tau蛋白的Ser-198/199/202位点和Ser-396/404位点的过度磷酸化只在anisomycin浓度为0.4 μg/mL时出现,三种浓度的anisomycin均未引起tau蛋白Ser-214位点磷酸化的改变;同时,GSK-3活性在anisomycin为0.1 μg/mL时没有明显变化,当anisomycin浓度升高到0.2 μg/mL和0.4 μg/mL时出现明显增高,而PKA的活性没有明显的改变.使用GSK-3的特异性抑制剂氯化锂(LiCl)则完全阻断MAPK被过度激活导致的tau蛋白磷酸化水平的增高,而同时MAPK活性不受影响.以上结果提示:过度激活MAPK可以导致tau蛋白Ser-198/199/202和Ser-396/404位点过度磷酸化,其机制可能涉及MAPK激活GSK-3的间接作用.     

RT-PCR法同时测定APP与tau mRNA的含量(英文)

β—淀粉样蛋白前体(APP)与tau蛋白是两种与Alzheimer病的病理改变相关的蛋白质,近来也发现它们或其修饰产物在包含体肌炎及慢性氯喹中毒的肌细胞中沉积。为更好地研究这两种相关蛋白质在肌细胞中的表达,我们建立了用逆转录-多聚酶链反应同时定量这两种基因转录产物的方法。本法选用甘油醛-3-磷酸脱氢酶(G_3PD)作为内标,用激光图像分析测定扩增产物。结果显示,PCR在很宽的范围内呈指数扩增,两种靶mRNA与内标mRNA扩增效率相当,因而可以G_3PD来校正APP与tau的测定结果。初步实验显示氯喹中毒首先促进肌肉细胞中APP与tau蛋白的表达,在第4周达到高峰,然后表达逐渐减少,在第10周转为明显抑制作用。     

1型糖尿病葡萄糖代谢与胰岛素信号传导途径异常导致大鼠海马Alzheimer样tau蛋白过度磷酸化修饰

Tau蛋白过度磷酸化是Alzheimer病(Alzheimer disease, AD)的一个重要病理特征.采用 I 型糖尿病大鼠模型,研究胰岛素信号传导途径及葡萄糖代谢失调对tau蛋白过度磷酸化的形成机制进行探讨.以同龄Wistar大鼠做对照（CTL）,胰腺大部分切除造低胰岛素组（PX）,STZ较大剂量一次性注射造1型糖尿病模型即低胰岛素高血糖组（T1DM）.葡萄糖氧化酶法检测血浆血糖,放免法检测血浆胰岛素,蛋白质印迹分析海马内总tau蛋白及tau蛋白上部分位点（Ser199、Thr212、Ser214、Ser396及Ser422）的磷酸化及神经细胞膜上葡萄糖转运子3（Glucose transport 3,GLUT3）水平.γ-32P-ATP和特异性底物肽检测海马内胰岛素信号传导系统中的关键酶糖原合成酶激酶-3β（Glycogen synthase kinase-3β, GSK-3β）活性.发现3组大鼠海马回总tau蛋白水平无显著差异,但以高血糖、低胰岛素血症为特征的T1DM组在tau蛋白Ser199、Thr212、Ser214、Ser396及Ser422位点上,呈现过度磷酸化状态,以低胰岛素血症为特征而血糖正常的PX组在位点Ser199、Thr212及Ser396上磷酸化程度比CTL组显著上升, 在位点Ser214及 Ser422上的磷酸化程度的改变不显著;T1DM及PX组大鼠海马 GSK-3β活性显著高于CTL组, 而GLUT3水平在T1DM和PX组均降低, 尤以T1DM组降低更显著.研究结果显示,胰岛素水平低下可能通过激活GSK-3β和下调细胞内葡萄糖代谢的双重作用引起脑内tau蛋白过度磷酸化.     

蛋白磷酸酯酶抑制剂冈田酸对人神经母细胞瘤 SK-N-SH细胞系tau蛋白磷酸化水平的影响

观察蛋白磷酸酯酶-1和蛋白磷酸酯酶-2A的抑制剂冈田酸(okadaicacid,OA)对人神经母细胞瘤系SK-N-SH细胞tau蛋白磷酸化水平的变化,确定tau蛋白过度磷酸化细胞模型的合适剂量和时间。用不同剂量OA与SK-N-SH细胞共温育不同时间,用显微镜观察细胞形态变化,用Western印迹法检测磷酸化tau蛋白和非磷酸化tau蛋白在Ser202位点和Ser404位点磷酸化水平的变化。10～160nmol/LOA与SK-N-SH神经细胞温育3～24h,可引起细胞形态损伤呈剂量依赖性和时间依赖性的变化,起效剂量和时间为10nmol/L和3h。10nmol/LOA与SK-N-SH细胞温育6～24h,磷酸化tau蛋白Ser199/Ser202位点和Ser404位点的表达明显增高,非磷酸化tau蛋白Ser202位点和Ser404位点的表达明显降低,总tau蛋白含量无明显变化。OA可以作为很好的研究tau蛋白过度磷酸化的工具药,10nmol/LOA与SK-N-SH神经细胞共温育6h可以作为制备细胞模型的适宜条件。     

Rapamycin decreases tau phosphorylation at Ser214 through regulation of cAMP-dependent kinase

Preventing or reducing tau hyperphosphorylation is considered to be a therapeutic strategy in the treatment of Alzheimer’s disease (AD). Rapamycin may be a potential therapeutic agent for AD, because the rapamycin-induced autophagy may enhance the clearance of the hyperphosphorylated tau. However, recent rodent studies show that the protective effect of rapamycin may not be limited in the autophagic clearance of the hyperphosphorylated tau. Because some tau-related kinases are targets of the mammalian target of rapamycin (mTOR), we assume that rapamycin may regulate tau phosphorylation by regulating these kinases. Our results showed that in human neuroblastoma SH-SY5Y cells, treatment with rapamycin induced phosphorylation of the type IIα regulatory (RIIα) subunit of cAMP-dependent kinase (PKA). Rapamycin also induced nuclear translocation of the catalytic subunits (Cat) of PKA and decreases in tau phosphorylation at Ser214 (pS214). The above effects of rapamycin were prevented by pretreatment with the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibitor U0126. In addition, these effects of rapamycin might not depend on the level of tau expression, because similar results were obtained in both the non-tau-expressing wild type human embryonic kidney 293 (HEK293) cells and HEK293 cells stably transfected with the longest isoform of recombinant human tau (tau441; HEK293/tau441). These findings suggest that rapamycin decreases pS214 via regulation of PKA. Because tau phosphorylation at Ser214 may prime tau for further phosphorylation by other kinases, our findings provide a novel possible mechanism by which rapamycin reduces or prevents tau hyperphosphorylation.     

Tau phosphorylation and μ-calpain activation mediate the dexamethasone-induced inhibition on the insulin-stimulated Akt phosphorylation

Evidence has suggested that insulin resistance (IR) or high levels of glucocorticoids (GCs) may be linked with the pathogenesis and/or progression of Alzheimer's disease (AD). Although studies have shown that a high level of GCs results in IR, little is known about the molecular details that link GCs and IR in the context of AD. Abnormal phosphorylation of tau and activation of μ-calpain are two key events in the pathology of AD. Importantly, these two events are also related with GCs and IR. We therefore speculate that tau phosphorylation and μ-calpain activation may mediate the GCs-induced IR. Akt phosphorylation at Ser-473 (pAkt) is commonly used as a marker for assessing IR. We employed two cell lines, wild-type HEK293 cells and HEK293 cells stably expressing the longest human tau isoform (tau-441; HEK293/tau441 cells). We examined whether DEX, a synthetic GCs, induces tau phosphorylation and μ-calpain activation. If so, we examined whether the DEX-induced tau phosphorylation and μ-calpain activation mediate the DEX-induced inhibition on the insulin-stimulated Akt phosphorylation. The results showed that DEX increased tau phosphorylation and induced tau-mediated μ-calpain activation. Furthermore, pre-treatment with LiCl prevented the effects of DEX on tau phosphorylation and μ-calpain activation. Finally, both LiCl pre-treatment and calpain inhibition prevented the DEX-induced inhibition on the insulin-stimulated Akt phosphorylation. In conclusion, our study suggests that the tau phosphorylation and μ-calpain activation mediate the DEX-induced inhibition on the insulin-stimulated Akt phosphorylation.     

Effects of tau phosphorylation on proteasome activity

Dysfunction of proteasome contributes to the accumulation of the abnormally hyperphosphorylated tau in Alzheimer's disease. However, whether tau hyperphosphorylation and accumulation affect the activity of proteasome is elusive. Here we found that a moderate tau phosphorylation activated the trypsin-like activity of proteasome, whereas further phosphorylation of tau inhibited the activity of the protease in HEK293 cells stably expressing tau441. Furthermore, tau hyperphosphorylation could partially reverse lactacystin-induced inhibition of proteasome. These results suggest that phosphorylation of tau plays a dual role in modulating the activity of proteasome.     

Expression of the hyperphosphorylated tau attenuates ER stress-induced apoptosis with upregulation of unfolded protein response

The neural dysfunction in Alzheimer's disease (AD) could arise from endoplasmic reticulum (ER) stress and deficits of the unfolded protein response (UPR). To explore whether tau hyperphosphorylation, a hallmark of AD brain pathologies, plays a role in ER stress-induced alterations of cell viability, we established cell lines with stable expression of human tau (HEK293/tau) or the vector (HEK293/vec) and treated the cells with thapsigargin (TG), an ER stress inducer. We observed that the HEK293/tau cells were more resistant than the HEK293/vec cells to the TG-induced apoptosis, importantly, a time dependent increase of tau phosphorylation at Thr205 and Thr231 sites was positively correlated with the inhibition of apoptosis. We also observed that expression of tau upregulated phosphorylation of PERK, eIF2 and IRE1 with an increased cleavage of ATF6 and ATF4. The potentiation of UPR was also detected in HEK293/tau cells treated with other ER stress inducers, including staurosporine, camptothecin and hydrogen peroxide, in which a suppressed apoptosis was also shown. Our data suggest that tau hyperphosphorylation could attenuate the ER stress-induced apoptosis with the mechanism involving upregulation of UPR system.     

Human Tau may Modify Glucocorticoids-Mediated Regulation of cAMP-dependent Kinase and Phosphorylated cAMP Response Element Binding Protein

Phosphorylation of the cAMP response element binding protein (CREB) by cAMP-dependent kinase (PKA) is critical to memory formation. However, activation of PKA can also increase tau phosphorylation, which may contribute to memory impairment. Therefore, the regulation of PKA may be part of the mechanism by which glucocorticoids (GCs) influence memory. Additionally, the cellular response to GCs may be affected by the presence of human tau. The goal of this paper was to study GCs-mediated regulation of PKA as well as CREB and tau phosphorylation in wild-type HEK293 cells and HEK293 cells stably expressing human tau441 (HEK293/tau441 cells). By using dexamethasone (DEX) as GCs, we found that DEX induced a tau-dependent selective decrease in the level of PKA RIIβ subunit protein. The observed decrease in RIIβ expression was not due to alterations of mRNA levels and was reversed by inhibiting the proteasome with lactacystin. Moreover, the decrease in RIIβ did not diminish the co-localization of the catalytic subunit of PKA with tau and might contribute to the DEX-induced increase in tau phosphorylation at Ser-214. DEX also induced a tau-dependent decrease in CREB phosphorylation that could not be reversed by activating PKA with forskolin. Taken together, these results show that human tau protein may alter the GCs-mediated regulation of PKA activity and CREB phosphorylation.     

Direct interaction of soluble human recombinant tau protein with Abeta 1-42 results in tau aggregation and hyperphosphorylation by tau protein kinase II

We report here that aggregated beta-amyloid (Abeta) 1-42 promotes tau aggregation in vitro in a dose-dependent manner. When Abeta-mediated aggregated tau was used as a substrate for tau protein kinase II (TPK II), an 8-fold increase in the rate of TPK II-mediated tau phosphorylation was observed. The extent of TPK II-dependent tau phosphorylation increased as a function of time and Abeta 1-42 concentration, and hyperphosphorylated tau was found to be decorated with an Alzheimer's disease-related phosphoepitope (P-Thr-231). In HEK 293 cells co-expressing CT-100 amyloid precursor protein and tau, the release of Abeta 1-42 from these cells was impaired. Taken together, these in vitro results suggest that Abeta 1-42 promotes both tau aggregation and hyperphosphorylation.     

Nitric oxide induces tau hyperphosphorylation via glycogen synthase kinase-3beta activation

Nitric oxide is associated with neurofibrillary tangle, which is composed mainly of hyperphosphorylated tau in the brain of Alzheimer's disease (AD). However, the role of nitric oxide in tau hyperphosphorylation is unclear. Here we show that nitric oxide produced by sodium nitroprusside (SNP), a recognized donor of nitric oxide, induces tau hyperphosphorylation at Ser396/404 and Ser262 in HEK293/tau441 cells with a simultaneous activation of glycogen synthase kinase-3beta (GSK-3beta). Pretreatment of the cells with 10 mM lithium chloride (LiCl), an inhibitor of GSK-3, 1 h before SNP administration inhibits GSK-3beta activation and prevents tau from hyperphosphorylation. This is the first direct evidence demonstrating that nitric oxide induces AD-like tau hyperphosphorylation in vitro, and GSK-3beta activation is partially responsible for the nitric oxide-induced tau hyperphosphorylation. It is suggested that nitric oxide may be an upstream element of tau abnormal hyperphosphorylation in AD.     

蝙蝠葛碱拮抗缓激肽引起的钙稳态改变 及细胞骨架蛋白的异常磷酸

为研究蝙蝠葛碱 (dauricine , Dau) 拮抗缓激肽 (bradykinin , BK) 诱导的 Alzheimer 样钙稳态失衡及细胞骨架蛋白异常磷酸化的作用,采用双波长荧光分光光度计测定细胞内钙离子浓度 ([Ca2+] i) ,用 MTT 法检测细胞代谢水平,用免疫组织化学方法观察 tau 蛋白表达和磷酸化 . 结果表明,Dau (3 μmol/L , 6 μmol/L) 可抑制 BK 诱导的 [Ca2+]i 升高,保护 BK 引起的神经元代谢降低,拮抗 BK 引起的 tau 蛋白异常磷酸化和聚集 . 结果提示： Dau 可拮抗 BK 诱导的 Alzheimer 样钙稳态失衡及细胞骨架蛋白异常磷酸化的作用 .     

The involvement of glycogen synthase kinase-3 and protein phosphatase-2A in lactacystin-induced tau accumulation

Here, we demonstrated that lactacystin inhibited proteasome dose-dependently in HEK293 cells stably expressing tau. Simultaneously, it induces accumulation of both non-phosphorylated and hyperphosphorylated tau and decreases the binding of tau to the taxol-stabilized microtubules. Lactacystin activates glycogen synthase kinsase-3 (GSK-3) and decreases the phosphorylation of GSK-3 at serine-9. LiCl inhibits GSK-3 and thus reverses the lactacystin-induced accumulation of the phosphorylated tau. Lactacystin also inhibits protein phosphase-2A (PP-2A) and it significantly increases the level of inhibitor 1 of PP-2A. These results suggest that inhibition of proteasome by lactacystin induces tau accumulation and activation of GSK-3 and inhibition of PP-2A are involved.     

Synthesis and conformational properties of phosphopeptides related to the human tau protein

In the brains of Alzheimer's disease patients, the tau protein dissociates from the axonal microtubule and abnormally aggregates to form a paired helical filament (PHF). One of the priorities in Alzheimer research is to determine the effects of abnormal phosphorylation on the local structure. A series of peptides corresponding to isolated regions of tau protein have been successfully synthesized using Fmoc-based chemistry and their conformations were determined by 1H NMR spectroscopy and circular dichroism (CD) spectroscopy. Immunodominant peptides corresponding to tau-(256-273), tau-(350-367) and two phosphorylated derivatives in which a single Ser was phosphorylated at positions 262 and 356, respectively, were the main focus of the study. A direct alteration of the local structure after phosphorylation constitutes a new strategy through which control of biological activity can be enforced. In our study on Ser262 in R1 peptide and Ser356 in R4 peptide, phosphorylation modifies both the negative charge and the local conformation nearby the phosphorylation sites. Together, these structural changes indicate that phosphorylation may act as a conformational switch in the binding domain of tau protein to alter specificity and affinity of binding to microtubule, particularly in response to the abnormal phosphorylation events associated with Alzheimer's disease.