钙蛋白酶-细胞周期依赖性蛋白激酶5通路参与β淀粉样蛋白25-35诱导的tau蛋白过度磷酸化

目的探讨凝聚态β淀粉样蛋白25-35片段(Aβ25-35)对胎鼠皮质神经元中性钙蛋白酶-细胞周期依赖性蛋白激酶5(calpain-CDK5)通路的影响及其对tau蛋白的过度磷酸化和骨架稳定性的影响。方法用荧光酶标仪测定荧光强度来反映calpain活性;用Western-blot和(或)免疫细胞化学检测CDK5的激活蛋白p25/p35的蛋白水平和tau蛋白Thr205/Ser404位点磷酸化情况来体现CDK5活性;用电镜技术观察微管结构的变化来显示细胞骨架的改变情况。结果20μmol/L的凝聚态Aβ25-35作用于皮质神经元12 h后,检测反映calpain活性的荧光强度(每微克蛋白荧光强度)高达680.25±37.77,与空白对照组167.25±11.67相比,差异有统计学意义(P<0.05);对CDK5有活化作用的p25蛋白水平比空白对照组升高(2.07±0.20)倍,差异也有统计学意义(P<0.05);CDK5的作用底物tau蛋白Thr205和Ser404位点磷酸化程度也显著增加,分别比空白对照组升高(1.80±0.27)和(1.83±0.14)倍,差异均有统计学意义(P<0.05);同时出现神经元微管骨架排列紊乱。结论凝聚态Aβ25-35通过活化皮质神经元的calpain,使p35降解p25来激活CDK5,促使tau蛋白过度磷酸化,破坏了微管骨架的稳定性。     

海马内注射纤丝状Aβ42诱导tau异常磷酸化的研究

目的　观察海马内注射纤丝状Aβ42 后神经元Ser 2 0 2位点磷酸化的tau蛋白 (PS2 0 2 tau)的表达 ,探讨Aβ42 与tau蛋白超磷酸化的关系。方法　应用立体定向技术对老年大鼠进行海马内注射纤丝状Aβ42 ,采用免疫组织化学染色方法 ,显示PS2 0 2 tau的表达情况 ,并进行图像分析。结果　纤丝状Aβ42 注射组双侧海马PS2 0 2 tau的表达明显高于正常组和双蒸水注射组 ,两侧海马PS2 0 2 tau的表达没有明显差异。结论　纤丝状Aβ42 能使PS2 0 2 tau的表达增加 ,提示它具有诱导tau蛋白超磷酸化的效应。     

胰岛素抑制糖尿病大鼠皮质tau蛋白磷酸化作用的研究

目的探讨胰岛素抑制糖尿病大鼠皮质tau蛋白磷酸化、改善记忆障碍的可能机制。方法用链脲佐菌素(STZ)建立糖尿病大鼠模型,用32P放射性标记法检测对照组、DM(Diabetes mellitus)组及Insulin组糖原合酶激酶-3(glycogen synthase kinase-3,GSK-3)的活性,用蛋白印迹法检测tau蛋白磷酸化水平,用电跳台试验检测大鼠的保留记忆。结果与对照组相比,DM组的GSK-3活性明显增加(P<0.01),tau蛋白在Ser198/Ser199/Ser202位点出磷酸化程度明显升高(P<0.01,),且伴有记忆障碍。与DM组相比较,Insulin组GSK-3活性降低(P<0.01),tau蛋白在Ser198/Ser199/Ser202位点磷酸化降低,(P<0.05),记忆障碍改善,与对照组比较无统计学差异(P>0.05)。结论糖尿病大鼠脑皮质GSK-3活性升高,tau蛋白过度磷酸化增加,胰岛素通过抑制GSK-3活性,降低tau蛋白过度磷酸化,改善糖尿病大鼠的记忆障碍。     

慢性不可预见性温和应激后大鼠行为及微管相关蛋白tau磷酸化的改变

目的研究慢性不可预见性温和应激所致的动物行为学改变及微管相关蛋白tau磷酸化的改变。方法将大鼠随机分为慢性不可预见性温和应激抑郁（CUMS）组和对照组,对模型组大鼠进行连续21d的慢性不可预见性应激。进行行为学观察,使用western blotting检测总tau,tau磷酸化（Ser356,Thr231）水平的改变。结果CUMS组大鼠慢性应激后糖水偏好及自主活动显著减低,与对照组比较差异有统计学意义（P〈0．05）;CUMS组大鼠慢性应激后海马tau磷酸化表达升高,与对照组比较差异有统计学意义（P〈0．05）。结论慢性应激后微管相关蛋白tau磷酸化水平升高,神经可塑性受损,提示了新的抑郁症和阿尔茨海默病联系的生化机制。     

海马内注射纤丝状Aβ42诱导tau异常磷酸化的研究

目的观察海马内注射纤丝状Aβ42后神经元Ser 202位点磷酸化的tau蛋白(PS202-tau)的表达,探讨Aβ42与tau蛋白超磷酸化的关系.方法应用立体定向技术对老年大鼠进行海马内注射纤丝状Aβ42,采用免疫组织化学染色方法,显示PS202-tau的表达情况,并进行图像分析.结果纤丝状Aβ42注射组双侧海马PS202-tau的表达明显高于正常组和双蒸水注射组,两侧海马PS202-tau的表达没有明显差异.结论纤丝状Aβ42能使PS202-tau的表达增加,提示它具有诱导tau蛋白超磷酸化的效应.     

MAPK信号通路与阿尔茨海默病中tau蛋白磷酸化的关系

阿尔茨海默病(AD)是一类神经退行性疾病,tau蛋白过度磷酸化形成的神经原纤维缠结为其主要病理特征之一,是AD发病的重要因素.促分裂原活化蛋白激酶(MAPK)是一类脯氨酸依赖的蛋白激酶,在AD病人体内参与诱导tau蛋白的过度磷酸化.MAPK的三条途径ERK、JNK、p38都参与诱导tau蛋白过度磷酸化,且与Aβ、氧化应激、炎性因子及蛋白磷酸酯酶等因素相关,由此阐述MAPK在AD进程中的重要作用,并提示MAPK可成为AD治疗中的新靶点.     

叶酸和维生素B12对老年大鼠海马中tau蛋白磷酸化的影响

目的研究叶酸和维生素B12与tau蛋白磷酸化的关系，及叶酸和维生素B12在阿尔茨海默病（Alzheimer disease’s，AD）的发病机制中的可能作用。方法用免疫印迹和免疫组织化学的方法观察和比较2个月龄和40个月龄大鼠海马中tau蛋白的磷酸化情况，以及用叶酸和维生素B12处理后的40个月龄大鼠海马中tau蛋白的磷酸化水平。结果发现40个月龄老年大鼠脑中tau蛋白Ser396／404位点磷酸化水平比2个月龄大鼠高97％。同时发现用叶酸和维生素B12处理后，40个月龄大鼠海马中tau蛋白Ser396／404位点的过度磷酸化水平降低了27％。结论叶酸和维生素B12对神经骨架蛋白tau有一定的保护作用。     

髓样细胞触发受体 2与阿尔茨海默病关系的研究进展

阿尔茨海默病(AD)是老年期痴呆最常见的类型,是一种复杂的、不可逆的神经退行性疾病.其特征性的病理改变是细胞外出现β淀粉样蛋白(Aβ)沉积形成的淀粉样斑块(SP)、细胞内过度磷酸化的tau蛋白形成的纤维缠结及显著的突触丢失、脑萎缩和神经元细胞的死亡.     

基于Aβ与tau蛋白过度磷酸化损伤机制的阿尔茨海默病治疗靶点

阿尔茨海默病(Alzheimer's disease,AD)的发病机制主要包括Aβ蛋白表达增高在脑内聚集形成老年斑和tau蛋白过度磷酸化在胞内形成神经原纤维缠结.尽管Aβ与tau蛋白的损伤机制一直是AD研究的重点,但目前仍未找到能有效治疗AD的药物.本文主要概述了Aβ蛋白聚集与tau蛋白过度磷酸化对大脑损伤作用的分子机...     

Wnt/β-连环蛋白信号通路参与调节糖皮质激素诱导的阿尔茨海默病样病变

目的 探讨Wnt/β-连环蛋白(catenin)信号通路在糖皮质激素(glucocorticoids,GC)诱导的阿尔茨海默病样病变中的信号调节机制.方法 地塞米松(dexamethasone,DEX)分别处理转染人tau441的人胚肾293细胞(HEK293/tau)和野生型的人胚肾293细胞(HEK293/wt),采用细胞计数试剂盒( CCK-8)法检测DEX对细胞活力的影响,Western blot研究两组细胞tau蛋白磷酸化(p-T205,Tau-1)、β-catenin、p-β-catenin、B细胞淋巴瘤/白血病-2蛋白(Bcl-2)及其上游激酶糖原合成激酶-3β(GSK-3β)、ps9-GSK-3β水平的变化,并加用GSK-3β的抑制剂氯化锂(LiCI),观察其对相关蛋白相应的逆转作用.结果 CCK-8细胞活力检测结果显示,1μmol/L DEX处理细胞48h,HEK293/wt组细胞活力(存活率)下降到95.5％±3.2％,HEK293/tau组下降到77.8％±4.4％,两者差异有统计学意义(t =6.60,P＜0.05);Western blot结果显示,1μmoL/L DEX处理两组细胞48 h,使得HEK293/tau细胞ps9-GSK3β、Tau -I、β-catenin、Bcl-2水平分别下降到对照组的47.8％±10.4％、53.9％±11.7％、50.9％±7.6％、48.4％±6.5％,差异均有统计学意义(t=7.01、3.86、7.09、7.30,均P＜0.05),而p-T205、p-β-catenin相对磷酸化水平分别增加到对照组的180.5％±22.2％、201.3％±27.6％,差异均有统计学意义(t=5.51、5.27,均P＜0.05);LiCI可以相应逆转上述改变.结论 在人tau存在的前提下,GC通过抑制Wnt/β-catenin信号转导通路,促进了HEK293/tau细胞的阿尔茨海默病样病变.     

Site-specific effects of tau phosphorylation on its microtubule assembly activity and self-aggregation

Microtubule-associated protein tau is abnormally hyperphosphorylated and aggregated into neurofibrillary tangles in brains with Alzheimer's disease. The phosphorylation sites of tau are mainly localized in the proline-rich (residues 172–251) and C-terminal tail (residues 368–441) regions, which flank the microtubule-binding repeats. Here, we investigated the effects of tau phosphorylation at these distinct sites/regions on its activity of stimulating microtubule assembly and its self-aggregation. We found that tau phosphorylation at the proline-rich region by dual-specificity tyrosine-phosphorylated and -regulated kinase 1A inhibited its microtubule assembly activity moderately and promoted its self-aggregation slightly. Tau phosphorylation at the C-terminal tail region by glycogen synthase kinase-3β increased its activity and promoted its self-aggregation markedly. Tau phosphorylation at both regions plus the microtubule-binding region by cAMP-dependent protein kinase diminished its activity (∼70% inhibition) and disrupted microtubules. These studies reveal the differential regulation of tau's biological activity and self-aggregation by phosphorylation at various sites/regions.     

突触蛋白I片段促进α-突触核蛋白的聚集

目的 探讨突触蛋白I(Synapsin I,Syn I)被剪切后形成的C83片段对α-突触核蛋白聚集的影响。方法 在稳定表达α-突触核蛋白的HEK293细胞、小鼠原代神经元以及Tau P301S转基因小鼠体内分别过表达Syn I全长及其C83片段,通过细胞免疫荧光染色、蛋白免疫印迹技术以及免疫组织化学染色的方法观察Syn I全长及其C83片段对α-突触核蛋白磷酸化及聚集的影响。结果 Syn I C83片段促进HEK293细胞中α-突触核蛋白的磷酸化、泛素化以及聚集,并诱导小鼠原代神经元以及Tau P301S转基因小鼠体内α-突触核蛋白的磷酸化和聚集。结论 在细胞和动物模型中Syn I C83片段均可以促进α-突触核蛋白的聚集,这可能是Syn I C83片段导致认知功能障碍的重要原因之一。     

Interleukin-1 induces tau phosphorylation and morphological changes in cultured human astrocytes

Tau phosphorylation is associated with neurite outgrowth and morphogenesis in neurons. Since inflammatory stimuli induce marked morphological changes in astrocytes, we examined the effect of interleukin 1alpha (IL-1alpha), an inflammatory cytokine, on the expression and phosphorylation of tau protein in cultured human astrocytes. Western blot analysis showed a rapid and transient increase (3-30 min) of tau phosphorylation at the AT8 epitope. The cells began to extend processes from 24 h after IL-1alpha stimulation. It is known that tau phosphorylation at the AT8 epitope reduces its ability to bind to, and stabilize, microtubules. The early tau phosphorylation induced by IL-1alpha might create a plastic environment for morphological changes in astrocytes.     

Proteasome Inhibition Drives HDAC6-Dependent Recruitment of Tau to Aggresomes

Lesions containing aggregated and hyperphosphorylated tau protein are characteristic of neurodegenerative tauopathies. We have developed a cellular model of pathological tau deposition and clearance by overexpressing wild type human tau in HEK293 cells. When proteasome activity is inhibited, HEK293/tau cells accumulate tau protein in structures that bear many of the hallmarks of aggresomes. These include recruitment of tau into large spherical inclusions, accumulation of the retrograde motor protein dynein at the centrosome, formation of an intermediate filament cage around inclusions, and clustering of mitochondria at the aggresome. Tau aggresomes form rapidly and can be cleared upon relief of proteasome inhibition. We observe recruitment of pathological misfolded phospho-tau species to aggresomes. Immunoblotting reveals accumulation of detergent insoluble aggregated tau species. Knockdown of histone deacetylase 6, a protein known to interact with tau, reveals a requirement for HDAC6 activity in tau aggresome formation. Direct observation of the accumulation and clearance of abnormal tau species will allow us to dissect the cellular and molecular mechanisms at work in clearing aggresomal tau and its similarity to disease relevant pathological tau clearance mechanisms.     

Divergent phosphorylation pattern of tau in P301L tau transgenic mice

Aggregates of hyperphosphorylated tau are prominent in brains of patients with Alzheimer's disease or frontotemporal dementia (FTD). They have been reproduced in animal models following the identification of tau mutations in familial cases of FTD. This includes our previously generated transgenic model, pR5, which expresses FTD (P301L) mutant tau in neurons. The mice are characterized by tau aggregation including tangle (NFT) formation, memory impairment and mitochondrial dysfunction. In 8-month-old mice, S422 phosphorylation of tau is linked to NFT formation, however, a detailed analysis of tau solubility, phosphorylation and aggregation has not been done nor have the mice been monitored until a high age. Here, we undertook an analysis by immunohistochemistry, Gallyas impregnation and Western blotting of brains from 3 month- up to 20 month-old mice. NFTs first appeared at 6 months in the amygdala, followed by the CA1 region of the hippocampus. As the mice get older, the solubility of tau is decreased as determined by sequential extractions. Histological analysis revealed increased phosphorylation at the AT180, AT270 and 12E8 epitopes with ageing. The numbers of AT8-positive neurons increased from 3 to 6 months old. However, whereas S422 appeared only late and concomitantly with NFT formation, the only neurons left with AT8-reactivity at 20 months were those that had undergone NFT formation. As hyperphosphorylated tau continued to accumulate, the lack of AT8-reactivity suggests regulatory mechanisms in specifically dephosphorylating the AT8 epitope in the remaining neurons. Thus, differential regulation of phosphorylation is important for NFT formation in neurodegenerative diseases with tau pathology.     

Hyperphosphorylation of tau is mediated by ERK activation during anticancer drug-induced apoptosis in neuroblastoma cells

Phosphorylated tau protein is the major component of paired helical filaments in Alzheimer disease (AD). We have previously shown that abnormal tau phosphorylation was induced in neuroblastoma SK-N-SH cells by the anticancer drug, paclitaxel, during apoptosis [Guise et al., 1999: Apoptosis 4:47-58]. In the present study, we first demonstrated a shift from fetal tau to hyperphosphorylated tau after incubation with paclitaxel, that showed some similarities with the hyperphosphorylated tau in AD, by using several tau antibodies, N-Term, Tau-1 and AT-8. Tau phosphorylation occurred independently of caspase-3 activation. We next showed that a sustained activation of ERK (extracellular signal-regulated kinase) induced both tau phosphorylation and apoptosis during paclitaxel treatment (1 microM). The inhibition of ERK activation by using the pharmacological MEK1/2 inhibitor, PD98059 (50 microM), or an antisense strategy, reduced tau phosphorylation and neuronal apoptosis (P < 0.001), indicating a link between ERK activation, tau phosphorylation and apoptosis. Doxorubicin (0.2 microM), an anticancer drug whose mechanism of action is independent of microtubules, also induced ERK activation, tau phosphorylation and apoptosis. Moreover, doxorubicin induced some morphological features of neurodegeneration such as loss of neurites and disorganization of the cytoskeleton in apoptotic neuroblastoma cells. Altogether, our results suggest that tau phosphorylation plays a significant role in apoptosis enhancing disruption of microtubules that in turn leads to formation of apoptotic bodies, suggesting that neurodegeneration and apoptosis are related.     

Pseudo-phosphorylation of tau at Ser202 and Thr205 affects tau filament formation

The microtubule-associated protein tau aggregates into insoluble filaments in numerous neurodegenerative diseases, most common of which is Alzheimer's disease. Tau aggregation in Alzheimer's disease appears to follow a continuum from soluble monomer to an end point of insoluble extracellular tangles with a strong correlation between the amount of fibrillar tau and dementia. The phosphorylation of amino acids S202 and T205 in the tau molecule is recognized by the phosphorylation-specific monoclonal antibody, AT8, and has been observed by a number of researchers to be an early step in the progression of monomer to filaments. In addition, these amino acids are located in a proline-rich region containing a set of five phosphorylation sites (one being S202), that when phosphorylated, were reported to alter several properties of tau, including filament formation. Considering these observations, we have investigated the role of S202 and T205 phosphorylation in the in vitro polymerization of tau. Pseudo-phosphorylation mutants were constructed by site-directed mutagenesis in which amino acids S202 and T205 were changed to negatively charged glutamic acids mimicking post-translational phosphorylation. These pseudo-phosphorylated, mutant tau proteins were then assayed in vitro for changes in structure, polymerization into filaments, and microtubule binding. Phosphorylation at the AT8 site does not appear to influence either SDS-resistant structure nor microtubule binding. However, in regard to filament formation, phosphorylation at S202 appears to enhance polymerization; and phosphorylation at both sites not only enhances polymerization but also makes filament formation more sensitive to small changes in tau concentration.     

Phosphorylated serine422 on tau proteins is a pathological epitope found in several diseases with neurofibrillary degeneration

Neuronal inclusions with bundles of abnormal filaments made of tau polymers are found in numerous diseases with neurofibrillary degeneration. Tau proteins are the basic components of paired helical filaments (PHF) in Alzheimer’s disease (AD), and are abnormally phosphorylated. A disease-specific phosphorylation site at serine422 was demonstrated on PHF, but not on tau proteins from biopsy-derived brain samples. In the present study, we report the characterization of a polyclonal antibody (988) against the serine422 phosphorylation site. By using biochemical and immunohistochemical methods, we confirmed that it is not found on tau proteins from biopsy- or autopsy-derived control samples, and we investigated the presence of this epitope on tau proteins in several neurodegenerative disorders, including AD, Down syndrome (DS), Guamanian amyotrophic lateral sclerosis/Parkinsonism-dementia complex (ALS/PDC), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), postencephalitic parkinsonism (PEP) and Pick’s disease (PiD). By Western blotting, antibody 988 labeled the characteristic tau triplet (tau 55, 64, 69) in AD, DS, Guamanian ALS/PDC and PEP. PSP and CBD exhibited their typical tau doublet (tau 64, 69), whereas the doublet tau 55 and 64 was detected in PiD. In all of these neurodegenerative disorders, antibody 988 clearly labeled NFT and dystrophic neurites, as well as Pick bodies in PiD cases, whereas no staining was observed in control cases. These data indicate that phosphorylation of serine422 on tau proteins is a common feature among neurodegenerative disorders and is therefore not specific of AD. Moreover, phosphorylation of this epitope permits the distinction between normal tau proteins and pathological tau proteins. Received: 24 July 1998 / Revised: 8 September 1998 / Accepted: 14 September 1998     

Phosphorylated tau in neuritic plaques of APP<Superscript>sw</Superscript>/Tau<Superscript>vlw</Superscript> transgenic mice and Alzheimer disease

We have previously reported that double-transgenic APP(SW)/Tau(VLW) mice show enhanced amyloid deposition, stronger tau hyperphosphorylation, increased sarkosyl tau polymers, and wider tau filaments when compared to simple mutant models. To validate these transgenic mice as models of Alzheimer disease pathology, in the present study we analyze tau phosphorylation at 12E8 and AT-8 epitopes in amyloid plaques. In APP(SW) mice, phospho-tau in plaque-associated neurites suggests a local direct effect of plaque-amyloid (and/or APP(SW)) on tau phosphorylation. In vitro, attempts to identify which kinases are induced by fibrillar amyloid reveal to Protein Kinase C as responsible for phosphorylation at the 12E8 epitope. Tau(VLW) mice, without plaques, show increased tau phosphorylation at the 12E8 epitope, particularly in pyramidal neurons. APP(SW)/Tau(VLW) mice show earlier and stronger 12E8 tau phosphorylation. Ultrastructurally, the same two types of neurites are found in plaques from APP(SW)/Tau(VLW) and Alzheimer disease (AD) brains: (a) dystrophic giant neurites filled with degenerating organelles and/or phospho-tau-positive filaments and (b) non-dystrophic phospho-tau-positive small punctiform neurites. Both types of plaque-associated neurites are AT-8 positive in APP(SW)/Tau(VLW) mice and AD, but 12E8-positive dystrophic neurites are only detected in AD. We conclude that the simultaneous presence of human mutated Tau(VLW) and plaque-amyloid (and/or APP(SW)) potentiates and anticipates tau phosphorylation at the 12E8 epitope, intensifying pyramidal neuron immunostaining and tau filament formation in this double-transgenic model. Thus, the APP(SW)/Tau(VLW) mouse is a useful model to study neuritic plaques, since they reproduce most of the characteristics that these structures have in AD.