Tau蛋白在神经元细胞质和细胞核内的不同功能及其磷酸化对阿兹海默病的影响

Tau蛋白的功能,现有的研究结果已经比较清晰,涉及多种胞内异常,包括自噬、轴质运输、神经极性的紊乱,以及胞质Tau蛋白分泌到胞外所引起的一系列变化。Tau蛋白在细胞质和细胞核内具有不同功能,在胞质中以微管相关蛋白(microtubule-associated proteins,MAPs)形式承担运输、细胞支架和细胞运动等功能;在核内大量存在于核仁中,并作为端粒成分保护核内DNA;糖原合酶激酶-3(glycogen synthase kinase-3,GSK-3)、磷酸腺苷活化蛋白激酶(adenosine-monophosphate activated protein kinase,AMPK)、微管亲和调节蛋白激酶(microtubule-affinity regulating kinases,MARKs)和腺苷酸环化酶依赖的蛋白激酶(cyclic AMP-dependent protein kinase,PKA)对Tau蛋白异常磷酸化的影响,并由此讨论Tau蛋白在老年痴呆等神经退行性疾病中的可能作用。     

鉴定免疫细胞中DNA疫苗结合蛋白

为研究DNA疫苗从细胞质到细胞核的过程,对免疫细胞中DNA疫苗的结合蛋白进行初步鉴定。 提取小鼠脾脏免疫细胞的细胞质蛋白,将细胞质蛋白分别与 DNA 疫苗 pVAX-OVA 和空载体 pVAX 共孵育,孵育后首先由琼脂糖凝胶电泳分离与DNA结合的蛋白,然后通过SDS-PAGE进行分离和纯化,最后应用质谱技术分析其蛋白组分。质谱结果初步鉴定了免疫细胞中pVAX-OVA 结合的蛋白有IQ motif containing F4 等。免疫细胞中与 pVAX 结合的蛋白有Foxl2,SUV420H2 和 gamma actin 等。Foxl2具有核定位序列,DNA结合区域和与核转运蛋白相互作用的特点,可能对DNA疫苗的进入具有促进作用。这些蛋白质是否可以影响DNA疫苗有效进入细胞核进行基因表达需要进一步研究。     

运动与阿尔茨海默病：基于Tau蛋白翻译后修饰视角的研究述评

阿尔茨海默病（Alzheimer’s disease,AD）是一种与年龄有关的神经退行性疾病,严重危害老年人的身心健康,给社会带来巨大的经济压力。但目前其发病机制尚不完全明确,临床仍无根治的有效方法。Tau蛋白是一种微管相关蛋白质,能够参与维持微管相关结构稳定,具有可溶性且不会聚集。在AD病理状态下,病人脑内Tau蛋白结构和功能异常。异常的Tau蛋白聚集成不可溶的神经纤维缠结,损害微管运输能力,导致病人认知功能障碍。Tau蛋白结构和功能的改变是由多种翻译后修饰过程来调控的,即将特定的化学修饰基团与Tau蛋白N-端或C-端结合,直接改变蛋白质的性质和功能。AD病人脑内Tau蛋白的磷酸化、糖基化、乙酰化及SUMO化等多种翻译后修饰异常,与Tau蛋白的降解和毒性物质的聚集密切相关。本文综述近年来的研究后发现,运动可以通过改善Tau蛋白翻译后的某些异常修饰来预防和改善AD,主要作用方式如下：(1）运动可通过抑制GSK 3β和MAPK等蛋白激酶活性来抑制Tau蛋白的过度磷酸化,可能通过上调PP2A活性来促进Tau蛋白去磷酸化;(2）运动可通过提高GLUT1和GLUT3蛋白质水平,可能通过调节OGA和OGT活性平衡,提高蛋白质O-GlcNAc糖基化水平;(3）运动可能通过AMPK/mTORC1途径抑制p300以及激活SIRT1,降低Tau蛋白乙酰化水平;同时运动还可能通过抑制HDAC6,改善Tau蛋白KXGS基序异常乙酰化程度;(4）运动可能通过调节磷酸化与SUMO化共定位点,改善Tau蛋白异常SUMO化水平。     

综述:环境因素与Tau蛋白的异常磷酸化

神经纤维缠结是阿尔茨海默病(Alzheimer's disease,AD)的重要病理特征之一,其发生发展因素一直是相关研究领域的热点问题．神经纤维缠结由异常磷酸化的Tau蛋白错误折叠、聚集所形成,因此对诱发Tau蛋白异常磷酸化的因素进行探究显得尤为重要．Tau蛋白异常磷酸化逐渐形成神经缠结是AD早期发生的病理过程之一,本文就Tau蛋白异常磷酸化发生的环境影响因素进行了讨论．     

Tau蛋白过度磷酸化的致病机制及其在创伤性脑损伤中的作用

作为微管相关蛋白的一员,Tau蛋白结合、稳定微管及其过度磷酸化时参与神经退行性疾病的机制已被广泛研究。近年来许多研究发现Tau蛋白的异常过度磷酸化状态及其神经毒性作用是部分创伤性脑损伤患者远期认知功能障碍的重要致病机制之一。本文首先总结了对Tau蛋白生理功能及其致病机制的研究进展,并结合最新研究发现分析了Tau蛋白如何参与创伤性脑损伤后远期认知功能障碍。     

阿尔兹海默病中tau蛋白磷酸化调节

细胞内高度磷酸化tau蛋白形成的神经纤维缠结是阿尔兹海默病的主要病理特征之一。过度磷酸化的tau蛋白将引起细胞内微管的紊乱,从而造成神经元突触连接的丢失。Tau蛋白的磷酸化受到多种因素的影响,这些因素的失常将会导致tau蛋白的异常磷酸化。Tau蛋白的基本功能和结构、翻译后的主要修饰以及蛋白激酶和磷酸酯酶的调节,在阿尔兹海默病理以及预防治疗中发挥重要作用。     

甲醛诱导Tau蛋白形成“孔道样”聚集结构

尽管Lin等(University of California, Santa Barbara)就蛋白构象病中细胞死亡的机制提出了“非特异性淀粉样离子通道”(aspecific amyloid ion channels)学说,但到目前为止,尚未发现神经Tau蛋白能形成“孔道样”聚集结构,也未寻找到可以导致蛋白质形成“孔道样”聚集结构的诱导剂．依据本实验室提出的“散发性老年痴呆发生发展中的内源性甲醛慢性损伤”假说,采用一定浓度的甲醛与Tau蛋白进行温育,观察到甲醛可以明显诱导Tau蛋白分子聚集并形成淀粉样沉积物,同时也观察到了Tau蛋白“孔道样”聚集结构．上述结果为探索甲醛诱导Tau蛋白错误折叠形成的产物导致细胞代谢障碍和死亡的机制提供了新的研究思路．     

抑制褪黑素生物合成对大鼠Tau蛋白磷酸化的影响

目的 研究抑制褪黑素的生物合成对大鼠海马Tau蛋白磷酸化的影响。方法 侧脑室注射氟哌啶醇并腹腔注射加强,利用免疫组化检测大鼠海马区域Tau蛋白磷酸化情况;HPLC检测血清中褪黑素水平。结果 模型组大鼠海马Tau蛋白在Ser199/Ser202和Ser396/Ser404位点均发生异常过度磷酸化,褪黑素治疗组较模型组的磷酸化程度轻。结论 褪黑素水平的降低可能与AD样Tau蛋白异常过度磷酸化相关,外源性补充褪黑素可以减轻Tau蛋白的异常过度磷酸化。     

DEK蛋白与细胞凋亡的研究进展

DEK蛋白是一种广泛存在于细胞核内的可磷酸化的核蛋白.研究认为DEK蛋白与DNA的特异性结合能够改变与其结合的DNA的拓扑结构,进而影响基因的活性.细胞应激反应与DEK蛋白有潜在的关系,在死亡受体介导的细胞凋亡过程中,DEK蛋白的磷酸化状态发生了变化.DEK蛋白的磷酸化、多聚腺苷化均能促使DEK蛋白从染色质上释放下来并最终到达细胞外成为导致某些自身免疫性疾病的抗原.近来研究显示,DEK蛋白与细胞凋亡密切相关,DEK蛋白的过表达对细胞凋亡的作用具有双重性,即促进或抑制细胞凋亡.     

综述:Tau蛋白过度磷酸化机制及其在阿尔茨海默病神经元变性中的作用

Tau蛋白是神经元中含量最高的微管相关蛋白,其经典生物学功能是促进微管组装和维持微管的稳定性．在阿尔茨海默病(Alzheimer's disease,AD)患者,异常过度磷酸化的Tau蛋白以配对螺旋丝结构形成神经原纤维缠结并在神经元内聚积．大量研究提示,Tau蛋白异常在AD患者神经变性和学习记忆障碍的发生发展中起重要作用．本课题组对Tau蛋白异常磷酸化的机制及其对细胞的影响进行了系列研究,发现Tau蛋白表达和磷酸化具有调节细胞生存命运的新功能,并由此对AD神经细胞变性的本质提出了新见解．本文主要综述作者实验室有关Tau蛋白的部分研究结果．     

Thermodynamics of the Interaction between Alzheimer's Disease Related Tau Protein and DNA

Tau hyperphosphorylation can be considered as one of the hallmarks of Alzheimer''s disease and other tauophaties. Besides its well-known role as a microtubule associated protein, Tau displays a key function as a protector of genomic integrity in stress situations. Phosphorylation has been proven to regulate multiple processes including nuclear translocation of Tau. In this contribution, we are addressing the physicochemical nature of DNA-Tau interaction including the plausible influence of phosphorylation. By means of surface plasmon resonance (SPR) we measured the equilibrium constant and the free energy, enthalpy and entropy changes associated to the Tau-DNA complex formation. Our results show that unphosphorylated Tau binding to DNA is reversible. This fact is in agreement with the protective role attributed to nuclear Tau, which stops binding to DNA once the insult is over. According to our thermodynamic data, oscillations in the concentration of dephosphorylated Tau available to DNA must be the variable determining the extent of Tau binding and DNA protection. In addition, thermodynamics of the interaction suggest that hydrophobicity must represent an important contribution to the stability of the Tau-DNA complex. SPR results together with those from Tau expression in HEK cells show that phosphorylation induces changes in Tau protein which prevent it from binding to DNA. The phosphorylation-dependent regulation of DNA binding is analogous to the Tau-microtubules binding inhibition induced by phosphorylation. Our results suggest that hydrophobicity may control Tau location and DNA interaction and that impairment of this Tau-DNA interaction, due to Tau hyperphosphorylation, could contribute to Alzheimer''s pathogenesis.     

Parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and alpha-synuclein mutations promote Tau protein phosphorylation at Ser262 and destabilize microtubule cytoskeleton in vitro

In Parkinson disease (PD) brain, a progressive loss of dopaminergic neurons leads to dopamine depletion in the striatum and reduced motor function. Lewy bodies, the characteristic neuropathological lesions found in the brain of PD patients, are composed mainly of α-synuclein protein. Three point mutations in the α-synuclein gene are associated with familial PD. In addition, genome-wide association studies indicate that α-synuclein and Tau protein synergistically increase disease susceptibility in the human population. To determine the mechanism by which α-synuclein and Tau act together, we have used PD-causing neurotoxin MPTP and pathogenic α-synuclein mutants A30P, E46K, and A53T as models. We found that exposure of human neuroblastoma M17 cells to MPTP enhances the intracellular α-synuclein protein level, stimulates Tau protein phosphorylation at Ser(262), and induces apoptosis. In mouse brain, ablation of α-synuclein function significantly suppresses Tau phosphorylation at Ser(262). In vitro, α-synuclein binds to phosphorylated Ser(214) of Tau and stimulates PKA-catalyzed Tau phosphorylation at Ser(262). PD-associated α-synuclein mutations increase α-synuclein binding to Tau and stimulate Tau phosphorylation at Ser(262). In HEK-293 cells, α-synuclein and its all PD-associated mutants destabilize the microtubule cytoskeleton in a similar extent. In contrast, when co-expressed with Tau, these PD-associated mutants destabilize microtubules with significantly higher potency than WT. Our results demonstrate that α-synuclein is an in vivo regulator of Tau protein phosphorylation at Ser(262) and suggest that PD-associated risk factors such as environmental toxins and α-synuclein mutations promote Tau phosphorylation at Ser(262), causing microtubule instability, which leads to loss of dopaminergic neurons in PD brain.     

Phosphorylation of Tau at Thr212, Thr231, and Ser262 Combined Causes Neurodegeneration

Abnormal hyperphosphorylation of the microtubule-associated protein Tau is a hallmark of Alzheimer disease and related diseases called tauopathies. As yet, the exact mechanism by which this pathology causes neurodegeneration is not understood. The present study provides direct evidence that Tau abnormal hyperphosphorylation causes its aggregation, breakdown of the microtubule network, and cell death and identifies phosphorylation sites involved in neurotoxicity. We generated pseudophosphorylated Tau proteins by mutating Ser/Thr to Glu and, as controls, to Ala. These mutations involved one, two, or three pathological phosphorylation sites by site-directed mutagenesis using as backbones the wild type or FTDP-17 mutant R406W Tau. Pseudophosphorylated and corresponding control Tau proteins were expressed transiently in PC12 and CHO cells. We found that a single phosphorylation site alone had little influence on the biological activity of Tau, except Thr²¹², which, upon mutation to Glu in the R406W background, induced Tau aggregation in cells, suggesting phosphorylation at this site along with a modification on the C-terminal of the protein facilitates self-assembly of Tau. The expression of R406W Tau pseudophosphorylated at Thr²¹², Thr²³¹, and Ser²⁶² triggered caspase-3 activation in as much as 85% of the transfected cells, whereas the corresponding value for wild type pseudophosphorylated Tau was 30%. Cells transfected with pseudophosphorylated Tau became TUNEL-positive.     

The binding and phosphorylation of Thr231 is critical for Tau's hyperphosphorylation and functional regulation by glycogen synthase kinase 3beta

Neuropathological hallmarks of Alzheimer's disease are extracellular senile plaques and intracellular neurofibrillary lesions. The neurofibrillary lesions mainly consist of the hyperphosphorylated microtubule-associated protein Tau predominantly expressed in the axon of CNS neurons. Hyperphosphorylation of Tau negatively affects its binding to tubulin and decreases the capacity to promote microtubule assembly. Among a number of proline-directed kinases capable of phosphorylating paired helical filament-Tau, glycogen synthase kinase 3beta (GSK3beta) was first identified as a Tau protein kinase I and has been demonstrated to phosphorylate Tau both in vivo and in vitro. However, the phosphorylation mechanism of Tau by GSK3beta remained unclear. In this study, we show that the T231 is the primary phosphorylation site for GSK3beta and the Tau227-237 (AVVRTPPKSPS) derived from Tau containing T231P232 motif is identified as the GSK3beta binding site with high affinity of a Kd value 0.82 +/- 0.16 mumol/L. Our results suggest that direct binding and phosphorylation of T231P232 motif by GSK3beta induces conformational change of Tau and consequentially alters the inhibitory activity of its N-terminus that allows the phosphorylation of C-terminus of Tau by GSK3beta. Furthermore, hyperphosphorylation reduces Tau's ability to promote tubulin assembly and to form bundles in N18 cells. T231A mutant completely abolishes Tau phosphorylation by GSK3beta and retains the ability to promote tubulin polymerization and bundle formation. Taken together, these results suggest that phosphorylation of T231 by GSK3beta may play an important role in Tau's hyperphosphorylation and functional regulation.     

Down-regulation of WW domain-containing oxidoreductase induces Tau phosphorylation in vitro. A potential role in Alzheimer's disease

Numerous enzymes hyperphosphorylate Tau in vivo, leading to the formation of neurofibrillary tangles (NFTs) in the neurons of Alzheimer's disease (AD). Compared with age-matched normal controls, we demonstrated here that the protein levels of WW domain-containing oxidoreductase WOX1 (also known as WWOX or FOR), its Tyr33-phosphorylated form, and WOX2 were significantly down-regulated in the neurons of AD hippocampi. Remarkably knock-down of WOX1 expression by small interfering RNA in neuroblastoma SK-N-SH cells spontaneously induced Tau phosphorylation at Thr212/Thr231 and Ser515/Ser516, enhanced phosphorylation of glycogen synthase kinase 3beta (GSK-3beta) and ERK, and enhanced NFT formation. Also an increased binding of phospho-GSK-3beta with phospho-Tau was observed in these WOX1 knock-down cells. In comparison, increased phosphorylation of Tau, GSK-3beta, and ERK, as well as NFT formation, was observed in the AD hippocampi. Activation of JNK1 by anisomycin further increased Tau phosphorylation, and SP600125 (a JNK inhibitor) and PD-98059 (an MEK1/2 inhibitor) blocked Tau phosphorylation and NFT formation in these WOX1 knock-down cells. Ectopic or endogenous WOX1 colocalized with Tau, JNK1, and GSK-3beta in neurons and cultured cells. 17Beta-estradiol, a neuronal protective hormone, increased the binding of WOX1 and GSK-3beta with Tau. Mapping analysis showed that WOX1 bound Tau via its COOH-terminal short-chain alcohol dehydrogenase/reductase domain. Together WOX1 binds Tau via its short-chain alcohol dehydrogenase/reductase domain and is likely to play a critical role in regulating Tau hyperphosphorylation and NFT formation in vivo.     

Cul5 mediates taurine-stimulated mTOR mRNA expression and proliferation of mouse mammary epithelial cells

Cullin5 (Cul5) protein can regulate multiple signaling pathways; however, it is still largely unknown the role and molecule mechanism of Cul5 in regulation of the mTOR signaling. In this study, we determined the effect of Cul5 on the proliferation of HC11 cells, a mouse mammary epithelial cell line, and explored the corresponding molecular mechanism. We found that Cul5 was highly expressed in mammary gland tissues in the lactation stage compared with that in puberty and involution. Using gene knockdown and activation methods, we showed that Cul5 promoted proliferation of HC11 cells, mRNA expression and protein phosphorylation of mTOR. Taurine (Tau) affected Cul5 mRNA and protein levels in a dose-dependent manner. Cul5 localized to the nucleus and knockdown of Cul5 almost totally blocked the stimulation of Tau on mTOR mRNA expression and protein phosphorylation. PI3K inhibition almost totally abolished the stimulation of Tau on Cul5 expression. In summary, our data uncover that Cul5 is a positive regulator of proliferation of HC11 cells, and mediates the stimulation of Tau on mRNA expression and subsequent protein phosphorylation of mTOR. Our data lay a new theoretical foundation for regulating mammary cell proliferation and promoting milk yield.

     

Nuclear tau, a key player in neuronal DNA protection

Tau, a neuronal protein involved in neurodegenerative disorders such as Alzheimer disease, which is primarily described as a microtubule-associated protein, has also been observed in the nuclei of neuronal and non-neuronal cells. However, the function of the nuclear form of Tau in neurons has not yet been elucidated. In this work, we demonstrate that acute oxidative stress and mild heat stress (HS) induce the accumulation of dephosphorylated Tau in neuronal nuclei. Using chromatin immunoprecipitation assays, we demonstrate that the capacity of endogenous Tau to interact with neuronal DNA increased following HS. Comet assays performed on both wild-type and Tau-deficient neuronal cultures showed that Tau fully protected neuronal genomic DNA against HS-induced damage. Interestingly, HS-induced DNA damage observed in Tau-deficient cells was completely rescued after the overexpression of human Tau targeted to the nucleus. These results highlight a novel role for nuclear Tau as a key player in early stress response.     

γ-Aminobutyric acid type A (GABAA) receptor activation modulates tau phosphorylation

Abnormal phosphorylation and aggregation of the microtubule-associated protein Tau are hallmarks of various neurodegenerative diseases, such as Alzheimer disease. Molecular mechanisms that regulate Tau phosphorylation are complex and currently incompletely understood. We have developed a novel live cell reporter system based on protein-fragment complementation assay to study dynamic changes in Tau phosphorylation status. In this assay, fusion proteins of Tau and Pin1 (peptidyl-prolyl cis-trans-isomerase 1) carrying complementary fragments of a luciferase protein serve as a sensor of altered protein-protein interaction between Tau and Pin1, a critical regulator of Tau dephosphorylation at several disease-associated proline-directed phosphorylation sites. Using this system, we identified several structurally distinct GABA(A) receptor modulators as novel regulators of Tau phosphorylation in a chemical library screen. GABA(A) receptor activation promoted specific phosphorylation of Tau at the AT8 epitope (Ser-199/Ser-202/Thr-205) in cultures of mature cortical neurons. Increased Tau phosphorylation by GABA(A) receptor activity was associated with reduced Tau binding to protein phosphatase 2A and was dependent on Cdk5 but not GSK3β kinase activity.     

The role of tau phosphorylation in transfected COS-1 cells

Tau cDNAs from each of the six human isoforms were transfected into COS- 1 cells and, in every case, more than one peptide was observed. The diversity of expressed isoforms was due to different levels of tau phosphorylation. Tau phosphorylation results in a decrease of the protein electrophoretic mobility. The major contribution to this mobility shift is due to the phosphorylation at the at the C-terminus of the molecule, as inferred from the expression of tau fragments. Phosphorylation takes place in some of the sites modified in neural cells and in the basis of AD patients. Copolymerization studies indicate that the level of phosphorylation, as well as the localization of the modified residues, may affect the binding of the protein to microtubules. These results indicate that phosphorylation regulates tau function inside the cell.     

Oligomer Formation of Tau Protein Hyperphosphorylated in Cells

Abnormal phosphorylation (“hyperphosphorylation”) and aggregation of Tau protein are hallmarks of Alzheimer disease and other tauopathies, but their causative connection is still a matter of debate. Tau with Alzheimer-like phosphorylation is also present in hibernating animals, mitosis, or during embryonic development, without leading to pathophysiology or neurodegeneration. Thus, the role of phosphorylation and the distinction between physiological and pathological phosphorylation needs to be further refined. So far, the systematic investigation of highly phosphorylated Tau was difficult because a reliable method of preparing reproducible quantities was not available. Here, we generated full-length Tau (2N4R) in Sf9 cells in a well defined phosphorylation state containing up to ∼20 phosphates as judged by mass spectrometry and Western blotting with phospho-specific antibodies. Despite the high concentration in living Sf9 cells (estimated ∼230 μm) and high phosphorylation, the protein was not aggregated. However, after purification, the highly phosphorylated protein readily formed oligomers, whereas fibrils were observed only rarely. Exposure of mature primary neuronal cultures to oligomeric phospho-Tau caused reduction of spine density on dendrites but did not change the overall cell viability.