Tau蛋白基因突变与神经退行性疾病

Ｔａｕ蛋白是神经细胞中含量最高的微管相关蛋白,其正常功能是促进微管蛋白（ｔｕｂｕｌｉｎ）组装成微管（ｍｉｃｒｏｔｕｂｕｌｅ）,并维持已形成微管的稳定性。Ｔａｕ蛋白的翻译后异常修饰与阿尔茨海默病（Ａｌｚｈｅｉｍｅｒｄｉｓｅａｓｅ,ＡＤ）的神经原纤维退化有关［１］。本文综述最近有关Ｔａｕ蛋白基因突变,ＴａｕｍＲＮＡ剪接改变导致Ｔａｕ蛋白组成、结构和功能异常的机制,及其与几种神经退行性疾病的关系的研究。１．Ｔａｕ蛋白基因结构及其表达产物Ｔａｕ蛋白基因位于１７号染色体（１７ｐ２１．１１）,由１７个外显子…     

β淀粉样蛋白与Alzheimer病

Ａｌｚｈｅｉｍｅｒ病是当今颇受人们关注的疾病,近年来对其发病的分子机制俐较为深入。目前的研究热点集中在β淀粉样蛋白ｔａｕ蛋白和ＡＤ的发病关系上。本文就β淀粉样蛋白的来淅,其前体蛋白的基因突变,代谢加工途径,神经毒性作用以及转基因动物诸方面与ＡＤ的发病关系作一综述。     

淀粉样前体蛋白和Alzheimer‘s病

淀粉样前体蛋白（ＡＰＰ）与Ａｌｚｈｅｉｍｅｒ＇ｓ病（ＡＤ）的发病机制关系密切,本文综述了近年来对ＡＰＰ的分子研究,包括ＡＰＰ的分子特性,生理功能和代谢过程,ＡＰＰ与β－淀粉样蛋白（β／Ａ４）的关系,ＡＰＰ异常代谢及其与ＡＤ的关联等。     

HeLa、HEK293、SH-SY5Y细胞中的Tau蛋白

通过间接免疫荧光测定了HeLa、HEK-293、SH-SY5Y细胞内Tau蛋白的分布,观察到在细胞间期单克隆抗体Tau-1的荧光信号分布于细胞质和胞核中．特别是HeLa细胞,其胞核内具有相对较高的Tau蛋白免疫荧光信号．通过分离SH-SY5Y的细胞核,更为清楚地显示了Tau蛋白在细胞核中的分布,并且免疫荧光信号与DNA的Hoechst33258染色信号相重合．Western blotting的测定结果进一步证明了SH-SY5Y细胞的胞质和胞核中均含有Tau蛋白的不同异构体．以上结果提示,Tau蛋白不仅存在于神经、肌肉等细胞内,也存在于肿瘤细胞系,并且分布于间期的胞核中．     

大脑皮质神经干细胞定向分化为神经元过程中Tau蛋白表达的研究

目的探讨Tau蛋白在新生大鼠大脑皮质神经干细胞定向分化为神经元过程中的表达及意义.方法采用细胞培养、免疫细胞化学方法(SABC法)观察及计算机图像分析技术测定Tau蛋白在神经干细胞定向分化为神经元过程中不同时段的表达情况.结果在神经干细胞定向分化为神经元的过程中,Tau蛋白由核周淡染分布渐至在胞体与突起中密集均匀分布,随着突起伸展而不断地延伸,并且Tau蛋白的表达量逐渐增加.结论新生大鼠大脑皮质神经干细胞定向分化为神经元过程中,Tau蛋白的时空表达与神经干细胞定向分化的神经元形态变化有一定的相关性并在此过程中发挥着重要的作用.     

副粘病毒血凝素-神经氨酸酶蛋白结构与功能的研究进展

副粘病毒的血凝素-神经氨酸酶和融合蛋白具有重要的生物学活性,其中前者具有受体识别活性、神经氨酸酶活性和促进融合蛋白的细胞融合作用.本文对近年来血凝素-神经氨酸酶结构和功能方面的研究进展进行了综述.     

Tau蛋白高度磷酸化致阿尔茨海默病动物模型研究进展

阿尔茨海默病（Alzheimer＇sdisease,AD）是老年人中最常见的神经退行性疾病,以过度磷酸化tau蛋白为核心形成的神经原纤维缠结为AD的主要病理特征之一。近年来对tau蛋白磷酸化的研究备受关注。在AD的实验研究中,探索理想的AD动物模型对于明确AD的病因、发病机制及药物的研发等方面起关键作用。本文对Tau蛋白磷酸化致AD主要动物模型的研究进展进行了综述,包括Tau转基因动物模型、激酶和磷酸化酶系统失衡致Tau蛋白过度磷酸化损伤模型、降低Tau蛋白糖基化致Tau过度磷酸化模型等。     

SmpB蛋白结构与功能研究进展

SmpB是一类普遍存在于细菌中的小RNA结合蛋白。研究表明SmpB除了在反式翻译中起着辅助tmRNA分子拯救滞留核糖体的作用,其也可以作为RNA分子伴侣调节体内RpoS的表达,以及具有直接调控RNase R及双组份系统的功能。SmpB参与的调控作用对于细菌蛋白质合成质量控制、致病菌中毒力系统调控、维持机体正常生长及发育等过程具有关键作用。本综述主要从SmpB蛋白结构及其对RNA、蛋白质调控功能等方面进行论述,以期对发掘细菌性疾病治疗靶点,研发新型抗生素,提供新的方向和思路。     

Tau蛋白在阿尔茨海默病中的作用

阿尔茨海默病(AD)是非常普遍的神经变性性疾病并且是老年人痴呆的主要原因。AD患者的症状特点包括进行性的认知障碍、记忆丧失和行为障碍,与大脑中的病理变化密切相关。AD现成为全球最严重的健康和社会经济问题。在AD患者脑中神经纤维网或神经营养障碍的过程中存在tau蛋白的异常。tau蛋白丧失其促微管组装的生物学功能,导致细胞骨架的破坏、丝状物形成和神经缠结,轴突运输损害,进而导致突触蛋白失去功能和神经退行性病变。其数量和结构的改变将会影响其功能而且会出现异常聚集。调节Tau蛋白的异常聚集的分子机制主要是一些翻译后修饰使其结构及构象发生变化。因此,异常磷酸化和截断的tau蛋白作为tau蛋白病理过程的关键机制而引起学者关注。本文描述了tau蛋白的结构和功能及其在AD中的主要病理变化,同时在本文中还涉及到磷酸化的tau蛋白是神经元对氧化应激的代偿反应这一观点。对tau蛋白进行更加全面的解读。     

Characterization of In Vitro Glycation Sites of Tau

Abstract: Tau is a microtubule-associated protein that loses microtubule binding activity and aggregates into paired helical filaments (PHFs) in Alzheimer's disease. Nonenzymic glycation is one of the posttranslational modifications detected in PHF-tau, but not in normal tau. PHF-tau has reduced ability to bind to microtubules. To determine whether glycation of tau occurs in its microtubule binding domains, we have characterized in vitro glycation sites of the longest isoform of tau, which has four microtubule binding domains (Tau-4). The identified glycation sites are Lys-87, 132, 150, 163, 174, 225, 234, 259, 280, 281, 347, 353, and 369. We have also studied glycation of another isoform of tau, which has only three microtubule binding domains (Tau-3). This isoform is modified by glucose 15–20% more slowly than Tau-4. However, the glycation sites appear to be the same in both isoforms, except for Lys-280 and 281; these are located in the second microtubule binding domain, which is missing in Tau-3. Lys-150, 163, and 174 are located within or proximal to the sequence of tau that is involved in the microtubule nucleation activity, and Lys-259, 280, 281, 347, 353, and 369 are located in the microtubule binding domains. Glycation at these sites can affect the functional properties of tau, and advanced glycation at these sites might lead to the formation of insoluble aggregates similar to the ones seen in Alzheimer's disease.     

Degradation of Tau by Lysosomal Enzyme Cathepsin D: Implication for Alzheimer Neurofibrillary Degeneration

Abstract: The degradation of different isoforms of human recombinant tau (R-tau; T39, T40, and T44) and fetal tau (F-tau) by cathepsin D (CD) was investigated. Gel electrophoresis and Coomassie Blue staining of different R-tau species digested at pH 3.5 showed very little differences in CD susceptibility. Immunoblotting analyses revealed that amino and carboxy termini of tau were cleaved before other regions. F-tau was most vulnerable to proteolysis at both termini. Digestion of R-tau with 0.01 unit of CD/ml at pH 3.5 resulted in cleavage between Phe⁸-Glu⁹, Met⁴¹⁹-Val⁴²⁰, Thr⁴²⁷-Leu⁴²⁸-Ala⁴²⁹, and Leu⁴³⁶-Ala⁴³⁷ as determined by amino acid sequencing and mass spectroscopy (numbering of amino acids was based on T40). With higher concentrations of CD (1 unit/ml), additional sites of digestion were detected between amino acids 34–161, 200–257, and 267–358. The cleavage sites at amino acids 34–161 and 267–358 were observed at pH 3.5, whereas that at amino acids 200–257 was detected at pH 7.0. Our results suggest that CD cleavage of tau could generate tau fragments with intact microtubule binding domains, which could have a role in the pathogenesis of paired helical filaments (PHFs) in Alzheimer's disease. Such proteolysis might also contribute to the changes of PHF phenotype observed in intracellular and extracellular tangles.     

Amyloidogenesis of Tau protein

The role of microtubule‐associated protein Tau in neurodegeneration has been extensively investigated since the discovery of Tau amyloid aggregates in the brains of patients with Alzheimer's disease (AD). The process of formation of amyloid fibrils is known as amyloidogenesis and attracts much attention as a potential target in the prevention and treatment of neurodegenerative conditions linked to protein aggregation. Cerebral deposition of amyloid aggregates of Tau is observed not only in AD but also in numerous other tauopathies and prion diseases. Amyloidogenesis of intrinsically unstructured monomers of Tau can be triggered by mutations in the Tau gene, post‐translational modifications, or interactions with polyanionic molecules and aggregation‐prone proteins/peptides. The self‐assembly of amyloid fibrils of Tau shares a number of characteristic features with amyloidogenesis of other proteins involved in neurodegenerative diseases. For example, in vitro experiments have demonstrated that the nucleation phase, which is the rate‐limiting stage of Tau amyloidogenesis, is shortened in the presence of fragmented preformed Tau fibrils acting as aggregation templates (“seeds”). Accordingly, Tau aggregates released by tauopathy‐affected neurons can spread the neurodegenerative process in the brain through a prion‐like mechanism, originally described for the pathogenic form of prion protein. Moreover, Tau has been shown to form amyloid strains—structurally diverse self‐propagating aggregates of potentially various pathological effects, resembling in this respect prion strains. Here, we review the current literature on Tau aggregation and discuss mechanisms of propagation of Tau amyloid in the light of the prion‐like paradigm.     

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．     

Abnormal Tau phosphorylation of the Alzheimer-type also occurs during mitosis

In Alzheimer's disease, neurofibrillary degeneration results from the aggregation of abnormally phosphorylated Tau proteins into filaments and it may be related to the reactivation of mitotic mechanisms. In order to investigate the link between Tau phosphorylation and mitosis, Xenopus laevis oocytes in which most of the M-phase regulators have been discovered were used as a cell model. The human Tau isoform htau412 (2+3-10+) was microinjected into prophase I oocytes that were then stimulated by progesterone that activate cyclin-dependent kinase pathways. Hyperphosphorylation of the Tau isoform, which is characterized by a decrease of its electrophoretic mobility and its labelling by a number of phosphorylation-dependent antibodies, was observed at the time of germinal vesicle breakdown. Surprisingly, Tau immunoreactivity, considered as typical of Alzheimer's pathology (AT100 and phospho-Ser422), was observed in meiosis II. Because meiosis II is considered as a mitosis-like phase, we investigated if our observation was also relevant to a neurone-like model. Abnormal Tau phosphorylation was detected in mitotic human neuroblastoma SY5Y cells overexpressing Tau. Regarding AT100-immunoreactivity and phospho-Ser422, we suggest that phosphatase 2A inhibition and a phosphorylation combination of mitotic kinases may lead to this Alzheimer-type phosphorylation. Our results not only demonstrate the involvement of mitotic kinases in Alzheimer-type Tau phosphorylation but also indicate that Xenopus oocyte could be a useful model to identify the kinases involved in this process.     

人参皂甙Rb1减轻冈田酸诱导的大鼠海马神经元Tau蛋白过度磷酸化

为研究人参皂甙Rb1(ginsenoside Rb1)对冈田酸(okadaic acid,OA)诱导的大鼠海马神经元Tau蛋白过度磷酸化的影响及其可能机制,实验随机分为正常组、溶媒对照组、OA模型组和Rb1预处理组。正常组不作任何处理;Rb1预处理组大鼠分别用5、10、20 mg/kg的Rb1预处理,每天一次,共14 d,于第13天向海马背侧注射1.5μl OA[0.483 μl,溶于10% 二甲基亚砜(dimethysulphoxide,DMSO)];OA模型组大鼠于第13天时海马背侧注射OA,溶媒对照组则注射等体积的生理盐水。各组均于第15天收取标本。通过Biescbowski’s染色、免疫组化和Western blot,分别观察大鼠海马神经元胞体和突起内神经原纤维的改变和磷酸化Tau蛋白的表达水平,同时检测蛋白磷酸酯酶2A(protein phosphatase-2A,PP2A)活性以探讨其作用机制。结果显示:(1)OA模型组与溶媒对照组及正常组比较,海马神经元胞体和突起着色较深,染色不均匀;神经元中Thr231和Sei396位点磷酸化的Tau蛋白和总Tau含量增多;PP2A活性则明显下降(P<0.01):(2)Rb1预处理组大鼠海马神经元胞体和突起染色均匀,神经原纤维走行规则;海马神经元中Thr231和Ser396位点磷酸化的Tau蛋白和总Tau 含量较OA模型组减少,而PP2A活性明显增高(P<0.01)。以上观察结果表明,人参皂甙Rb1可以减轻OA诱导的大鼠海马神经元Tau蛋白过度磷酸化,其机制可能与提高PP2A活性有关。     

Corticosterone Exacerbates Kainate-Induced Alterations in Hippocampal Tau Immunoreactivity and Spectrin Proteolysis In Vivo

Abstract: Aberrant elevations in intracellular calcium levels, promoted by the excitatory amino acid glutamate, may be a final common mediator of the neuronal damage that occurs in hypoxic-ischemic and seizure disorders. Glutamate and altered neuronal calcium homeostasis have also been proposed to play roles in more chronic neurodegenerative disorders, including Alzheimer's disease. Any extrinsic factors that may augment calcium levels during such disorders may significantly exacerbate the resulting damage. Glucocorticoids (GCs), the adrenal steroid hormones released during stress, may represent one such extrinsic factor. GCs can exacerbate hippocampal damage induced by excitotoxic seizures and hypoxia-ischemia, and we have observed recently that GCs elevate intracellular calcium levels in hippocampal neurons. We now report that the excitotoxin kainic acid (KA) can elicit antigenic changes in the microtubule-associated protein tau similar to those seen in the neurofibrillary tangles of Alzheimer's disease. KA induced a transient increase in the immunoreactivity of hippocampal CA3 neurons towards antibodies that recognize aberrant forms of tau (5E2 and Alz-50). The tau immunoreactivity appeared within 3h of KA injection, preceded extensive neuronal damage, and subsequently disappeared as neurons degenerated. KA also caused spectrin breakdown, indicating the involvement of calcium-dependent proteases. Physiological concentrations of corticosterone (the species-typical GC of rats) enhanced the neuronal damage induced by KA and, critically, enhanced the intensity of tau immunoreactivity and spectrin breakdown. Moreover, the GC enhancement of spectrin proteolysis was prevented by energy supplementation, supporting the hypothesis that GC disruption of calcium homeostasis in the hippocampus is energetic in nature. Taken together, these findings demonstrate that neurofibrillary tangle-like alterations in tau, and spectrin breakdown, can be induced by excitatory amino acids and exacerbated by GCs in vivo.     

Chaperone-Like Manner of Human Neuronal Tau Towards Lactate Dehydrogenase

In our experiments, inactivation of lactate dehydrogenase (LDH, EC1.1.1.27) in the presence of human microtubule-associated tau is observably suppressed during thermal and guanidine hydrochloride (GdnHCl) denaturation. Kinetic studies show tau can prevent LDH from self-aggregation monitored by light scattering during thermal denaturation. On the other hand, neuronal tau promotes reactivation of LDH and suppresses self-aggregation of non-native LDH when GdnHCl solution is diluted. Furthermore, the reactivation yield of LDH decreases significantly with delayed addition of tau. All experiments were completed in the reducing buffer with 1 mM DTT to avoid between tau and LDH forming the covalent bonds during unfolding and refolding. Thus, Tau prevents proteins from misfolding and aggregating into insoluble, nonfunctional inclusions and assists them to refold to reach the stable native state by binding to the exposed hydrophobic patches on proteins instead of by forming or breaking covalent bonds. Additionally, tau remarkably enhances reactivation of GDH (glutamic dehydrogenase, EC 1.4.1.3), another carbohydrate metabolic enzyme, also showing a chaperone-like manner. It suggests that neuronal tau non-specifically functions a chaperone-like protein towards the enzymes of carbohydrate metabolism.