磷酸酶和糖苷酶Alzheimer神经原纤维缠结的作用

目的 探讨Ａｌｚｈｅｉｍｅｒ病（ＡＤ）神经原纤维缠结形成的可能机制及逆转ＡＤ神经元变性的可能性和途径。方法 去磷酸化，去糖基化，负染电子显微 放免印迹图像定量分析技术。结果（１）磷酸酶－２Ａ、－２Ｂ的去磷酸化作用可使ＡＤ神经原纤维缠结中的Ⅱ型双螺旋丝（ＰＨＦⅡ）结构松解，释放游离ｔａｕ蛋白的量比对照组高２５％，去磷酸化可恢复ＰＨＦⅡ－ｔａｕ促微管组装的生物学功能；（２）糖苷酶泊去糖基化作用则使ＰＨ     

雌激素对神经瘤细胞磷酸化的保护作用

ｌｚｈｅｉｍｅｒ病（ＡＤ）脑组织中最明显的生化异常是微管相关蛋白ｔａｕ的异常过度磷酸化，异常磷酸化的ｔａｕ蛋白是双螺旋丝（ＰＨＦ）的主要成分，并以这种形式在神经元核周围形成神经原纤维缠结。在ＳＨＳＹ５Ｙ细胞系中，８０％的ｔａｕ蛋白在ｔａｕ１表位有...     

微管相关蛋白tau与Alzheimer病神经原纤维退变

Ａｌｚｈｅｉｍｅｒ病（ＡＤ）是成人痴呆症中最常见的一种。神经细胞内的神经原纤维缠结（ＮＦＴ）是其特征性脑损伤之一。ＮＦＴ的主要组成成分是异常过度磷酸化的微管相关蛋白ｔａｕ，这种ｔａｕ蛋白以聚集的双螺旋丝（ＰＨＦ）存在。越来越多的实验证明：ＮＦＴ引起的...     

Alzheimer病患者脑脊液中诊断性生化标志物

迄今,Ａｌｚｈｅｉｍｅｒ病（ＡＤ）的最终确诊仍需靠脑组织病理检查。脑脊液（ＣＳＦ）中的某些生化标志物的检测在一定程度上可以反映脑组织的变化。为此,我们对ＡＤ患者ＣＳＦ中ｔａｕ蛋白（ＣＳＦ－ｔａｕ）、淀粉样β蛋白（Ａβ）、载脂蛋白Ｅ（ａｐｏＥ）、天冬氨酸转氨酶（ＡＳＴ）和糖基化的乙酰胆碱酯酶作一综述。以探讨其对ＡＤ患者的诊断价值。１　ｔａｕ蛋白　　ＣＳＦ中ｔａｕ蛋白有两种形式：一种是正常的ｔａｕ蛋白（未磷酸化）,一种是异常磷酸化的ｔａｕ蛋白（ＰＨＦ－ｔａｕ）。ｔａｕ蛋白被称之为微管相关蛋白,高度磷…     

糖苷酶Alzheimer病神经原纤维缠结结构的影响

内切糖苷酶Ｆ／Ｎ－糖苷酶Ｆ可使Ａｌｚｈｅｉｍｅｒ病神经原纤维缠结选择性反糖基化，去糖基化作用使神经原纤维缠结的螺旋消失，在电子显微镜下呈直径为（２．５±０．５）ｎｍ的线性纤维丝结构，这些线性纤维丝常以束存在，单纯去糖基化作用并不引起ｔａｕ蛋白从缠结结构的释放明显增高，但去糖基化处理可明显增高蛋白磷酸酯酶从缠结结构中释放游离ｔａｕ蛋白。这些结果提示：ｔａｕ蛋白的糖基化作用与Ａｌｚｈｅｉｍｅｒ病患者病     

L-和S-型髓磷脂相关糖蛋白(MAG)在成年大鼠前髓帆的少突胶质细胞单位表型中的不同表达

本实验将雌性Ｗｉｓｔａｒ大鼠用４％的多聚甲醛灌注、固定,在脑干及小脑处分离得到前髓帆（ＡＭＶ）,用磷酸酐酶Ⅱ（ＣＡⅡ）免疫细胞化学染色及Ｒｉｐ免疫同位素双标记髓磷脂碱性蛋白（ＭＢＰ）,蛋白脂质蛋白（ＰＬＰ）,ＬＭＡＧ和ＳＭＡＧ,最后用荧光显微镜观察。结果显示：ＡＭＶ中少突胶质细胞可分为Ⅰ～Ⅳ型：Ⅰ／Ⅱ型小直径纤维表达ＬＭＡＧ＋／ＳＭＡＧ－／ＣＡＩＩ＋;Ⅱ／Ⅲ型表达ＬＭＡＧ＋／ＳＭＡＧ＋／ＣＡＩＩ＋;Ⅲ／Ⅳ型大直径纤维表达ＬＭＡＧ＋／ＳＭＡＧ＋／ＣＡＩＩ－。Ｒｉｐ和髓磷脂少突胶质细…     

急性脑梗死和Alzheimer病脑脊液高水平髓鞘素抗原B细胞免疫应答

目的确认急性脑梗死（ＡＣＩ）和Ａｌｚｈｅｉｍｅｒ病（ＡＤ）对髓鞘素碱性蛋白（ＭＢＰ）、髓鞘素结合糖蛋白（ＭＡＧ）和含脂质蛋白（ＰＬＰ）的Ｂ细胞免疫应答。方法采用酶联免疫斑点技术检测了ＡＣＩ、临床可能ＡＤ和其它神经疾病（ＯＮＤ）对照组患者外周血和脑脊液（ＣＳＦ）中ＭＢＰ、ＭＡＧ和ＰＬＰ抗体分泌细胞。结果ＡＣＩ、ＡＤ和ＯＮＤ患者外周血中均可检出ＩｇＧ、ＩｇＡ、ＩｇＭ三种表型ＭＢＰ、ＭＡＧ、ＰＬＰ抗体分泌细胞，无显著差异。但ＡＣＩ和ＡＤ患者ＣＳＦ中ＩｇＧ型ＭＢＰ、ＭＡＧ和ＰＬＰ抗体分泌细胞均呈显著性增高。结论ＡＣＩ急性脑缺血损伤和ＡＤ神经变性可能导致体内Ｂ细胞激活及ＣＮＳ内髓鞘素反应性Ｂ细胞免疫应答，其病理意义有待探讨。     

阿尔茨海默病患者脑液tau蛋白含量的检测

目的 探讨ＣＳＦ中ｔａｕ蛋白浓度对于诊断ＡＤ的意义，同时分析其与载旧白Ｅ（ＡＰＯＥ）的不同等位基因型之间的关系。方法 阿尔茨海默病（ＡＤ）组纳入４５例，多发性梗塞性痴呆（ＭＩＤ）组２９例，精神分裂症组３７例，其它神经系统疾病（ＯＮＤ）组２６例，正常脑脊液（ＣＳＦ）对照组（正常组）１２例。采用酶联免疫吸附分析法检测ｔａｕ蛋白浓度，采用聚合酶链式反应方法检测ＡＰＯＥ４ 基因型。结果 ＡＤ组ＣＳＦ中ｔａ     

脑卒中患者客观生活质量及其影响因素的研究

目的：探索脑卒中幸存者的客观生活质量及其影响因素。方法：采用横断面调查的方法对２０５例脑卒中幸存者及２１６例正常对照以ＦＡＩ、ＡＮＦＤ、ＳＤＳ及ＤＩＳ、ＳＡＳ、ＵＬＳ、ＳＳＲＳ、ＬＳＩＡ、ＬＳＩＢ、ＦＡＣＥＳＩＩ－ＣＶ、ＴＳＢＩＡ及ＴＳＢＩＢ、补充综合问卷等为工具进行客观生活质量及有关影响因素的研究。结果：发现脑卒中幸存者的ＦＡＩ明显低于对照人群,其主要影响因素依次为ＡＮＦＤ、ＳＳＲＳ、ＳＤＳ、Ｄ     

重症肌无力患者外周血乙酰胆碱受体特异性T细胞sIL-2R、IFN-γ、IL-4的表达和转录

目的了解ＭＧ患者的乙酰胆碱受体（ＡＣｈＲ）特异性细胞免疫应答。方法采用酶联免疫吸附试验（ＥＬＩＳＡ）检测３０例ＭＧ患者和２０名健康对照者经ＡＣｈＲ刺激后外周血单核细胞（ＰＢＭＣ），辅助性Ｔ细胞１（Ｔｈ１）相关的干扰素（ＩＦＮ）γ、辅助性Ｔ细胞２（Ｔｈ２）相关的白细胞介素（ＩＬ）４及与细胞免疫活化密切相关的可溶性白细胞介素２受体（ｓＩＬ２Ｒ）的分泌，用逆转录聚合酶链反应（ＲＴＰＣＲ）结合狭缝印迹杂交检测ＩＦＮγ、ＩＬ４的信息核糖核酸（ｍＲＮＡ）转录。结果总ＭＧ患者组ＩＦＮγ显著高于健康对照组，尤以急性ＭＧ患者组（１４例）更明显，且其升高与其ｍＲＮＡ转录水平一致。虽然这两组ＭＧ患者的ｓＩＬ２Ｒ亦明显高于健康对照组，但患者组的ＩＬ４表达及其ｍＲＮＡ转录与健康对照组相比差异无显著意义。结论ＭＧ患者有Ｔｈ１和Ｔｈ２的失衡，ＭＧ患者有ＡＣｈＲ特异性细胞免疫活化，Ｔｈ１的细胞因子ＩＦＮγ可能作为效应因子参与了ＭＧ的发病。     

PP2B isolated from human brain preferentially dephosphorylates Ser-262 and Ser-396 of the Alzheimer disease abnormally hyperphosphorylated tau

Summary. PP2B is one of the major serine/threonine phosphatases in the brain. We quantitated the dephosphorylation of various sites of Alzheimer disease abnormally hyperphosphorylated tau by PP2B purified from six (three Alzheimer and three control) autopsied human brains. The purified PP2B was essentially homogenous holoenzyme as determined by SDS-PAGE, Western blot analyses and biochemical characterization. Purified PP2B from all six brains efficiently dephosphorylated ³²P-tau with specific activities ranging from 684–1286 pmol ³²Pi/mg/min. Estimated by dot-blot analyses, the purified PP2B (on average from six brains) dephosphorylated Alzheimer tau at pS199, pT217, pS262, pS396 and pS422 by 38%, 32%, 63%, 78%, and 32%, respectively. Dephosphorylation of tau at pT181, pS202, pT205, pT212, pS214, and pS404 by PP2B was undetectable. The preferential dephosphorylation of Ser262 and Ser396 by PP2B suggests a possible involvement of this phosphatase in Alzheimer neurofibrillary degeneration.     

Contributions of protein phosphatases PP1, PP2A, PP2B and PP5 to the regulation of tau phosphorylation

Abnormal hyperphosphorylation of tau is believed to lead to neurofibrillary degeneration in Alzheimer's disease (AD) and other tauopathies. Recent studies have shown that protein phosphatases (PPs) PP1, PP2A, PP2B and PP5 dephosphorylate tau in vitro, but the exact role of each of these phosphatases in the regulation of site-specific phosphorylation of tau in the human brain was unknown. Hence, we investigated the contributions of these PPs to the regulation of tau phosphorylation quantitatively. We found that these four phosphatases all dephosphorylated tau at Ser199, Ser202, Thr205, Thr212, Ser214, Ser235, Ser262, Ser396, Ser404 and Ser409, but with different efficiencies toward different sites. The K(m) values of tau dephosphorylation catalysed by PP1, PP2A and PP5 were 8-12 microm, similar to the intraneuronal tau concentration of human brain, whereas the K(m) of PP2B was fivefold higher. PP2A, PP1, PP5 and PP2B accounted for approximately 71%, approximately 11%, approximately 10% and approximately 7%, respectively, of the total tau phosphatase activity of human brain. The total phosphatase activity and the activities of PP2A and PP5 toward tau were significantly decreased, whereas that of PP2B was increased in AD brain. PP2A activity negatively correlated to the level of tau phosphorylation at the most phosphorylation sites in human brains. Our findings indicate that PP2A is the major tau phosphatase that regulates its phosphorylation at multiple sites in human brain. The abnormal hyperphosphorylation of tau is partially due to a downregulation of PP2A activity in AD brain.     

Pharmacological targets to inhibit Alzheimer neurofibrillary degeneration

Neurofibrillary degeneration appears to be required for the clinical expression of Alzheimer disease (AD) and related tauopathies. Given the polyetiology of these diseases and the pivotal involvement of neurofibrillary degeneration in their pathogenesis, inhibition of this lesion offers a promising therapeutic target. Studies from our laboratories have shown that there is a protein phosphorylation/dephosphorylation imbalance and that the microtubule associated protein tau is abnormally hyperphosphorylated in the brain of patients with AD and in this form it is the major protein subunit of paired helical filaments/neurofibrillary tangles (PHF/NFT). The abnormal tau which is polymerized into PHF/NFT neither promotes or inhibits in vitro microtubule assembly. In contrast the cytosolic abnormally hyperphosphorylated tau from AD brain, the AD P-tau neither associates with tubulin nor promotes in vitro microtubule assembly but instead it sequesters normal tau, MAP1 and MAP2 and inhibits microtubule assembly. The AD P-tau readily self-assembles in vitro into tangles of PHF/straight filaments under physiological conditions of protein concentration, pH, ionic strength and reducing conditions and this self assembly requires the abnormal hyperphosphorylation of this protein. The activity of phosphoseryl/phosphothreonyl protein phosphatase (PP)-2A which regulates the phosphorylation of tau, is compromised in AD brain. Thus, modulation of the activities of protein phosphatase-2A and tau kinases and inhibition of the sequestration of normal MAPs by AD P-tau offer promising therapeutic opportunities to inhibit neurofibrillary degeneration and the diseases characterized by this lesion.     

Alzheimer neurofibrillary degeneration

Neurofibrillary degeneration has primary and pivotal involvement in the pathogenesis of Alzheimer disease (AD) and other tauopathies. The inhibition of this lesion offers a promising therapeutic approach. The microtubule- associated protein (MAP) tau is abnormally hyperphosphorylated in the brain of patients with AD, and in this form it is the major protein subunit of paired helical filaments/neurofibrillary tangles (PHF/NFT). The abnormal tau that is polymerized into PHF/NFT is apparently inert and has no effect on microtubule assembly in vitro. The cytosolic abnormally hyperphosphorylated tau from AD brain, the AD P-tau, does not promote in vitro microtubule assembly but, instead, sequesters normal tau, MAP1, and MAP2 and inhibits microtubule assembly. The AD P-tau readily self-assembles in vitro into tangles of PHF/straight filaments, and this self-assembly requires the abnormal hyperphosphorylation of this protein. Although, to date, an up-regulation of the activity of a tau kinase has not been established, the activity of phosphoseryl/ phosphothreonyl protein phosphatase (PP)-2A, which regulates the phosphorylation of tau, is compromised in AD brain. Thus, modulation of the activities of pp-2A and one or more tau kinases and inhibition of the sequestration of normal MAPs by AD P-tau offer promising therapeutic opportunities to inhibit neurofibrillary degeneration and the diseases characterized by this lesion. Development of high-throughput screening assays for potential drugs aimed at these therapeutic targets is currently under way.     

Activation of protein phosphatase 2B and hyperphosphorylation of Tau in Alzheimer's disease

Protein phosphatase 2B (PP2B) is one of the major brain phosphatases and can dephosphorylate tau at several phosphorylation sites in vitro. Previous studies that measured PP2B activity in human brain crude extracts showed that PP2B activity was either unchanged or decreased in Alzheimer's disease (AD) brain. These results led to the speculation that PP2B might regulate tau phosphorylation and that a down-regulation of PP2B might contribute to abnormal hyperphosphorylation of tau. In this study, we immunoprecipitated PP2B from brains of six AD subjects and seven postmortem- and age-matched controls and then measured the phosphatase activity. We found a three-fold increase in PP2B activity in AD brain as compared with control brains. The activation was due to the partial cleavage of PP2B by calpain I that was activated in AD brain. The truncation of PP2B appeared to alter its intracellular distribution in the brain. In human brains, PP2B activity correlated positively, rather than negatively, to the levels of tau phosphorylation at several sites that can be dephosphorylated by PP2B in vitro. Truncation of PP2B in the frontal cortex was more than in the temporal cortex, and tau phosphorylation was also more in the frontal cortex. Taken together, these results indicate that truncation of PP2B by calpain I elevates its activity but does not counteract the abnormal hyperphosphorylation tau in AD brain.     

Pharmacological approaches of neurofibrillary degeneration

Alzheimer disease (AD) and related tauopathies are all characterized histopathologically by neurofibrillary degeneration. The neurofibrillary changes, whether of paired helical filaments (PHF), twisted ribbons or straight filaments (SF) are made up of abnormally hyperphosphorylated tau. Unlike normal tau which promotes assembly and maintains structure of microtubules, the abnormal tau not only lacks these functions but also sequesters normal tau, MAP1 and MAP2, and causes disassembly of microtubules. This toxic behavior of the abnormal tau is solely due to its hyperphosphorylation because dephosphorylation restores it into a normal-like protein. The abnormal hyperphosphorylation also promotes the self-assembly of tau into PHF/SF. The state of phosphorylation of a phosphoprotein is the function of the activities of protein kinases and as well as of protein phosphatases that regulate the level of phosphorylation. A cause of the abnormal hyperphosphorylation in AD brain is a decrease in the activity of protein phosphatase (PP)-2A, a major regulator of the phosphorylation of tau. A decrease in PP-2A activity results in the abnormal hyperphosphorylation of tau not only by decreased dephosphorylation of tau but also by stimulating the activities of tau kinases like CaMKII, PKA and MAP kinases which are regulated by PP-2A. Thus, the abnormal hyperphosphorylation can be inhibited both by inhibition of the activity/s of a tau protein kinase and as well as by restoration of the activity/s of a tau protein phosphatase. The development of drugs that inhibit neurofibrillary degeneration is a very promising and feasible therapeutic approach to inhibit the progression of AD and related tauopathies.     

Mechanisms of tau-induced neurodegeneration

Alzheimer disease (AD) and related tauopathies are histopathologically characterized by a specific type of slow and progressive neurodegeneration, which involves the abnormal hyperphosphorylation of the microtubule associated protein (MAP) tau. This hallmark, called neurofibrillary degeneration, is seen as neurofibrillary tangles, neuropil threads, and dystrophic neurites and is apparently required for the clinical expression of AD, and in related tauopathies it leads to dementia in the absence of amyloid plaques. While normal tau promotes assembly and stabilizes microtubules, the non-fibrillized, abnormally hyperphosphorylated tau sequesters normal tau, MAP1 and MAP2, and disrupts microtubules. The abnormal hyperphosphorylation of tau, which can be generated by catalysis of several different combinations of protein kinases, also promotes its misfolding, decrease in turnover, and self-assembly into tangles of paired helical and or straight filaments. Some of the abnormally hyperphosphorylated tau ends up both amino and C-terminally truncated. Disruption of microtubules by the non-fibrillized abnormally hyperphosphorylated tau as well as its aggregation as neurofibrillary tangles probably impair axoplasmic flow and lead to slow progressive retrograde degeneration and loss of connectivity of the affected neurons. Among the phosphatases, which regulate the phosphorylation of tau, protein phosphatase-2A (PP2A), the activity of which is down-regulated in AD brain, is by far the major enzyme. The two inhibitors of PP-2A, I₁^PP2A and I₂^PP2A, which are overexpressed in AD, might be responsible for the decreased phosphatase activity. AD is multifactorial and heterogeneous and involves more than one etiopathogenic mechanism.     

PP2A and Alzheimer disease

Phosphorylation and, therefore, binding capacity of microtubule-associated protein tau is regulated by specific kinases and phosphatases. Activation of tau kinases plays a crucial role in tau- hyper-phosphorylation in Alzheimer disease (AD) and related tauopathies. Among phosphatases, protein phosphatase 2A, PP2A, is a principal tau dephosphorylating enzyme in the brain. PP2A acts as trimer composed of a catalytic (PP2A C), a scaffolding (PP2A A) and a regulatory (PP2 AB; B55α) subunit. Several abnormalities of PP2A have been reported in AD, including decreased mRNA and protein levels of the PP2A C (not replicated by other studies); decreased protein levels of the PP2A A and B55α; reduced PP2A C methylation at Leu309 due to impaired function methyltransferase type IV; increased PP2A C phosphorylation at Tyr307; up-regulation of the PP2A inhibitors I1 and I2; and loss of enzymatic activity. These observations indicate that PP2A is a putative target of therapeutic intervention considering that enhancing PP2A activity would decrease tau hyper-phosphorylation in AD. In spite of these achievements further studies are needed to replicate the reported individual different alterations converging in PP2A in AD.     

Microtubule assembly competence analysis of freshly-biopsied human tau,dephosphorylated tau,and Alzheimer tau

Phosphorylation of the microtubule-associated protein tau regulates its binding to microtubules; highly phosphorylated tau is also a prime component of paired helical filaments (PHFs) of Alzheimer's disease (AD). Tau from freshly biopsied human, monkey, and rat brain share similar electrophoretic mobility patterns and overlapping phosphorylated epitopes when compared to AD tau isolated from AD brain. We compared the microtubule reassembly competence of fresh isolates of phosphorylated tau to that of maximally dephosphorylated tau and tau from AD brain. A rapid procedure was developed which permitted the enrichment of phosphorylated and dephosphorylated tau from human biopsies in the absence of protein kinase and phosphatase activity. Microtubule assembly assays, using a spectrophotometric measure and purified bovine brain tubulin, were used to correlate assembly competence with states of tau electrophoretic mobility. Maximally dephosphorylated human biopsy-derived tau and monkey tau were assembly competent; tau from AD brain was virtually unable to direct microtubule assembly. Unmodified, biopsy-derived tau from non-AD brain was intermediate in assembly competence relative to AD tau and dephosphorylated tau. Several lines of evidence were used to correlate phosphorylation states of tau with microtubule assembly. First, in vitro dephosphorylation of human biopsy-derived tau with either PP2A or PP2B alone or in combination led to increasing assembly competence as the electrophoretic mobility of tau increased. Second, addition of the protein phosphatase inhibitor okadaic acid (10 μM) to brain-slice preparations slowed electrophoretic mobility of tau and decreased binding competence. We suggest that tau derived from freshly-biopsied brain exists in a range of phosphorylated states, and that dephosphorylation by PP2A and/or PP2B increases microtubules assembly competence. © 1996 Wiley-Liss, Inc.     

Decrease of protein phosphatase 2A and its association with accumulation and hyperphosphorylation of tau in Down syndrome

Virtually all individuals with Down syndrome (DS) develop neurofibrillary tangles, a characteristic brain lesion of Alzheimer's disease (AD), when they reach the fourth decade of life. In AD, neurofibrillary tangles are thought to result from abnormal hyperphosphorylation of tau protein, which, in turn, can result from down-regulation of protein phosphatase (PP) 2A, a major brain tau phosphatase. The abnormal hyperphosphorylation of tau in DS had not yet been characterized, and its causes were not understood. In this study, by using quantitative Western blot analysis, we found that the level of the catalytic subunit of PP2A, but not of PP1, PP2B or PP5, was dramatically decreased. The decrease of PP2A level correlated negatively to tau level and tau phosphorylation at several abnormal hyperphosphorylation sites, including Ser199, Thr205, Thr212, Ser262, Ser396 and Ser422. Our results indicate that PP2A is down-regulated in DS brain and suggest that this down-regulation might be involved in the abnormal hyperphosphorylation and accumulation of tau.