Phosphatase Activity Toward Abnormally Phosphorylated τ: Decrease in Alzheimer Disease Brain

Abstract: Microtubule-associated protein τ is abnormally hyperphosphorylated and aggregated in affected neurons of Alzheimer disease brain. This hyperphosphorylated τ can be dephosphorylated at some of the abnormal phosphorylated sites by purified protein phosphatase-1, 2A, and 2B in vitro. In the present study, we have developed an assay to measure protein phosphatase activity toward τ-1 sites (Ser¹⁹⁹/Ser²⁰²) using the hyperphosphorylated τ isolated from Alzheimer disease brain as substrate. Using this assay, we have identified that in normal brain, protein phosphatase-2A and 2B and, to a lesser extent, 1 are involved in the dephosphorylation of τ. The K _m values of dephosphorylation of the hyperphosphorylated τ by protein phosphatase-2A and 2B are similar. The τ phosphatase activity is decreased by ∼30% in brain of Alzheimer disease patients compared with those of age-matched controls. These findings suggest that a defect of protein phosphatase could be the cause of the abnormal hyperphosphorylation of τ in Alzheimer disease.     

阿尔次海默病tau蛋白异常修饰与其功能的关系

阿尔次海默病易溶型胞浆tau和难溶型双螺旋丝中的tau均被异常磷酸化和异常糖基化修饰.异常修饰的tau丧失其促微管组装活性,用不同蛋白磷酸酯酶对难溶型双螺旋丝中的tau去磷酸化处理后可不同程度恢复其促微管组装生物学活性.单纯去糖基化处理只在很小限度恢复tau的功能,但去糖基化预处理可增强去磷酸化对tau上述活性的恢复.提示:a.tau的异常磷酸化是导致其功能活性丧失的直接因素,而糖基化修饰可能通过对其结构的影响而间接对tau功能活性发挥作用;b.蛋白磷酸酯酶可部分抑制和逆转阿尔次海默病的脑病理损伤.     

建立超敏ELISA-双酶底物循环法检测tau蛋白

利用酶联免疫吸附法(ELISA)的高特异性和双酶底物循环的高灵敏度,建立了ELISA-双酶底物循环扩增检测法.用传统ELISA测定纯化tau蛋白和阿尔茨海默病异常磷酸化tau蛋白的范围值分别为1～32 ng和0.2～10 ng,而此法的测定范围值分别为0.75～200 pg和0.5～50 pg,比传统ELISA的灵敏度分别提高1 300倍和400倍,可测定范围值亦分别扩大了8.5倍和2倍.可准确测定阿尔茨海默病患者脑脊液样品中的微量tau蛋白和异常磷酸化tau蛋白,为阿尔茨海默病的早期诊断和鉴别诊断提供了新技术.     

Alzheimer neurofibrillary degeneration: significance, etiopathogenesis, therapeutics and prevention

Alzheimer disease (AD) is multi-factorial and heterogeneous. Independent of the aetiology, this disease is characterized clinically by chronic and progressive dementia and histopathologically by neurofibrillary degeneration of abnormally hyperphosphorylated tau seen as intraneuronal neurofibrillary tangles, neuropil threads and dystrophic neurites, and by neuritic (senile) plaques of beta-amyloid. The neurofibrillary degeneration 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 abnormally hyperphosphorylated tau sequesters normal tau, MAP1 and MAP2, and disrupts microtubules. The abnormal hyperphosphorylation of tau also promotes its self-assembly into tangles of paired helical and or straight filaments. Tau is phosphorylated by a number of protein kinases. Glycogen synthase kinase-3 (GSK-3) and cyclin dependent protein kinase 5 (cdk5) are among the kinases most implicated in the abnormal hyperphosphorylation of tau. Among the phosphatases which regulate the phosphorylation of tau, protein phosphatase-2A (PP-2A), the activity of which is down-regulated in AD brain, is by far the major enzyme. The inhibition of abnormal hyperphosphorylation of tau is one of the most promising therapeutic targets for the development of disease modifying drugs. A great advantage of inhibiting neurofibrillary degeneration is that it can be monitored by evaluating the levels of total tau and tau phosphorylated at various known abnormally hyperphosphorylated sites in the cerebrospinal fluid of patients, obtained by lumbar puncture. There are at least five subgroups of AD, each is probably caused by a different etiopathogenic mechanism. The AD subgroup identification of patients can help increase the success of clinical trials and the development of specific and potent disease modifying drugs.     

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。     

Dephosphorylation of microtubule-associated protein tau by protein phosphatase 5

Protein phosphatase 5 (PP5) is a 58-kDa novel phosphoseryl/phosphothreonyl protein phosphatase. It is ubiquitously expressed in all mammalian tissues examined, with a high level in the brain, but little is known about its physiological substrates. We found that this phosphatase dephosphorylated recombinant tau phosphorylated with cAMP-dependent protein kinase and glycogen synthase kinase-3beta, as well as abnormally hyperphosphorylated tau isolated from brains of patients with Alzheimer's disease. The specific activity of PP5 toward tau was comparable to those reported with other protein substrates examined to date. The PP5 activity toward tau was stimulated by arachidonic acid by 30- to 45-fold. Immunostaining demonstrated that PP5 was primarily cytoplasmic in PC12 cells and in neurons of postmortem human brain tissue. A small pool of PP5 associated with microtubules. Expression of active PP5 in PC12 cells resulted in reduced phosphorylation of tau, suggesting that PP5 can also dephosphorylate tau in cells. These results suggest that PP5 plays a role in the dephosphorylation of tau and might be involved in the molecular pathogenesis of Alzheimer's disease.     

14-3-3 Protein, Total Tau and Phosphorylated Tau in Cerebrospinal Fluid of Patients with Creutzfeldt-Jakob Disease and Neurodegenerative Disease in Japan

1.Sporadic Creutzfeldt-Jakob disease (CJD) is a rapidly progressive and fatal disease. Patients with CJD usually become akinetic mutism within approximately 6 months. In addition, clinical signs and symptoms at early stage of sporadic CJD may not be easy to distinguish from other neurodegenerative diseases by neurological findings. However, diagnostic biochemical parameters including 14-3-3 protein, S100, neuron-specific enorase in cerebrospinal fluid (CSF) have been used as diagnostic markers, elevated titers of these markers can also be observed in CSF in other neurodegenerative diseases. Therefore, we examined other biochemical markers to discriminate CJD from other neurodegenerative diseases in CSF. 2.We analyzed CSF samples derived from 100 patients with various neurodegenerative disorders by Western blot of 14-3-3 protein, quantification of total tau (t-tau) protein, and phosphorylated tau (p-tau) protein. All patients with CJD in this study showed positive 14-3-3 protein and elevated t-tau protein (>1000 pg/mL) in CSF. We also detected positive 14-3-3 protein bands in two patients in non-CJD group (patients with dementia of Alzheimer's type; DAT) and also detected elevated t-tau protein in three patients in non-CJD group. Elevated t-tau protein levels were observed in two patients with DAT and in one patient with cerevrovascular disease in acute phase. 3.To distinguish patients with CJD from non-CJD patients with elevated t-tau protein in CSF, we compared the ratio of p-tau and t-tau proteins. The p-/t-tau ratio was dramatically and significantly higher in DAT patients rather than in CJD patients. 4.Therefore, we concluded that the assay of t-tau protein may be useful as 1st screening and the ratio of p-tau protein/t-tau protein would be useful as 2nd screening to discriminate CJD from other neurodegenerative diseases.     

蛋白磷酸酯酶对Alzheimer神经原纤维缠结的松解作用

神经原纤维缠结是Ａｌｚｈｅｉｍｅｒ患者的特征性脑病理损伤,其形成机制至今不明．根据神经原纤维缠结的基本组分是异常磷酸化ｔａｕ蛋白的聚集形式双螺旋丝（ｐａｉｒｅｄｈｅｌｉｃａｌｆｉｌａｍｅｎｔｓ,ＰＨＦ）的研究结果,推测蛋白磷酸酯酶与蛋白激酶的失衡可能与ＰＨＦ的形成有关．将蛋白磷酸酯酶ＰＰ－２Ａ和ＰＰ－２Ｂ与ＰＨＦ一起在３７℃保温３０ｍｉｎ可使ＰＨＦ缠结结构松解,成为单个ＰＨＦ原纤维,延长去磷酸化反应时间至３ｈ可使ＰＨＦ结构进一步松解,释放一些游离ＰＨＦ原纤维片段．放免印迹定量分析结果表明：ＰＰ－２Ａ处理的ＰＨＦ样品比对照者释放游离ｔａｕ蛋白的量增加２５％．此外,ＰＰ－２Ａ和ＰＰ－２Ｂ去磷酸化的ＰＨＦ对脑中钙激活的中性蛋白水解酶的抗性降低．这些研究资料从结构上显示了Ａｌｚｈｅｉｍｅｒ病脑病理损伤的可逆性,为Ａｌｚｈｅｉｍｅｒ病治疗的可能性提供了实验依据     

Tau蛋白在阿尔兹海默病治疗中的研究进展

阿尔兹海默病中一个重要的病理特点是神经原纤维缠结(NFTs),其与Tau蛋白具有密切的关系。Tau蛋白是含量最高的微管相关蛋白。正常Tau蛋白可与微管蛋白结合促进其有效聚合形成微管。而过度磷酸化的Tau蛋白则会自我聚集,进而导致NFTs的形成。近年来,国内外就Tau为主要靶点治疗AD有了很大进展。本文就Tau蛋白在AD中的病理学意义,与Aβ蛋白的相互作用以及以Tau为主要切入点对AD进行治疗的方法作一综述。     

Co-immunoprecipitation with Tau Isoform-specific Antibodies Reveals Distinct Protein Interactions and Highlights a Putative Role for 2N Tau in Disease

Alternative splicing generates multiple isoforms of the microtubule-associated protein Tau, but little is known about their specific function. In the adult mouse brain, three Tau isoforms are expressed that contain either 0, 1, or 2 N-terminal inserts (0N, 1N, and 2N). We generated Tau isoform-specific antibodies and performed co-immunoprecipitations followed by tandem mass tag multiplexed quantitative mass spectrometry. We identified novel Tau-interacting proteins of which one-half comprised membrane-bound proteins, localized to the plasma membrane, mitochondria, and other organelles. Tau was also found to interact with proteins involved in presynaptic signal transduction. MetaCore analysis revealed one major Tau interaction cluster that contained 33 Tau pulldown proteins. To explore the pathways in which these proteins are involved, we conducted an ingenuity pathway analysis that revealed two significant overlapping pathways, “cell-to-cell signaling and interaction” and “neurological disease.” The functional enrichment tool DAVID showed that in particular the 2N Tau-interacting proteins were specifically associated with neurological disease. Finally, for a subset of Tau interactions (apolipoprotein A1 (apoA1), apoE, mitochondrial creatine kinase U-type, β-synuclein, synaptogyrin-3, synaptophysin, syntaxin 1B, synaptotagmin, and synapsin 1), we performed reverse co-immunoprecipitations, confirming the preferential interaction of specific isoforms. For example, apoA1 displayed a 5-fold preference for the interaction with 2N, whereas β-synuclein showed preference for 0N. Remarkably, a reverse immunoprecipitation with apoA1 detected only the 2N isoform. This highlights distinct protein interactions of the different Tau isoforms, suggesting that they execute different functions in brain tissue.     

微管相关蛋白tau在动物死亡后被快速去磷酸化

tau蛋白是神经细胞中主要的微管相关蛋白, 它的异常过度磷酸化被认为是阿尔茨海默病 (AD) 致病过程中的关键因素. 由于法律、社会、家庭等诸多因素使得获取的人脑组织标本常常在死亡后2~3 h以上,因此了解死亡不同时间后tau蛋白磷酸化的改变,对研究tau蛋白的功能及在AD致病过程中作用显得十分重要. 用位点特异的、磷酸化依赖的抗tau蛋白抗体检测正常大鼠脑中tau蛋白磷酸化程度及死亡后其磷酸化的变化情况,再用非同位素的点印迹技术测定鼠脑中tau蛋白激酶、磷酸酶在不同温度下的活性. 结果发现,正常鼠脑中tau蛋白除了Ser262,Ser409,Ser422外,在Thr181,Ser199,Ser202,Thr205,Thr212,Ser214,Thr217,Ser396和Ser404存在不同程度的磷酸化,并且在死亡后3 h,出现tau的多位点的去磷酸化及tau蛋白迁移加快,6 h后更为明显,但tau蛋白水平即使在大鼠死亡后6 h,仍未见有明显的改变. 用点印迹测定蛋白激酶和磷酸酶活性结果显示,tau蛋白激酶、磷酸酶活性均有温度依赖性降低,在25℃时激酶活性降低远大于磷酸酶活性的降低,tau蛋白在死亡后的快速去磷酸化与相对高的磷酸酶作用有关.     

钙蛋白酶对兔脑皮质中细胞骨架蛋白tau的降解作用

钙蛋白酶 (calpain)是钙依赖性中性蛋白酶 ,根据其对钙敏感性的不同 ,可分为m 和 μ 钙蛋白酶两型。分别用不同浓度CaCl2 溶液温育Wistar大鼠脑皮质匀浆液 ,并用Western印迹和定量图像分析技术检测不同亚型钙蛋白酶对tau蛋白的降解作用。发现 :在 37℃用 1mmol LCa2 温育底物 15min ,即出现大量分子量为 2 9kD的tau蛋白降解片段 ;当Ca2 浓度为 5mmol L时 ,tau蛋白几乎全部被降解 ;这种tau蛋白降解可被特异性的钙蛋白酶抑制剂完全逆转。进一步的研究发现 ,分别用 μ 钙蛋白酶抑制剂 ( 0 .0 5 μmol Lcalpastatin) ,m 钙蛋白酶抑制剂 ( 10 0 μmol LcalpaininhibitorIV)或总钙蛋白酶抑制剂 ( 5 5 2 μmol Lcalpeptin)与 1mmol LCa2 共同温育Wistar大鼠脑皮质匀浆液 ,1mmol LCa2 激活的tau蛋白降解分别被抑制 8.6 %、92 .5 %和 97.8%。该研究结果表明 ,一定浓度的Ca2 可同时激活 μ 钙蛋白酶和m 钙蛋白酶 ,这两种亚型均参与降解tau蛋白 ,但m 钙蛋白酶的作用比 μ 钙蛋白酶更强     

Dephosphorylation of DBC1 by Protein Phosphatase 4 Is Important for p53-Mediated Cellular Functions

Deleted in breast cancer-1 (DBC1) contributes to the regulation of cell survival and apoptosis. Recent studies demonstrated that DBC is phosphorylated at Thr454 by ATM/ATR kinases in response to DNA damage, which is a critical event for p53 activation and apoptosis. However, how DBC1 phosphorylation is regulated has not been studied. Here we show that protein phosphatase 4 (PP4) dephosphorylates DBC1, regulating its role in DNA damage response. PP4R2, a regulatory subunit of PP4, mediates the interaction between DBC1 and PP4C, a catalytic subunit. PP4C efficiently dephosphorylates pThr454 on DBC1 in vitro, and the depletion of PP4C/PP4R2 in cells alters the kinetics of DBC1 phosphorylation and p53 activation, and increases apoptosis in response to DNA damage, which are compatible with the expression of the phosphomimetic DBC-1 mutant (T454E). These suggest that the PP4-mediated dephosphorylation of DBC1 is necessary for efficient damage responses in cells.     

Protein restriction cycles reduce IGF‐1 and phosphorylated Tau,and improve behavioral performance in an Alzheimer's disease mouse model

In laboratory animals, calorie restriction (CR) protects against aging, oxidative stress, and neurodegenerative pathologies. Reduced levels of growth hormone and IGF‐1, which mediate some of the protective effects of CR, can also extend longevity and/or protect against age‐related diseases in rodents and humans. However, severely restricted diets are difficult to maintain and are associated with chronically low weight and other major side effects. Here we show that 4 months of periodic protein restriction cycles (PRCs) with supplementation of nonessential amino acids in mice already displaying significant cognitive impairment and Alzheimer's disease (AD)‐like pathology reduced circulating IGF‐1 levels by 30–70% and caused an 8‐fold increase in IGFBP‐1. Whereas PRCs did not affect the levels of β amyloid (Aβ), they decreased tau phosphorylation in the hippocampus and alleviated the age‐dependent impairment in cognitive performance. These results indicate that periodic protein restriction cycles without CR can promote changes in circulating growth factors and tau phosphorylation associated with protection against age‐related neuropathologies.     

Ubiquilin-1 and protein quality control in Alzheimer disease

Single nucleotide polymorphisms in the ubiquilin-1 gene may confer risk for late-onset Alzheimer disease (AD). We have shown previously that ubiquilin-1 functions as a molecular chaperone for the amyloid precursor protein (APP) and that protein levels of ubiquilin-1 are decreased in the brains of AD patients. We have recently found that ubiquilin-1 regulates APP trafficking and subsequent secretase processing by stimulating non-degradative ubiquitination of a single lysine residue in the cytosolic domain of APP. Thus, ubiquilin-1 plays a central role in regulating APP biosynthesis, trafficking and ultimately toxicity. As ubiquilin-1 and other ubiquilin family members have now been implicated in the pathogenesis of numerous neurodegenerative diseases, these findings provide mechanistic insights into the central role of ubiquilin proteins in maintaining neuronal proteostasis.     

Regulation of Secretion of Alzheimer Amyloid Precursor Protein by the Mitogen-Activated Protein Kinase Cascade

Abstract: Activation of protein kinase C (PKC) regulates the processing of Alzheimer amyloid precursor protein (APP) into its soluble form (sAPP) and amyloid β-peptide (Aβ). However, little is known about the intermediate steps between PKC activation and modulation of APP metabolism. Using a specific inhibitor of mitogen-activated protein (MAP) kinase kinase activation (PD 98059), as well as a dominant negative mutant of MAP kinase kinase, we show in various cell lines that stimulation of PKC by phorbol ester rapidly induces sAPP secretion through a mechanism involving activation of the MAP kinase cascade. In PC12-M1 cells, activation of MAP kinase by nerve growth factor was associated with stimulation of sAPP release. Conversely, M1 muscarinic receptor stimulation, which is known to act in part through a PKC-independent pathway, increased sAPP secretion mainly through a MAP kinase-independent pathway. Aβ secretion and its regulation by PKC were not affected by PD 98059, supporting the concept of distinct secretory pathways for Aβ and sAPP formation.     

Structural and biochemical characterization of inhibitor-1alpha

Inhibitor-1alpha is one of the isoforms of human protein phosphatase inhibitor-1. It is a product of alternative splicing of inhibitor-1 gene and lacks 51 internal amino acids from residue 84 to 134 of inhibitor-1. Here we have characterized the structural and biochemical properties of inhibitor-1alpha. Structural analysis of recombinant inhibitor-1alpha by NMR spectroscopy revealed that inhibitor-1alpha adopts a predominantly random coil conformation. Excluding the region from residue 84 to 134 of inhibitor-1, the structural features of inhibitor-1 and inhibitor-1alpha are almost the same as each other. The IC(50) value of inhibitor-1alpha in inhibition of Protein phosphatase-1 (PP1) is comparable to that of inhibitor-1, indicating that inhibitor-1alpha is a potent inhibitor of PP1 when Thr-35 is phosphorylated by PKA. For phosphorylation by PKA and dephosphorylation by protein phosphatase-1, -2A, and -2B, the measured kinetic parameters of inhibitor-1alpha are very close to those of inhibitor-1. Taken together, these results suggest that inhibitor-1alpha preserves the structure of inhibitor-1, the PP1 inhibitory activity and the functional specificities toward phosphorylation by PKA and dephosphorylation by protein phosphatase-1, -2A, and -2B.     

The Structure,Role, and Regulation of Type 1 Protein Phosphatases

Abstract

Type 1 protein phosphatases (PP-1) comprise a group of widely distributed enzymes that specifically dephosphorylate serine and threonine residues of certain phosphoproteins. They all contain an isoform of the same catalytic subunit, which has an extremely conserved primary structure. One of the properties of PP-1 that allows one to distinguish them from other serine/threonine protein phosphatases is their sensitivity to inhibition by two proteins, termed inhibitor 1 and inhibitor 2, or modulator. The latter protein can also form a 1:1 complex with the catalytic subunit that slowly inactivates upon incubation. This complex is reactivated in vitro by incubation with MgATP and protein kinase F_A/GSK-3. In the cell the type 1 catalytic subunit is associated with noncatalytic subunits that determine the activity, the substrate specificity, and the subcellular location of the phosphatase. PP-1 plays an essential role in glycogen metabolism, calcium transport, muscle contraction, intracellular transport, protein synthesis, and cell division. The activity of PP-1 is regulated by hormones like insulin, glucagon, α- and β-adrenergic agonists, glucocorticoids, and thyroid hormones.