体外聚集测定使用tau蛋白的蛋白质
Summary
未修饰的和磷酸化的tau蛋白以两种体外聚集测定中使用以显示磷酸化依赖快速聚合动力学。这些测定铺路用于可调节tau蛋白的倾向，以形成背后阿尔茨海默氏病的进展纤丝化合物未来画面。
Abstract
Alzheimer’s disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of hyperphosphorylated tau protein in the form of neurofibrillary tangles (NFTs). The density of NFT correlates well with cognitive impairment and other neurodegenerative symptoms, thus prompting the endeavor of developing tau aggregation-based therapeutics. Thus far, however, tau aggregation assays use recombinant or synthetic tau that is devoid of the pathology-related phosphorylation marks. Here we describe two assays using recombinant, hyperphosphorylated tau as the subject. These assays can be scaled up for high-throughput screens for compounds that can modulate the kinetics or stability of hyperphosphorylated tau aggregates. Novel therapeutics for Alzheimer’s disease and other tauopathies can potentially be discovered using hyperphosphorylated tau isoforms.
Introduction
阿尔茨海默氏病（AD）是一个大的集合称为tau蛋白病的神经变性疾病中的一种。典型的病理Tau蛋白病的基本是神经原纤维缠结，在神经元，星形胶质细胞和小胶质细胞1-4。在NFT密度与认知功能障碍3,5和神经元丢失6。 NFT主要含有高度磷酸化tau蛋白（简称为“P-tau蛋白”此后），其形成直链或成对螺旋纤丝（PHF）7,8。头被认为是有利于轴突运输的是神经信号和贩运9,10必不可少的微管相关蛋白。每个tau蛋白分子含有2〜3磷酸盐在正常脑，而是由几个折叠的磷含量的增加在τ病变的患者11。多激酶可能有助于tau蛋白包括GSK3β（糖原合酶激酶3β）和CDK5（细胞周期素德依赖激酶5）12,13，但直接触发的病理磷酸化仍然遥遥无期14。异常磷酸化中或附近的微管结合基序上解离的tau从微管15，并且使头误定位到somatodendritic隔室，其中p-tau蛋白寡聚成直链或成对螺旋丝，可以最终聚合成NFT的内含物。 tau蛋白磷酸化，NFT形成与神经变性之间的紧密联系导致普遍的假设是p-tau蛋白缠结引起的凋亡和其他细胞毒性反应，因此是对神经变性τ病变16,17的根本原因。基于这个前提药物屏幕和早期临床试验已经启动18。然而，这种假设面临的挑战19,20。例如，圣克鲁斯等人表明，转基因小鼠的认知功能可以通过抑制突变体的表达来改进人类tau蛋白，即使NFTS继续形成从现有的tau蛋白分子21。在果蝇模型，NFT显示出螯合的有毒胞质tau蛋白以保护下面的神经元细胞22,23。显然，NFT的发病中的作用，如果有的话，将极大地影响τ病变治疗剂的发展方向。
在高浓度的，重组的或正常脑τ蛋白自发但慢慢地聚合成PHF状结构体外 ，通过几个β折叠优选荧光染料，电子显微镜和光散射光谱24-27的结合所指示的。添加肝素或花生四烯酸，丰富的脂肪酸在人类的大脑，大大加速了PHF形成头isoform-和诱导浓度依赖性28-32。有趣的是，tau蛋白纯化从AD大脑或体外磷酸化反应制得面面俱到一ggregates更快，更高效地26,33-35。这些结果与对 – tau蛋白的病理性作用良好的协议。 体外基于对tau的聚集系统可以因此用作一个强大的工具用于AD药物筛选。
定在发现的药物可控制的tau聚集在上升的兴趣的tau聚集和AD的渐进的神经变性之间的密切联系，以及在药物开发的最新故障靶向Aβ斑块，公元36-38的另一个重要的组织学标志物。事实上，几个小组已经在不同的通量开始药物屏幕，使用体外的tau聚集反应作为主要测定法。被发现的一些化学品的展示在体外 39-42对tau聚集的抑制或逆转的活动。但是，目前所有的tau蛋白聚集调节屏幕使用未经修改的头说错过荧光粉的主要病理标记ylation，提高用于在公元治疗使用这些化合物的特异性和功效的关注。
一个发展中的聚集试验的生化特性和AD药物筛选的主要障碍是生产足够数量的病理生理学相关tau蛋白的蛋白质。使用拉链辅助催化系统，其中tau蛋白的同种型1N4R和GSK-3β激酶共表达在大肠杆菌中的大肠杆菌亮氨酸拉链融合蛋白，我们已经克服了这个挑战（穗等人 ，提交;请参阅tau蛋白和P-tau蛋白的最终产品图1;另见43 P-tau蛋白的初步质谱鉴定）。从特异性针对tau的不同磷酸化位点9的抗体的面板，阳性信号出现在八个位置（数据未示出）。下面，我们描述的协议及仪器能够区分的聚集动力学ð未修改的tau蛋白和P-tau蛋白物种之间ifferences。这些检测是从公布的协议，可测得硫代黄素T（ThT的）或硫代黄素S（THS）的荧光的增加对淀粉样蛋白（tau聚集）结合26修改。在第一个“终端”，无染料的方法，聚合反应被组装并孵育在不存在淀粉样蛋白染料。在不同时间点，每个反应的等分试样被去除，并与含的ThT缓冲器的等体积混合，以阻止聚集和允许的ThT结合tau聚集。荧光是由IAP FluoroMax-2荧光计测量。在第二“与染料”持续监控测定法，或ThT的THS被包括在聚合反应。荧光可以连续地在整个实验手动测量或使用多板读数器。此外，我们描述了在连续测量莫使用的tau和对tau的一个近生理浓度的聚集的测定德。磷酸化的效果仍然易被察觉。下面，我们将描述一步一步的操作的程序，并显示这些分析的代表性结果。讨论一些每种方法的利弊，以及潜在的药物筛选的应用将随之而来。
以高浓度，tau蛋白聚集成淀粉样蛋白样结构自发。然而，在实验室中，tau蛋白纤维化典型地通过例如诱导肝素（平均分子量6000克/摩尔）和花生四烯酸加速。本文所示的例子包括30μM肝素。 tau蛋白的淀粉样蛋白聚集体的形成是通过从淀粉通过硫磺素T（ThT的）或硫磺素S（THS）结合而产生的荧光进行监测。结合后tau聚集，ThT的表现出红移荧光（激发：450纳米;峰值发射：485纳米）。 THS，而另一方面，具有弱发射在之前结合淀粉样蛋白为510nm（激发在450nm处），但这种fluorescencë增加显著中淀粉样蛋白的存在，如聚集的τ44。这两种染料的检测tau蛋白和P-tau蛋白聚集工作。因为的ThT（ 见图2）的强和相对宽的发光峰的，只有30在荧光单元减少在510nm％。为方便起见，我们使用的激发/发射波长（ 即 450纳米/ 510纳米）同样的组合来监控头聚集在使用任何染料。
头聚合所用的染料的存在或不存在下进行，这取决于测定的目的和τ蛋白的可用性。反应的两种模式如下所示。此外，我们证明了两种不同的仪器操作 – 单样本荧光计（ISA-SPEX FluoroMax-2）和多板酶标仪（Spectramax M2）。读者应该能够适应这些协议，以满足他们的特定需求和仪器的可用性。
Protocol
Representative Results
Discussion
该协议证明了不同的检测条件和检测磷酸化依赖的快速tau蛋白的聚集动力学的仪器。在终端测定中，荧光染料的ThT加到从主混合物在每个时间点除去反应的一部分。结合淀粉样蛋白诱导的荧光，然后测量26。在第二，与染料模式，tau蛋白聚集是在的ThT或THS的存在下，使这种类型的适合的tau聚集物的生长的实时自动评估反应。每种方法都有其优点和缺点。
终端模式反应?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This work was supported by the National Institute on Aging (AG039768) to MHK. We thank Drs. Thomas Sharkey and Honggao Yan for generously providing the instruments, as well Sean Weise and Yan Wu for technical assistance.
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| Trizma base | Sigma | T1503 | |
| NaCl | Macron Fine Chemicals | MAL-7581-06 | |
| Ethylenediaminetetraacetic Acid (EDTA) | Invitrogen | 15576-028 | |
| Thioflavin T | Sigma | T3516 | Stored in dark |
| Thioflavin S | Sigma | T1892 | Stored in dark |
| heparin | Sigma | H3393 | |
| DL-Dithiothreitol (DTT) | Sigma | D9779 | Stored at 4°C |
| 96-well plate | Corning | 3917 | |
| ISA SPEX FluoroMax-2 | Horiba | ||
| SpectraMax M2 Multi-Mode Microlate Reader | Molecular Devices | ||
| Mouse Anti-Tau Monoclonal Antibody | R&D Systems | MAB3494 | Stored at –80° |
Riferimenti
- Lee, V. M., Goedert, M., Trojanowski, J. Q. Neurodegenerative tauopathies. Annu Rev Neurosci. 24, 1121-1159 (2001).
- Ballatore, C., Lee, V. M., Trojanowski, J. Q. Tau-mediated neurodegeneration in Alzheimer’s disease and related disorders. Nat Rev Neurosci. 8 (9), 663-672 (2007).
- Arriagada, P. V., Marzloff, K., Hyman, B. T. Distribution of Alzheimer-type pathologic changes in nondemented elderly individuals matches the pattern in Alzheimer’s disease. Neurology. 42 (9), 1681-1688 (1992).
- Arriagada, P. V., Growdon, J. H., Hedley-Whyte, E. T., Hyman, B. T. Neurofibrillary tangles but not senile plaques parallel duration and severity of Alzheimer’s disease. Neurology. 42 (3 Pt 1), 631-639 (1992).
- Bancher, C., Braak, H., Fischer, P., Jellinger, K. A. Neuropathological staging of Alzheimer lesions and intellectual status in Alzheimer’s and Parkinson’s disease patients. Neurosci Lett. 162 (1-2), 179-182 (1993).
- Guillozet, A. L., Weintraub, S., Mash, D. C., Mesulam, M. M. Neurofibrillary tangles, amyloid, and memory in aging and mild cognitive impairment. Arch Neurol. 60 (5), 729-736 (2003).
- Hasegawa, M., et al. Protein sequence and mass spectrometric analyses of tau in the Alzheimer’s disease brain. J Biol Chem. 267 (24), 17047-17054 (1992).
- Matsuo, E. S., et al. Biopsy-derived adult human brain tau is phosphorylated at many of the same sites as Alzheimer’s disease paired helical filament tau. Neuron. 13 (4), 989-1002 (1994).
- Bamburg, J. R., Bloom, G. S. Cytoskeletal pathologies of Alzheimer disease. Cell Motil Cytoskeleton. 66 (8), 635-649 (2009).
- Denk, F., Wade-Martins, R. Knock-out and transgenic mouse models of tauopathies. Neurobiol Aging. 30 (1), 1-13 (2009).
- Gong, C. X., Iqbal, K. Hyperphosphorylation of microtubule-associated protein tau: a promising therapeutic target for Alzheimer disease. Curr Med Chem. 15 (23), 2321-2328 (2008).
- Mazanetz, M. P., Fischer, P. M. Untangling tau hyperphosphorylation in drug design for neurodegenerative diseases. Nat Rev Drug Discov. 6 (6), 464-479 (2007).
- Brunden, K. R., Trojanowski, J. Q., Lee, V. M. Advances in tau-focused drug discovery for Alzheimer’s disease and related tauopathies. Nat Rev Drug Discov. 8 (10), 783-793 (2009).
- Querfurth, H. W., LaFerla, F. M. Alzheimer’s disease. N Engl J Med. 362 (4), 329-344 (2010).
- Buee, L., Bussiere, T., Buee-Scherrer, V., Delacourte, A., Hof, P. R. Tau protein isoforms, phosphorylation and role in neurodegenerative disorders. Brain Res Brain Res Rev. 33 (1), 95-130 (2000).
- Lee, V. M., Brunden, K. R., Hutton, M., Trojanowski, J. Q. Developing therapeutic approaches to tau, selected kinases, and related neuronal protein targets. Cold Spring Harb Perspect Med. 1 (1), 006437 (2011).
- Mandelkow, E. M., Mandelkow, E. Biochemistry and cell biology of tau protein in neurofibrillary degeneration. Cold Spring Harb Perspect Med. 2 (7), a006247 (2012).
- Bulic, B., Pickhardt, M., Mandelkow, E. Progress and Developments in Tau Aggregation Inhibitors for Alzheimer Disease. J Med Chem. 56 (11), 4135-4155 (2013).
- Cowan, C. M., Quraishe, S., Mudher, A. What is the pathological significance of tau oligomers. Biochem Soc Trans. 40 (4), 693-697 (2012).
- Spires-Jones, T. L., Kopeikina, K. J., Koffie, R. M., de Calignon, A., Hyman, B. T. Are tangles as toxic as they look. J Mol Neurosci. 45 (3), 438-444 (2011).
- SantaCruz, K., et al. Tau suppression in a neurodegenerative mouse model improves memory function. Science. 309 (5733), 476-481 (2005).
- Wittmann, C. W., et al. Tauopathy in Drosophila: neurodegeneration without neurofibrillary tangles. Science. 293 (5530), 711-714 (2001).
- Shulman, J. M., Feany, M. B. Genetic modifiers of tauopathy in Drosophila. Genetica. 165 (3), 1233-1242 (2003).
- Wille, H., Drewes, G., Biernat, J., Mandelkow, E. M., Mandelkow, E. Alzheimer-like paired helical filaments and antiparallel dimers formed from microtubule-associated protein tau in vitro. J Cell Biol. 118 (3), 573-584 (1992).
- Alonso, A., Zaidi, T., Novak, M., Grundke-Iqbal, I., Iqbal, K. Hyperphosphorylation induces self-assembly of tau into tangles of paired helical filaments/straight filaments. Proc Natl Acad Sci U S A. 98 (12), 6923-6928 (2001).
- Barghorn, S., Biernat, J., Mandelkow, E. Purification of recombinant tau protein and preparation of Alzheimer-paired helical filaments in vitro. Methods Mol Biol. 299, 35-51 (2005).
- Wilson, D. M., Binder, L. I. Polymerization of microtubule-associated protein tau under near-physiological conditions. J Biol Chem. 270 (41), 24306-24314 (1995).
- Wilson, D. M., Binder, L. I. Free fatty acids stimulate the polymerization of tau and amyloid beta peptides. In vitro evidence for a common effector of pathogenesis in Alzheimer’s disease. Am J Pathol. 150 (6), 2181-2195 (1997).
- Perez, M., Valpuesta, J. M., Medina, M., Montejo de Garcini, E., Avila, J. Polymerization of tau into filaments in the presence of heparin: the minimal sequence required for tau-tau interaction. J Neurochem. 67 (3), 1183-1190 (1996).
- Carlson, S. W., et al. A complex mechanism for inducer mediated tau polymerization. Biochimica. 46 (30), 8838-8849 (2007).
- Goedert, M., et al. Assembly of microtubule-associated protein tau into Alzheimer-like filaments induced by sulphated glycosaminoglycans. Nature. 383 (6600), 550-553 (1996).
- King, M. E., Gamblin, T. C., Kuret, J., Binder, L. I. Differential assembly of human tau isoforms in the presence of arachidonic acid. J Neurochem. 74 (4), 1749-1757 (2000).
- Rankin, C. A., Sun, Q., Gamblin, T. C. Pseudo-phosphorylation of tau at Ser202 and Thr205 affects tau filament formation. Brain Res Mol Brain Res. 138 (1), 84-93 (2005).
- Rankin, C. A., Sun, Q., Gamblin, T. C. Pre-assembled tau filaments phosphorylated by GSK-3b form large tangle-like structures. Neurobiol Dis. 31 (3), 368-377 (2008).
- Grundke-Iqbal, I., et al. Abnormal phosphorylation of the microtubule-associated protein tau (tau) in Alzheimer cytoskeletal pathology. Proc Natl Acad Sci U S A. 83 (13), 4913-4917 (1986).
- Castellani, R. J., Perry, G. Pathogenesis and disease-modifying therapy in Alzheimer’s disease: the flat line of progress. Arch Med Res. 43 (8), 694-698 (2012).
- Green, R. C., et al. Effect of tarenflurbil on cognitive decline and activities of daily living in patients with mild Alzheimer disease: a randomized controlled trial. JAMA. 302 (23), 2557-2564 (2009).
- Gauthier, S., et al. Effect of tramiprosate in patients with mild-to-moderate Alzheimer’s disease: exploratory analyses of the MRI sub-group of the Alphase study. J Nutr Health Aging. 13 (6), 550-557 (2009).
- Pickhardt, M., et al. Anthraquinones inhibit tau aggregation and dissolve Alzheimer’s paired helical filaments in vitro and in cells. J Biol Chem. 280 (5), 3628-3635 (2005).
- Crowe, A., Ballatore, C., Hyde, E., Trojanowski, J. Q., Lee, V. M. High throughput screening for small molecule inhibitors of heparin-induced tau fibril formation. Biochem Biophys Res Commun. 358 (1), 1-6 (2007).
- Taniguchi, S., et al. Inhibition of heparin-induced tau filament formation by phenothiazines, polyphenols, and porphyrins. J Biol Chem. 280 (9), 7614-7623 (2005).
- Sigurdsson, E. M. Tau-focused immunotherapy for Alzheimer’s disease and related tauopathies. Curr Alzheimer Res. 6 (5), 446-450 (2009).
- Tan, Y. J., et al. Phosphopeptide Enrichment with TiO-Modified Membranes and Investigation of Tau Protein Phosphorylation. Anal Chem. 85 (12), 5699-5706 (2013).
- Santa-Maria, I., Perez, M., Hernandez, F., Avila, J., Moreno, F. J. Characteristics of the binding of thioflavin S to tau paired helical filaments. J Alzheimers Dis. 9 (3), 279-285 (2006).
- Lira-De Leon, K. I., et al. Molecular mechanism of tau aggregation induced by anionic and cationic dyes. J Alzheimers Dis. 35 (2), 319-334 (2013).
- DiNitto, J. P., Wang, L., Wu, J. C. Continuous fluorescence-based method for assessing dicer cleavage efficiency reveals 3′ overhang nucleotide preference. BioTechniques. 48, 303-311 (2010).
- Maeda, S., et al. Granular tau oligomers as intermediates of tau filaments. Biochimica. 46 (12), 3856-3861 (2007).
- Pickhardt, M., et al. Phenylthiazolyl-hydrazide and its derivatives are potent inhibitors of tau aggregation and toxicity in vitro and in cells. Biochimica. 46 (35), 10016-10023 (2007).
- Rankin, C. A., Sun, Q., Gamblin, T. C. Tau phosphorylation by GSK-3beta promotes tangle-like filament morphology. Mol Neurodegener. 2, 12 (2007).
- McKee, A. C., et al. Chronic traumatic encephalopathy in athletes: progressive tauopathy after repetitive head injury. J Neuropathol Exp Neurol. 68 (7), 709-735 (2009).
- Herrup, K. Reimagining Alzheimer’s disease–an age-based hypothesis. J Neurosci. 30 (50), 16755-16762 (2010).
- Gavett, B. E., Stern, R. A., McKee, A. C. Chronic traumatic encephalopathy: a potential late effect of sport-related concussive and subconcussive head trauma. Clin Sports Med. 30 (1), 179-188 (2011).
- Tsitsopoulos, P. P., Marklund, N. Amyloid-beta Peptides and Tau Protein as Biomarkers in Cerebrospinal and Interstitial Fluid Following Traumatic Brain Injury: A Review of Experimental and Clinical Studies. Front Neurol. 4, 79 (2013).
