Tranthyretin's Regulatory Role in Beta-Amyloid Aggregation and Toxicity

Tranthyretin 在 β-淀粉样蛋白聚集和毒性中的调节作用

• 批准号: 8288509
• 负责人: Jeffrey A Johnson
• 金额: $ 39.47万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8288509
• 关键词: APP-PS1; Acute; Address; Adenoviruses; Affect; Alanine; Alzheimer' s Disease; Amyloid beta-Protein; Animals; Astrocytes; Binding; Binding Sites; Biological; Biological Assay; Blood; Brain; Carrier Proteins; Cell Culture Techniques; Cerebrospinal Fluid; Cessation of life; Characteristics; Chemicals; Coupled; Data; Deposition; Disease; Glial Fibrillary Acidic Protein; Goals; Human; In Vitro; Incubated; Injection of therapeutic agent; Investigation; Kinetics; Label; Lead; Libraries; Ligand Binding; Ligands; Link; Mass Spectrum Analysis; Measures; Mediating; Methods; Modeling; Modification; Molecular; Morphology; Mus; Mutagenesis; Mutation; Nature; Neuronal Dysfunction; Neurons; Pathologic; Patients; Peptide Library; Peptides; Plasma; Prealbumin; Protein Precursors; Proteins; Recombinants; Research; Retinol Binding Proteins; Role; Screening procedure; Senile Plaques; Site; Staging; Structure; Testing; Thyroxine; Toxic effect; Toxicity Tests; Transgenic Animals; Transgenic Mice; Validation; Variant; Work; abeta accumulation; age related; aggregation pathway; base; dentate gyrus; design; in vitro Assay; in vitro testing; in vivo; in vivo Model; light scattering; mutant; nanoparticle; neuron loss; neurotoxic; neurotoxicity; novel; overexpression; oxidation; prevent; programs; promoter; protective effect; research study; restoration

DESCRIPTION (provided by applicant): Beta-amyloid (A?) is the primary protein component of Alzheimer's disease (AD) amyloid plaques, and there is substantial evidence to support the hypothesis that soluble A? aggregates are neurotoxic. Transgenic mice expressing the Swedish mutation of the human A? precursor protein (APPSw) produce high levels of A? and develop amyloid plaques, but surprisingly they do not suffer the extensive neuronal cell death characteristic of AD. Recent studies have uncovered a possible explanation: APPSw mice upregulate synthesis of transthyretin (TTR), a transport protein found in plasma and cerebrospinal fluid, and TTR appears to protect the mice from the neurotoxic effects of A?. The long-term goals of this project are to answer three questions that arise from this intriguing discovery: (1) how does TTR exert its protective activity? (2) why does this natural protective activity fail in AD? (3) can it be restored or replaced? In aim 1, the specific residues on TTR involved with binding to A? will be identified. From mass spectrometry analysis coupled with peptide array binding studies, residues on the G strand and near the EF helix of TTR were implicated. Further definition of the binding site will be obtained by screening for A? binding to peptide library derived from overlapping sequences of TTR, and by targeted alanine mutagenesis. Compounds that mimic the TTR binding sites will be synthesized and tested for A? binding as well as inhibition of in vitro toxicity. In aim 2, the effect of TTR and variants on ? aggregation will be characterized. Preliminary data show that TTR quaternary structure and stability, oxidation, and binding of natural ligands all influence the extent of A? binding to TTR. The greatest A? binding is observed at intermediate aggregation states. Put another way, TTR may be a natural scavenger for the most toxic A? aggregates. Aggregation of A? in the presence of TTR will be characterized by a combination of dynamic and static light scattering, and nanoparticle tracking. These complementary methods, which are particularly suited for examining soluble aggregates, will yield data on aggregate size, size distribution, number, morphology, and aggregation rate. Synthetic mimics, developed in aim 1, will also be characterized for their effect on A? aggregation. In aim 3, further validation of TTR's neuroprotective action will be sought. Astrocytes will be transfected to secrete TTR (wt and monomeric), and inhibition of A? toxicity will be tested in vitro with mixed cortical cultures, or y adding medium from secreting astrocytes to highly enriched cortical neurons. Transgenic mice that overexpress TTR in astrocytes on an APP/PS1 background will be generated, and pathological endpoints will be evaluated to ascertain the level of in vivo protection afforded by TTR. Finally, initial screening of promising TTR mimics from Aim 1 will be tested in a stereotactic injection assay. These studies, which integrate chemical, biophysical, and biological approaches, will provide a rational basis for developing novel pharmacological approaches to preventing AD by enhancement of TTR's natural defenses. PUBLIC HEALTH RELEVANCE: Alzheimer's disease has been linked to deposition of beta-amyloid plaques in the brain and subsequent death of neurons. Studies with transgenic mice suggest that a normal protein, transthyretin may provide some protection against the neuronal cell death caused by beta-amyloid. This investigation will provide a rational basis for elucidating why this protective action of TTR is lost and for developing novel pharmacological approaches to preventing Alzheimer's disease by restoration or replacement of the natural defenses provided by transthyretin.

描述（由申请人提供）：β-淀粉样蛋白（A？）是阿尔茨海默氏病（AD）淀粉样蛋白斑块的主要蛋白质成分，并且有大量证据可以支持可溶于A的假设？聚集体是神经毒性的。表达人类瑞典突变的转基因小鼠？前体蛋白（APPSW）产生高水平的A？并开发淀粉样蛋白斑，但令人惊讶的是，它们不会遭受广泛的AD神经元细胞死亡特征。最近的研究发现了一个可能的解释：APPSW小鼠上调了甲状腺素蛋白（TTR）的合成，这是一种在血浆和脑脊液中发现的转运蛋白，TTR似乎可以保护小鼠免受A？的神经毒性影响。该项目的长期目标是回答这一有趣的发现引起的三个问题：（1）TTR如何发挥其保护活动？ （2）为什么这种自然保护活动在AD中失败？ （3）可以恢复或更换吗？在AIM 1中，TTR上的特定残基与A与A结合涉及？将被确定。从质谱分析与肽阵列结合研究结合的质谱分析中，与TTR的EF螺旋和EF螺旋附近的残基有关。结合位点的进一步定义将通过筛选A来获得？与TTR重叠序列和靶向丙氨酸诱变得出的肽文库结合。模拟TTR结合位点的化合物将合成并测试A？结合以及抑制体外毒性。在AIM 2中，TTR和变体的影响？聚集将被表征。初步数据表明，TTR四级结构和稳定性，氧化和自然配体的结合都会影响A的程度？与TTR结合。 最大的A？在中间聚集状态下观察到结合。换句话说，TTR可能是最有毒的天然清真寺？聚合。 A的聚合？在存在的情况下，TTR的特征是动态和静态光散射以及纳米颗粒跟踪的结合。这些互补方法特别适合检查可溶性聚集体，将产生有关骨料大小，大小分布，数量，形态和聚集率的数据。在AIM 1中开发的合成模拟物也将因其对A的影响而被特征。聚合。在AIM 3中，将寻求对TTR神经保护作用的进一步验证。星形胶质细胞将被转染至分泌TTR（WT和单体），并抑制A？毒性将在体外用混合皮质培养物进行测试，或将介质分泌到高度富集的皮质神经元中。将生成APP/PS1背景中星形胶质细胞中过表达TTR的转基因小鼠，并将评估病理终点以确定TTR提供的体内保护水平。最后，将在立体定向注射测定法中测试对AIM 1的有希望的TTR模拟物的初步筛选。这些研究结合化学，生物物理和生物学方法的研究将为开发新型的药理学方法提供合理的基础，以通过增强TTR的自然防御能力来预防AD。 公共卫生相关性：阿尔茨海默氏病与大脑中β-淀粉样蛋白斑块的沉积以及随后的神经元死亡有关。对转基因小鼠的研究表明，正常蛋白质，经胸蛋白可以提供一些保护，以防止β-淀粉样蛋白引起的神经元细胞死亡。这项调查将为阐明 为什么丢失了TTR的这种保护作用，以及通过恢复或替换Transthyretin提供的自然防御能力来预防阿尔茨海默氏病的新型药理学方法。