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: 8661656
• 负责人: Jeffrey A Johnson
• 金额: $ 37.38万
• 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/8661656
• 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; 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; 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; screening

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.

描述（由申请人提供）：β-淀粉样蛋白（Aβ）是阿尔茨海默病（AD）淀粉样斑块的主要蛋白质成分，并且有大量证据支持可溶性Aβ的假设。聚集体具有神经毒性。表达人类 A? 瑞典突变体的转基因小鼠前体蛋白 (APPSw) 产生高水平的 A?并形成淀粉样斑块，但令人惊讶的是，它们并没有遭受 AD 所特有的广泛神经元细胞死亡的影响。最近的研究发现了一个可能的解释：APPSw 小鼠上调甲状腺素运载蛋白 (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 Aβ 的神经毒性作用。该项目的长期目标是回答这一有趣发现所引发的三个问题：（1）TTR 如何发挥其保护活性？ (2) 为什么这种自然保护活性在 AD 中失效？ （3）可以恢复或更换吗？在目标 1 中，TTR 上的特定残基涉及与 A？将被识别。通过质谱分析和肽阵列结合研究，发现 G 链上和 TTR EF 螺旋附近的残基受到影响。结合位点的进一步定义将通过筛选A？与源自 TTR 重叠序列的肽库结合，并通过定向丙氨酸诱变。将合成模拟 TTR 结合位点的化合物并测试 A？结合以及抑制体外毒性。在目标 2 中，TTR 和变体对 ? 的影响？聚合将被表征。初步数据表明，TTR四级结构和稳定性、氧化以及天然配体的结合都会影响A？与 TTR 结合。 最伟大的A？在中间聚集状态下观察到结合。换句话说，TTR 可能是毒性最强的 A 的天然清除剂？聚合体。 A 的聚合？在存在 TTR 的情况下，其特征将是动态和静态光散射以及纳米粒子跟踪的组合。这些补充方法特别适合检查可溶性聚集体，将产生有关聚集体尺寸、尺寸分布、数量、形态和聚集率的数据。目标 1 中开发的合成模拟物也将因其对 A? 的影响而被表征。聚合。在目标 3 中，将寻求进一步验证 TTR 的神经保护作用。星形胶质细胞将被转染以分泌TTR（野生型和单体型），并抑制A？将使用混合皮质培养物在体外测试毒性，或者添加来自分泌星形胶质细胞的培养基至高度富集的皮质神经元。将生成在 APP/PS1 背景下星形胶质细胞中过度表达 TTR 的转基因小鼠，并评估病理终点以确定 TTR 提供的体内保护水平。最后，将通过立体定向注射测定对 Aim 1 中有希望的 TTR 模拟物进行初步筛选。这些研究整合了化学、生物物理和生物学方法，将为开发通过增强 TTR 自然防御来预防 AD 的新药理学方法提供合理的基础。