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

运甲状腺素蛋白在 β-淀粉样蛋白聚集和毒性中的调节作用

• 批准号: 7896768
• 负责人: Jeffrey A Johnson
• 金额: $ 43.12万
• 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 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7896768
• 关键词: Acids; Address; Adenoviruses; Affect; Alzheimer' s Disease; Amyloid; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Amyloid deposition; Amyloidosis; Animal Model; Asses; Astrocytes; Behavior; Benzoxazoles; Biochemical; Biological Assay; Blood; Brain; Carrier Proteins; Cell Culture Techniques; Cells; Cerebral cortex; Cerebrospinal Fluid; Cessation of life; Characteristics; Circular Dichroism; Complex; Data; Defect; Deposition; Diflunisal; Disease; Dissociation; Electron Microscopy; Engineering; Evaluation; Fluorescence; Fluorescence Resonance Energy Transfer; Goals; Heart; Hippocampus (Brain); Human; In Vitro; Infection; Inherited; Injection of therapeutic agent; Investigation; Kinetics; Ligand Binding; Ligands; Link; Maps; Mass Spectrum Analysis; Measures; Modeling; Modification; Molecular; Molecular Sieve Chromatography; Mus; Mutation; Nature; Neurofibrillary Tangles; Neurons; Pathology; Patients; Plasma; Prealbumin; Presenile Alzheimer Dementia; Production; Protein Precursors; Proteins; Recombinants; Resistance; Resveratrol; Role; Senile Plaques; Structure; Surface Plasmon Resonance; System; Testing; Tg2576; Thyroxine; Tissues; Toxic effect; Transgenic Mice; Variant; abeta accumulation; age related; amyloidogenesis; base; cell injury; crosslink; dentate gyrus; dimer; gel electrophoresis; in vivo; in vivo Model; insight; interest; lateral ventricle; light scattering; monomer; mouse model; mutant; neuron loss; neurotoxic; neurotoxicity; novel; overexpression; prevent; protein folding; research study; sound; tool

Transthyretin's Regulatory Role in Beta-Amyloid Aggregation and Toxicity Alzheimer’s disease (AD) has been linked to deposition of beta-amyloid (Aβ) as amyloid plaques in the brain. Transgenic mice expressing the human Aβ precursor protein (APP) produce high levels of Aβ and develop amyloid plaques, but they do not suffer the extensive neuronal cell death that is characteristic of AD. Recent studies have uncovered a possible explanation: these transgenic mice greatly increase the synthesis of the transport protein transthyretin (TTR), and TTR appears to protect the mice from the neurotoxic effects of Aβ. The long-term goals of this project are to answer three questions that arise from these intriguing results: (1) how does TTR exert its protective activity? (2) why does this natural protective activity fail in AD? (3) can it be restored? Our hypothesis is that subtle changes in TTR tertiary and/or quaternary structure strongly modulate TTR- Aβ interactions. In specific aim 1, several TTR mutants that differ in their tertiary and/or quaternary structure and stability will be produced and characterized. Each mutant will be screened for its ability to interfere with Aβ aggregation, and to inhibit Aβ toxicity in an in vitro cell culture model. The data will be analyzed to identify correlations between TTR structure and stability with its ability to alter Aβ aggregation and toxicity. In specific aim 2, a detailed examination of the interaction between TTR (both wildtype and selected mutants) and Aβ will be undertaken. Biophysical and biochemical tools such as circular dichroism, fluorescence, static and dynamic light scattering, crosslinking, and kinetic modeling will be employed. From these data will emerge molecular-level mechanistic insights into the nature of TTR-Aβ association and the means by which TTR affects Aβ aggregation kinetics. The goal of specific aim 3 is to identify small ligands that stabilize TTR and determine their influence on TTR’s ability to modulate Aβ aggregation and toxicity. In aim 4, TTR (wildtype and mutants) along with TTR-binding ligands will be tested for protection against Aβ toxicity in ex vivo and in vivo mouse models. This will be achieved by using a newly developed assay in which stereotactic injection of Aβ into mouse brain leads to loss of CA1 and dentate gyrus neurons, and by infection of astrocytes with adenovirus-TTR constructs. The project spans from characterization of the structure and stability of TTR (and mutants), through in vitro assessment of TTR’s effect on Aβ aggregation and toxicity, to in vivo evaluation of TTR efficacy at preventing neuronal cell death. These studies will provide a sound and rational basis for developing novel pharmacological approaches to preventing AD by enhancement of the natural defenses provided by TTR.

转蛋白在β-淀粉样蛋白聚集和毒性阿尔茨海默氏病（AD）中的调节作用已与大脑中β-淀粉样蛋白（Aβ）的沉积联系在一起。表达人Aβ前体蛋白（APP）的转基因小鼠产生了高水平的Aβ并产生淀粉样蛋白斑块，但它们并未遭受广泛的AD特征神经元细胞死亡。最近的研究发现了一个可能的解释：这些转基因小鼠大大增加了转运蛋白转甲切林（TTR）的合成，而TTR似乎可以保护小鼠免受Aβ的神经毒性影响。该项目的长期目标是回答从这些有趣的结果引起的三个问题：（1）TTR如何执行其受保护的活动？ （2）为什么这种自然保护活动在AD中失败？ （3）可以恢复吗？我们的假设是，TTR第三纪和/或第四纪结构的细微变化强烈调节TTR-Aβ相互作用。在特定的目标1中，将产生和表征几个在第三纪和/或第四纪结构和稳定性上不同的TTR突变体。将筛选每个突变体的能力，以干扰Aβ聚集，并在体外细胞培养模型中抑制Aβ毒性。将分析数据，以确定TTR结构与稳定性之间的相关性与改变Aβ聚集和毒性的能力。在特定的目标2中，将对TTR（Wildtype和选定突变体）和Aβ之间的相互作用进行详细检查。将进行生物物理和生化工具，例如圆形二色性，荧光，静态和动态光散射，交联和动力学建模。从这些数据中，将出现对TTR-Aβ关联性质的分子级机械见解以及TTR影响Aβ聚集动力学的手段。特定目标3的目的是确定稳定TTR的小配体，并确定其对TTR调节Aβ聚集和毒性能力的影响。在AIM 4中，将测试TTR（WildType和突变体）以及TTR结合配体，以保护离体和体内小鼠模型中的Aβ毒性。这将通过使用新开发的测定法实现，在这种测定中，立体定向注射Aβ在小鼠脑中会导致CA1和齿状回神经元的丧失，以及通过腺病毒-TTR构建体的星形胶质细胞感染。该项目涵盖了TTR（和突变体）的结构和稳定性的表征，通过对TTR对Aβ聚集和毒性的影响的体外评估，以及在预防神经元细胞死亡方面的TTR有效性的体内评估。这些研究将为开发新型的药理学方法提供合理和合理的基础，以通过增强TTR提供的自然防御能力来防止AD。