BETA AMYLOID FREE RADICAL PRODUCTION AND NEUROTOXICITY--RELEVANCE TO AD

β 淀粉样蛋白自由基的产生和神经毒性——与 AD 的相关性

• 批准号: 6097996
• 负责人: D. Allan Butterfield
• 金额: $ 13.15万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6097996
• 关键词: Alzheimer's disease; amyloid proteins; electron spin resonance spectroscopy; free radicals; gerbil /jird; laboratory rat; neurotoxins; nitrone; oxidative stress; pathologic process; protein structure function

One of the key pathologic findings of Alzheimer's disease (AD) is senile (amyloid) plaques, composed of aggregated amyloid surrounded by dystrophic neurites and other moieties. Beta-amyloid (Abeta) is a 39-43 amino acid peptide that is toxic to neurons only after an "aging" period. In contrast, the 11 amino acid fragment of Abeta containing residues 25- 35, Abeta (25-35), is highly toxic immediately upon solubilization. Previously, we reported strong evidence for an O2-dependent, but metal- independent, free-radical-based mechanism for abeta aggregation and neurotoxicity. Abeta generates free radicals after an "aging" period while Abeta(25-35) developed free radicals immediately upon solubilization. We developed a model to explain the neurotoxicity of abeta to neuronal and glial membranes and have provided experimental evidence to support this model. A free radical-based mechanism of AD is consistent with the myriad of membrane protein and lipid alterations reported in AD. Insight into the mechanism by which these Abeta-induced free radicals arise is needed. The proposed research will provide this insight. In Specific Aim # 1, we will define systematically the compositional and structural determinants of Abeta(25-35) that lead to free radical production and neurotoxicity. The hypothesis to be tested is that key amino acids in, and the structure of, Abeta peptides are essential to their free radical generating nd neurotoxic properties. It is unclear whether the PBN-trapped radical is oxygen-centered or carbon- centered. Consequently, in Specific Aim # 2, we will use 17O2, which has a nuclear spin, to determine this point. In Specific Aim # 3, based on insights gained in Specific Aims # 1 and 2, we will use specific substitutions of key amino acids in Abeta(1-40) to assess their importance in the free radical generating and neurotoxic properties of Abeta(1-40). In Specific Aim # 4, we will use EPR to test a prediction of the model we developed, namely, that Abeta(1-40) [and more rapidly Abeta(25-35)] will produce membrane damage to critical components of cortical neuronal and glial membranes. The hypothesis to be tested is that Abeta(1-40), Abeta(25-35), and Abeta peptides produced from Specific Aims 1-3, that produce a 3-line EPR spectrum with the spin trap PBN, will produce regional damage within the neocortical synaptosomal membrane. In each Aim, we will correlate EPR results with Abeta peptide-induced neurotoxicity, reactive oxygen species production, protein oxidation, Ca2+ flux, glutamate uptake, changes in peptide structure assessed by CD, amino acid alterations (e.g., met sulfoxide formation), the ability to form fibrils as assessed by EM, and abrogation of these free radical effects by appropriate free radical scavengers. Insight into the molecular basis of AD and potential therapeutic intervention is envisaged.

阿尔茨海默氏病（AD）的关键病理发现之一是老年 （淀粉样蛋白）斑块，由包围的聚集淀粉样蛋白组成 营养不良的神经突和其他部分。 β-淀粉样蛋白（ABETA）是39-43 仅在“衰老”时期，对神经元有毒的氨基酸肽。 相反，含有残基的11个氨基酸片段25- 35，Abeta（25-35）在溶解后立即剧毒。 以前，我们报道了有力的证据表明O2依赖性但金属 - 独立的，基于自由基的ABETA聚集机制和 神经毒性。 ABETA在“衰老”时期产生自由基 而Abeta（25-35）立即在 溶解化。 我们开发了一个模型来解释神经毒性 Abeta至神经元和神经胶质膜，并提供了实验性 支持此模型的证据。 基于自由基的AD机制是 与无数的膜蛋白和脂质改变一致 在广告中报告。 深入了解这些Abeta诱导的机制 需要自由基。 拟议的研究将为这提供 洞察力。 在特定的目标＃1中，我们将系统地定义 Abeta的组成和结构决定因素（25-35）导致 自由基生产和神经毒性。 要检验的假设 那是关键的氨基酸，而Abeta肽的结构是 它们的自由基生成ND神经毒性特性至关重要。 它 尚不清楚PBN捕获的自由基是以氧气为中心还是碳 集中。 因此，在特定的目标＃2中，我们将使用17o2，其中 核自旋，以确定这一点。 在特定的目标＃3中，基于 在特定目标＃1和2中获得的见解，我们将使用特定的 在Abeta中取代关键氨基酸（1-40）以评估其 在自由基产生和神经毒性特性中的重要性 Abeta（1-40）。 在特定目标4中，我们将使用EPR测试预测 在我们开发的模型中，即Abeta（1-40）[更快地 Abeta（25-35）]将对关键组件的关键组件造成膜损害 皮质神经元和神经胶质膜。 要检验的假设是 那个Abeta（1-40），Abeta（25-35）和Abeta肽是由特定的 AIM 1-3，与自旋陷阱PBN产生3线EPR光谱 在新皮质突触体膜内产生区域损伤。 在每个目标中，我们都将EPR结果与Abeta肽诱导的 神经毒性，活性氧的产生，蛋白质氧化， Ca2+通量，谷氨酸摄取，通过CD评估的肽结构的变化， 氨基酸的改变（例如，遇到硫氧化物形成）， 由EM评估的原纤维形成原纤维，废除这些自由基 适当的自由基清除剂的影响。 深入了解 AD的分子基础和潜在的治疗干预是 设想。