ABETA FIBRILS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 老年痴呆症的一个病理特征老年痴呆症（AD）是大脑内老年斑中聚集的淀粉样β肽（A？）的积累。 A？1-42是在老年斑中发现的主要肽，具有自聚集成褶状片构象以形成纤维状淀粉样蛋白（fA）的显著倾向。致密的老年斑由fA？的致密聚集体组成;这种积累被广泛认为是AD发病机制中的基本事件，启动了一系列有害过程，包括诱导氧化应激、炎症和神经毒性。 过去十年的研究表明，多酚，在各种植物中发现的具有可变酚结构的天然物质， 健康食品， 具有有效的抗氧化特性，似乎在预防多种疾病中发挥作用。 几个研究小组最近发现，一些多酚能够抑制合成A ²在体外聚集成纤维和低聚物。 研究得最好的是姜黄素，一种用于咖喱的香料（AD在印度的发病率是美国的一半），通过硫代嘌呤-T（ThT）荧光和透射电子显微镜（TEM）检测，抑制了A的原纤维化。 此外，AD小鼠模型（Tg 2576小鼠）从17至22个月的年龄喂养姜黄素有减少淀粉样蛋白斑块负荷相比，未经处理的小鼠，这表明对斑块发病机制的影响。许多其他多酚（包括单宁酸、杨梅素、桑色素、阿魏酸等）也已显示出体外抗淀粉样蛋白生成活性。虽然主要的焦点一直在抑制A？聚集，初步但间接的证据表明，多酚可能会促进体外淀粉样纤维解聚。 聚集和解聚的抑制之间的这种区别是重要的，因为其对疾病停滞与逆转的影响。 我们建议使用冷冻电子显微镜（cryo-EM），以确定姜黄素是否诱导淀粉样纤维结构的变化。先前的报道已经证明，合成的淀粉样蛋白原纤维可以使用冷冻电镜直接可视化，并揭示了一种可以观察到的跨纤维条纹的<$-片层结构（尽管由A <$11 -25形成的原纤维比A <$1 -42具有更好的成像特征）。 在存在或不存在姜黄素的情况下（在添加后的不同时间），使用cryo-EM和刚果红（其结合但不诱导原纤维解聚）对淀粉样原纤维进行成像。 图像分析将包括原纤维直径、条纹之间的距离（代表半片之间的距离）和原丝缠绕的节距的测量。 我们还建议使用冷冻EM进一步表征姜黄素孵育的分解产物。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A pathological hallmark of Alzheimers Disease (AD) is the accumulation of aggregated amyloid-beta peptides (A¿) in senile plaques within the brain. A¿1-42, the predominant peptide found in senile plaques, has a striking propensity to self-aggregate into a ¿-pleated sheet conformation to form fibrillar amyloid (fA¿). Compact senile plaques are comprised of dense aggregates of fA¿; this accumulation is widely believed to be a fundamental event in the pathogenesis of AD, initiating a cascade of deleterious process including the induction of oxidative stress, inflammation, and neurotoxicity. Studies over the last decade have suggested that polyphenols, natural substances with variable phenolic structures found in various health foods, possess potent antioxidant properties and appear to play a role in the prevention of a variety of diseases. Several groups have recently found that some polyphenols are capable of inhibiting the aggregation of synthetic A¿ into fibrils and oligomers in vitro. The best studied, curcumin, the active ingredient in tumerica spice used in curry (the incidence of AD in India is ¿ that in the US), inhibited the fibrilization of A¿ as detected by thioflavine-T (ThT) fluorescence, and transmission electron microscopy (TEM). Furthermore, an AD mouse model (Tg2576 mice) fed curcumin from 17 to 22 months of age had decreased amyloid plaque burden compared to untreated mice, suggesting an effect on plaque pathogenesis. A host of other polyphenols (including tannic acid, myricetin, morin, ferulic acid, amongst others) has also been shown to demonstrate anti-amyloidogenic activity in vitro. While the primary focus has been on the inhibition of A¿ aggregation, preliminary but indirect evidence suggests that polyphenols may promote amyloid fibril disaggregation in vitro. This distinction between the inhibition of aggregation and disaggregation is important because of its implications for disease arrest vs. reversal. We propose to use cryo-electron microscopy (cryo-EM) to determine if curcumin induces changes in the structure of the amyloid fibril. Previous reports have demonstrated that synthetic amyloid fibrils can be directly visualized using cryo-EM, and reveal a ¿-sheet structure that is observable as striations across the fibers (though fibrils formed from A¿11-25 had better imaging characteristics than A¿1-42). Amyloid fibrils will be imaged using cryo-EM in the presence or absence of curcumin (at varying times after addition), and with Congo Red, which binds but does not induce fibril disaggregation. Image analysis will include measurements of fibril diameter, distance between striations (representing distance between ¿-sheets), and the pitch of protofilament wrapping. We also propose to use cryo-EM to further characterize breakdown products of curcumin incubation.