ABETA FIBRILS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。阿尔茨海默病S病(AD)的一个病理特征是聚集在脑内老年斑块中的淀粉样β-多肽(A？)。A？1-42是老年斑中发现的主要多肽，具有显著的自我聚集成折叠片状构象以形成纤维淀粉样蛋白(FA)的倾向。致密的老年斑由致密的FA聚集体组成；这种聚集被广泛认为是AD发病机制中的一个基本事件，启动了一系列有害的过程，包括诱导氧化应激、炎症和神经毒性。过去十年的研究表明，多酚是在各种保健食品中发现的具有可变酚类结构的天然物质，具有强大的抗氧化性能，似乎在预防各种疾病中发挥作用。几个研究小组最近发现，在体外，一些多酚能够抑制合成的Aβ聚集成纤维和低聚物。研究得最好的是，姜黄素，咖喱中使用的肿瘤香料的有效成分(AD在印度的发病率是美国的)，通过硫代黄素-T(THT)荧光和透射电子显微镜(TEM)检测，可以抑制A？的纤化。此外，17至22个月龄喂食姜黄素的AD小鼠模型(Tg2576小鼠)与未治疗的小鼠相比，淀粉样斑块负荷减少，这表明在斑块发病机制中有作用。许多其他多酚(包括单宁酸、杨梅素、桑色素、阿魏酸等)在体外也显示出抗淀粉样蛋白生成的活性。虽然主要的焦点一直是抑制A？聚集，但初步的间接证据表明，多酚可能在体外促进淀粉样原纤维的解聚。这种抑制聚集和解聚之间的区别是重要的，因为它意味着疾病的阻止和逆转。我们建议使用冷冻电子显微镜(CRYO-EM)来确定姜黄素是否导致淀粉样原纤维结构的改变。以前的报告已经证明，合成的淀粉样原纤维可以使用冷冻-EM直接可视化，并揭示出一种可观察到的横跨纤维的片状结构(尽管由A？11-25形成的原纤维具有比A？1-42更好的成像特性)。淀粉样纤维将在姜黄素存在或不存在的情况下(在添加后的不同时间)和刚果红进行冷冻-EM成像，刚果红结合但不会诱导纤维解聚。图像分析将包括测量原丝直径、条纹之间的距离(代表片材之间的距离)和原丝包裹的节距。我们还建议使用冷冻-EM来进一步表征姜黄素孵化的分解产物。

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 Alzheimer¿s 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 tumeric¿a 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.