Structural Studies of Alzheimer's beta-Amyloid Fibrils

于二零一九财政年度，本集团在以下方面取得进展： (1)冷冻电子显微镜下的淀粉样蛋白-β原纤维结构：我们已经获得了40个残基的淀粉样蛋白-β（Ab 40）原纤维的高质量冷冻电子显微镜图像，这些原纤维是通过接种来自人脑组织的富含淀粉样蛋白的提取物获得的。 我们已经修改了标准的图像处理软件，以包括来自给定显微照片中相同原纤维的原纤维片段（“颗粒”）之间的取向相关性。 通过这种修改，我们已经获得了高分辨率的密度图（3.0埃报告的分辨率），允许明确的分子结构测定。 冷冻-EM密度揭示了一个令人惊讶的结构，由两个中心交叉β层组成，其中Ab 40分子完全延伸，而不是采用在不同原纤维多晶型物的早期研究中看到的U形或S形构象。 除了两个中心层之外，两个另外的层显示归因于Ab 40“半分子”的密度。 我们尝试性地将这些“半分子”层解释为由具有β-发夹构象的分子形成的交叉β片层。 β-发夹构象的确认将取决于目前正在进行的固态NMR测量。 我们预期于二零二零财政年度初公布该等冷冻电镜研究的结果。 (2)非痴呆个体的淀粉样β纤维多态性脑组织的表征：在我们实验室的早期工作中（Qiang等人，Nature 2017），我们使用固态NMR光谱来表征阿尔茨海默病（AD）患者脑组织中的淀粉样蛋白-β原纤维多态性。 脑源性原纤维的2D固态NMR谱显示40-残基肽（Ab 40）的单一主要多晶型物和42-残基肽（Ab 42）的两种主要多晶型物。 我们现在将同样的方法应用于死亡时认知正常的个体的脑组织，但发现他们的大脑皮层中有高水平的淀粉样蛋白沉积。 用于这些实验的组织获自拉什阿尔茨海默病中心的宗教秩序研究。 到目前为止，我们已经研究了Ab 40纤维来源于种子生长的淀粉样蛋白富集提取物的额叶皮质样品的八个人。 我们已经发现，这些原纤维的2D固态NMR光谱与我们早期工作中来自AD患者的皮质组织的Ab 40原纤维的光谱没有显著不同。 因此，认知障碍不是唯一与Ab 40原纤维多态性相关的。 42-残基肽原纤维（Ab 42）的固态NMR测量将于2020财年初进行。

Progress in FY2019 has been in the following areas: (1) AMYLOID-BETA FIBRIL STRUCTURES FROM CRYO-ELECTRON MICROSCOPY: We have obtained high-quality cryo-EM images of 40-residue amyloid-beta (Ab40) fibrils obtained by seeding with amyloid-enriched extract from human brain tissue. We have modified standard image-processing software to include orientational correlations among fibril segments ("particles") that come from the same fibrils in a given micrograph. With this modification, we have obtained high-resolution density maps (3.0 Angstrom reported resolution) that allow unambiguous molecular structure determination. The cryo-EM density reveals a surprising structure, consisting of two central cross-beta layers in which Ab40 molecules are fully extended, rather than adopting the U-shaped or S-shaped conformations seen in earlier studies of different fibril polymorphs. In addition to the two central layers, two additional layers show density attributable to Ab40 "half-molecules". We tentatively interpret these "half-molecule" layers to cross-beta sheets formed by molecules with beta-hairpin conformations. Confirmation of beta-hairpin conformations will depend on solid state NMR measurements, which are currently in progress. We expect to publish the results of these cryo-EM investigations in early FY2020. (2) CHARACTERIZATION OF AMYLOID-BETA FIBRIL POLYMORPHS BRAIN TISSUE OF NON-DEMENTED INDIVIDUALS: In earlier work from our lab (Qiang et al., Nature 2017), we used solid state NMR spectroscopy to characterize amyloid-beta fibril polymorphisms in brain tissue of Alzheimer's disease (AD) patients. 2D solid state NMR spectra of brain-derived fibrils showed a single predominant polymorph for the 40-residue peptide (Ab40) and two predominant polymorphs for the 42-residue peptide (Ab42). We are now applying the same methods to brain tissue of individuals who were cognitively normal at the time of death, but who were found to have high levels of amyloid deposition in their cerebral cortex. Tissue for these experiments was obtained from the Religious Orders Study of the Rush Alzheimer's Disease Center. So far, we have examined Ab40 fibrils derived by seeded growth from amyloid-enriched extract of frontal cortex samples of eight individuals. We have found that the 2D solid state NMR spectra of these fibrils are not significantly different from spectra of Ab40 fibrils that were derived from cortical tissue of AD patients in our earlier work. Thus, cognitive impairment is not uniquely correlated with Ab40 fibril polymorphisms. Solid state NMR measurements on 42-residue peptide fibrils (Ab42) will be performed in early FY2020.