Degenerative and Dementing Diseases of Aging

在四个科学项目和三个核心中，我们提议研究Aβ和tau是如何成为导致神经退行性疾病的普恩的。Prion一词最初被定义为Prion蛋白质(PrP)，在PrP中，主要是α螺旋的细胞异构体经历结构转变为富含β-折叠的形式，该形式能够自我催化细胞PrP异构体内的构象转变。现已了解到，蛋白质构象繁殖是一项基本的生物学原理，它的功能与酵母的表型特征以及人类的突触可塑性和记忆有关。这也是一系列与包括阿尔茨海默病(AD)在内的神经退行性疾病相关的蛋白质能够通过宿主的大脑传播的一般机制。阻止这种传播可能会带来一个新的治疗领域，减缓或阻止AD和相关的神经退行性疾病(NDS)。在此次P01更新申请中，我们计划研究Aβ和tau蛋白的分子生物学、生物物理学和结构。在项目1中，我们建议扩大我们对阿尔茨海默病和唐氏综合症(DS)患者大脑的Prion生物分析。DS研究提供的大脑来自比AD患者年轻得多的患者，这可能会减少与年龄相关的人工制品。我们计划确定Aβ和tau蛋白的菌株如何产生不同的疾病结果，以及不同的遗传修饰物如何影响神经发病机制。我们还将在一系列新型转基因(TG)小鼠和人类胚胎干细胞模型中实验测试这些发现。根据我们最近对AD和DS脑中Aβ和tau蛋白的发现，我们可能能够开发一种针对AD的血液检测方法。在项目2中，除了研究与AD相关的蛋白质突变如何影响Aβ分离和处理外，我们还将应用多种技术来进一步确定Pron菌株差异的分子基础，并更好地了解它们的繁殖保真度。项目2还将提供广泛的生物物理和化学生物学方法，以生成结构信息，以便整合到项目4中。项目3将使用冷冻电子显微镜(低温电子显微镜)进行结构研究，以提供Aβ和tau蛋白以及与相关蛋白质的复合体的高分辨率数据。这些都将通过使用重组蛋白质和合成肽在体外产生的样品，以及从体内来源提纯的蛋白质和复合体来完成。项目4将使用小角X射线散射和软X射线层析成像生成数据，并将这些数据与项目1-3的所有结构、生化和生物物理数据相结合，以生成从原子到细胞尺度的结构模型。这些模型将通过与项目1-3的迭代进一步完善。这些项目将得到一个协调研究人员之间互动的管理核心、一个蛋白质组学和生物物理学核心(包括对质谱学的专家支持)和一个动物核心(将提供最高水平的动物生产、程序和护理)的支持。这些项目和核心将相互作用，以比任何单独的小组希望单独实现的更快的速度进行研究。

In four scientific Projects and three Cores, we propose to study how Aβ and tau become prions causing neurodegenerative diseases. The term “prion” was originally defined for the prion protein (PrP), in which a largely α-helical cellular isoform undergoes a structural transition to a β-sheet rich form that is able to self-catalyze a conformational transition within the cellular PrP isoform. It is now understood that protein conformational propagation is a fundamental biological principle that underlies functions as diverse as phenotypic traits in yeast as well as synaptic plasticity and memory in humans. It is also the general mechanism by which a range of proteins associated with neurodegenerative diseases, including Alzheimer’s disease (AD), are able to spread through the brain of the host. Stopping this propagation could lead to a new field of therapeutics that slow or halt AD and related neurodegenerative diseases (NDs). In this P01 renewal application, we plan to study the molecular biology, biophysics and structure of Aβ and tau prions. In Project 1, we propose to expand our prion bioassays of brains from AD and Down syndrome (DS) patients. DS studies offer brains from much younger patients than those from AD, which will likely diminish age-dependent artifacts. We plan to determine how strains of Aβ and tau prions produce different disease outcomes, and how different genetic modifiers impact neuropathogenesis. We will also test these findings experimentally in a range of novel transgenic (Tg) mouse and human embryonic stem cell models. Based on our recent findings of Aβ and tau prions in AD and DS brains, we may be able to develop a blood test for AD. In Project 2 we will apply multiple techniques to further determine the molecular basis of prion strain differences and better understand the fidelity of their propagation, in addition to studying how mutations in proteins associated with AD impact Aβ sequestration and processing. Project 2 will also provide broad-ranging biophysical and chemical biological approaches to generate structural information for integration in Project 4. Structural studies using cryo-electron microscopy (cryo-EM) will be performed in Project 3 to provide high-resolution data of Aβ and tau prions, and complexes with associated proteins. These will be done both with samples generated in vitro using recombinant proteins and synthetic peptides, and from proteins and complexes purified from in vivo sources. Project 4 will generate data using small-angle X-ray scattering and soft X-ray tomography, and integrate these data with all structural, biochemical and biophysical data from Projects 1–3, to generate structural models from the atomic to cellular scale. These models will then be further refined via iteration with Projects 1–3. The Projects will be supported by an Administrative Core to coordinate interactions among investigators, a Proteomics & Biophysics Core including expert support for mass spectrometry, and an Animals Core that will provide the highest level of animal production, procedures and care. Together the Projects and Cores will interact to proceed with research more rapidly than any individual group could hope to achieve alone.