Degenerative and Dementing Diseases of Aging

在四个科学项目和三个核心中，我们建议研究Aβ和tau如何成为导致神经退行性疾病的朊病毒。术语“朊病毒”最初定义为朊病毒蛋白（PrP），其中大部分α-螺旋细胞同种型经历结构转变为富含β-折叠的形式，其能够自催化细胞PrP同种型内的构象转变。现在理解，蛋白质构象繁殖是一个基本的生物学原理，其作为酵母中的表型性状以及人类中的突触可塑性和记忆等多种功能的基础。它也是一系列与神经退行性疾病（包括阿尔茨海默病（AD））相关的蛋白质能够通过宿主大脑传播的一般机制。阻止这种传播可能会导致一个新的治疗领域，减缓或停止AD和相关的神经退行性疾病（ND）。在本次P01更新申请中，我们计划研究Aβ和tau朊病毒的分子生物学、生物物理学和结构。在项目1中，我们建议扩大我们的朊病毒的AD和唐氏综合征（DS）患者的大脑生物测定。DS研究提供的大脑来自比AD患者年轻得多的患者，这可能会减少年龄依赖性伪影。我们计划确定Aβ和tau朊病毒株如何产生不同的疾病结果，以及不同的遗传修饰剂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对这些发现进行实验测试。基于我们最近在AD和DS大脑中发现的Aβ和tau朊病毒，我们可能能够开发出AD的血液检测。在项目2中，我们将应用多种技术进一步确定朊病毒菌株差异的分子基础，并更好地了解其繁殖的保真度，此外还将研究与AD相关的蛋白质突变如何影响Aβ的隔离和加工。项目2还将提供广泛的生物物理和化学生物学方法，以生成用于项目4的结构信息。将在项目3中使用冷冻电子显微镜（cryo-EM）进行结构研究，以提供Aβ和tau朊病毒以及与相关蛋白质的复合物的高分辨率数据。这些将使用使用重组蛋白和合成肽在体外产生的样品以及从体内来源纯化的蛋白和复合物进行。项目4将使用小角度X射线散射和软X射线断层扫描生成数据，并将这些数据与项目1-3的所有结构，生物化学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。这些模型将通过项目1-3的迭代进一步完善。这些项目将得到一个行政核心的支持，以协调研究人员之间的互动，一个蛋白质组学和生物物理学核心，包括质谱分析的专家支持，以及一个动物核心，将提供最高水平的动物生产，程序和护理。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.