Structural characterization of A-beta strain variation in AD mouse models

摘要 阿尔茨海默病(AD)中结构不同的淀粉样β蛋白(Aβ)聚集体(株)可以 潜在地解释了疾病进展和严重程度上的差异。由人造纤维形成的纤维 体外培养的Aβ和从患者脑提取液中播种的Aβ纤维导致了各种Aβ菌株。之前的研究包括 AD脑植入材料导致了Aβ结构的变化，这种结构随患者和疾病阶段的不同而变化，提示 这种特异的Aβ菌株不仅可能影响AD的进展，还可能影响潜在的治疗方法。然而，a 使用患者种子合成Aβ的偏见是，种子可能会选择种子最高的Aβ菌株 潜在的掩盖了其他可能在疾病中发挥重要作用的菌株。我们的长期目标是了解 神经退行性疾病中重要的几种病理蛋白的菌株变异基础，如 α-突触核蛋白、亨廷顿蛋白和Aβ。这项应用的目的是确定体内产生的 在单个淀粉样鼠模型内和之间发现的Aβ菌株的结构，这将回答 以下问题：1)在单个小鼠身上发现的β斑块主要由一种菌株或 混合菌株？菌株是否取决于性别、大脑区域或所检查的小鼠模型？菌株怎么样了？ 被用人脑纤维播种的小鼠影响吗？2)播种实验捕捉到了 在AD大脑中发现的多样性，还是偏向于最有种子能力的物种？3)什么是 这些菌株之间的结构差异？我们将从以下三个具体目标来解决这些问题： 在目标1中，我们将直接检测Aβ株在淀粉样变性小鼠模型中的变化和分布。 这将通过测量从15N标记的脑提取液中提纯的固态核磁共振谱来实现 APPKINL-F、APPKINL-G-F和5XFAD小鼠。一组5XFAD小鼠将被接种AD患者脑提取液。 在目标2中，我们将从淀粉样蛋白小鼠模型中确定Aβ菌株的种子潜能。我们将播种 淀粉样变性小鼠模型脑提取液重组A-β的种植动力学研究 不同菌株的核磁共振谱，并与原始小鼠脑提取液和Aβ的核磁共振谱进行比较 从人类阿尔茨海默病脑提取液中提取。在目标3中，我们将确定Aβ菌株的碱基集合的结构 淀粉样蛋白小鼠模型。我们将使用创新的固态核磁共振和EPR方法来确定高- 捕获短期和远程订单详细信息的β的解析结构。这些措施的预期结果是什么 AIMS的目标是我们将在活体生成的Aβ聚集体上开创核磁共振波谱的先河。我们将绘制分布图 Aβ菌株在整个大脑中的分布，并确定菌株对大脑区域、性别、年龄和 老鼠模型。我们将通过组织学和生物化学将核磁共振结构与脑病理联系起来。我们 将开发一种EPR和固态核磁共振相结合的方法来确定纤维结构。这些结果 将使我们能够设计一种依赖于β菌株的AD诊断和治疗方法。

Abstract Structurally dissimilar aggregates (strains) of the amyloid beta peptide (Aβ) in Alzheimer's Disease (AD) can potentially explain differences seen in the progression and severity of the disease. Fibrils formed by synthetic Aβ in vitro and Aβ fibrils seeded from patient brain extracts led to a variety of Aβ strains. Previous research with AD brain seeded material led to Aβ structures that varied with patients and with the stage of disease, suggesting that specific Aβ strains not only could affect the progression of AD but also potential treatment. However, a bias using patient seeded synthetic Aβ is that seeding might select for those Aβ strains with the highest seeding potential masking other strains that could be important in the disease. Our long-term goal is to understand the basis of strain variation for several pathological proteins important in neurodegenerative disease such as tau, α-synuclein, huntingtin, and Aβ. The objective of this application is to determine the in vivo-generated structures of Aβ strains found within and between individual amyloid mouse models, which will answer the following questions: 1) Are Aβ plaques found in individual mice composed predominantly of one strain or mixtures of strains? Do strains depend on gender, brain region, or mouse model examined? How are strains impacted by seeding mice with fibrils from human brains? 2) Are seeding experiments capturing the structural variety found in AD brains or are they biased towards the most seeding competent species? 3) what are the structural differences between these strains? We will address these questions in the following 3 specific aims: In Aim 1, we will directly detect the Aβ strain variety and distribution in mouse models of amyloid pathology. This will be accomplished by measuring solid-state NMR spectra on brain extracts purified from 15N labeled APPKINL-F, APPKINL-G-F, and 5XFAD mice. A subset of 5XFAD mice will be seeded with AD patient brain extract. In Aim 2, we will determine the seeding potential of Aβ strains from amyloid mouse models. We will seed recombinant Aβ with brain extract from amyloid pathology mouse models, measure the seeding kinetics of different strains, and compare their NMR spectra to those of the original mouse brain extract and those of Aβ seeded from human AD brain extract. In Aim 3 we will determine the structures of a basis set of Aβ strains from amyloid mouse models. We will use an innovative solid-state NMR and EPR approach to determine high- resolution structures of Aβ that capture short and long-range order details. The expected outcome of these aims is that we will pioneer NMR spectroscopy on vivo-generated Aβ aggregates. We will map the distribution of Aβ strains throughout the brain, and determine the dependence of strains on brain region, gender, age, and mouse model. We will correlate the NMR structures with brain pathology by histology and biochemistry. We will develop a combined EPR and solid-state NMR approach for fibril structure determination. These outcomes will enable us to design Aβ strain dependent diagnostics and treatment for AD.