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

摘要 阿尔茨海默病（AD）中淀粉样β肽（Aβ）的结构上不同的聚集体（菌株）可以 这可能解释了疾病进展和严重程度的差异。合成纤维形成的原纤维 体外Aβ和从患者脑提取物中接种的Aβ原纤维导致多种Aβ菌株。以前的研究与 AD脑种植材料导致Aβ结构随患者和疾病阶段而变化，表明 特异性Aβ菌株不仅可以影响AD的进展，而且可能影响AD的治疗。但 使用患者接种的合成Aβ的偏倚是，接种可能选择接种量最高的Aβ菌株 可能掩盖了在疾病中可能重要的其他菌株。我们的长期目标是了解 在神经变性疾病中重要的几种病理蛋白质的菌株变异的基础， tau、α-突触核蛋白、亨廷顿蛋白和Aβ。本申请的目的是确定体内产生的 在单个淀粉样蛋白小鼠模型内部和之间发现的Aβ菌株的结构，这将回答 以下问题：1）在个体小鼠中发现的Aβ斑块是否主要由一种菌株组成， 菌株的混合物？品系是否取决于性别、大脑区域或所检查的小鼠模型？菌株怎么样 用人类大脑的纤维植入小鼠会产生什么影响2)播种实验是否捕捉到了 在AD大脑中发现的多样性，或者它们偏向于最有播种能力的物种？3)有哪些 这些菌株之间的结构差异我们将在以下三个具体目标中解决这些问题： 目的1：直接检测淀粉样蛋白病理模型小鼠中Aβ菌株的种类和分布。 这将通过测量从15 N标记的脑提取物中纯化的脑提取物的固态NMR光谱来实现。 APPKINL-F、APPKINL-G-F和5XFAD小鼠。5XFAD小鼠的子集将接种AD患者脑提取物。 在目标2中，我们将确定来自淀粉样蛋白小鼠模型的Aβ菌株的接种潜力。我们将播种 用来自淀粉样蛋白病理学小鼠模型的脑提取物重组Aβ，测量 不同的菌株，并将它们的NMR谱与原始小鼠脑提取物和Aβ的NMR谱进行比较 从人类AD脑提取物中接种。在目标3中，我们将确定Aβ菌株基组的结构， 淀粉样蛋白小鼠模型。我们将使用创新的固态NMR和EPR方法来确定高- Aβ的分辨率结构，捕获短和长范围的顺序细节。这些活动的预期成果 目的是我们将开创体内产生的Aβ聚集体的NMR光谱学。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 小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学联系起来。我们 将开发一种结合EPR和固态核磁共振的方法来确定原纤维结构。这些结果 将使我们能够设计Aβ菌株依赖性AD诊断和治疗。

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.