High-throughput mapping of selectively vulnerable cell types and projections in aging and Alzheimer's Disease

摘要 该应用程序的长期目标是使用具有成本效益的高吞吐量方法来识别细胞类型 以及地图预测，这些预测在衰老和阿尔茨海默病(AD)的进展中选择性地脆弱。 它是为了检验这样一种假设，即在神经解剖学和神经学中存在同时或连续的脆弱性 衰老和AD发展过程中关键阶段的蛋白质相互作用网络/信号通路。大号 大量证据表明，阿尔茨海默病是一种异质性、多因素的疾病，它选择性地影响 大脑的某些区域，如内嗅皮层(EC)，而其他区域，如小脑，保留 不受影响。最近对AD神经病理学分期的研究表明，AD相关的神经病理始于 蓝斑(LC)或EC，然后是海马区(HC)，然后是前额叶皮质(PFC)。 但细胞类型及其相关的投射和分子/信号通路选择性地易受 单个神经元的水平还没有被很好地理解。衰老是AD的主要危险因素。因此，重要的是要 了解进展过程中是否存在不同、相似或重叠的选择性漏洞 在衰老和阿尔茨海默病之间。神经元投射仅使用整体标记法进行系统的标测 技术，例如染料示踪剂，它模糊了混合的单个神经元的不同投射。这一大堆 获得全脑连接的标记法需要大量的动物，而且成本低 生产量大、劳动强度大、成本高。最近，MAPseq(多路投影分析) 来自一个或多个脑核/来源的测序)方法已被开发来绘制 通过用随机RNA序列标记大组神经元来标记数千甚至数百万个单个神经元 (“条形码”)在一个大脑中。轴突被填充条形码mRNA，每个假定的投射区域被解剖， 并提取条形码mRNA并进行测序。此外，每个条形码神经元还标有 GFP，使单个神经元的荧光激活细胞分选能够获得单细胞(NC)RNAseq 转录信息，允许细胞类型分类和生化/信号分析 小路。对这一MAPseq和scRNAseq的纵向研究将提供前所未有的数据 告知引起或促成衰老和阿尔茨海默病恶化的原因。多源 将使用MAPseq和NC-RNAseq来绘制投影图并在四个区域中识别细胞类型 脑区，即LC、EC、HC和PFC。LC全程提供主要的去甲肾上腺素能输入。 整个大脑。EC向HC提供关键的皮质输入，而HC在学习记忆中是必不可少的。PFC 对包括学习和记忆在内的各种高阶功能提供自上而下的调节。都是男性 雌性野生型小鼠和AD小鼠的APPNLF系，它携带敲门人瑞典人和伊比利亚人 淀粉样前体蛋白的突变，重要的是，表达生理水平的Aβ，并表现出 将使用与年龄相关的神经病理和认知障碍，模拟晚发性AD。

Abstract The long-term goal of this application is to use a cost effective high-throughput approach to identify cell types and map projections that are selectively vulnerable in the progression of aging and Alzheimer's Disease (AD). It is to test the hypothesis that there are concurrent or sequential vulnerabilities in neuroanatomical and protein-interacting network/signaling pathways at critical stages in the progression of aging and AD. A large body of evidence demonstrates that AD is a heterogeneous, multifactorial disease that selectively affects certain regions of the brain, e.g. the entorhinal cortex (EC), while other areas, such as the cerebellum, remain unaffected. Recent studies on the staging of AD neuropathology showed AD-related neuropathology begins in the locus coeruleus (LC) or the EC, followed by the hippocampus (HC) and then the prefrontal cortex (PFC). But cell types, their associated projections and molecular/signaling pathways that are selectively vulnerable at the single neurons level are not well understood. Aging is a major risk factor for AD. Thus, it is important to understand whether there are distinct, similar or overlapping selective vulnerabilities in the progression between aging and AD. Neuronal projections have only been systematically mapped using bulk labeling techniques, e.g., dye tracers, which obscure the diverse projections of intermingled single neurons. This bulk labeling approach to obtain whole brain connectomes requires a large number of animals and is low throughput, labor intensive and expensive. Recently, the MAPseq (Multiplexed Analysis of Projections by Sequencing) approach from one or multiple brain nuclei/sources has been developed to map the projections of thousands or even millions of single neurons by labeling large sets of neurons with random RNA sequences (``barcodes'') in a single brain. Axons are filled with barcode mRNA, each putative projection area is dissected, and the barcode mRNA is extracted and sequenced. Furthermore, each barcoded neuron is also labeled with GFP, enabling fluorescence-activated cell sorting of individual neurons for single cell (nc) RNAseq to obtain transcriptomic information that allows cell type classification and the analysis of biochemical/signaling pathways. A longitudinal study with this MAPseq coupled with scRNAseq will provide unprecedented data informing the cause for the initiation of or contribution to the exacerbation of aging and AD. The multiple source MAPseq coupled with nc-RNAseq will be employed to map the projections and to identify cell types in four brain regions, namely the LC, EC, HC and PFC. The LC provides the major noradrenergic inputs throughout the entire brain. The EC provides key cortical inputs to the HC, which is essential in learning memory. The PFC provides the top-down regulation on various higher order functions, including learning and memory. Both male and female wild-type mice and the APPNLF line of AD mouse, which carries knockin human Swedish and Iberia mutations in the amyloid precursor protein and, importantly, expresses physiological levels of Aβ and exhibited age-related neuropathology and cognitive impairment, mimicking late onset AD, will be used.