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

摘要 本申请的长期目标是使用具有成本效益的高通量方法来鉴定细胞类型 并绘制在衰老和阿尔茨海默病（AD）进展中选择性脆弱的预测。 这是为了检验假设，有并发或顺序的弱点，在神经解剖和 蛋白质相互作用网络/信号通路在衰老和AD进展的关键阶段。大 大量证据表明，AD是一种异质性、多因素疾病， 大脑的某些区域，例如内嗅皮层（EC），而其他区域，例如小脑，仍然存在 不受影响最近关于AD神经病理学分期的研究表明，AD相关的神经病理学开始于 蓝斑（LC）或EC，接着是海马（HC），然后是前额叶皮质（PFC）。 但是，细胞类型，它们的相关投射和分子/信号通路， 单个神经元水平还没有很好地理解。衰老是AD的主要危险因素。因此必须 了解是否有不同的，类似的或重叠的选择性弱点，在进展 和AD之间的联系神经元投射仅使用批量标记系统地映射 技术，例如，染料示踪剂，掩盖了混杂的单个神经元的不同投射。该散装 获得全脑连接体的标记方法需要大量的动物， 生产量大、劳动密集且昂贵。MAPseq（Multiplexed Analysis of Projections） 已经开发了来自一个或多个脑核/源的神经元（例如，神经元、神经元、细胞核/细胞核/ 通过用随机RNA序列标记大量神经元， （"条码“）在一个单一的大脑。轴突充满条形码mRNA，解剖每个假定的投射区域， 提取条码mRNA并测序。此外，每个条形码化神经元还标记有 GFP，使得单个神经元的荧光激活细胞分选能够用于单细胞（nc）RNAseq，以获得 转录组信息，允许细胞类型分类和生化/信号转导分析 途径。这种MAPseq与scRNAseq结合的纵向研究将提供前所未有的数据 告知引发或促成衰老和AD恶化的原因。多源 MAPseq与nc-RNAseq结合将用于绘制投影图，并在四个细胞中鉴定细胞类型。 脑区，即LC，EC，HC和PFC。LC提供整个过程中的主要去甲肾上腺素能输入 整个大脑。EC为HC提供关键的皮层输入，这在学习记忆中是必不可少的。的PFC 提供了自上而下的调节各种高阶功能，包括学习和记忆。男性 雌性野生型小鼠和AD小鼠的APPNLF系，其携带敲入的人Swedish和伊比利亚 淀粉样前体蛋白的突变，重要的是，表达生理水平的Aβ， 将使用与年龄相关的神经病理学和认知障碍，模拟迟发性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.