Mapping Cellular Resolution Connectopathies in Aging and Alzheimer's Disease

项目摘要/摘要 这项名为“绘制衰老和阿尔茨海默氏病的细胞分辨率连接病变图”的计划将 系统和全面地表征细胞类型、特定的解剖和分子表型 内嗅皮层衰老与阿尔茨海默病(AD)：AD病理学的起点。我们会 对耳鼻喉科投射神经元的细胞分辨率、年龄相关的形态分子表型进行分析 2M、9M、18M APPSAA-(KI/KI)雄性和雌性小鼠的单核RNA测序和甲基组分析。 新的遗传稀疏标记将用于标记和表征大鼠耳鼻咽喉锥体神经元的形态 APPSAA-(KI/KI)/MORF3/Cux2-Creer和APPSAA-(KI/KI)/MORF3/ETVF1-Creer小鼠的不同皮层。 这些研究将提供关于分子定义的耳鼻咽喉管神经细胞类型如何与 年龄、性别和β病理导致进行性转录/表观基因组、形态和突触 体内的缺陷。此外，我们还将绘制与年龄相关的ENT投射的形态分子表型图 人源化Tau模型中的神经元MAPT(H1)-GR*N279K及其MAPT(H1)对照。一个组合的单人- 核转录和全基因组染色质可及性分析将应用于MAPT(H1)- GR*N279K和MAPT(H1)雄性和雌性小鼠在2M、9M和18M定义整合 转录/表观基因组ENT神经细胞类型，并鉴定经历年龄依赖的神经细胞亚群 突变的人源化Tau小鼠模型中的多模式分子失调。RNAScope现场多路传输 将进行杂交和GeoMX数字空间剖面图分析，以确定形态分子类型 MAPT(H1)-GR*N279K敲入小鼠与MAPT(H1)小鼠耳鼻喉部受影响最大的神经元的比较 在衰老过程中。为了识别与年龄相关的连接漏洞并绘制AD中的连接中断情况，我们 将系统地量化由遗传和连接定义的耳鼻喉癌引起的轴突输出的变化 使用2M、9M、18M雄性和雌性Cux2-Creer和ETV1-Creer小鼠的细胞类型。特定于单元类型的连接 中断也将在两个下一代AD鼠标模型中进行检查，APP敲入(APPSAA-KI/KI WITH 野生型对照)和MAPT(H1)-GR*N279K[有MAPT(H1)对照]，不同年龄和性别。小说 病毒稀疏标记将用于表征年龄和AD相关的轴突营养不良，而遗传稀疏 在新产生的MORF3小鼠系中进行标记将有助于识别耳鼻喉癌细胞的局部形态变化 类型。将研究与年龄和AD相关的对ENT输入神经元的形态损害以及它们的 突触对不同类型的耳鼻喉癌细胞的破坏。最后，我们将建立一个基于云的可视化平台 将衰老和阿尔茨海默病的完整分子解剖回路缺陷映射到Allen公共坐标 为数据的传播和分析提供便利的框架。尽管当前项目的重点是 Ent，为数据生产、收集和分析建立的管道可以向上扩展以识别全脑范围的 衰老和其他迟发性阿尔茨海默病小鼠模型中的细胞型特定解剖-分子缺陷。

PROJECT SUMMARY/ABSTRACT The proposed project, “Mapping Cellular Resolution Connectopathies in Aging and Alzheimer's Disease,” will systematically and comprehensively characterize cell-type specific anatomical and molecular phenotypes across aging and Alzheimer’s disease (AD) in the entorhinal cortex (ENT): the ground zero of AD pathology. We will map cellular resolution, age-dependent morpho-molecular phenotypes of ENT projection neurons by performing single-nucleus RNA-sequencing and methylomic analysis in 2m, 9m, 18m APPSAA-(KI/KI) male and female mice. Novel genetic sparse labeling will be used to label and characterize morphology of ENT pyramidal neurons in different cortical layers in APPSAA-(KI/KI)/MORF3/Cux2-CreER and APPSAA-(KI/KI)/MORF3/Etv1-CreER mice. These studies will provide comprehensive data on how molecularly defined ENT neuronal cell types interact with age-, sex- and Aβ pathology to confer progressive transcriptomic/epigenomic, morphological, and synaptic deficits in vivo. In addition, we will map the age-dependent morpho-molecular phenotypes of ENT projection neurons in humanized Tau models, MAPT(H1)-GR*N279K and their MAPT(H1) controls. A combined single- nucleus transcriptomics and genome-wide chromatin accessibility assays will be applied to MAPT(H1)- GR*N279K and MAPT(H1) male and female mice at 2m, 9m, and 18m to define integrated transcriptomic/epigenomic ENT neuronal cell types, and to identify neuronal subsets undergoing age-dependent multi-modal molecular dysregulation in mutant “humanized” Tau mouse models. RNAscope multiplex in situ hybridization and GeoMX digital spatial profiling analyses will be performed to identify morpho-molecular types of neurons in ENT that are most affected in MAPT(H1)-GR*N279K knock-in mice compared to MAPT(H1) mice during aging. To identify age-related connectional vulnerabilities and to map connectivity disruptions in AD, we will systematically quantify changes of axonal outputs arising from genetically and connectionally defined ENT cell types using 2m, 9m, 18m male and female Cux2-CreER and Etv1-CreER mice. Cell-type specific connectivity disruptions also will be examined in two next generation AD mouse models, APP knock-in (APPSAA-KI/KI with wildtype controls) and MAPT(H1)-GR*N279K [with MAPT(H1) controls], across ages and in both sexes. Novel viral sparse labeling will be used to characterize age- and AD-related axonal dystrophy, while genetic sparse labeling in newly generated MORF3 mouse lines will help to identify local morphological changes in ENT cell types. Age- and AD-related morphological compromises to ENT input neurons will be studied along with their synaptic disruptions onto different ENT cell types. Finally, we will establish a cloud-based visualization platform to map the integrated molecular-anatomic circuit deficits of aging and AD to the Allen Common Coordinate Framework to facilitate dissemination and analysis of the data. Although the focus of the current project is the ENT, the pipelines established for data production, collection, and analysis can be scaled up to identify brainwide cell-type specific anatomic-molecular deficits in aging and other late-onset AD mouse models.