Mapping Cellular Resolution Connectopathies in Aging and Alzheimer's Disease

项目概要/摘要 拟议中的项目，"映射衰老和阿尔茨海默病中的细胞分辨率连接病"，将 系统、全面地表征细胞类型特异性解剖和分子表型 内嗅皮层（ENT）中的衰老和阿尔茨海默病（AD）：AD病理学的起点。我们将 绘制ENT投射神经元的细胞分辨率、年龄依赖性形态分子表型， 在2m、9m、18m APPSAA-（KI/KI）雄性和雌性小鼠中进行单核RNA测序和甲基化组学分析。 新的遗传稀疏标记将用于标记和表征ENT锥体神经元的形态， APPSAA-（KI/KI）/MORF3/Cux2-CreER和APPSAA-（KI/KI）/MORF3/Etv1-CreER小鼠的不同皮质层。 这些研究将提供关于分子定义的ENT神经元细胞类型如何与 年龄、性别和A β病理学赋予进行性转录组学/表观基因组学、形态学和突触 体内缺陷。此外，我们将绘制年龄依赖性的ENT投射的形态分子表型 在人源化Tau模型、MAPT（H1）-GR * N279K及其MAPT（H1）对照中的神经元中的神经元。一个组合的单- 核转录组学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2个月、9个月和18个月时， 转录组/表观基因组ENT神经元细胞类型，并鉴定经历年龄依赖性的神经元亚群 在突变型"人源化" Tau小鼠模型中的多模式分子失调。原位多重RNAscope 将进行杂交和GeoMX数字空间剖面分析，以确定形态分子类型 与MAPT（H1）小鼠相比，MAPT（H1）-GR * N279 K基因敲入小鼠中ENT神经元受影响最大 在老化过程中。为了识别与年龄相关的连接漏洞并绘制AD中的连接中断，我们 将系统地量化由遗传和连接定义的ENT引起的轴突输出的变化 细胞类型，使用2m、9m、18m雄性和雌性Cux2-CreER和Etv1-CreER小鼠。细胞类型特异性连接 还将在两种下一代AD小鼠模型中检查破坏，APP敲入（APPSAA-KI/KI， 野生型对照）和MAPT（H1）-GR * N279 K [与MAPT（H1）对照]的差异。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，而遗传稀疏标记将用于表征年龄和AD相关的轴突营养不良。 在新产生的MORF 3小鼠系中进行标记将有助于鉴定ENT细胞中的局部形态学变化 类型将沿着研究年龄和AD相关的对ENT输入神经元的形态学损害， 不同类型的ENT细胞上的突触破坏。最后，我们将建立一个基于云的可视化平台 将衰老和AD的整合分子解剖回路缺陷映射到艾伦共同坐标 便利数据传播和分析的框架。虽然当前项目的重点是 ENT，为数据生产、收集和分析建立的管道可以扩大规模，以识别全脑范围的 细胞类型特异性解剖分子缺陷在衰老和其他迟发性AD小鼠模型。

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.