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.

PROJECT NARRATIVE Alzheimer’s Disease (AD) is the most common source of late-life dementia, as well as the third leading cause of death in the United States, and currently has no effective treatment. Leveraging the most advanced BRAIN Initiative neurotechnologies in circuit tracing, sparse neuronal labeling, single-cell molecular profiling, and informatics, we will identify cell-type-specific connectional and molecular disruptions across age and in two AD mouse models, and will develop an integrated informatics pipeline and web-based visualization portal for data sharing. Together, our study will provide a rich resource to study selective molecular, cellular, and circuitry vulnerabilities in AD, and to provide novel readouts in two next-generation AD mouse models to study disease mechanisms and to test candidate therapeutics.