We aim to uncover metabolic and protein signaling pathways contributing to the regional vulnerability of neocortical pyramidal neurons in Alzheimer's disease and to identify novel targets for detection and intervention. We will compare the prefrontal cortex, a neocortical brain area afflicted by neuropathology early in Alzheimer's disease, with the primary visual cortex, a brain area that is relatively spared. We will use mass spectrometric imaging in combination with segmentation analyses, to identify spatial changes of small molecules and proteins in postmortem brain sections prepared from these neocortical areas from donors at various clinical and pathological stages of Alzheimer's disease compared to controls. Then, we will apply multiplexed immunofluorescence imaging on sequential sections from the same specimens, to obtain information on cellular and microenvironment changes in and around vulnerable neocortical neurons during disease progression, correlated with the clinical severity, degree and location of neuropathological changes, and the risk genotype. Further, we will register the data from both imaging techniques to identify metabolic pathways and protein signaling changes at the regional, laminar and cellular level and to locate covariation in molecular and cellular phenotypes contributing to Alzheimer's disease vulnerability. In addition to generating a comprehensive dataset of the cellular and molecular changes at various stages of Alzheimer's disease, these studies will involve the development, validation, and dissemination of novel tools for analysis of large datasets generated using two powerful imaging tools, one that detects hundreds of analytes with the possibility of detecting previously unknown contributors to disease and the other that provides higher resolution with a select set of known markers. In the long term, the data generated from these studies could provide the basis for testing novel disease- modifying treatments by cell-type specific targeting of identified metabolic pathways using experimental models, such as brain organoids to replicate cortical lamination with human neurons or humanized mouse chimeras to model interactions between neurons and non-neuronal cell types.