Summary Alzheimer’s disease (AD) and other dementias are a looming public health crisis in the US which is expected to generate catastrophic healthcare and economic impacts over the next decades. Despite enormous efforts made in AD research, current treatments provide only marginal benefits in the clinic. Many studies on early AD have found certain regions of the brain are more vulnerable to degeneration than others. But the investigation into the molecular mechanisms behind such selective degeneration is challenging and is hampered by the complexity of cellular machinery and heterogeneity. Although the sequencing based single-cell transcriptomics can address this challenge to certain degree, the conclusions usually still need validation at the protein level. Since most cellular functions, drug targets, and clinical diagnosis are based on protein signaling and biomarkers, the development of a counterpart functional proteomics technology would be beneficial to provide another perspective more directly relevant to therapeutics. Besides, surveying a large panel of proteins at the omics level is necessary since the cohort of proteins critical to AD pathogenesis remains elusive. In this project, we will develop a single-cell functional proteomics tool that could complement single-cell sequencing by measuring 300 critical proteins relevant to neurodegeneration. This novel technology will increase the coverage of functional proteome by 10-100 times over prevailing technologies, and it will take biomedical research broadly to a new level. This tool, in tandem with in vivo microPET imaging and in vitro specimen imaging, will facilitate identification of the biomarkers and regulatory networks pertinent to the subpopulations of brain cells that are vulnerable or resistant to neurodegeneration and amyloid beta toxicity. The proposed aims are (1) Establish single-cell reiterative MIST technology for analyzing 300 intracellular and surface proteins, and optimize the assay conditions by testing on a mouse cell line, and (2) Determine the molecular markers and regulatory networks of vulnerable versus resistant brain cells using single-cell reiterative MIST technology, microPET and degeneration imaging on an AD mouse model. The success of this project will generate innovative technology and methods that enable deep investigation of AD pathogenesis from a new, clinically important perspective, and it will lay the foundation for further brain-wide study of AD development and identification of drug targets.