Alzheimer's disease (AD) is a major threat to health of older individuals and is a looming public health disaster. Promised solutions in diagnostics and therapeutics will come only through research. Project 2 proposes to gain biological and medical insights into the proteins discovered in the Center by multiplex mapping to specific CSF extracellular vesicles (EV) and lipoproteins (LP), and to specific cells and subcellular structures in specific regions of brain. Proteins in CSF remain the best performing biomarkers for AD. CSF proteins exist in various forms: free in solution, on a unique class of LP, and on EV that include exosomes, microvesicles, and apoptotic bodies. LP and EV have varying mechanisms of production and biological functions. Localization and relative quantification of proteins of interest discovered in Project 1 to each class of CSF particles will provide powerful insight into cell of origin, cellular biology, and potential medical meaning. Further biological and medical insights can be gained by carefully mapping proteins in human brain. Indeed, the power of multiplexed imaging is its ability to reveal co-localization or mutual exclusivity, and to infer regulatory roles and gain mechanistic insight. For example, we think very differently about the biological roles of proteins restricted in their expression to synapses vs. mitochondria, glutamatergic vs. GABA-ergic neurons, or hippocampal pyramidal neurons vs. striatal medium spiny neurons. In pathologic states, co-localization with hallmark pathologic structures (major protein) highlights pathways of injury and response to injury: senile plaques (amyloid beta peptides), neurofibrillary degeneration (paired helical filament-tau), Lewy bodies and neurites (phospho-alpha-synuclein), and phospho-TDP-43 inclusions. We will test the hypothesis that multiplex analysis of CSF particles as well as subcellular, cellular, and regional mapping will provide key biological and medical insights into CSF proteins discovered by de novo proteomic analysis of CSF in Project 1. The same probes will be applied to CSF LP and EV using flow cytometry, and to brain regions using Multiplexed Ion Beam Imaging (MIBI).