PROJECT SUMMARY/ABSTRACT Alzheimer’s disease (AD) afflicts millions of Americans, yet no effective treatments exist. Iron has been shown to be involved in key AD pathologic processes, including amyloid and tau aggregation, inflammation, oxidative stress, and cell death mechanisms. Despite this growing evidence, it is challenging to ascertain alterations in iron metabolism in vivo, limiting potential translation to biomarkers and novel therapies. Exosomes are nanometer-sized vesicles shed by cells to transport proteins, nucleic acids, metals, lipids or metabolites. While exosomes reflect cellular processes and can reveal disease-related pathologies in human tissues and biofluids, iron abnormalities in AD exosomes have not yet been investigated. We will address this knowledge gap through state-of-the-art exosome isolation technology combined with advanced iron imaging, protein quantification and next generation sequencing methods. Our goal is to investigate iron dysregulation in exosomes from post- mortem AD brains, in order to unveil AD-specific biomarkers and facilitate the development of novel therapies. The project aims are: (1) To determine whether the quantity, oxidation state, and cellular origin of exosomal iron is altered in AD. Using MRI and synchrotron X-ray microscopy, we will quantify tissue iron content and oxidation state in human AD and control hippocampal specimens. We will then use our novel exosome isolation platform, ExoTIC, to isolate exosomes from regions of high hippocampal iron content in the same specimens. Using antibodies that target cell-surface proteins, we will enrich the isolated exosomes based on their cellular origin (e.g. neurons, microglia, etc.). We will quantify exosomal iron content from each cell type using mass spectrometry (ICP-MS), and measure exosomal iron oxidation state using electron microscopy. Taken together, we will determine whether iron content and oxidation state are altered in Alzheimer’s exosomes compared to controls, in particular in exosomes originating in microglia, the brain’s immune cells. (2) Detect dysregulation of iron-related proteins and RNAs in AD exosomes. Using Western blotting on the enriched exosomes, we will determine whether levels of proteins that play a role in iron metabolism are altered in AD compared to controls. Because exosomes are generally rich in microRNAs that are known to regulate gene expression, we will use RNA-Seq to determine whether exosomal microRNAs regulating these same iron-related proteins are also altered in AD. Machine learning algorithms will enable the creation of an atlas of microRNAs linking iron, iron-related proteins, and neuropathology, which should provide a deeper understanding of AD biology. Characterization of exosome content in the AD brain should result in cell-specific signatures of iron dysregulation associated with neurodegeneration. This approach may elucidate novel aspects of AD biology, lead to novel assays to detect early AD, and fa...