Distinguishing Microglial Exosomes from Monocytes & Macrophages to Support a Novel Approach for Detecting Neuroinflammation

Neuroinflammation contributes significantly to the pathogenesis of Alzheimer’s and Parkinson’s diseases. However, the inflammatory pathways contributing to neurodegeneration are not well understood. Moreover, there is a need to identify changes in inflammatory signaling that may occur early in disease progression to identify potential targets for therapeutic intervention. An important step towards addressing this need is understanding how the extracellular vesicles (EVs) released by microglia can be detected in the periphery. For microglia, phagocytic macrophages, and CD 14+ monocytes share many genes and membrane- bound proteins, and there is currently no method to distinguish microglia EVs from those generated by macrophages or monocytes. Therefore, this study aims to identify membrane-bound proteins unique to microglia EVs to enable their reliable isolation. Liquid-chromatography tandem mass spectrometry analysis was used to detect proteins in the EVs from both normal and disease-associated human stem-cell differentiated microglia (iMGL), and human induced pluripotent stem cell-derived CD 14+ monocytes and macrophages. We identified 23 proteins unique to the microglial EVs, eight of which localize to the membrane and may be potential targets for isolation. This investigation also used RNA sequencing to gain insight into the contents of DAM-like and control iMGL EVs and of microglia and white blood cells in Alzheimer’s disease. We propose that the contents of microglial EVs isolated from peripheral compartments will provide crucial insight for understanding the current inflammatory state of CNS microglia. This approach could provide a means to track changes in microglial activation over time, which is critical for understanding the progression of neuroinflammatory diseases like Alzheimer's and Parkinson's. Additionally, it may offer insights into potential therapeutic targets for modulating neuroinflammation.