Identification of state‐specific proteomic characteristics of microglia‐derived exosomes

Alzheimer's disease (AD) is the most common neurodegenerative disorder defined by progressive pathological protein aggregation (amyloid-beta and tau) and deterioration of cognitive function. Microglia-mediated neuroinflammation is a key pathological component of AD; however, there are critical gaps in our understanding of how microglia perpetuate AD pathology. One proposed mechanism of microglia-mediated neuroinflammation and neurodegeneration is exosome release because of their critical role in the transport of macromolecules between cells to facilitate intercellular communication. Thus, it is possible that microglia-derived exosomes transfer pathogenic cargo which could perpetuate AD pathology. The proteomic profiles and influence of different microglia-derived exosomal populations on AD pathology remain unknown. We hypothesize that different microglia states determine the molecular composition of exosomes.We treated a murine microglia cell line, BV2 cells, with various cytokines to polarize them and collect their exosomes for downstream mass spectrometry (MS) analyses. Three groups of BV2 cells (n=4/group) were treated with either lipopolysaccharide (LPS) to polarize to a pro-inflammatory state, interleukin 10 (IL-10) to polarize to an anti-inflammatory state, or transforming growth factor beta (TGF-β) to polarize to a homeostatic state. Untreated BV2 cells served as a control group. Following 72 hours of treatment, BV2 cells were lysed and cell culture media was collected for exosome isolation.Transmission electron microscopy images and western blotting for exosomal marker, CD9, confirmed exosome purification by our isolation method. In MS studies, we identified 533 proteins in exosome fractions and 1,866 proteins in BV2 cell proteomes. We found that known exosome related proteins, Sdcbp and Igsf8, were significantly increased in the exosomal proteome compared to the whole cell proteome. We identified proteins that are differentially expressed across polarization with unique proteins increased in exosomes derived from LPS, IL-10, and TGF-β treated BV2 cells. Validation studies of these top hits are ongoing, and studies are underway to replicate these findings in primary microglia.Our results indicate that exosomes derived from microglia adopt distinct state-associated protein profiles which may have differential effects on other cell types. This work will guide future studies concerning the role of exosomal cargo in perpetuating AD pathology.