Novel method for collecting hippocampal interstitial fluid extracellular vesicles (EV^ISF) reveals sex-dependent changes in microglial EV proteome in response to Aβ pathology

Brain-derived extracellular vesicles (EVs) play an active role in Alzheimer's disease (AD), relaying important physiological information about their host tissues. The internal cargo of EVs is protected from degradation, making EVs attractive AD biomarkers. However, it is unclear how circulating EVs relate to EVs isolated from disease-vulnerable brain regions. We developed a novel method for collecting EVs from the hippocampal interstitial fluid (ISF) of live mice. EVs (EV^ISF) were isolated via ultracentrifugation and characterized by nanoparticle tracking analysis, immunogold labelling, and flow cytometry. Mass spectrometry and proteomic analyses were performed on EV^ISF cargo. EV^ISF were 40–150 nm in size and expressed CD63, CD9, and CD81. Using a model of cerebral amyloidosis (e.g., APPswe, PSEN1dE9 mice), we found protein concentration increased but protein diversity decreased with Aβ deposition. Genotype, age, and Aβ deposition modulated proteostasis- and immunometabolic-related pathways. Changes in the microglial EV^ISF proteome were sexually dimorphic and associated with a differential response of plaque associated microglia. We found that female APP/PS1 mice have more amyloid plaques, less plaque associated microglia, and a less robust- and diverse- EV^ISF microglial proteome. Thus, in vivo microdialysis is a novel technique for collecting EV^ISF and offers a unique opportunity to explore the role of EVs in AD.