NOVEL METHOD FOR COLLECTING HIPPOCAMPAL INTERSTITIAL FLUID EXTRACELLULAR VESICLES (EVISF) IN A MOUSE MODEL OF ALZHEIMER’S DISEASE

Abstract

Extracellular vesicles (EVs) serve as promising biomarkers of neurodegenerative disorders, such as Alzheimer’s disease (AD), by revealing changes in proteostasis, neuroinflammation, and metabolism. Here, a novel method is described for collecting hippocampal interstitial fluid (ISF) EVs (EVISF) using in vivo microdialysis in the presence and absence of AD-related amyloid-β (Aβ) pathology. In vivo microdialysis was used to collect hippocampal ISF from 3- and 9-month-old, unanaesthetized, unrestrained, APPswe/PS1ΔE9 (APP/PS1), a mouse model of Aβ overexpression, and B6C3 wildtype (WT) mice. EVISF were isolated via ultracentrifugation then underwent nanoparticle tracking analysis, flow cytometry, and immunogold labeling. Mass spectrometry and proteomic analysis were performed on EVISF cargo and surface proteins. Hippocampal EVISF collected via in vivo microdialysis from APP/PS1 and WT mice were 40-150nm and CD63- and CD9-positive. While a subset of previously published EV protein datasets were present in EVISF, we identified novel proteins within this specific pool of EVs. We found sex-dependent changes in EVISF due to both age, genotype, and Aβ pathology. While we observed an increase in protein concentration within EVISF, we saw a marked decrease in EVISF protein diversity in both the core and on the surface of the EVs with Aβ deposition. Pathway analyses revealed changes in proteostasis, metabolism, and inflammation in EVISF with age and with the presence of Aβ pathology. Cell-type specific proteins were sex-dependently modulated in the core of EVISF and correlated with ISF Aβ40, ISF Aβ42, Aβ pathology, and glial activation. We developed a novel method for collecting hippocampal EVISF by utilizing in vivo microdialysis. Age, genotype, and Aβ pathology alter EVISF and their contents, most notably by modulating protein diversity. Further, cell-type specific proteins are altered in EVISF of APP/PS1 mice compared to WT. In vivo EVISF offer a unique opportunity to identify novel brain-derived AD biomarkers as well as to shed light on the role of EVISF in the AD-pathological cascade.