Background: Brain tissue-derived extracellular vesicles (bdEVs) play neurodegenerative and protective roles, including in Alzheimer’s disease (AD). Extracellular vesicles (EVs) may also leave the brain to betray the state of the CNS in the periphery. Only a few studies have profiled the proteome of bdEVs and source brain tissue. Additionally, studies focusing on bdEV cell type-specific surface markers are rare. Objective: We aimed to reveal the pathological mechanisms inside the brain by profiling the tissue and bdEV proteomes in AD patients. In addition, to indicate targets for capturing and molecular profiling of bdEVs in the periphery, CNS cell-specific markers were profiled on the intact bdEV surface. Methods: bdEVs were separated and followed by EV counting and sizing. Brain tissue and bdEVs from age-matched AD patients and controls were then proteomically profiled. Total tau (t-tau), phosphorylated tau (p-tau), and antioxidant peroxiredoxins (PRDX) 1 and 6 were measured by immunoassay in an independent bdEV separation. Neuron, microglia, astrocyte, and endothelia markers were detected on intact EVs by multiplexed ELISA. Results: Overall, concentration of recovered bdEVs was not affected by AD. Proteome differences between AD and control were more pronounced for bdEVs than for brain tissue. Levels of t-tau, p-tau, PRDX1, and PRDX6 were significantly elevated in AD bdEVs compared with controls. Release of certain cell-specific bdEV markers was increased in AD. Conclusion: Several bdEV proteins are involved in AD mechanisms and may be used for disease monitoring. The identified CNS cell markers may be useful tools for peripheral bdEV capture.

Background and ObjectivesVariants of the apolipoprotein E(APOE)gene are the greatest known risk factors for sporadic Alzheimer disease (AD). Three majorAPOEisoform alleles,ε2, ε3, andε4, encode and produce proteins that differ by only 1–2 amino acids but have different binding partner interactions. WhereasAPOE ε2is protective against AD relative toε3, ε4is associated with an increased risk for AD development. However, the role ofAPOEin gene regulation in AD pathogenesis has remained largely undetermined. Extracellular vesicles (EVs) are lipid bilayer–delimited particles released by cells to dispose of unwanted materials and mediate intercellular communication, and they are implicated in AD pathophysiology. Brain-derived EVs (bdEVs) could act locally in the tissue and reflect cellular changes. To reveal whetherAPOEgenotype affects EV components in AD brains, bdEVs were separated from patients with AD with differentAPOEgenotypes for parallel small RNA and protein profile.MethodsbdEVs from late-stage AD brains (BRAAK stages 5–6) from patients withAPOEgenotypesε2/3(n = 5),ε3/3(n = 5),ε3/4(n = 6), andε4/4(n = 6) were separated using our published protocol into a 10,000gpelleted extracellular fraction (10K) and a further purified EV fraction. Counting, sizing, and multiomic characterization by small RNA sequencing and proteomic analysis were performed for 10K, EVs, and source tissue.ResultsComparingAPOEgenotypes, no significant differences in bdEV total particle concentration or morphology were observed. Overall small RNA and protein profiles of 10K, EVs, and source tissue also did not differ substantially between differentAPOEgenotypes. However, several differences in individual RNAs (including miRNAs and tRNAs) and proteins in 10K and EVs were observed when comparing the highest and lowest risk groups(ε4/4andε2/3). Bioinformatic analysis and previous publications indicate a potential regulatory role of these molecules in AD.DiscussionFor patients with late-stage AD in this study, only a few moderate differences were observed for small RNA and protein profiles betweenAPOEgenotypes. Among these, several newly identified 10K and EV-associated molecules may play roles in AD progression. Possibly, larger genotype-related differences exist and are more apparent in or before earlier disease stages.