Relationships of APOE Genotypes With Small RNA and Protein Cargo of Brain Tissue Extracellular Vesicles From Patients With Late-Stage AD

Abstract

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.

International Society for Extracellular Vesicles workshop. QuantitatEVs: Multiscale analyses, from bulk to single extracellular vesicle

“…During the discussion, it emerged that, despite the presence of various ATLASes dedicated to the EV field (EV‐TRACK Consortium et al., 2017; Griffiths‐Jones, 2006; Kalra et al., 2012; Keerthikumar et al., 2016; Kim et al., 2015; S. Li et al., 2018; J. R. Li et al., 2019; Liu et al., 2019; Lötvall et al., 2012; Murillo et al., 2019; Pisitkun et al., 2004; Russo et al., 2018; Wang et al., 2021; Hulstaert et al., 2020; also summarized and discussed in Mugoni et al., 2022. Additional ATLASes are presented in Huang et al., 2023; Lai et al., 2022; Xie et al., 2021; Bockorny et al., 2023; Cheung et al., 2016; Subramanian et al., 2015; Xu et al., 2020; Russo et al., 2012; Chandy et al., 2020) the lack of detailed annotation, updating, and standardization in such databases should be overcome to better interpret the results of bulk analyses on different material sources. This can be facilitated by detailed metadata standardization and annotation.…”

Survey of organ‐derived small extracellular vesicles and particles (sEVPs) to identify selective protein markers in mouse serum

“…To begin the isolation of sEVPs, the supernatant was centrifuged at 10,000 × g for 1 h at 4 • C to remove apoptotic bodies and microvesicles, and then passed through a 0.22-μm filter (Millipore Sigma, SLGS033SS) to remove debris. We used ultracentrifugation to isolate sEVPs after the removal of tissue debris and spun the supernatant at 100,000 × g for 2 h at 4 • C Crescitelli et al, 2021;Huang, Driedonks, Cheng, Rajapaksha, Turchinovich, et al, 2022;Saludas et al, 2022). The sEVPs in the resulting pellet were washed with filtered phosphate-buffered saline (FPBS) and re-ultracentrifuged at 100,000 × g for 1 h at 4 • C. It is important to note that further sorting of the isolated sEVPs (Crescitelli et al, 2021) by density gradient or size-exclusion chromatography was not performed to preserve the yield of EVPs for mass spectrometry (MS) analysis.…”

RNA landscapes of brain tissue and brain tissue-derived extracellular vesicles in simian immunodeficiency virus (SIV) infection and SIV-related central nervous system pathology

“…bdEVs were separated from frozen occipital cortex tissues using our published protocol with minor modifications (Huang et al, 2022a(Huang et al, , 2022b(Huang et al, , 2020a. Before extraction, a small (∼50 mg) piece of tissue was stored at -80°C for later RNA extraction from brain homogenate (BH).…”