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Background and Objectives: Variants of the apolipoprotein E (APOE) gene are the greatest known risk factors for sporadic Alzheimer disease (AD). Three major APOE isoform alleles, ϵ2, ϵ3, and ϵ4, encode and produce proteins that differ by only 1-2 amino acids but have different binding partner interactions. Whereas APOE ϵ2 is protective against AD relative to ϵ3, ϵ4 is associated with an increased risk for AD development. However, the role of APOE in 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 whether APOE genotype affects EV components in AD brains, bdEVs were separated from patients with AD with different APOE genotypes for parallel small RNA and protein profile. Methods: bdEVs from late-stage AD brains (BRAAK stages 5-6) from patients with APOE genotypes ϵ2/3 (n = 5), ϵ3/3 (n = 5), ϵ3/4 (n = 6), and ϵ4/4 (n = 6) were separated using our published protocol into a 10,000g pelleted 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. Results: Comparing APOE genotypes, 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 different APOE genotypes. 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 (ϵ4/4 and ϵ2/3). Bioinformatic analysis and previous publications indicate a potential regulatory role of these molecules in AD. Discussion: For patients with late-stage AD in this study, only a few moderate differences were observed for small RNA and protein profiles between APOE genotypes. 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.

RNA-seq is increasingly employed for quantitative profiling of small RNAs (e.g., microRNAs, piRNAs, snoRNAs) in diverse sample types including isolated cells, tissues and cell-free biofluids. The accuracy and reproducibility of the multiple small RNA-seq library preparation methods in use, however, have not been systematically assessed. We report systematic results obtained by a consortium of nine labs that independently sequenced reference, ‘ground truth’, samples of synthetic small RNAs and human plasma-derived RNA. Three commercially available library preparation methods employing adapters of defined sequence and six methods using adapters with degenerate bases were assessed. Both protocol- and sequence-specific biases were identified, including biases that reduce the ability of small RNA-seq to accurately measure adenosine-to-inosine editing in microRNAs. We report that these biases were mitigated by library preparation methods that incorporate adapters with degenerate bases. MicroRNA relative quantification between samples using small RNA-seq was found to be accurate and reproducible across laboratories and methods.

© 2018, © 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of The International Society for Extracellular Vesicles. The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.