Plasma Exosomal miRNAs in Persons with and without Alzheimer Disease: Altered Expression and Prospects for Biomarkers

Abstract

To assess the value of exosomal miRNAs as biomarkers for Alzheimer disease (AD), the expression of microRNAs was measured in a plasma fraction enriched in exosomes by differential centrifugation, using Illumina deep sequencing. Samples from 35 persons with a clinical diagnosis of AD dementia were compared to 35 age and sex matched controls. Although these samples contained less than 0.1 microgram of total RNA, deep sequencing gave reliable and informative results. Twenty miRNAs showed significant differences in the AD group in initial screening (miR-23b-3p, miR-24-3p, miR-29b-3p, miR-125b-5p, miR-138-5p, miR-139-5p, miR-141-3p, miR-150-5p, miR-152-3p, miR-185-5p, miR-338-3p, miR-342-3p, miR-342-5p, miR-548at-5p, miR-659-5p, miR-3065-5p, miR-3613-3p, miR-3916, miR-4772-3p, miR-5001-3p), many of which satisfied additional biological and statistical criteria, and among which a panel of seven miRNAs were highly informative in a machine learning model for predicting AD status of individual samples with 83-89% accuracy. This performance is not due to over-fitting, because a) we used separate samples for training and testing, and b) similar performance was achieved when tested on technical replicate data. Perhaps the most interesting single miRNA was miR-342-3p, which was a) expressed in the AD group at about 60% of control levels, b) highly correlated with several of the other miRNAs that were significantly down-regulated in AD, and c) was also reported to be down-regulated in AD in two previous studies. The findings warrant replication and follow-up with a larger cohort of patients and controls who have been carefully characterized in terms of cognitive and imaging data, other biomarkers (e.g., CSF amyloid and tau levels) and risk factors (e.g., apoE4 status), and who are sampled repeatedly over time. Integrating miRNA expression data with other data is likely to provide informative and robust biomarkers in Alzheimer disease.

Fig 1. Expression of Alix in whole plasma vs. P3 fraction.

Fractions were prepared from normal mouse and human plasma, and equal amounts of protein were loaded for immunoblotting using anti-Alix antibody (see Methods). The P3 fractions were positive for Alix and were enriched relative to whole plasma.

Fig 2. Distribution of fold-changes across mature miRNA loci.

This figure shows the mean fold-change (i.e., the AD/control ratio) for mature miRNA loci in the filtered dataset (all sequences aligning to a given locus were summed up to give one value per locus). Shown are only those miRNAs which expressed mean counts of at least 3.6 in the control group (this threshold removes the lowest 25% of loci; low expressing miRNAs were removed to reduce noise and ensure that the ratios are robust). The distribution roughly follows a normal curve, with approximately equal numbers of miRNAs up and down across groups, and most miRNAs showing fold-changes of 2-fold or less. Note that the fold-change is displayed on a log scale (e.g., a value of 1 represents a 10-fold increase and a value of -1 represents a 10-fold decrease).