Low-Sample Input miRNA Profiling in Extracellular Vesicles-Enriched Plasma Fractions from Older Adults With/out Cognitive Impairment

The RNA cargo of extracellular vesicles (EVs) has been investigated to search for circulating biomarkers possibly associated with a given disease. In this direction, vesicular small RNAs like microRNAs (miRNAs) showed great potential e.g., in monitoring neurodegenerative diseases and their clinical signs. In this exploratory cross-sectional study, we looked for miRNAs possibly associated with age-related cognitive impairment. Using a high processivity and sensitive OpenArray technology, we analyzed the expression of 223 miRNAs in plasmatic total EV-enriched fractions from 43 subjects older than 65 years who received neuropsychological examination. In a smaller cohort of 17 subjects, we verified any miRNA discrepancies between EV-associated and unfractionated plasma samples. A set of candidate miRNAs showed reduced expression in EV-associated fractions from cognitively impaired (n. 12) compared to cognitively preserved (n. 23) subjects. In particular, the expression of 15 of them (miR-106b-5p, miR-128-3p, miR-15a-5p, miR-16-5p, miR-17-5p, miR-191-5p, miR-192-5p, miR-199a-3p, miR-221-3p, miR-222-3p, miR-223-3p, miR-22-3p, miR-26a-5p, miR-335-5p) correlated with the MMSE score. Pathways of neurodegeneration (ND), such as PI3K-Akt, Ras/MAPK, and Wnt signaling, resulted significantly involved. Furthermore, 18 miRNAs were implicated in metal ion binding processes, thus supporting the key role of this molecular mechanism in ND-associated cognitive dysfunction. The paired analysis of EV-plasma samples reported the overall upregulation of most of the 223 miRNAs in plasma samples, whereas miR-425-5p, miR-5035-3p, miR-192-5p, miR-181a-3p, miR-338-3p and miR-1271-5p were almost stable in the two fractions and miR-205-5p and miR-28-5p were significantly upregulated in EVs compared to plasma. In our view, these findings support the potential relevance of vesicular-associated miRNAs as candidates of age-related cognitive dysfunctions, although for clinical practice some methodological issues may arise, i.e., the feasibility of total EVs extraction. Further confirmations are needed, e.g., the complete characterization of EVs’ cargo and origin, and validation in larger samples.