Abstract
Although an aging population has led to a growing socioeconomic burden of neurodegenerative diseases such as Alzheimer’s disease (AD), inadequate techniques for early diagnosis is a significant impediment to current understanding of pathophysiology and the development of effective treatments. Thus, there is a great need to discover non-invasive diagnostic biomarkers that can facilitate early intervention and improve prognosis for AD. Small extracellular vesicles (EVs), such as exosomes, are crucial mediators of intercellular communication present in peripheral circulation which may provide a more sensitive source of biomarkers to detect early stages of the disease or monitor disease progression. Micro-RNA (miRNA) cargo of EVs, mediators of gene expression that are dysregulated in disease, represent a promising target for biomarker discovery.
However, the difficulty in characterising and isolating specific EVs has limited the reproducibility, and the clinical utility of miRNA from EV's. In this study, a workflow was developed for selective isolation of circulating EVs using size exclusion chromatography (SEC) to assess expression of EV miRNA derived from plasma of males with Alzheimer's disease (AD) and cognitively healthy age-matched controls from a New Zealand cohort (Otago-AD, n=10), to assess whether this could be a non-invasive marker of AD.
Characterisation to establish the identity of vesicle preparations (dynamic light scattering, cryogenic electron microscopy, immunoblot analysis) confirmed that this optimized SEC protocol effectively isolated a population of homogenous small (60-70 nm) EVs that exhibited properties consistent with characteristic features of exosomes (size, morphology, biochemical composition). RNA cargo was then extracted from the EVs using a solid-phase separation column and miRNA profiling was performed to assess the relative expression of candidate miRNA, normalized to U6 snRNA.
Among six miRNAs evaluated by RT-qPCR, the expression of miR-342-3p was significantly downregulated in plasma-derived sEVs of AD patients. Interestingly, bioinformatic analysis revealed involvement of this miRNA in the p53 pathway, a critical signaling hub for the cellular stress response, that is altered in AD. This study provides crucial confirmation of miR-342-3p down-regulation in AD circulating sEVs and supports previous findings that the p53 pathway is impaired in AD. These findings not only contribute to our understanding of key components of AD pathology, but also provide a potential biomarker for diagnosis and a possible target for disease-modifying interventions, providing a foundation for subsequent research.