Immuno-digital invasive cleavage assay for analyzing Alzheimer’s amyloid ß-bound extracellular vesicles

Abstract

Background: The protracted preclinical stage of Alzheimer’s disease (AD) provides the opportunity for early intervention to prevent the disease; however, the lack of minimally invasive and easily detectable biomarkers and their measurement technologies remain unresolved. Extracellular vesicles (EVs) are nanosized membrane vesicles released from a variety of cells and play important roles in cell–cell communication. Neuron-derived and ganglioside-enriched EVs capture amyloid-ß protein, a major AD agent, and transport it into glial cells for degradation; this suggests that EVs influence Aß accumulation in the brain. EV heterogeneity, however, requires the use of a highly sensitive technique for measuring specific EVs in biofluid. In this study, immuno-digital invasive cleavage assay (idICA) was developed for quantitating target-intact EVs. Methods: EVs were captured onto ganglioside GM1-specific cholera toxin B subunit (CTB)-conjugated magnetic beads and detected with a DNA oligonucleotide-labeled Aß antibody. Fluorescence signals for individual EVs were then counted using an invasive cleavage assay (ICA). This idICA examines the Aß-bound and GM1-containing EVs isolated from the culture supernatant of human APP-overexpressing N2a (APP-N2a) cells and APP transgenic mice sera. Results: The idICA quantitatively detected Aß-bound and GM1-containing EVs isolated from culture supernatants of APP-N2a cells and sera of AD model mice. The idICA levels of Aß-associated EVs in blood gradually increased from 3- to 12-month-old mice, corresponding to the progression of Aß accumulations in the brain of AD model mice. Conclusions: The present findings suggest that peripheral EVs harboring Aß and GM1 reflect Aß burden in mice. The idICA is a valuable tool for easy quantitative detection of EVs as an accessible biomarker for preclinical AD diagnosis.