Extracellular vesicles (EVs), including small EVs (sEVs) such as exosomes, exhibit great potential for the diagnosis and treatment of brain disorders, representing a valuable tool for precision medicine. The latter demands high-quality human biospecimens, especially in complex disorders in which pathological and specimen heterogeneity, as well as diverse individual clinical profile, often complicate the development of precision therapeutic schemes and patient-tailored treatments. Thus, the collection and characterization of physiologically relevant sEVs are of the utmost importance. However, standard brain EV isolation approaches rely on tissue dissociation, which can contaminate EV fractions with intracellular vesicles.
Based on multiscale analytical platforms such as cryo-EM, label-free proteomics, advanced flow cytometry, and ExoView analyses, University of Minho researchers compared and characterized the EV fraction isolated with this novel method with a classical digestion-based EV isolation procedure. Moreover, EV biogenesis was pharmacologically manipulated with either GW4869 or picrotoxin to assess the validity of the spontaneous-release method, while the injection of labelled-EVs into the mouse brain further supported the integrity of the isolated vesicles.
The researchers present an efficient purification method that captures a sEV-enriched population spontaneously released by mouse and human brain tissue. In addition, they tested the significance of the release method under conditions where biogenesis/secretion of sEVs was pharmacologically manipulated, as well as under animals’ exposure to chronic stress, a clinically relevant precipitant of brain pathologies, such as depression and Alzheimer’s disease. These findings show that the released method monitors the drug-evoked inhibition or enhancement of sEVs secretion while chronic stress induces the secretion of brain exosomes accompanied by memory loss and mood deficits suggesting a potential role of sEVs in the brain response to stress and related stress-driven brain pathology.
Biogenesis and isolation of extracellular vesicles from brain tissue
a Schematic representation of the biogenesis of three types of extracellular vesicles (EVs): microvesicles, apoptotic bodies, and exosomes. On the one hand, microvesicles arise from the budding of the plasma membrane, while apoptotic bodies originate from cell blebbing. On the other hand, exosomes are released upon multivesicular bodies (MVB) fusion with the plasma membrane. MVBs develop following invagination of early endosomes with intraluminal vesicles (ILVs). These ILVs can be degraded upon fusion with lysosomes or released as exosomes. MVB-derived exosome biogenesis can involve the ESCRT machinery or occur via an ESCRT-independent pathway. b Schematic representation of the main idea of the 2 methods compared in the current studies: the standard method which includes chemical and mechanical (e.g., papain) digestion of the brain tissue and the release method for brain sEVs collection, which isolates EVs that are spontaneously released by the brain tissue
Overall, the spontaneous release method of sEV yield may contribute to the characterization and biomarker profile of physiologically relevant brain-derived sEVs in brain function and pathology.