Extracellular Vesicles (EVs) as “A Window to the Brain”: Potential, Challenges and Future Perspectives

Extracellular vesicles (EVs) are spherical, membrane-bound, nano-sized particles released from nearly all cell types under physiological and pathological conditions and are known to mediate intercellular crosstalk. These vesicles are enclosed by a phospholipid bilayer membrane and may range in size between 30–1000 nm [1]. Depending on the size, EVs are classified as exosomes, microvesicles, and apoptotic bodies. The knowledge behind the secretion of EVs was unknown until studies in the 1980s reported the discovery of a complex pathway in which intracellularly formed vesicles were secreted through multivesicular endosomes (MVE) [1]. This finding led to a series of studies that eventually improved the knowledge of the biogenesis of EVs. It is now known that the combination of early endosomes leads to the formation of late endosomes via the maturation process. Then intraluminal vesicles are formed due to luminal invagination of the endosomal membrane, leading to the formation of MVEs. These MVEs fuse with the plasma membrane to release the intraluminal vesicles as exosomes. Microvesicles are smaller EVs derived from the plasma membrane through budding and fission [1].

EVs can be secreted in huge numbers by every type of brain cell, including neurons, astrocytes, oligodendrocytes and microglia. They mediate interneuronal and glial-neuron crosstalk, modulate synaptic plasticity and survival of neurons, regulate myelination, and manage immune and stress responses [2]. The EV cargo comprises of diverse biomolecules like proteins, nucleic acids (mRNA, miRNAs, ncRNAs, DNA) and metabolites. The composition and contents of EVs may change with diseases, which can provide useful information on the communication of brain with other organs and tissues [3]. The cargo diversity and the ability of EVs to diffuse from release site and freely transfer across blood–brain-barrier (BBB) on either side have made them ideal candidates to be explored for their potential as non-invasive, circulating biomarkers of psychiatric disorders. Extracellular vesicles can thus be considered as “a window to the brain”. With this background, recent studies have tried to implement multi-omics profiling approaches on extracellular vesicles, isolated from various cohorts of psychiatric patients suffering from mood disorders, to unravel molecular signatures that may help in patient stratification, treatment prediction and identification of drug targets.

Although several research studies have focussed on identifying biomarkers of mood disorders and have reported association of several markers; but, a candidate biomarker(s) that can aid in the diagnosis, treatment and prognosis of the conditions remains unidentified. Few studies in recent years have suggested the potential role of EV protein cargo as novel biomarkers of Major Depressive Disorder (MDD). Hayakawa et al. (2018) observed increased IL34/CD81 in Neuron Derived Extracellular Vesicles (NDEVs) of MDD patients and recommended it as a biomarker (4). In another study, the levels of Brain derived Neurotrophic Factor (BDNF) and pro-BDNF within EVs obtained from serum of MDD patients were lower and higher respectively, when compared to controls, which reversed with antidepressant treatment. The authors suggested that EVs may help in the modulation of BDNF function by aiding in its movement across BBB [5]. Higher levels of Insulin Receptor Substrate-1 (IRS-1) in brain derived EVs of MDD patients compared to control subjects have been observed which may provide a link towards understanding the occurrence of Insulin resistance and defects of insulin signalling in MDD patients [6]. Differential protein expression in circulating EVs are also being increasingly studied in other mood disorders. Ranganathan et al. (2022) demonstrated elevated levels of glial fibrillary acid protein and decreased alpha-II-spectrin expression in the plasma-derived exosomes of schizophrenia (SCZ) patients, indicating an underlying astrocytic pathology in such patients [7]. Studies have investigated the role of brain insulin signalling pathways and insulin resistance as an underlying pathophysiological mechanism causing development of SCZ and bipolar disorder (BD). Markers of insulin pathway from plasma-derived neuronal EVs can thus be used for early diagnosis and therapeutic intervention in these disorders [8].Among the nucleic acid cargo, EV miRNAs have been explored as potential markers of MDD. The miR-139-5p has been identified as a top differentially expressed miRNA from genome-wide miRNA expression profiling of EVs derived from the blood of naïve MDD patients and healthy controls. This miRNA also exhibited good performance in discriminating between MDD and SCZ [9]. These findings have been replicated by Xie et al. (2021), who also reported higher levels of miR-139-5p in serum derived EVs of MDD patients, indicating its potential role as a biomarker of MDD [10]. Similarly, miR-206 was identified as the top differentially expressed miRNA from serum-derived EVs of SCZ patients, upregulation of which was linked to BDNF malfunction [11]. Many independent studies explored other miRs such as – 137, – 497, – 223, etc. in relation to SCZ. The miR-185-5p and -29c (upregulated), mir-484 and -142-3p (downregulated) are found to be differentially expressed in the RNA cargo of circulating EVs in BD patients [11]. So far, very few studies have assessed the metabolite cargo alterations in EVs of psychiatric patients, an area which requires urgent attention especially when findings suggests their potential as markers of other psychiatric disorders [12].

The technological advancements which have enabled studying the EV contents have opened several avenues for further research. However, basic EV research is still in its infancy. Studying of brain cell specific EVs and their cargo offers promising insights into brain physiology and pathology, however, its isolation and characterisation are a great challenge. International Society for Extracellular Vesicles laid down a minimum criteria that serve as a guidance when reporting EVs in the form of Minimal information for studies of extracellular vesicles (MISEV) 2018 guidelines. These guidelines helped to bring some uniformity and clarity into this comparatively novel field [13]. However, lack of defined protocols for cell-specific EV extraction, very small quantities of it circulating in body fluids, presence of similar character impurities, absence of normalisation and standardisation techniques, are a few key aspects among others that pose a significant challenge in establishing EVs as a potential marker for diseases.

The initial findings from studies that focussed on the potential of EVs and their cargo as markers of psychiatric disorders are quite promising. Future studies need to provide evidence that will help in understanding the specific roles of EVs in pathogenesis of such diseases. Intensive research is already been carried on neuron specific EVs that can determine neurodegenerative pathology years before its manifestation [14]. Researchers can focus on isolating, characterising, and studying brain derived EVs, that directly reflects the diseased state of the brain identifying specific molecular pathways and help in decreasing their global burden. Usage of more advanced technologies for EV cargo analysis may unravel novel information which may establish the role of EVs as novel biomarkers of brain diseases. Further, study of brain derived EVs from saliva, a non-invasive procedure, would be more compliant and can revolutionise management of such diseases [15]. Additionally, the integration of findings obtained from EV research from different –omics technologies with findings of neuroimaging and psychiatric evaluations along with genetic variation may hold immense potential towards the initiation of an era of extracellular vesicle (EV)-based brain “liquid biopsy” in precision medicine.