The construction of the blood–brain barrier (BBB), which is a natural barrier for maintaining brain homeostasis, is the result of a meticulous organisation in space and time of cell–cell communication processes between the endothelial cells that carry the BBB phenotype, the brain pericytes, the glial cells (mainly the astrocytes), and the neurons. The importance of these communications for the establishment, maturation and maintenance of this unique phenotype had already been suggested in the pioneering work to identify and demonstrate the BBB. As for the history of the BBB, the evolution of analytical techniques has allowed knowledge to evolve on the cell–cell communication pathways involved, as well as on the role played by the cells constituting the neurovascular unit in the maintenance of the BBB phenotype, and more particularly the brain pericytes. This review summarises the key points of the history of the BBB, from its origin to the current knowledge of its physiology, as well as the cell–cell communication pathways identified so far during its development, maintenance, and pathophysiological alteration.

Vesicular trafficking is essential for the cell to internalize useful proteins and soluble substances, for cell signaling or for the degradation of pathogenic elements such as bacteria or viruses. This vesicular trafficking also enables the cell to engage in secretory processes for the elimination of waste products or for the emission of intercellular communication vectors such as cytokines, chemokines and extracellular vesicles. Ras-related proteins (Rab) and their effector(s) are of crucial importance in all of these processes, and mutations/alterations to them have serious pathophysiological consequences. This review presents a non-exhaustive overview of the role of the major Rab involved in vesicular trafficking, with particular emphasis on their involvement in the biogenesis and secretion of extracellular vesicles, and on the role of Rab27 in various pathophysiological processes. Therefore, Rab and their effector(s) are central therapeutic targets, given their involvement in vesicular trafficking and their importance for cell physiology.

Within the neurovascular unit, brain pericytes (BPs) are of major importance for the induction and maintenance of the properties of the blood-brain barrier (BBB) carried by the brain microvessel endothelial cells (ECs). Throughout barriergenesis, ECs take advantage of soluble elements or contact with BPs to maintain BBB integrity and the regulation of their cellular homeostasis. However, very few studies have focused on the role of ECs in the maturation of BPs. The aim of this study is to shed light on the proteome of BPs solocultured (hBP-solo) or cocultured with ECs (hBP-coc) to model the human BBB in a non-contact manner. We first generated protein libraries for each condition and identified 2233 proteins in hBP-solo versus 2492 in hBP-coc and 2035 common proteins. We performed a quantification of the enriched proteins in each condition by sequential window acquisition of all theoretical mass spectra (SWATH) analysis. We found 51 proteins enriched in hBP-solo related to cell proliferation, contractility, adhesion and extracellular matrix element production, a protein pattern related to an immature cell. In contrast, 90 proteins are enriched in hBP-coc associated with a reduction in contractile activities as observed in vivo in ‘mature’ BPs, and a significant gain in different metabolic functions, particularly related to mitochondrial activities and sterol metabolism. This study highlights that BPs take advantage of ECs during barriergenesis to make a metabolic switch in favor of BBB homeostasis in vitro.

The concept of protein corona formation around extracellular vesicles (EVs) has given birth to new insights into how cells may recognize EVs by proteins presented at the EV surface. Here we present spatially resolved proteomics using the biotin ligase TurboID to map proteins interacting at the EV surface, without a need for physical isolation of the EV‐corona complexes. TurboID promiscuously biotinylates nearby proteins within a few nm distance from the fused ‘bait’ protein. We genetically engineered EVs by modifying CD63 as the bait protein to which TurboID was fused facing outward to map EV corona proteins. Biotinylated proteins were then analysed by Western blotting and liquid chromatography‐mass spectrometry. Western blots revealed protein patterns that are distinct depending on the localization of the fused TurboID. The mass spectrometry analysis identified many of the serum proteins commonly known to form a corona around a synthetic solid nanoparticle, also supporting those previously reported through the physical isolation approaches. Rather striking is, however, the EV corona footprint of endogenous proteins that tells us about the EV biogenesis and what the intrinsic endogenous corona might look like. This approach, which we coined EV‐SPEC (Spatial Proteomics of Endogenous Corona), has thus the potential to revolutionize our understanding of EV biology by shifting the focus from the EVs themselves to the proteins that make up the corona, or how the cell ‘sees’ them, in analogy with the biomolecular corona extensively characterized for synthetic nanoparticles.

La barrière hémato-encéphalique (BHE) protège le cerveau des éléments pathogènes et autres xénobiotiques circulants, et assure l’homéostasie cérébrale. Dès les premiers temps du développement embryonnaire, différents vecteurs de communication intercellulaire, chronologiques et hiérarchisés, s’installent entre (i) les progéniteurs endothéliaux qui porteront le futur phénotype BHE, et (ii) les péricytes cérébraux, principaux inducteurs de ce phénotype. Parmi ces vecteurs figurent les facteurs solubles sécrétés par les cellules, et les exosomes pour véhiculer des molécules inductrices. L’objectif de ce projet de thèse est de caractériser dans un premier temps la part et les mécanismes d’induction du phénotype BHE in vitro par les exosomes de PCs en contexte physiologique. La composition de ces exosomes sera analysée par approches omiques. De plus, étant donné que la composition des exosomes varie en contexte pathologique, des exosomes provenant de PCs traités avec diverses cytokines pro-inflammatoires, connues pour prendre part aux processus pathologiques de maladies neurodégénératives, seront également caractérisés en termes de composition et d’impact sur le phénotype BHE. Ces travaux devraient permettre de mettre en évidence la communication bidirectionnelle entre les cellules endothéliales et les péricytes cérébraux, ainsi que d’envisager les exosomes de péricytes cérébraux comme régulateurs de la BHE en contexte physiopathologique, et comme potentiels biomarqueurs pathologiques.