Extracellular vesicle transport across the BBB

The brain is a highly sensitive organ, which is protected from noxious agents circulating in the blood stream by the so-called blood-brain barrier (BBB). The BBB also ensures a steady and adequate supply of the required nutrients to the brain cells. The main component of the BBB in most vertebrates and in humans are the brain endothelial cells, which line the inside of the blood capillaries in the brain. This specialized cell layer is characterized by extremely tight cell-cell junctions and strictly regulates the passage of ions, molecules, vesicles, pathogens and (immune) cells from the bloodstream into the brain. The control and regulation of these transport mechanisms is essential for brain function, and aberrations of them can be a trigger or consequence of common diseases such as Alzheimer`s disease and stroke. The molecular mechanisms that transport key molecules such as glucose, amino acids or transferrin across the BBB have been studied in recent decades. Significant efforts were taken to exploit these transport mechanisms for the delivery of drugs into the central nervous system. Nonetheless, most pharmaceutical compounds fail to reach the diseased target area in the brain in sufficient quantities due the restrictions set by the BBB. A transport pathway which remains elusive yet mediates the exchange of extracellular vesicles (EVs) between blood stream and brain. Since several years there is evidence that EVs are able to cross the BBB in both directions, but no molecular mechanisms or determinants have been identified so far. In the present project, we will use a variety of different experimental approaches to identify the molecular mechanisms and associated cellular factors involved in EV transport across the BBB. Among others, we will employ sophisticated in vitro cell culture models of the BBB based on human induced pluripotent stem cells, high-resolution microscopy techniques and state-of-the-art proteomic analysis. To investigate disease-relevant changes in EV transport mechanisms, EVs derived from selected tumor cells or stroke models will also be applied and studied. The obtained data will expand the overall understanding of BBB function, and may have relevance to brain diseases, as well as future diagnostic and therapeutic developments.

The blood-brain barrier (BBB) is essential for brain function and physiology. Specialized endothelial cells regulate the transport of nutrients, metabolites and other molecules between the blood stream and the brain. In addition the BBB represents a protective layer for the central nervous system, preventing that noxious agents or pathogens reach the sensitive neuronal cells. Many cell types in the human body release small bubbles - so called extracellular vesicles (EVs) - which contain bio-active molecules for cellular communication. Several studies have proposed that EVs can cross the BBB in both directions, but no details about the mechanisms have been revealed yet. In this study in vitro BBB cell culture models were used to determine the quantity of EVs crossing the BBB and to uncover potential molecular pathways mediating the transport of EVs. In an extensive series of experiments, including a systematic test of numerous parameters and conditions in different BBB model systems, EV transport across intact BBB layers remained below detection limits, although the uptake of EVs by endothelial cells was confirmed by different methods, including flow cytometry and high resolution microscopy. The uptake of EVs could be blocked with pathway-specific agents, indicating that EVs are indeed internalized by BBB cells via distinct molecular mechanisms, i.e. macropinocytosis and caveolae-mediated endocytosis. Time series and live cell microscopy revealed that the vast majority of internalized EVs is targeted to lysomal structures and most EVs likely get degraded by the cells. Whether the cargo of the EVs is degraded as well, or released in the cell, repackaged or released from the cells is currently unknown. The latter could explain the evidence of detected brain-derived EV cargo in the peripheral blood stream, and vice versa, whereas permeation of intact EVs across the BBB remains unconfirmed. Additional BBB setups combining brain endothelial cells and astrocytes, were used to simulate ischemic stroke conditions by hypoxic treatment and depletion of glucose. Under such stress conditions, which led to gene expression changes and BBB leakage, an increase of EV secretion was observed, but transport of vesicles across the BBB was undetectable. In order to gain more insights into the molecular composition of a selected panel of tumor cell EVs and to identify potential interaction partners mediating the uptake of EVs by brain endothelial cells, a novel in vitro biotinylation technique was established. Mass spectrometry identified a large number of common and tumor-specific proteins present in EVs, which are subject of further investigations. Overall, in this study it could be shown that EVs released by various cell types can be internalized by brain endothelial cells, but rather than being transported across the BBB, the majority of EVs seem to be degraded in the lysomal pathway of the cells.