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International audience; In vertebrates, intercellular communication is largely mediated by connexins (Cx), a family of structurally related transmembrane proteins that assemble to form hemichannels (HCs) at the plasma membrane. HCs are upregulated in different brain disorders and represent innovative therapeutic targets. Identifying modulators of Cx-based HCs is of great interest to better understand their function and define new treatments. In this study, we developed automated versions of two different cell-based assays to identify new pharmacological modulators of Cx43-HCs. As HCs remain mostly closed under physiological conditions in cell culture, depletion of extracellular Ca 2+ was used to increase the probability of opening of HCs. The first assay follows the incorporation of a fluorescent dye, Yo-Pro, by real-time imaging, while the second is based on the quenching of a fluorescent protein, YFP QL , by iodide after iodide uptake. These assays were then used to screen a collection of 2242 approved drugs and compounds under development. This study led to the identification of 11 candidate hits blocking Cx43-HC, active in the two assays, with 5 drugs active on HC but not on gap junction (GJ) activities. To our knowledge, this is the first screening on HC activity and our results suggest the potential of a new use of already approved drugs in central nervous system disorders with HC impairments.

Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU) disease, is unique amongst human pathogens in its capacity to produce a lipid toxin called mycolactone. While previous studies have demonstrated that bacterially-released mycolactone diffuses beyond infection foci, the spatiotemporal distribution of mycolactone remained largely unknown. Here, we used the zebrafish model to provide the first global kinetic analysis of mycolactone’s diffusion in vivo, and multicellular co-culture systems to address the critical question of the toxin’s access to the brain. Zebrafish larvae were injected with a fluorescent-derivative of mycolactone to visualize the in vivo diffusion of the toxin from the peripheral circulation. A rapid, body-wide distribution of mycolactone was observed, with selective accumulation in tissues near the injection site and brain, together with an important excretion through the gastro-intestinal tract. Our conclusion that mycolactone reached the central nervous system was reinforced by an in cellulo model of human blood brain barrier and a mouse model of M. ulcerans-infection. Here we show that mycolactone has a broad but heterogenous profile of distribution in vivo. Our investigations in vitro and in vivo support the view that a fraction of bacterially-produced mycolactone gains access to the central nervous system. The relative persistence of mycolactone in the bloodstream suggests that assays of circulating mycolactone are relevant for BU disease monitoring and treatment optimization., Author summary Mycolactone is the major virulence factor of Mycobacterium ulcerans, the human pathogen causing Buruli ulcer (BU) disease. While it is now established that mycolactone is able to diffuse from infected tissues to exert immunomodulatory and analgesic effects at the systemic level, the in vivo spatiotemporal distribution of mycolactone remained largely unknown. Here, using the zebrafish larva, we describe the spatiotemporal distribution of a fluorescent derivative of mycolactone in vivo. We show that fluorescent mycolactone quickly diffuses from the blood circulation into various organs, and accumulates in muscles and brain. Mycolactone’s diffusion into the central nervous system was further confirmed in the mouse model of M. ulcerans infection. We reported previously that mycolactone suppresses the production of inflammatory mediators by primary microglia at nanomolar concentrations in vitro [1]. In the present study, we provide evidence suggesting that bacterially-produced mycolactone is able to reach the brain. While additional in vivo investigations will be required, we can speculate that the amount of mycolactone reaching the brain in the context of M. ulcerans infection is sufficient to modulate inflammation and pain transmission.

TFEB is a mammalian transcription factor that binds directly to the CLEAR consensus sequence (5'-GTCACGTGAC-3') present in the regulatory regions of genes inducing autophagosome formation, autophagosome-lysosome fusion, hydrolase enzyme expression, and lysosomal exocytosis. By modulating these activities, TFEB coordinates on-demand control over each cell's degradation pathway. Thus, a nuclear signaling pathway regulates cellular energy metabolism through TFEB. Our growing understanding of the role of TFEB and CLEAR in the promotion of healthy clearance together with in vitro and in vivo preclinical findings in various animal models of disease supports the conclusion that the pharmacological activation of TFEB could clear toxic proteins to treat both rare and common forms of neurodegenerative disease.

Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in South East Asia. It has been suggested that JEV gets access to the central nervous system (CNS) as a consequence of a preceding inflammatory process which leads to the blood-brain barrier (BBB) disruption and viral neuroinvasion. However, what happens at early times of JEV contact with the BBB is poorly understood. In the present work, we evaluated the ability of both a virulent and a vaccine strain of JEV (JEV RP9 and SA14-14-2, respectively) to cross anin cellulohuman BBB model consisting of hCMEC/D3 human endothelial cells cultivated on permeable inserts above SK-N-SH human neuroblastoma cells. Using this system, we demonstrated that both JEV RP9 and SA14-14-2 are able to cross the BBB without disrupting it at early times post-addition. Furthermore, this BBB model was able to discriminate between the virulent RP9 and the vaccine SA14-14-2 strains, as demonstrated by the presence of almost 10 times more RP9 infectious particles that crossed the BBB than SA14-14 particles at a high MOI. Besides contributing to the understanding of early events in JEV neuroinvasion, thisin celluloBBB model represents a suitable and useful system to study the viral determinants of JEV neuroinvasiveness and the molecular mechanisms by which this flavivirus crosses the BBB at early times of neuroinvasion.

International audience; The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the barrier and the brain parenchyma is still an unmet need. To fill this gap, we designed a 2D in cellulo model, the BBB-Minibrain, by combining a polyester porous membrane culture insert human BBB model with a Minibrain formed by a tri-culture of human brain cells (neurons, astrocytes and microglial cells). The BBB-Minibrain allowed us to test the transport of a neuroprotective drug candidate (e.g., Neurovita), through the BBB, to determine the specific targeting of this molecule to neurons and to show that the neuroprotective property of the drug was preserved after the drug had crossed the BBB. We have also demonstrated that BBB-Minibrain constitutes an interesting model to detect the passage of virus particles across the endothelial cells barrier and to monitor the infection of the Minibrain by neuroinvasive virus particles. The BBB-Minibrain is a reliable system, easy to handle for researcher trained in cell culture technology and predictive of the brain cells phenotypes after treatment or insult. The interest of such in cellulo testing would be twofold: introducing derisking steps early in the drug development on the one hand and reducing the use of animal testing on the other hand.

The intracellular calcium concentration ([Ca(2+)](i)) is an important factor determining the permeability of endothelial barriers including the blood-brain barrier (BBB). However, nothing is known concerning the effect of spatially propagated intercellular Ca(2+) waves (ICWs). The propagation of ICWs relies in large part on channels formed by connexins that are present in endothelia. We hypothesized that ICWs may result in a strong disturbance of endothelial function, because the [Ca(2+)](i) changes are coordinated and involve multiple cells. Thus, we aimed to investigate the effect of ICWs on endothelial permeability. ICW activity was triggered in immortalized and primary brain endothelial cells by lowering the extracellular Ca(2+) concentration. Low extracellular Ca(2+) increased the endothelial permeability and this was significantly suppressed by buffering [Ca(2+)](i) with BAPTA-AM, indicating a central role of [Ca(2+)](i) changes. The endothelial permeability increase was furthermore inhibited by the connexin channel blocking peptide Gap27, which also blocked the ICWs, and by inhibiting protein kinase C (PKC), Ca(2+)/calmodulin-dependent kinase II (CaMKII) and actomyosin contraction. We compared these observations with the [Ca(2+)](i) changes and permeability alterations provoked by the inflammatory agent bradykinin (BK), which triggers oscillatory [Ca(2+)](i) changes without wave activity. BK-associated [Ca(2+)](i) changes and the endothelial permeability increase were significantly smaller than those associated with ICWs, and the permeability increase was not influenced by inhibition of PKC, CaMKII or actomyosin contraction. We conclude that ICWs significantly increase endothelial permeability and therefore, the connexins that underlie wave propagation form an interesting target to limit BBB alterations. This article is part of a Special Issue entitled Electrical Synapses.

International audience; Located at the level of brain capillaries, the blood–brain barrier (BBB) is a crucial component of the neurovascular unit, since its highly regulated properties are needed to maintain optimal conditions for proper neuronal and glial functions. By modelling the BBB it is possible to make predictions about whether a compound’s interaction with the BBB is likely to compromise its functionality. A dysfunctional BBB may either affect brain entry of an agent or indirectly generate unwanted effects on neurons and glial cells by disturbing the brain homeostasis.Since the BBB controls the exchanges between the blood and brain compartments modelling the BBB in vitro can also help to investigate the ability of compounds to cross the BBB. In this chapter, the plethora of in vitro BBB models that exist today is discussed and several methods needed to set up and use of these in vitro models in the framework of in vitro toxicity study is detailed.

International audience; Historically, the focus has been to use in vitro BBB models to optimize rate of drug delivery to the CNS, whereas total in vivo brain/plasma ratios have been used for optimizing extent. However, these two parameters do not necessarily show good correlations with receptor occupancy data or other pharmacological readouts. In line with the free drug hypothesis, the use of unbound brain concentrations (Cu,br) has been shown to provide the best correlations with pharmacological data. However, typically the determination of this parameter requires microdialysis, a technique not ideally suited for screening in early drug development. Alternative, and less resource-demanding methodologies to determine Cu,br employ either equilibrium dialysis of brain homogenates or incubations of brain slices in buffer to determine fraction unbound brain (fu,br), which is subsequently multiplied by the total brain concentration to yield Cu,br. To determine Cu,br/Cu,pl ratios this way, still requires both in vitro and in vivo experiments that are quite time consuming. The main objective of this study was to explore the possibility to directly generate Cu,br/Cu,pl ratios in a single in vitro model of the BBB, using a co-culture of brain capillary endothelial and glial cells in an attempt to mimick the in vivo situation, thereby greatly simplifying existing experimental procedures. Comparison to microdialysis brain concentration profiles demonstrates the possibility to estimate brain exposure over time in the BBB model. A stronger correlation was found between in vitro Cu,br/Cu,pl ratios and in vivo Cu,br/Cu,pl obtained using fu,br from brain slice than with fu,br from brain homogenate for a set of 30 drugs. Overall, Cu,br/Cu,pl ratios were successfully predicted in vitro for 88% of the 92 studied compounds. This result supports the possibility to use this methodology for identifying compounds with a desirable in vivo response in the CNS early on in the drug discovery process.

The cytoplasmic Ca2+ concentration ([Ca2+]i) is an important factor determining the functional state of blood-brain barrier (BBB) endothelial cells but little is known on the effect of dynamic [Ca2+]i changes on BBB function. We applied different agonists that trigger [Ca2+]i oscillations and determined the involvement of connexin channels and subsequent effects on endothelial permeability in immortalized and primary brain endothelial cells. The inflammatory peptide bradykinin (BK) triggered [Ca2+]i oscillations and increased endothelial permeability. The latter was prevented by buffering [Ca2+]i with BAPTA, indicating that [Ca2+]i oscillations are crucial in the permeability changes. Bradykinin-triggered [Ca2+]i oscillations were inhibited by interfering with connexin channels, making use of carbenoxolone, Gap27, a peptide blocker of connexin channels, and Cx37/43 knockdown. Gap27 inhibition of the oscillations was rapid (within minutes) and work with connexin hemichannel-permeable dyes indicated hemichannel opening and purinergic signaling in response to stimulation with BK. Moreover, Gap27 inhibited the BK-triggered endothelial permeability increase in in vitro and in vivo experiments. By contrast, [Ca2+]i oscillations provoked by exposure to adenosine 5’ triphosphate (ATP) were not affected by carbenoxolone or Gap27 and ATP did not disturb endothelial permeability. We conclude that interfering with endothelial connexin hemichannels is a novel approach to limiting BBB-permeability alterations.