LSR/angulin-1 is a tricellular tight junction protein involved in blood-brain barrier formation.

The blood-brain barrier (BBB) is a term used to describe the unique properties of central nervous system (CNS) blood vessels. One important BBB property is the formation of a paracellular barrier made by tight junctions (TJs) between CNS endothelial cells (ECs). Here, we show that Lipolysis-stimulated lipoprotein receptor (LSR), a component of paracellular junctions at points in which three cell membranes meet, is greatly enriched in CNS ECs compared with ECs in other nonneural tissues. We demonstrate that LSR is specifically expressed at tricellular junctions and that its expression correlates with the onset of BBB formation during embryogenesis. We further demonstrate that the BBB does not seal during embryogenesis in Lsr knockout mice with a leakage to small molecules. Finally, in mouse models in which BBB was disrupted, including an experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis and a middle cerebral artery occlusion (MCAO) model of stroke, LSR was down-regulated, linking loss of LSR and pathological BBB leakage

Profiling the mouse brain endothelial transcriptome in health and disease models reveals a core blood-brain barrier dysfunction module.

Blood vessels in the CNS form a specialized and critical structure, the blood-brain barrier (BBB). We present a resource to understand the molecular mechanisms that regulate BBB function in health and dysfunction during disease. Using endothelial cell enrichment and RNA sequencing, we analyzed the gene expression of endothelial cells in mice, comparing brain endothelial cells with peripheral endothelial cells. We also assessed the regulation of CNS endothelial gene expression in models of stroke, multiple sclerosis, traumatic brain injury and seizure, each having profound BBB disruption. We found that although each is caused by a distinct trigger, they exhibit strikingly similar endothelial gene expression changes during BBB disruption, comprising a core BBB dysfunction module that shifts the CNS endothelial cells into a peripheral endothelial cell-like state. The identification of a common pathway for BBB dysfunction suggests that targeting therapeutic agents to limit it may be effective across multiple neurological disorders

Régulation de l'adressage et de la fonction du transporteur d'ammonium RhBG par phosphorylation et liaison à l'ankyrine G.

The human RhBG protein is a member of the Rh (Rhesus) family, first characterized gas channel in mammals, and hence of the Amt/Mep/Rh superfamily of ammonium transporters. RhBG has been shown to facilitate NH3 transport and to be anchored to the basolateral plasma membrane of kidney epithelial cells, via ankyrin-G. We showed here that mutation of the ankyrin-G binding site, localized in the C-terminal cytoplasmic domain, delayed cell surface expression, decreased plasma membrane stability and abolished NH3 transport function of RhBG in epithelial cell lines. We also demonstrated that tyrosine Y429, which belongs to the YED basolateral targeting signal of RhBG, was phosphorylated in vitro using purified Src and Syk kinases. Furthermore, incubation of HEK293 cells with an inhibitor of protein-tyrosine phosphatases, pervanadate, strongly decreased transport activity of native RhBG protein but had no effect on a Y429A mutant (blocking its phosphorylation). This result shows that Y429 is the only RhBG tyrosine residue to be involved in NH3 transport regulation, through phosphorylation, and that RhBG is phosphorylated ex vivo on this tyrosine. Then, we showed that Y429D and Y429E mutations, mimicking constitutive phosphorylation, abolished NH3 transport and enhanced solubilization of RhBG from the cell membrane. In contrast, the nonphosphorylated/ nonphosphorylatable Y429A and Y429F mutants behaved the same as wild-type RhBG. Conversely, confocal microscopy studies showed that Y429A or Y429F but not Y429E or Y429D mutations of residue 429 abolished the exclusive basolateral localization of RhBG in polarized epithelial cells. All these results demonstrate that targeting, anchoring to the membrane skeleton and ammonium transport activity of RhBG are regulated by both phosphorylation and membrane skeleton binding of its C-terminal cytoplasmic domain, and lead to a model in which phosphorylation of Y429, which belongs to the YED signal, would be necessary to RhBG targeting in polarized epithelial cells, whereas dephosphorylation of this tyrosine would allow RhBG anchorage to the spectrin-based skeleton, via ankyrin-G, and activation of NH3 channel through a possible conformational change of the C-terminal domain.La protéine RhBG humaine est un membre de la famille des protéines Rh (Rhésus), premiers canaux à gaz caractérisés chez les mammifères et par conséquent, de la superfamille Amt/Mep/Rh des transporteurs d'ammonium. Elle facilite le transport de la forme neutre de l'ammonium (NH3) et est ancrée à la membrane plasmique basolatérale de cellules épithéliales rénales, via l'ankyrine G, protéine adaptatrice du squelette membranaire dépendant de la spectrine. Nous avons montré que la mutation du motif d'ancrage à l'ankyrine G, localisé dans l'extrémité C-terminale intracytoplasmique de RhBG, retarde l'adressage, diminue la stabilité à la surface et abolit la fonction de transport de NH3 de la protéine RhBG dans les cellules épithéliales HEK293. Nous avons également montré que la tyrosine Y429, qui appartient au signal d'adressage basolatéral YED de RhBG, est phosphorylée in vitro par les kinases Src et Syk purifiées. D'autre part, le traitement de cellules HEK293 par le pervanadate, un inhibiteur de phosphatases sur phosphotyrosine (donc un activateur indirect des tyrosine kinases) diminue fortement l'activité de transport de la protéine native mais pas celle d'un mutant Y429A (bloquant sa phosphorylation). Ce résultat montre d'une part que Y429 est le seul résidu tyrosine de RhBG impliqué dans la régulation du transport de NH3, via une phosphorylation, d'autre part que la protéine RhBG est phosphorylée ex vivo sur cette tyrosine. Des mutations Y429D et Y429E, mimant une phosphorylation constitutive, abolissent le transport de NH3 et favorisent la solubilisation du RhBG membranaire. À l'inverse, des mutants Y429A et Y429F non phosphorylés et non phosphorylables se comportent comme la protéine RhBG sauvage. En revanche, des études en microscopie confocale montrent que seules les mutations Y429A et Y429F entraînent une dépolarisation de l'expression de RhBG dans des cellules MDCK polarisées, les mutants Y429D et Y429E étant normalement localisés dans le domaine basolatéral des cellules. L'ensemble de ces résultats démontre que l'adressage, l'ancrage au squelette sous-membranaire et la fonction de transport d'ammonium de RhBG sont régulés à la fois par la phosphorylation et la liaison au squelette membranaire de son domaine C-terminal cytoplasmique et nous a conduit à proposer un modèle dans lequel la phosphorylation de la tyrosine Y429 faisant partie du motif YED, serait nécessaire à l'adressage de RhBG vers la membrane basolatérale de cellules épithéliales polarisées, alors que la déphosphorylation de cette tyrosine permettrait l'ancrage de la protéine au squelette membranaire, via l'ankyrine G, et l'activation du canal à NH3 par un changement conformationnel potentiel de l'extrémité C-terminale

Genetic mouse models to study blood–brain barrier development and function

Abstract The blood–brain barrier (BBB) is a complex physiological structure formed by the blood vessels of the central nervous system (CNS) that tightly regulates the movement of substances between the blood and the neural tissue. Recently, the generation and analysis of different genetic mouse models has allowed for greater understanding of BBB development, how the barrier is regulated during health and its response to disease. Here we discuss: 1) Genetic mouse models that have been used to study the BBB, 2) Available mouse genetic tools that can aid in the study of the BBB, and 3) Potential tools that if generated could greatly aid in our understanding of the BBB

Régulation de l'adressage et de la fonction du transporteur d'ammonium RhBG par phosphorylation et liaison à l'ankyrine G

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Peripheral and central neuronal ATF3 precedes CD4+ T-cell infiltration in EAE.

Experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis produced by immunization with myelin oligodendrocyte glycoprotein (MOG) and adjuvants, results from profound T-cell mediated CNS demyelination. EAE is characterized by progressive, ascending motor dysfunction and symptoms of ongoing pain and hypersensitivity, in some cases preceding or concomitant with the motor deficits. In this regard, the EAE model mimics major features of multiple sclerosis, where a central neuropathic pain state is common. Although the latter condition is presumed to arise from a CNS loss of inhibitory controls secondary to the demyelination, dysfunction of sensory neurons may also contribute. Based on our previous studies that demonstrated the utility of monitoring expression of activating transcription factor 3 (ATF3), a sensitive marker of injured sensory neurons, here we followed both ATF3 and CD4+ T cells invasion of sensory ganglia (as well as the CNS) at different stages of the EAE model. We found that ATF3 is induced in peripheral sensory ganglia and brainstem well before the appearance of motor deficits. Unexpectedly, the ATF3 induction always preceded T cell infiltration, typically in adjacent, but non-overlapping regions. Surprisingly, control administration of the pertussis toxin and/or Complete Freund's adjuvants, without MOG, induced ATF3 in sensory neurons. In contrast, T cell infiltration only occurred with MOG. Taken together, our results suggest that the clinical manifestations in the EAE result not only from central demyelination but also from neuronal stress and subsequent pathophysiology of sensory neurons

Tissue kallikrein permits early renal adaptation to potassium load

Tissue kallikrein (TK) is a serine protease synthetized in renal tubular cells located upstream from the collecting duct where renal potassium balance is regulated. Because secretion of TK is promoted by K+ intake, we hypothesized that this enzyme might regulate plasma K+ concentration ([K+]). We showed in wild-type mice that renal K+ and TK excretion increase in parallel after a single meal, representing an acute K+ load, whereas aldosterone secretion is not modified. Using aldosterone synthase-deficient mice, we confirmed that the control of TK secretion is aldosterone-independent. Mice with TK gene disruption (TK−/−) were used to assess the impact of the enzyme on plasma [K+]. A single large feeding did not lead to any significant change in plasma [K+] in TK+/+, whereas TK−/− mice became hyperkalemic. We next examined the impact of TK disruption on K+ transport in isolated cortical collecting ducts (CCDs) microperfused in vitro. We found that CCDs isolated from TK−/− mice exhibit net transepithelial K+ absorption because of abnormal activation of the colonic H+,K+-ATPase in the intercalated cells. Finally, in CCDs isolated from TK−/− mice and microperfused in vitro, the addition of TK to the perfusate but not to the peritubular bath caused a 70% inhibition of H+,K+-ATPase activity. In conclusion, we have identified the serine protease TK as a unique kalliuretic factor that protects against hyperkalemia after a dietary K+ load