Bruno Saubaméa

Deletion of Astroglial Connexins Weakens the Blood–Brain Barrier

Astrocytes, the most prominent glial cell type in the brain, send specialized processes named endfeet, which enwrap blood vessels and express a large molecular repertoire dedicated to the physiology of the vascular system. One of the most striking properties of astrocyte endfeet is their enrichment in gap junction protein connexins 43 and 30 (Cx43 and Cx30) allowing for direct intercellular trafficking of ions and small signaling molecules through perivascular astroglial networks. The contribution of astroglial connexins to the physiology of the brain vascular system has never been addressed. Here, we show that Cx43 and Cx30 expression at the level of perivascular endfeet starts from postnatal days 2 and 12 and is fully mature at postnatal days 15 and 20, respectively, indicating that astroglial perivascular connectivity occurs and develops during postnatal blood–brain barrier (BBB) maturation. We demonstrate that mice lacking Cx30 and Cx43 in GFAP (glial fibrillary acidic protein)-positive cells display astrocyte endfeet edema and a partial loss of the astroglial water channel aquaporin-4 and β-dystroglycan, a transmembrane receptor anchoring astrocyte endfeet to the perivascular basal lamina. Furthermore, the absence of astroglial connexins weakens the BBB, which opens upon increased hydrostatic vascular pressure and shear stress. These results demonstrate that astroglial connexins are necessary to maintain BBB integrity.

Heterogeneity in the rat brain vasculature revealed by quantitative confocal analysis of endothelial barrier antigen and P-glycoprotein expression

While phenotypic endothelial heterogeneity is well documented in peripheral organs, it is only now being explored in the brain. We used confocal imaging of thick sections of rat brain to qualitatively and quantitatively examine the expression of two key markers of the blood—brain barrier (BBB) in the rat, P-glycoprotein (P-gp), and endothelial barrier antigen (EBA). We found that these markers were not uniformly distributed throughout the whole vasculature of the cortex and hippocampus. P-glycoprotein displayed a gradient of expression from an almost undetectable level in large penetrating arterioles to a high and uniform level in capillaries and venules. While EBA was lacking in all cerebral arterioles, regardless of their size, its expression varied greatly among endothelial cells in capillaries and venules, yielding a striking mosaic pattern. A detailed quantitative analysis of the distribution of these markers at the single cell level in capillaries is provided. These results challenge the view of a uniform BBB and suggest that regulatory mechanisms might differentially modulate BBB features not only among arterioles/capillaries/venules but also at the single cell level within the capillaries. Hypotheses are made regarding the underlying mechanisms and physiopathological consequences of this heterogeneity.

Opioid Transport by ATP-Binding Cassette Transporters at the Blood-Brain Barrier: Implications for Neuropsychopharmacology

Some of the ATP-binding cassette (ABC) transporters like P-glycoprotein (P-gp; ABCB1, MDR1), BCRP (ABCG2) and MRPs (ABCCs) that are present at the blood-brain barrier (BBB) influence the brain pharmacokinetics (PK) of their substrates by restricting their uptake or enhancing their clearance from the brain into the blood, which has consequences for their CNS pharmacodynamics (PD). Opioid drugs have been invaluable tools for understanding the PK-PD relationships of these ABC-transporters. The effects of morphine, methadone and loperamide on the CNS are modulated by P-gp. This review examines the ways in which other opioid drugs and some of their active metabolites interact with ABC transporters and suggests new mechanisms that may be involved in the variability of the response of the CNS to these drugs like carrier-mediated system belonging to the solute carrier (SLC) superfamily. Exposure to opioids may also alter the expression of ABC transporters. P-gp can be overproduced during morphine treatment, suggesting that the drug has a direct or, more likely, an indirect action. Variations in cerebral neurotransmitters during exposure to opioids and the release of cytokines during pain could be new endogenous stimuli affecting transporter synthesis. This review concludes with an analysis of the pharmacotherapeutic and clinical impacts of the interactions between ABC transporters and opioids.

In situ protein expression in tumour spheres: development of an immunostaining protocol for confocal microscopy

BackgroundMulticellular tumour sphere models have been shown to closely mimic phenotype characteristics of in vivo solid tumours, or to allow in vitro propagation of cancer stem cells (CSCs). CSCs are usually characterized by the expression of specific membrane markers using flow cytometry (FC) after enzymatic dissociation. Consequently, the spatial location of positive cells within spheres is not documented. Confocal microscopy is the best technique for the imaging of thick biological specimens after multi-labelling but suffers from poor antibody penetration. Thus, we describe here a new protocol for in situ confocal imaging of protein expression in intact spheroids.MethodsProtein expression in whole spheroids (150 μm in diameter) from two human colon cancer cell lines, HT29 and CT320X6, has been investigated with confocal immunostaining, then compared with profiles obtained through paraffin immunohistochemistry (pIHC) and FC. Target antigens, relevant for colon cancer and with different expression patterns, have been studied.ResultsWe first demonstrate that our procedure overcomes the well-known problem of antibody penetration in compact structures by performing immunostaining of EpCAM, a membrane protein expressed by all cells within our spheroids. EpCAM expression is detected in all cells, even the deepest ones. Likewise, antibody access is confirmed with CK20 and CD44 immunostaining. Confocal imaging shows that 100% of cells express β-catenin, mainly present in the plasma membrane with also cytoplasmic and nuclear staining, in agreement with FC and pIHC data. pIHC and confocal imaging show similar CA 19-9 cytoplasmic and membranar expression profile in a cell subpopulation. CA 19-9+ cell count confirms confocal imaging as a highly sensitive method (75%, 62% and 51%, for FC, confocal imaging and pIHC, respectively). Finally, confocal imaging reveals that the weak expression of CD133, a putative colon CSC marker, is restricted to the luminal cell surface of colorectal cancer acini, with CD133+ cellular debris into glandular lumina.ConclusionThe present protocol enables in situ visualization of protein expression in compact three-dimensional models by whole mount confocal imaging, allowing the accurate localization and quantification of cells expressing specific markers. It should prove useful to study rare events like CSCs within tumour spheres.

Ezrin-Radixin-Moesin-Binding Phosphoprotein (EBP50), an Estrogen-Inducible Scaffold Protein, Contributes to Biliary Epithelial Cell Proliferation

Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) anchors and regulates apical membrane proteins in epithelia. EBP50 is inducible by estrogen and may affect cell proliferation, although this latter function remains unclear. The goal of this study was to determine whether EBP50 was implicated in the ductular reaction that occurs in liver disease. EBP50 expression was examined in normal human liver, in human cholangiopathies (ie, cystic fibrosis, primary biliary cirrhosis, and primary sclerosing cholangitis), and in rats subjected to bile-duct ligation. The regulation of EBP50 by estrogens and its impact on proliferation were assessed in both bile duct-ligated rats and Mz-Cha-1 human biliary epithelial cells. Analyses of cell isolates and immunohistochemical studies showed that in normal human liver, EBP50 is expressed in the canalicular membranes of hepatocytes and, together with ezrin and cystic fibrosis transmembrane conductance regulator, in the apical domains of cholangiocytes. In both human cholangiopathies and bile duct-ligated rats, EBP50 was redistributed to the cytoplasmic and nuclear compartments. EBP50 underwent a transient increase in rat cholangiocytes after bile-duct ligation, whereas such expression was down-regulated in ovariectomized rats. In addition, in Mz-Cha-1 cells, EBP50 underwent up-regulation and intracellular redistribution in response to 17beta-estradiol, whereas its proliferation was inhibited by siRNA-mediated EBP50 knockdown. These results indicate that both the expression and distribution of EBP50 are regulated by estrogens and contribute to the proliferative response in biliary epithelial cells.

Effect of chronic exposure to morphine on the rat blood-brain barrier : focus on the P-glycoprotein

Morphine may affect the properties of the blood–brain barrier (BBB) by modifying the expression of certain BBB markers. We have determined the effect of chronic morphine treatment on the expression and function of some BBB markers in the rat. The mRNAs of 19 selected genes encoding caveolins, endothelial transporters, receptors and tight junctions proteins in the total RNA of isolated cortex microvessels were assayed by quantitative RT‐PCR (qRT‐PCR). The expression of genes Mdr1a, Mrp1, Bcrp, Glut‐1 and Occludin, was slightly increased, while that of Flk‐1 was decreased in microvessels from morphine‐treated rats. The expression of the Mrd1a and Mdr1b genes encoding the P‐glycoprotein (P‐gp) also increased in the whole hippocampus and cortex of morphine‐treated rats. The Mdr1a gene induction (1.38‐fold) observed by qRT‐PCR was also confirmed using in situ hybridization technique (1.40‐fold). Immunoblotting revealed an increase in P‐gp expression in the hippocampus (1.8‐fold) and cortex (1.36‐fold) of morphine‐treated rats, but no effect in isolated microvessels. In contrast, morphine treatment increased by 1.48‐fold the expression of P‐gp in a large vessel‐enriched fraction. The integrity of the BBB, measured by in situ brain perfusion of [14C]‐sucrose, and the activity of P‐gp at the BBB, measured with the P‐gp substrate [3H]‐colchicine, were not modified by morphine. Immunohistofluorescence experiments revealed that P‐gp expression is restricted to large vessels and microvessels in control rats and that morphine treatment did not induce the expression of P‐gp in the brain parenchyma (astrocytes or neurons). Taken together, our results showed that chronic morphine treatment does not significantly alter BBB integrity or P‐gp activity. The impact of morphine‐mediated P‐gp induction observed in large vessels remains to be determined in terms of brain disposition of drugs that are P‐gp substrates.

Identification of plasma membrane autoantigens in autoimmune hepatitis type 1 using a proteomics tool

Autoimmune hepatitis (AIH) is a liver disease with circulating autoantibodies predominantly directed against widely held cellular components. Because AIH is a liver‐specific disease, autoantibodies against plasma membrane antigens may be involved in its pathogenesis and have been reported; however, no definite identification has been described. We thus investigated the fine specificity of anti‐hepatocyte plasma membrane autoantibodies in type 1 AIH (AIH‐1) using a proteomic tool. A plasma membrane–enriched fraction was validated using enzymatic activity and western blot analysis experiments. Sera from AIH‐1 patients (n = 65) and from 90 controls, that is, healthy blood donors (n = 40) and patients with systemic diseases (n = 20) or other liver diseases (n = 30), were studied by immunoblot performed with plasma membrane proteins resolved by either sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) or 2‐dimensional (2D) electrophoresis. Proteins contained in the immunoreactive spots were identified by sequences provided by ion‐trap mass spectrometry. Hepatocytes probed with sera were also studied using confocal immunofluorescence and immunoelectron microscopy. The more prominent bands stained by patient sera were located at 38 kDa, 48, 50, 52 kDa, 62 kDa, 70 kDa, and a 95‐kDa double band. Six proteins with known potential plasma membrane expression were identified: liver arginase (38 kDa), cytokeratins (CK) 8 and 18 (48‐52 kDa), heat shock proteins (HSP) of 60, 70, 90 kDa, and valosin‐containing protein (VCP) of 92 kDa. The presence of anti‐membrane antibodies was confirmed by immunofluorescence and immunoelectron microscopy. Conclusion: Overall, our data demonstrate that liver arginase, CK 8/18, HSP 60, HSP 70, HSP 90, and VCP represent potential candidate targets on liver membrane for autoantibodies in AIH‐1. (HEPATOLOGY 2008;47:937–948.)

Immune Quiescence of the Brain Is Set by Astroglial Connexin 43

In the normal brain, immune cell trafficking and immune responses are strictly controlled and limited. This unique homeostatic equilibrium, also called brain immune quiescence, is crucial to maintaining proper brain functions and is altered in various pathological processes, from chronic immunopathological disorders to cognitive and psychiatric impairments. To date, the precise nature of factors regulating the brain/immune system interrelationship is poorly understood. In the present study, we demonstrate that one of these regulating factors is Connexin 43 (Cx43), a gap junction protein highly expressed by astrocytes at the blood–brain barrier (BBB) interface. We show that, by setting the activated state of cerebral endothelium, astroglial Cx43 controls immune recruitment as well as antigen presentation mechanisms in the mouse brain. Consequently, in the absence of astroglial Cx43, recruited immune cells elaborate a specific humoral autoimmune response against the von Willebrand factor A domain-containing protein 5a, an extracellular matrix protein of the brain. Altogether, our results demonstrate that Cx43 is a new astroglial factor promoting the immune quiescence of the brain.

Transport of Biogenic Amine Neurotransmitters at the Mouse Blood–Retina and Blood–Brain Barriers by Uptake1 and Uptake2

Uptake1 and uptake2 transporters are involved in the extracellular clearance of biogenic amine neurotransmitters at synaptic clefts. We looked for them at the blood–brain barrier (BBB) and blood–retina barriers (BRB), where they could be involved in regulating the neurotransmitter concentration and modulate/terminate receptor-mediated effects within the neurovascular unit (NVU). Uptake2 (Oct1-3/Slc22a1-3, Pmat/Slc29a4) and Mate1/Slc47a1 transporters are also involved in the transport of xenobiotics. We used in situ carotid perfusion of prototypic substrates like [3H]-1-methyl-4-phenylpyridinium ([3H]-MPP+), [3H]-histamine, [3H]-serotonin, and [3H]-dopamine, changes in ionic composition and genetic deletion of Oct1-3 carriers to detect uptake1 and uptake2 at the BBB and BRB. We showed that uptake1 and uptake2 are involved in the transport of [3H]-dopamine and [3H]-MPP+ at the blood luminal BRB, but not at the BBB. These functional studies, together with quantitative RT-PCR and confocal imaging, suggest that the mouse BBB lacks uptake1 (Net/Slc6a2, Dat/Slc6a3, Sert/Slc6a4), uptake2, and Mate1 on both the luminal and abluminal sides. However, we found evidence for functional Net and Oct1 transporters at the luminal BRB. These heterogeneous transport properties of the brain and retina NVUs suggest that the BBB helps protect the brain against biogenic amine neurotransmitters in the plasma while the BRB has more of a metabolic/endocrine role.

Blood-brain and retinal barriers show dissimilar ABC transporter impacts and concealed effect of P-glycoprotein on a novel verapamil influx carrier.

The respective impact and interplay between ABC (P‐glycoprotein/P‐gp/Abcb1a, BCRP/ABCG2, MRP/ABCC) and SLC transporter functions at the blood–brain barrier (BBB) and blood–retinal barriers (BRB) are incompletely understood.