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

第16回千葉カルシウム代謝研究会

Gene ontology term enrichments for RNA-Seq data from differentiated TSC2 deletion cell lines and microarray data of patient SEGAs (related to Fig. 2f). (XLSX 27.7 kb

Amino acid transport across the murine blood-brain barrier

he brain microvascular endothelial cells (BMECs) of the blood-brain barrier (BBB) provide a diffusion barrier between blood and brain interstitial fluid (ISF). The BBB plays a role in protecting the brain from changes in the blood. Amino acid transport through the BBB is crucial for establishing and maintaining an asymmetry in amino acid concentration between blood and brain and for the distribution of drugs and diagnostic markers. Interestingly, the cerebrospinal fluid (CSF) amino acid concentration is only about 10% that of plasma, except for glutamine [1]. Characterizing the expression and localization of solute carrier (SLC) transporters is one of the first steps in understanding the mechanisms regulating BBB transendothelial transport. Our aim is to understand how the amino acid gradient is maintained by BMEC amino acid transporters. To address this aim, we first examined which (amino acid) transporters are expressed in total at the murine BBB on the mRNA level and how their expression is affected by a short culture step [2]. By mRNA microarray analysis using freshly isolated as well as single-cultured (5 days) and co-cultured (with glial cells in non-contact) BMECs, we have shown that as much as 60% of the known amino acid transporters are expressed in BMECs. In freshly isolated, noncultured, BMECs Lat1-4F2hc is most prominently expressed, followed by the sodium-dependent amino acid transporters Taut, Snat2, Snat5, and Eaat3. Although glial coculture is often used to mimic in vitro the BBB in vivo, levels of 73% of the amino acid transporter mRNAs were strongly altered by culture. In particular, for 78% of the transporters highly expressed in noncultured BMECs down-regulation was verified by qPCR (Lat1-4F2hc, Taut, Cat-1, Xpct, Snat3, and Snat5). In contrast, y+Lat2, xCT, and Snat1 are expressed at low levels in noncultured BMECs and are upregulated by culture. We hypothesize that down-regulation of transporter mRNA during culture is characteristic of transporters, such as Lat1-4F2hc, mediating transendothelial amino acid transport in vivo. Conversely, we postulate that transporter mRNA upregulated by culture do so to serve increased cellular amino acid demands for growth. To understand the functional organization of the BBB and the role of transporters in physiological and (neuro)pathophysiological states, many of them characterized by impaired brain amino acid concentrations, it is necessary to not only determine at the mRNA level which transporters are expressed at the BBB, but also whether the actual transporters are expressed at the luminal and/or abluminal membranes of BMECs, and how they are regulated. Therefore, we studied the expression of Lat1-4F2hc (Slc7a5-Slc3a2), Snat1 (Slc38a1), and Snat3 (Slc38a3) in vivo on mouse brain tissue sections and additionally by in vivo biotinylation of the mouse brain vascular lumen with subsequent Western blot analysis. By combining the results obtained by the two different approaches, we, for the first time, could show that the sodium-dependent amino acid transporter Snat3, along with the exchanger Lat1- 4F2hc, is localized to both the abluminal and the luminal membranes of BMECs. Furthermore, they are subject to posttranslational modifications. For example, Snat3 expressed in the luminal membrane has been found to be highly glycosylated, suggesting the possibility that glycosylation might play a role for its luminal membrane insertion. The finding of Snat3 present on the luminal membrane of the BBB challenges the current hypothesis that sodium-dependent amino acid transport is an exclusive feature of the abluminal membrane [1, 3-9], in a position to protect the brain from neurotoxic levels of amino acids. We also found Snat1, another sodium-dependent transporter, localized to the luminal membrane of vascular endothelial cells, with higher expression in the bigger vessels compared to brain microvessels. Therefore, we conclude that Snat1 seems not to play a pivotal role in the differentiated endothelial cells of the BBB, which stands in agreement with the finding that Snat1-mRNA shows very low expression in noncultured BMECs [2]. Taken together, we showed expression of a high number of not characterized amino acid transporters at the BBB, as well as their differing modulation by culture, implying functional differences. We also showed the unexpected localization of secondary active transport mechanisms at the luminal membrane, facing the blood with its high amino acid levels. Therefore, to be able to better understand the BBB transportome and its role in brain amino acid homeostasis, further studies and re-thinking of current hypotheses are needed. Zusammenfassung. Mikrovasculäre Endothelzellen (BMECs) der Bluthirnschranke (BBB) bilden eine Diffusionsbarriere zwischen Blut und Interstitialflüssigkeit (ISF) des Gehirns. Die BBB schützt das Gehirn vor Veränderungen im Blut. Transport von Aminosäuren über die BBB ist ein wichtiger Schritt um die asymmetrische Aminosäurekonzentration zwischen Blut und Gehirn herzustellen und aufrechtzuerhalten. Aminosäuretransporter sind aber auch wichtig um Medikamente und diagnostische Marker im Gehirn zu verteilen. Interessanterweise ist die Aminosäurekonzentration in der Cerebrospinalflüssigkeit (CSF) nur ca. 10% die des Plasmas, mit Ausnahme von Glutamin [1]. Die Expression und Lokalisierung von “solute carrier” (SLC) Transportern zu charakterisieren ist einer der ersten Schritte um den Mechanismus zu verstehen mit dem BBB transendothelialer Transport reguliert wird. Unser Ziel ist es zu verstehen wie der bestehende Aminosäuregradient durch Aminosäuretransporter der BMECs erhalten wird. Um dieses Ziel zu erreichen haben wir als erstes untersucht welche Transporter überhaupt in der BBB von Mäusen exprimiert sind (mRNA) und wie ihre Expression durch eine kurze Kultur der Endothelzellen beeinflusst wird [2]. Durch eine Microarray-basierte Analyse der frisch isolierten, single-kultivierten (für 5 Tage) und co-kultivierten (mit Gliazellen ohne direkten Kontakt) BMECs konnten wir zeigen dass ganze 60% der bekannten Aminosäurentransporter in BMECs exprimiert sind. In frisch isolierten, nicht-kultivierten, BMECs zeigt Lat1-4F2hc die höchste Expression, gefolgt von den Natrium-abhängigen Aminosäuretransportern Taut, Snat2, Snat5, und Eaat3. Obwohl Co-Kultur von Endothel- mit Gliazellen oft verwendet wird um in vitro die BBB in vivo nachzuahmen, wurde die Expression von 73% der Aminosäuretransporter durch die Kultur der Endothelzellen stark verändert. Es konnte durch qPCR gezeigt werden dass 78% der Transporter mit hoher Expression in nicht-kultivierten BMECs einen weit niedrigeren Expressionslevel nach der Zellkultur aufweisen (Lat1-4F2hc, Taut, Cat-1, Xpct, Snat3, and Snat5). Im Gegensatz dazu zeigen y+Lat2, xCT, and Snat1 einen niedrigen Expressionslevel in nicht-kultivierten BMECs und eine höhere Expression nach dem Zellkulturschritt. Unserer Hypothese folgend repräsentieren Transporter deren mRNA während der Kultur der Zellen runter-reguliert wurde solche Transporter, die in vivo für transendothelialen Aminosäuretransport verantwortlich sind, wie z.B. Lat1-4F2h. Im Gegensatz dazu vermuten wir dass Transporter, welche während der Kultur der Zellen hoch-reguliert wurden, dies tun um den durch das Zellwachstum erhöhten zellulären Aminosäurebedarf zu decken. Um die funktionale Organisation der BBB und die Funktion der Transporter in physiologischen und (neuro)pathophysiologischen Bedingungen zu verstehen, welche oft durch beeinträchtigte Aminosäurekonzentrationen charakterisiert werden können, ist es wichtig neben der mRNA-Expression auch die Expression der Transporter in der luminalen und /oder abluminalen Membran der BMECs und deren Regulation zu bestimmen. Deswegen haben wir die Expression von Lat1-4F2hc (Slc7a5-Slc3a2), Snat1 (Slc38a1), und Snat3 (Slc38a3) in vivo auf Gewebsschnitten von Mäusegehirnen und darüber hinaus durch in vivo Biotinylierung des vaskulären Lumen in Mäusegehirnen mit darauf folgender Western- Blot Analyse untersucht. Die Ergebnisse der beiden unterschiedlichen Ansätze erlaubte es uns zum ersten Mal zu zeigen dass der Natrium-abhängige Transporter Snat3, ebenso wie der Exchanger Lat1-4F2hc, auf der luminalen wie auch abluminalen Membran der BMECs lokalisiert ist. Darüber hinaus sind die Transporter Objekt posttranslationaler Modifikationen. Zum Beispiel ist luminal exprimiertes Snat3 stark glykosyliert, was die Möglichkeit eröffnet dass Glykosylierung unter Umständen für die Insertion von Snat3 in die luminale Membran wichtig ist. Das Snat3 in der luminalen Membran der BBB zu finden ist steht im Gegensatz zu der gegenwärtige Hypothese dass Natrium-abhängiger Aminosäuretransport ausschliesslich an der abluminalen Membran stattfindet [1, 3-9], von wo aus sie das Gehirn vor neurotoxischen Aminosäureleveln schützten können. Wir fanden auch Snat1, einen anderen Natrium-abhängigen Transporter, in der luminalen Membran von vaskulären Endothelzellen, mit einer höheren Expression in grösseren wie in Mikrovesseln. Daraus folgerten wir dass Snat1 scheinbar keine entscheidende Funktion in den differenzierten Endothelzellen der BBB zu spielen scheint, was sich mit der sehr niedrigen Snat1-mRNA Expression in nicht-kultivierten BMECs deckt [2]. Zusammengenommen haben wir gezeigt dass eine grosse Anzahl nicht-charakterisierter Aminosäuretransporter in BBB Endothelzellen exprimiert ist, und dass diese Transporter unterschiedliche Regulation durch einen Zellkulturschritt zeigen, was wiederum auf funktionelle Unterschiede schliessen lässt. Darüber hinaus haben wir überraschenderweise auch gezeigt dass sekundäraktive Transporter in der luminalen Membran lokalisiert sind, wo sie in Kontakt mit den hohen Aminosäurekonzentrationen im Blut stehen. Um das BBB Transportom und seine Funktion in der Regulation der Aminosäurehomöostase im Gehirn besser zu verstehen sind deswegen weitere Studien und ein Überdenken der gegenwärtig vorherrschenden Hypothesen erforderlich

Differential axial localization along the mouse brain vascular tree of luminal sodium-dependent glutamine transporters Snat1 and Snat3

A specialized brain vasculature is key for establishing and maintaining brain interstitial fluid homeostasis, which for most amino acids (AAs) are ∼10% plasma levels. Indeed, regulation of AA homeostasis seems critical for normal central nervous system functions, and disturbances in brain levels have both direct and indirect roles in several neuropathologies. One mechanism contributing to the plasma to brain AA gradients involves polarized expression of solute carrier (SLC) family transporters on blood–brain barrier (BBB) endothelial cells. Of particular interest is the localization of sodium-dependent transporters that can actively move substrates against their concentration gradient. In this study, the in vivo endothelial membrane localization of the sodium-dependent glutamine transporters Snat3 (Slc38a3) and Snat1 (Slc38a1) was investigated in the mouse brain microvasculature using immunofluorescent colocalization with cellular markers. In addition, luminal membrane expression was probed by in vivo biotinylation. A portion of both Snat3 and Snat1 vascular expressions was localized on luminal membranes. Importantly, Snat1 expression was restricted to larger cortical microvessels, whereas Snat3 was additionally expressed on BBB capillary membranes. This differential expression of system A (Snat1) versus system N (Snat3) transporters suggests distinct roles for Snats in the cerebral vasculature and is consistent with Snat3 involvement in net transendothelial BBB AA transport

Culture-induced changes in blood-brain barrier transcriptome: implications for amino-acid transporters in vivo

Tight homeostatic control of brain amino acids (AA) depends on transport by solute carrier family proteins expressed by the blood-brain barrier (BBB) microvascular endothelial cells (BMEC). To characterize the mouse BMEC transcriptome and probe culture-induced changes, microarray analyses of platelet endothelial cell adhesion molecule-1-positive (PECAM1(+)) endothelial cells (ppMBMECs) were compared with primary MBMECs (pMBMEC) cultured in the presence or absence of glial cells and with b.End5 endothelioma cell line. Selected cell marker and AA transporter mRNA levels were further verified by reverse transcription real-time PCR. Regardless of glial coculture, expression of a large subset of genes was strongly altered by a brief culture step. This is consistent with the known dependence of BMECs on in vivo interactions to maintain physiologic functions, for example, tight barrier formation, and their consequent dedifferentiation in culture. Seven (4F2hc, Lat1, Taut, Snat3, Snat5, Xpct, and Cat1) of nine AA transporter mRNAs highly expressed in freshly isolated ppMBMECs were strongly downregulated for all cultures and two (Snat2 and Eaat3) were variably regulated. In contrast, five AA transporter mRNAs with low expression in ppMBMECs, including y(+)Lat2, xCT, and Snat1, were upregulated by culture. We hypothesized that the AA transporters highly expressed in ppMBMECs and downregulated in culture have a major in vivo function for BBB transendothelial transport.Journal of Cerebral Blood Flow & Metabolism advance online publication, 3 June 2009; doi:10.1038/jcbfm.2009.72

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

Potent and Selective BACE-1 Peptide Inhibitors Lower Brain Aβ Levels Mediated by Brain Shuttle Transport

Therapeutic approaches to fight Alzheimer's disease include anti-Amyloidβ (Aβ) antibodies and secretase inhibitors. However, the blood-brain barrier (BBB) limits the brain exposure of biologics and the chemical space for small molecules to be BBB permeable. The Brain Shuttle (BS) technology is capable of shuttling large molecules into the brain. This allows for new types of therapeutic modalities engineered for optimal efficacy on the molecular target in the brain independent of brain penetrating properties. To this end, we designed BACE1 peptide inhibitors with varying lipid modifications with single-digit picomolar cellular potency. Secondly, we generated active-exosite peptides with structurally confirmed dual binding mode and improved potency. When fused to the BS via sortase coupling, these BACE1 inhibitors significantly reduced brain Aβ levels in mice after intravenous administration. In plasma, both BS and non-BS BACE1 inhibitor peptides induced a significant time- and dose-dependent decrease of Aβ. Our results demonstrate that the BS is essential for BACE1 peptide inhibitors to be efficacious in the brain and active-exosite design of BACE1 peptide inhibitors together with lipid modification may be of therapeutic relevance

Additional file 11: Table S8. of Genomic analysis of the molecular neuropathology of tuberous sclerosis using a human stem cell model

Gene sets and associated genes that show after mTOR treatment a reversal of the change in expression detected in untreated TSC2 deletion cells (related to Fig. 4b). (XLSX 25.8 kb

Additional file 10: Table S7. of Genomic analysis of the molecular neuropathology of tuberous sclerosis using a human stem cell model

RNA-Seq and ribosome profiling data of differentiated TSC2 wild-type and homozygous deletion cell lines treated with mTOR inhibitors (related to Fig. 4). (XLSB 7.52 mb