Identification of folate binding macromolecule in rabbit choroid plexus.

A macromolecular binder of folic acid and folic acid derivatives has been identified in the particulate fraction of homogenates of rabbit choroid plexus. Within the choroid plexus, there are 2.3 nmol of folate-binding activity (binder) per g of tissue. The molecular weight of the folate binder complex, separated from the particulate fraction after solubilization with Triton X-100, was 340,000 to 400,000 by Sephadex gel filtration. The partially purified binder, when freed of endogenous folates, bound equivalent amounts of both [3H]folic acid and [methyl-14C]methyltetrahydrofolic acid per mg of protein. Folic acid, homofolic acid, 5-methyltetrahydrofolic acid, and to a lesser degree, methotrexate, inhibited the binding of both [3H]folic acid and [14C]methyltetrahydrofolic acid. Binding activity, which decreased below pH = 7.0, was unaffected by pretreatment with ribonuclease but was eliminated completely by papain and a protease (Streptomyces griseus). Although dihydrofolate reductase was present in choroid plexus, the binder was distinct from dihydrofolate reductase as judged by gel filtration and methotrexate sensitivity. This high affinity binder of folates may be responsible, in part, for the rapid, saturable uptake of folic acid and methyltetrahydrofolic acid by rabbit choroid plexus in vitro.     

Partial purification and characterization of a folatebinding protein from human choroid plexus

A folate-binding protein (binder) from human choroid plexus was solubilized with Triton X-100 and partially purified in three steps: (1) affinity chromatography, (2) Sephadex G-200 column chromatography, and (3) polyacrylamide gel electrophoresis. When the partially purified binder was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the binding activity was located in the region of the gel with a molecular weight between 45,000 and 60,000. The specific activity of the binder after the three purification steps was 1.2 g folic acid/mg protein, a 316-fold purification. Binding activity of the partially purified binder decreased below pH 6.0 and above pH 8.0 was unaffected by treatment with ribonuclease or deoxyribonuclease, but was abolished with trypsin, chymotrypsin, or protease (Streptomyces griesus). The binding of folic acid to the human binder was inhibited by folate > H₄-folate > methyl-H₄-folate dihydrofolate pteroic acid methotrexate aminopterin.     

Sodium localization in the choroid plexus

Summary The localization of sodium ion in the cat choroid plexus was studied by use of potassium pyroantimonate. The precipitates formed by the potassium pyroantimonate occur mostly on the plasma membrane in the epithelial cell and occasionally in the perivascular space. The precipitates in the epithelial cell are most numerous at the apical surface, particularly on the microvilli, and least in number at the basal and lateral surfaces. In the endothelial cell, the dense precipitates are situated on the plasma membrane as well as on the limiting membrane of the pinocytotic vesicle. Although the dense precipitates are sometimes situated on the external surface of the plasma membrane of the epithelial cell, most of them are localized on the internal surface of the plasma membrane. A similar localization of the precipitates is to be seen on the plasma membrane of the erythrocyte. When the cerebrospinal fluid/plasma ion ratio and potential gradients across the choroid plexus are considered, the precipitates on the plasma membrane would suggest a localization of sodium needed for the activation of ATPase.

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Zusammenfassung Die Lokalisation des Natriumions im Plexus chorioideus der Katze wurde mit Hilfe von Kaliumpyroantimonat untersucht. Die durch Kaliumpyroantimonat gebildeten Niederschläge treten meistens an der Plasmamembran in den Epithelzellen und gelegentlich im perivaskulären Raum auf. In den Epithelzellen kommen die Niederschläge am zahlreichsten an der apikalen Oberfläche vor, besonders an den Mikrovilli, am geringsten an den basalen und lateralen Oberflächen. In der Endothelzelle liegen die dichten Niederschläge an der Plasmamembran und an der Grenzmembran der Pinozytosebläschen. Einige der dichten Niederschläge befinden sich an der äußeren Oberfläche der Plasmamembran der Epithelzellen, die meisten aber an der inneren Oberfläche der Plasmamembran. Eine ähnliche Lokalisation der Niedersschläge wurde an der Plasmamembran des Erythrozyten festgestellt. Wenn man das Liquor Plasma-Ionenverhältnis und die Potentialgradienten am Plexus chorioideus in Betracht zieht, liegt es nahe, die nachgewiesene Lokalisation des Natriums auf eine Aktivierung von ATPase zu beziehen.

     

Identification of conventional and novel endothelin receptors in sheep choroid plexus cells

Previous studies have demonstrated the presence of super-high affinity endothelin receptors with apparent K_d's on the order of pM in different brain tissues. This study was designed to characterize, in detail, the receptors present in SCP cells, a non-transformed sheep choroid plexus cell line. Competitive binding assays with receptor-selective ligands indicated the presence of at least three classes of binding sites: a conventional receptor of the ET_A subtype with a K_d = 0.4 nM that mediates an increase in intracellular levels of inositol 1,4,5-trisphosphate (IP₃) in response to ET-1 and two additional sites with much higher binding affinities. The latter two sites are not coupled to the common signal transduction pathways of IP₃, cAMP and cGMP. Northern blot analysis confirmed the presence of only the ET_A subtype mRNA in SCP cells. It remains to determined if the multiple binding sites are distinct gene products, multiple affinity states of a single receptor molecule or a result of cooperative association of one site with either the ligand or with other proteins.     

Thyroxine transport in choroid plexus

The role of the choroid plexus in thyroid hormone transport between body and brain, suggested by strong synthesis and secretion of transthyretin in this tissue, was investigated in in vitro and in vivo systems. Rat choroid plexus pieces incubated in vitro were found to accumulate thyroid hormones from surrounding medium in a non-saturable process. At equilibrium, the ratio of thyroid hormone concentration in choroid plexus pieces to that in medium decreased upon increasing the concentration of transthyretin in the medium. Fluorescence quenching of fluorophores located at different depths in liposome membranes showed maximal hormone accumulation in the middle of the phospholipid bilayer. Partition coefficients of thyroxine and triiodothyronine between lipid and aqueous phase were about 20,000. After intravenous injection of 125I-labeled thyroid hormones, choroid plexus and parts of the brain steadily accumulated 125I-thyroxine, but not [125I]triiodothyronine, for many hours. The accumulation of 125I-thyroxine in choroid plexus preceded that in brain. The amount of 125I-thyroxine in non-brain tissues and the [125I]triiodothyronine content of all tissues decreased steadily beginning immediately after injection. A model is proposed for thyroxine transport from the bloodstream into cerebrospinal fluid based on partitioning of thyroxine between choroid plexus and surrounding fluids and binding of thyroxine to transthyretin newly synthesized and secreted by choroid plexus.     

The permeability of the frog choroid plexus to nonelectrolytes

Summary Anin vitro preparation of the frog choroid plexus has been used to measure the permeability of the choroidal epithelium to 50 nonelectrolytes by an osmotic method. The method involves the measurement of nonelectrolyte reflection coefficients () by a rapid electrical procedure. For the majority of compounds tested, there was a good correlation between the rate of solute permeation and the solute's bulk-phase lipid: water partition coefficients; i.e., the higher the partition coefficient the greater the permeability. The membrane lipids of the choroid plexus differ from the membrane lipids of the gall bladder in at least three ways: (1) the lipids of the choroid plexus cannot distinguish between branched chain solutes and their straight chain isomers; (2) small polar solutes such as urea and acetamide permeate via the membrane lipids to a significant extent; and (3) the smaller selectivity ratios suggest that the lipids of the choroid plexus contain more hydrogen bonding sites (i.e., there are stronger solute: lipid intermolecular forces in the choroid plexus). The permeability characteristics of the choroid plexus are qualitatively similar to those of most other cell membranes. In addition, there is evidence for the presence of a special mechanism for the transport of sugar across this epithelium.     

Dichotomous development of the organic anion transport protein in liver and choroid plexus

Both adult liver and choroid plexus express the organic aniontransport protein (oatp1) and transport[³⁵S]bromosulfophthalein(BSP). Studies of the developing rat liver reveal that oatp1 mRNA andprotein do not begin to be expressed until 15 days postnatal and are atadult levels by 30 days. Uptake of[³⁵S]BSP follows thesame time course. In contrast, neonatal rat choroid plexus expressesoatp1 mRNA and protein. When quantified on a weight basis, the uptakeof [³⁵S]BSP in choroidplexus is lower in the adult than at earlier stages of development.Although fluorescence confocal microscopy of adult rat choroid plexusshows that oatp is localized to the apical surface, facing thecerebrospinal fluid, this method reveals an intracellular localizationof oatp1 in the neonate. Approximately 12 wk are required for theappearance of the adult pattern of distribution. Changes in thelocalization and activity of oatp1 during development could play animportant role in the pathobiology of maturation of the liver and thecentral nervous system.

     

Affinity of folic acid for the folate-binding protein of choroid plexus.

The affinity of folic acid for the folate-binding protein of rabbit choroid plexus was determined by equilibrium dialysis at 4 °C. All solutions contained 0.02% Triton X-100 to maintain the binder in solution. At pH 7.0, the apparent dissociation constant (K_a) at a binder concentration of 0.36 nm was 9.4 pm with slight positive cooperativity (Hill coefficient = 1.19). The K_a increased at pH 6.0 and when a higher concentration of binder (3.25 nm) was used to 30.1 and 46.0 pm, respectively. However, the maximal binding capacity per milligram of protein did not change. At pH 5.0, the K_a was greater than 20 nm. These results show that the affinity of the choroid plexus folate-binding protein (when solubilized in Triton X-100) for folic acid depends on both the concentration of binder and the pH.     

Altered gravity downregulates aquaporin-1 protein expression in choroid plexus.

Aquaporin-1 (AQP1) is a water channel expressed abundantly at the apical pole of choroidal epithelial cells. The protein expression was quantified by immunocytochemistry and confocal microscopy in adult rats adapted to altered gravity. AQP1 expression was decreased by 64% at the apical pole of choroidal cells in rats dissected 5.5-8 h after a 14-day spaceflight. AQP1 was significantly overexpressed in rats readapted for 2 days to Earth's gravity after an 11-day flight (48% overshoot, when compared with the value measured in control rats). In a ground-based model that simulates some effects of weightlessness and alters choroidal structures and functions, apical AQP1 expression was reduced by 44% in choroid plexus from rats suspended head down for 14 days and by 69% in rats suspended for 28 days. Apical AQP1 was rapidly enhanced in choroid plexus of rats dissected 6 h after a 14-day suspension (57% overshoot, in comparison with control rats) and restored to the control level when rats were dissected 2 days after the end of a 14-day suspension. Decreases in the apical expression of choroidal AQP1 were also noted in rats adapted to hypergravity in the NASA 24-ft centrifuge: AQP1 expression was reduced by 47% and 85% in rats adapted for 14 days to 2 G and 3 G, respectively. AQP1 is downregulated in the apical membrane of choroidal cells in response to altered gravity and is rapidly restored after readaptation to normal gravity. This suggests that water transport, which is partly involved in the choroidal production of cerebrospinal fluid, might be decreased during spaceflight and after chronic hypergravity.     

Folate transport in the choroid plexus.

The uptake of 5-methyltetrahydrofolic acid (5-MTHF) by the isolated choroid plexus of hog was studied and shown to be both temperature and time dependent. Uptake of 5-MTHF by the isolated choroid plexus was a saturable process and exhibited a K_t of 0.9 × 10⁻⁶M and Vmax of 1.39 nmole/gm dry wt/min. The system did not require the presence of sodium ion nor was it ouabain sensitive. The presence of metabolic inhibitors, e.g., 2,4-dinitrophenol, did not suppress the uptake rate. Deprivation of oxygen also did not affect the rate of 5-MTHF transport. Addition of folic acid to the incubating medium led to countertransport of intracellular 5-MTHF. Efflux studies also indicated that the majority of the intracellular 5-MTHF was rapidly exchangeable and therefore probably present in the cell water in a free state. Chromatographic analyses confirmed that 5-MTHF was not metabolically altered during the transport process. It is suggested that 5-methyltetrahydrofolic acid is transported in the isolated choroid plexus via a carrier-mediated process.     

Cell trafficking through the choroid plexus

The choroid plexus is a multifunctional organ that sits at the interface between the blood and cerebrospinal fluid (CSF). It serves as a gateway for immune cell trafficking into the CSF and is in an excellent position to provide continuous immune surveillance by CD4⁺ T cells, macrophages and dendritic cells and to regulate immune cell trafficking in response to disease and trauma. However, little is known about the mechanisms that control trafficking through this structure. Three cell types within the choroid plexus, in particular, may play prominent roles in controlling the development of immune responses within the nervous system: the epithelial cells, which form the blood-CSF barrier, and resident macrophages and dendritic cells in the stromal matrix. Adhesion molecule and chemokine expression by the epithelial cells allows substantial control over the selection of cells that transmigrate. Macrophages and dendritic cells can present antigen within the choroid plexus and/or transmigrate into the cerebral ventricles to serve a variety of possible immune functions. Studies to better understand the diverse functions of these cells are likely to reveal new insights that foster the development of novel pharmacological and macrophage-based interventions for the control of CNS immune responses.     

Cell trafficking through the choroid plexus

The choroid plexus is a multifunctional organ that sits at the interface between the blood and cerebrospinal fluid (CSF). It serves as a gateway for immune cell trafficking into the CSF and is in an excellent position to provide continuous immune surveillance by CD4⁺ T cells, macrophages and dendritic cells and to regulate immune cell trafficking in response to disease and trauma. However, little is known about the mechanisms that control trafficking through this structure. Three cell types within the choroid plexus, in particular, may play prominent roles in controlling the development of immune responses within the nervous system: the epithelial cells, which form the blood-CSF barrier, and resident macrophages and dendritic cells in the stromal matrix. Adhesion molecule and chemokine expression by the epithelial cells allows substantial control over the selection of cells that transmigrate. Macrophages and dendritic cells can present antigen within the choroid plexus and/or transmigrate into the cerebral ventricles to serve a variety of possible immune functions. Studies to better understand the diverse functions of these cells are likely to reveal new insights that foster the development of novel pharmacological and macrophage-based interventions for the control of CNS immune responses.     

Identification of the novel localization of tenascinX in the monkey choroid plexus and comparison with the mouse

Tenascin-X (Tn-X) belongs to the tenascin family of glycoproteins and has been reported to be significantly associated with schizophrenia in a single nucleotide polymorphism analysis in humans. This finding indicates an important role of Tn-X in the central nervous system (CNS). However, details of Tn-X localization are not clear in the primate CNS. Using immunohistochemical techniques, we found novel localizations of Tn-X in the interstitial connective tissue and around blood vessels in the choroid plexus (CP) in macaque monkeys. To verify the reliability of Tn-X localization, we compared the Tn-X localization with the tenascin-C (Tn-C) localization in corresponding regions using neighbouring sections. Localization of Tn-C was not observed in CP. This result indicated consistently restricted localization of Tn-X in CP. Comparative investigations using mouse tissues showed equivalent results. Our observations provide possible insight into specific roles of Tn-X in CP for mammalian CNS function.Key words: tenascin-X, choroid plexus, monkey, mouse, Ehlers-Danlos syndrome, schizophrenia.The tenascins (Tn) are a family of four glyco-protein members – tenascin-C (Tn-C), tenascin-R (Tn-R), tenascin-W (Tn-W) and tenascin-X (Tn-X) – found diversely in the extra-cellular matrix of vertebrate organs (Hsia and Schwarzbauer, 2005; Tucker and Chiquet-Ehrismann, 2009). Important functions of Tn have been investigated in developmental cell adhesion modulation and pathological conditions such as wound healing and tumourigenesis (Adams and Watt, 1993; Hsia and Schwarzbauer, 2005; Tucker and Chiquet-Ehrismann, 2009). Tn-C and Tn-R are prominent in the nervous system and play a role in the development of neurite outgrowth and postnatal synaptic plasticity (Yamaguchi, 2000; Chiquet-Ehrismann and Tucker, 2004; Dityatev and Schachner, 2006). Tn-W is found abundantly in the developing bone and stroma of certain tumours (Chiquet-Ehrismann and Tucker, 2004; Tucker and Chiquet-Ehrismann, 2009). Tn-X is the first tenascin member shown to be clearly associated with the human connective tissue disorder Ehlers–Danlos syndrome (EDS; Burch et al., 1997). Patients with a Tn-X deficiency suffer from skin hyperextensibility, joint hypermobility and poor wound healing ability (Bristow et al., 2005). These symptoms are caused by the occurrence of abnormal irregular collagen fibres. Tn-X plays a role in collagen fibrillogenesis by directly binding to collagen (Mao et al. 2002; Minamitani et al. 2004). Mice with a Tn-X deficiency also showed skin symptoms comparable with those of EDS (Mao et al., 2002).Interestingly, in an analysis of human single nucleotide polymorphisms, Tn-X was reported to be significantly associated with schizophrenia (Wei and Hemmings, 2004; Tochigi et al., 2007). However, thus far, there have been no neuroanatomical reports on the involvement of Tn-X in schizophrenia. In the mammalian central nervous system (CNS), Tn-X mRNA expression has only been shown in the rat meninges of the olfactory bulb (Deckner et al., 2000). Recently, we found novel Tn-X localizations in the adult mouse leptomeninges trabecula in the cerebral cortex and in the connective tissue in the lateral ventricle choroid plexus (CP; Imura and Sato, 2008). Our finding of Tn-X localization in CP, which produces cerebrospinal fluid (CSF), might be a key factor in the investigation of the association between CSF metabolism and enlarged ventricles in schizophrenia. Enlarged ventricles are typical structural abnormalities associated with schizophrenia (Staal et al., 1999). Furthermore, CP secretes biologically active molecules into the CSF for brain development, activity and protection (Strazielle and Ghersi-Egea, 2000; Brown et al., 2004; Thouvenot et al., 2006; Johanson et al., 2008). In these molecules, for instance, there is a brain-derived neurotrophic factor (BDNF), the gene expression level and polymorphism of which have been analysed in relation to the pathogenesis of schizophrenia (Buckley et al., 2007). One study reported that BDNF is able to stimulate Tn-X expression in vitro (Takeda et al., 2005).The validity and limitations of animal models (rodents and monkeys) for use in the study of schizophrenia have been discussed (Tordjman et al., 2007). The authors concluded that monkeys appear to be an interesting social interaction model, more so than rodents, because of their complex well-organized social structure. In addition to differences in social structure, the dopaminergic system of rats and monkeys is quite different (García-Cabezas et al., 2009), and dysfunction of the dopaminergic system is related to schizophrenia (Wang et al. 2008).The CSF outflow system has been studied in some animal models (Kapoor et al., 2008). An anatomical difference in arachnoid granulations has been shown between rodents and monkeys (Krisch, 1988). Arachnoid granulations in monkeys are structurally similar to those in humans (Cooper, 1958; Krisch, 1988). In contrast, arachnoid granulations in rodents are similar to those of cats and dogs (Krisch, 1988). It is possible that Tn-X localization in CP is different between rodents and monkeys.Therefore, details concerning Tn-X localization in monkey CP need to be clarified. In the present study, we compared the immunohistochemistry of Tn-X in monkey CP with that in mouse CP. Subsequently, to verify the reliability of Tn-X localization, we compared it with Tn-C localization in corresponding regions using neighbouring sections.