Multi-omics analyses of choroid plexus carcinoma cell lines reveal potential targetable pathways and alterations.

PURPOSE: Choroid plexus carcinomas (CPCs) are extremely rare brain tumors and carry a dismal prognosis. Treatment options are limited and there is an urgent need to develop models to further research. In the present study, we established two CPC cell lines and performed multi-omics analyses. These cell lines serve as valuable models to propose new treatments in these rare but deadly brain tumors. METHODS: Multi-omic profiling including, (i) methylation array (EPIC 850 K), (ii) whole genome sequencing (WGS), (iii) CANCERPLEX cancer genome panel testing, (iv) RNA sequencing (RNA-seq), and (v) proteomics analyses were performed in CCHE-45 and NGT131 cell lines. RESULTS: Both cell lines were classified as methylation class B. Both harbored pathogenic TP53 point mutations; CCHE-45 additionally displayed TP53 loss. Furthermore, alterations of the NOTCH and WNT pathways were also detected in both cell lines. Two protein-coding gene fusions, BZW2-URGCP, and CTTNBP2-ERBB4, mutations of two oncodrivers, GBP-4 and KRTAP-12-2, and several copy number alterations were observed in CCHE-45, but not NGT131. Transcriptome and proteome analysis identified shared and unique signatures, suggesting that variability in choroid plexus carcinoma tumors may exist. The discovered difference's importance and implications highlight the possible diversity of choroid plexus carcinoma and call for additional research to fully understand disease pathogenesis. CONCLUSION: Multi-omics analyses revealed that the two choroid plexus carcinoma cell lines shared TP53 mutations and other common pathway alterations and activation of NOTCH and WNT pathways. Noticeable differences were also observed. These cell lines can serve as valuable models to propose new treatments in these rare but deadly brain tumors.

25-Year Storage of Human Choroid Plexus in Methyl Salicylate Preserves Its Antigen Immunoreactivity.

OBJECTIVE: Immunohistochemical investigation of archival histological material is a serious problem, since long-term storage of biological tissues, most often in formalin, leads to a loss of antigenic properties. However, the biological material can also be stored in the clearing agent methyl salicylate. The aim of this study was to assess the antigenicity of the human choroid plexus after extra long-term storage in methyl salicylate. MATERIAL AND METHOD: The study was performed on samples of fixed human choroid plexus (occasionally with attached neighboring pineal gland) stored in either methyl salicylate or paraffin blocks for 25 years. Chromogenic and fluorescence immunohistochemistry of vimentin, GFAP, type IV collagen, ß-catenin, α-smooth muscle actin, von Willebrand factor, CD68, mast cell tryptase, TMEM119, and synaptophysin was carried out. RESULTS: The storage of human choroid plexus in methyl salicylate for 25 years does not impair its histomorphology and preserves the properties of all the antigens assessed, which makes their immunohistochemical visualization possible using both light and fluorescence microscopy. Additionally, we found that long-term storage of human choroid plexus in methyl salicylate does not cause an increase in autofluorescence. CONCLUSION: Methyl salicylate can be recommended as a medium for long-term storage of biological tissue, as it provides excellent brain tissue preservation and retains its antigenic properties for up to 25 years.

Central Nervous System Stimulants Limit Caffeine Transport at the Blood-Cerebrospinal Fluid Barrier.

Caffeine, a common ingredient in energy drinks, crosses the blood-brain barrier easily, but the kinetics of caffeine across the blood-cerebrospinal fluid barrier (BCSFB) has not been investigated. Therefore, 127 autopsy cases (Group A, 30 patients, stimulant-detected group; and Group B, 97 patients, no stimulant detected group) were examined. In addition, a BCSFB model was constructed using human vascular endothelial cells and human choroid plexus epithelial cells separated by a filter, and the kinetics of caffeine in the BCSFB and the effects of 4-aminopyridine (4-AP), a neuroexcitatory agent, were studied. Caffeine concentrations in right heart blood (Rs) and cerebrospinal fluid (CSF) were compared in the autopsy cases: caffeine concentrations were higher in Rs than CSF in Group A compared to Group B. In the BCSFB model, caffeine and 4-AP were added to the upper layer, and the concentration in the lower layer of choroid plexus epithelial cells was measured. The CSF caffeine concentration was suppressed, depending on the 4-AP concentration. Histomorphological examination suggested that choroid plexus epithelial cells were involved in inhibiting the efflux of caffeine to the CSF. Thus, the simultaneous presence of stimulants and caffeine inhibits caffeine transfer across the BCSFB.

Novel Human Insulin Isoforms and Cα-Peptide Product in Islets of Langerhans and Choroid Plexus.

Human insulin (INS) gene diverged from the ancestral genes of invertebrate and mammalian species millions of years ago. We previously found that mouse insulin gene (Ins2) isoforms are expressed in brain choroid plexus (ChP) epithelium cells, where insulin secretion is regulated by serotonin and not by glucose. We further compared human INS isoform expression in postmortem ChP and islets of Langerhans. We uncovered novel INS upstream open reading frame isoforms and their protein products. In addition, we found a novel alternatively spliced isoform that translates to a 74-amino acid (AA) proinsulin containing a shorter 19-AA C-peptide sequence, herein designated Cα-peptide. The middle portion of the conventional C-peptide contains ß-sheet (GQVEL) and hairpin (GGGPG) motifs that are not present in Cα-peptide. Islet amyloid polypeptide (IAPP) is not expressed in ChP, and its amyloid formation was inhibited in vitro more efficiently by Cα-peptide than by C-peptide. Of clinical relevance, the ratio of the 74-AA proinsulin to proconvertase-processed Cα-peptide was significantly increased in islets from type 2 diabetes mellitus autopsy donors. Intriguingly, 100 years after the discovery of insulin, we found that INS isoforms are present in ChP from insulin-deficient autopsy donors.

Iron accumulation in the choroid plexus, ependymal cells and CNS parenchyma in a rat strain with low-grade haemolysis of fragile macrocytic red blood cells.

Iron accumulation in the CNS is associated with many neurological diseases via amplification of inflammation and neurodegeneration. However, experimental studies on iron overload are challenging, since rodents hardly accumulate brain iron in contrast to humans. Here, we studied LEWzizi rats, which present with elevated CNS iron loads, aiming to characterise choroid plexus, ependymal, CSF and CNS parenchymal iron loads in conjunction with altered blood iron parameters and, thus, signifying non-classical entry sites for iron into the CNS. Non-haem iron in formalin-fixed paraffin-embedded tissue was detected via DAB-enhanced Turnbull Blue stainings. CSF iron levels were determined via atomic absorption spectroscopy. Ferroportin and aquaporin-1 expression was visualised using immunohistochemistry. The analysis of red blood cell indices and serum/plasma parameters was based on automated measurements; the fragility of red blood cells was manually determined by the osmotic challenge. Compared with wild-type animals, LEWzizi rats showed strongly increased iron accumulation in choroid plexus epithelial cells as well as in ependymal cells of the ventricle lining. Concurrently, red blood cell macrocytosis, low-grade haemolysis and significant haemoglobin liberation from red blood cells were apparent in the peripheral blood of LEWzizi rats. Interestingly, elevated iron accumulation was also evident in kidney proximal tubules, which share similarities with the blood-CSF barrier. Our data underscore the importance of iron gateways into the CNS other than the classical route across microvessels in the CNS parenchyma. Our findings of pronounced choroid plexus iron overload in conjunction with peripheral iron overload and increased RBC fragility in LEWzizi rats may be seminal for future studies of human diseases, in which similar constellations are found.

Immunoreactivities for hepcidin, ferroportin, and hephaestin in astrocytes and choroid plexus epithelium of human brains.

Iron plays essential roles in the central nervous system. However, how the iron level is regulated in brain cells including glia and neurons remains to be fully clarified. In this study, the localizations of hepcidin, ferroportin, and hephaestin, which are known to be involved in iron efflux, were immunohistochemically examined in autopsied human brains. Immunoreactivities for hepcidin and ferroportin were observed in granular structures within the cytoplasm of reactive astrocytes and epithelial cells of the choroid plexus. Granular structures showing immunoreactivities for hepcidin and ferroportin were also stained with antibodies for early endosome antigen 1 (EEA1). In addition, immunoreactivity for hephaestin was observed in the cytoplasm of epithelial cells of the choroid plexus as well as reactive astrocytes. Immunoreactivity for hephaestin in the cytoplasm of reactive astrocytes was occasionally colocalized with immunoreactivity for EEA1, while that of hephaestin was frequently observed in the cytoplasm showing no immunoreactivity for EEA1. These findings suggest that immunoreactivities for hepcidin and ferroportin are localized in close proximity to granular structures showing immunoreactivity for EEA1 in the cytoplasm of human brain astrocytes. They also suggest that immunoreactivity of hephaestin is localized in the cytoplasm of the choroid plexus epithelium as well as reactive astrocytes of human brains.

Choroid plexus-derived miR-204 regulates the number of quiescent neural stem cells in the adult brain.

Regulation of adult neural stem cell (NSC) number is critical for lifelong neurogenesis. Here, we identified a post-transcriptional control mechanism, centered around the microRNA 204 (miR-204), to control the maintenance of quiescent (q)NSCs. miR-204 regulates a spectrum of transcripts involved in cell cycle regulation, neuronal migration, and differentiation in qNSCs. Importantly, inhibition of miR-204 function reduced the number of qNSCs in the subependymal zone (SEZ) by inducing pre-mature activation and differentiation of NSCs without changing their neurogenic potential. Strikingly, we identified the choroid plexus of the mouse lateral ventricle as the major source of miR-204 that is released into the cerebrospinal fluid to control number of NSCs within the SEZ. Taken together, our results describe a novel mechanism to maintain adult somatic stem cells by a niche-specific miRNA repressing activation and differentiation of stem cells.

Distribution of FcRn Across Species and Tissues.

The neonatal Fc receptor (FcRn) is a major histocompatibility complex class I type molecule that binds to, transports, and recycles immunoglobulin G (IgG) and albumin, thereby protecting them from lysosomal degradation. Therefore, besides the knowledge of FcRn affinity, FcRn protein expression is critical in understanding the pharmacokinetic behavior of Fc-containing biotherapeutics such as monoclonal antibodies. The goal of this investigation was to achieve for the first time a comparative assessment of FcRn distribution across a variety of tissues and species. FcRn was mapped in about 20 tissues including placenta from human and the most frequently used species in non-clinical safety testing of monoclonal antibodies (mouse, rat, cynomolgus monkey). In addition, the FcRn expression pattern was characterized in two humanized transgenic mouse lines (Tg32 and Tg276) expressing human FcRn under different promoters, and in the severe combined immunodeficient (SCID) mouse. Consecutive sections were stained with specific markers, namely, anti-CD68 for macrophages and anti-von Willebrand Factor for endothelial cells. Overall, the FcRn expression pattern was comparable across species and tissues with consistent expression of FcRn in endothelial cells and interstitial macrophages, Kupffer cells, alveolar macrophages, enterocytes, and choroid plexus epithelium. The human FcRn transgenic mouse Tg276 showed a different and much more widespread staining pattern of FcRn. In addition, immunodeficiency and lack of IgG in SCID mice had no negative effect on FcRn expression compared with wild-type mice.

The V-ATPase is expressed in the choroid plexus and mediates cAMP-induced intracellular pH alterations.

The cerebrospinal fluid (CSF) pH influences brain interstitial pH and, therefore, brain function. We hypothesized that the choroid plexus epithelium (CPE) expresses the vacuolar H+-ATPase (V-ATPase) as an acid extrusion mechanism in the luminal membrane to counteract detrimental elevations in CSF pH. The expression of mRNA corresponding to several V-ATPase subunits was demonstrated by RT-PCR analysis of CPE cells (CPECs) isolated by fluorescence-activated cell sorting. Immunofluorescence and electron microscopy localized the V-ATPase primarily in intracellular vesicles with only a minor fraction in the luminal microvillus area. The vesicles did not translocate to the luminal membrane in two in vivo models of hypocapnia-induced alkalosis. The Na+-independent intracellular pH (pHi) recovery from acidification was studied in freshly isolated clusters of CPECs. At extracellular pH (pHo) 7.4, the cells failed to display significant concanamycin A-sensitive pHi recovery (i.e., V-ATPase activity). The recovery rate in the absence of Na+ amounted to <10% of the pHi recovery rate observed in the presence of Na+ Recovery of pHi was faster at pHo 7.8 and was abolished at pHo 7.0. The concanamycin A-sensitive pHi recovery was stimulated by cAMP at pH 7.4 in vitro, but intraventricular infusion of the membrane-permeant cAMP analog 8-CPT-cAMP did not result in trafficking of the V-ATPase. In conclusion, we find evidence for the expression of a minor fraction of V-ATPase in the luminal membrane of CPECs. This fraction does not contribute to enhanced acid extrusion at high extracellular pH, but seems to be activated by cAMP in a trafficking-independent manner.

Differential regional and cellular distribution of TFF3 peptide in the human brain.

TFF3 is a member of the trefoil factor family (TFF) predominantly secreted by mucous epithelia. Minute amounts are also expressed in the immune system and the brain. In the latter, particularly the hypothalamo-pituitary axis has been investigated in detail in the past. Functionally, cerebral TFF3 has been reported to be involved in several processes such as fear, depression, learning and object recognition, and opiate addiction. Furthermore, TFF3 has been linked with neurodegenerative and neuropsychiatric disorders (e.g., Alzheimer's disease, schizophrenia, and alcoholism). Here, using immunohistochemistry, a systematic survey of the TFF3 localization in the adult human brain is presented focusing on extrahypothalamic brain areas. In addition, the distribution of TFF3 in the developing human brain is described. Taken together, neurons were identified as the predominant cell type to express TFF3, but to different extent; TFF3 was particularly enriched in various midbrain and brain stem nuclei. Besides, TFF3 immunostaining staining was observed in oligodendroglia and the choroid plexus epithelium. The wide cerebral distribution should help to explain its multiple effects in the CNS.

Immunoreactivity of glucose transporter 5 is located in epithelial cells of the choroid plexus and ependymal cells.

High fructose intake is associated with increased plasma triglyceride concentration, hepatic steatosis, impaired glucose tolerance, insulin resistance, and high blood pressure. In addition, increased fructose intake has recently been supposed to be a risk factor for dementia. However, direct effects of fructose on the brain function remain to be clarified. The localization of glucose transporter 5 (Glut5), a representative transporter of fructose, was immunohistochemically examined in the brains of humans, rats, and mice to clarify whether fructose was transported from the blood into the brain. Glut5 immunoreactivity was demonstrated to be located in the epithelial cells of the choroid plexus and the ependymal cells in the brains of humans and rats using commercial antibodies for Glut5. In addition, mRNA expression of mouse Glut5 was confirmed in the brains of mice. Immunohistochemical examination using a custom-made antibody against two regions of amino acid sequences of mouse Glut5 revealed that Glut5 immunoreactivity was also seen in the epithelial cells of the choroid plexus and the ependymal cells in the brains of mice. These findings show that Glut5 immunoreactivity is located in the epithelial cells of the choroid plexus and the ependymal cells, suggesting the possibility of the direct transportation of intravascular fructose into the brain parenchyma.

Distribution and pharmacokinetics of double-radiolabeled endotoxin in the rat brain and peripheral organs.

The endotoxin, lipopolysaccharide (LPS), of Salmonella typhimurium was biosynthetically labeled with (3)H and (14)C incorporated into the fatty acyl chains and glucosamine residues, respectively. The radio-labeled LPS was isolated from the bacteria and then injected into Sprague-Dawley rats. The distribution of (14)C and (3)H-LPS in plasma and other organs was determined following intraperitoneal (IP) doses of (14)C and (3)H-LPS (200 µg/kg). Plasma concentrations of both fatty acyl chains and glucosamine residues were biphasic, with a relatively rapid decay followed by a slow decline for 48 h. Similar biphasic results were found in the peripheral organs (kidney and heart) and brain barrier tissues (meninges and choroid plexus). In other brain tissues (brain stem, caudate nucleus, hypothalamus, frontal cortex, cerebellum and hippocampus), the glucosamine residue was biphasic, whereas the fatty acyl chains showed accumulation. Highest concentrations of LPS were found in the plasma, spleen and the liver. In addition, in the liver, sustained elevations of (14)C-glucosamine and (3)H-fatty acyl chains were observed. This indicates LPS accumulation in the liver. By contrast, the spleen showed biphasic decay of glucosamine residues and accumulation of fatty acyl chains. In the brain barrier tissues, peak LPS concentrations were significantly reduced (about 70%) and were further reduced (about 95%) in other brain tissues. The high elevation of LPS in the spleen is considered indicative of an immune response. Our findings highlight the potential significant role of lipid A as shown with the sustained elevation of (3)H-fatty acyl chains in the brain.

Immunohistochemical and morphometric analysis of immunoglobulin light-chain immunoreactive amyloid in psammoma bodies of the human choroid plexus.

The aim of this research was to establish the presence of amyloid and to quantify immunohistochemical reactions of kappa and lambda light chains of psammoma bodies of the choroid plexus. Choroid plexus tissue obtained from 14 right lateral ventricles postmortem was processed histologically and stained with Congo red, thioflavin T, and monoclonal antibodies for kappa and lambda light chains. Morphological analysis was performed with a light microscope at lens magnifications of 4×, 10×, 20×, 25×, and 40×. The morphometric characteristics of psammoma bodies that were kappa and lambda positive and negative were analyzed with ImageJ. Histological analysis showed that the psammoma bodies, stromal blood vessel walls, and some epithelial cells reacted positively with Congo red and thioflavin T. Psammoma bodies were predominantly positive for lambda light chains. Lambda positivity was detected inside some stromal blood vessels, which pointed to a probable systemic origin for these light chains. Morphometric analysis showed that the mean optical densities of lambda- and kappa-positive psammoma bodies were significantly higher than those that gave a negative reaction. The percentage of lambda-positive psammoma bodies was significantly higher than the percentage of lambda-negative psammoma bodies in 80% of the cases, while the reaction with kappa light chains was negative in the majority of the cases. Linear regression analysis showed a significant increase in the percentage of lambda-positive psammoma bodies and their mean optical density with age. Finally, it can be concluded that the positive reaction of psammoma bodies in the choroid plexus with respect to amyloid and lambda light chains may point to the presence of light-chain amyloid in their structures.

Involvement of the choroid plexus in the inflammatory response after acute spinal cord injury in dogs: an immunohistochemical study.

The choroid plexus (CP) is increasingly recognized as an important contributor to central nervous system (CNS) inflammation by recruitment of inflammatory cells and release of inflammatory cytokines. Here we investigate the role of the CP epithelium (CPE) as a source of three pro-inflammatory molecules of potential importance in inflammation after acute spinal cord injury (SCI): IL-1ß, TNF-α, and hsp70. Immunohistochemical (IHC) staining for these three proteins was performed on 4th ventricular CPE from 4 dogs euthanized 12-48 h after spontaneous acute SCI, and from 4 neurologically normal dogs euthanized for other reasons. IHC staining was quantified using Aperio ImageScope software. IHC staining in the CPE of dogs with acute SCI was 2.2, 1.6 and 1.5 times higher than that of normal dogs, for IL-1ß, TNF-α, and hsp70, respectively. Increases were statistically significant (p<0.1) for IL-1ß and TNF-α, and closely approached significance for hsp70. These findings indicate that the CPE could serve as an important source of these inflammatory mediators after SCI. There was also an inverse correlation between IL-1ß and hsp70 staining and duration of clinical signs in acute SCI, suggesting that increased expression of these proteins by the CPE may be of particular importance in the immediate-early inflammatory response after acute SCI.

Changes in the brain accumulation of glucocorticoids in abcb1a-deficient CF-1 mice.

The multidrug resistance transporter, P-glycoprotein (P-gp), contributes to highly lipophilic molecules penetrating the brain from the blood at a much lower rate than expected, and has numerous substrates, inhibitors and modulators. The drug-transporting isoform of P-gp is coded by a single human gene, ABCB1, and shares 80% homology with the murine drug-transporting isoforms, abcb1a and abcb1b, which share 92% homology with each other. Although these murine isoforms are highly similar, there are known affinity differences between the isoforms, and the localisation of the two isoforms in the brain is also disputed. Studies using mice genetically modified to be deficient in one or both isoforms of P-gp have also resulted in conflicting data. The contribution of the abcb1a isoform, which is considered to contribute most to the central nervous system (CNS)-protective role of P-gp, is investigated in the present study using CF-1-abcb1a(-/-) mice and the well-established brain/choroid plexus perfusion technique. Twenty-minute in situ brain/choroid plexus perfusions in CF-1-abcb1a(-/-) mice indicated the increased accumulation of [(3) H]cortisol, [(3) H]corticosterone and [(3) H]dexamethasone in most of the brain regions examined compared to CF-1-abcb1a(+/+) mice. Taken together with our earlier published studies in abcb1a/b(-/-) mice, these data strongly suggest that the in vivo CNS accumulation of glucocorticoids obtained using single knockout strains [e.g. abcb1a(-/-)] cannot be directly compared with those obtained in double knockout strains [e.g. abcb1a/b(-/-)].

Angiogenesis of buffalo choroid plexuses: structural and immunocytochemical study.

Mammalian choroid plexuses (CPs) are vascularized structures involved in numerous exchange processes that supply nutrients and hormones to the brain, and that remove deleterious compounds and metabolites from the brain. Studies in the adult Mediterranean buffalo have investigated the morphology of CPs using histochemical and immunohistochemical techniques. To date, however, there have been no studies conducted on ruminants regarding this removal process which serves to repair functional vascular damage in the CPs. Each of these vascular repair processes is a very complex and none of these has not yet been completely understood. Then, the aim of the present study is to investigate the morphological processes during angiogenesis in the CPs of healthy adult buffaloes, utilizing transmission electron microscopy (TEM), scanning electron microscopy (SEM), and immunogold-labeling SEM analysis (biomarkers: angiopoietin-2 [Ang-2], vascular endothelial growth factor receptor-3 [VEGFR-3], and CD133). At TEM, the inner surface of the blood capillaries sometimes showed pillar-like cells, which in contact with endothelial cells formed prominences, which in turn formed neo-blood capillaries. With immunogold-labeling SEM analysis, the CP blood capillaries showed Ang-2 and VEGF-3, respectively, in positive particles and spheroid formations. In addition, the external surface of the blood capillaries showed spheroid formations that originated from the neo-vascular capillaries whose terminals formed a capillary network, positive to CD133. On the basis of these results, the following hypothesis can be made, namely, that these CPs are vascular structures which play a fundamental role in maintaining brain homeostasis and self-repairing of functional vascular damage, independently of the presence of rete mirabile in this species.

Regulation of brain copper homeostasis by the brain barrier systems: effects of Fe-overload and Fe-deficiency.

Maintaining brain Cu homeostasis is vital for normal brain function. The role of systemic Fe deficiency (FeD) or overload (FeO) due to metabolic diseases or environmental insults in Cu homeostasis in the cerebrospinal fluid (CSF) and brain tissues remains unknown. This study was designed to investigate how blood-brain barrier (BBB) and blood-SCF barrier (BCB) regulated Cu transport and how FeO or FeD altered brain Cu homeostasis. Rats received an Fe-enriched or Fe-depleted diet for 4 weeks. FeD and FeO treatment resulted in a significant increase (+55%) and decrease (-56%) in CSF Cu levels (p<0.05), respectively; however, neither treatment had any effect on CSF Fe levels. The FeD, but not FeO, led to significant increases in Cu levels in brain parenchyma and the choroid plexus. In situ brain perfusion studies demonstrated that the rate of Cu transport into the brain parenchyma was significantly faster in FeD rats (+92%) and significantly slower (-53%) in FeO rats than in controls. In vitro two chamber Transwell transepithelial transport studies using primary choroidal epithelial cells revealed a predominant efflux of Cu from the CSF to blood compartment by the BCB. Further ventriculo-cisternal perfusion studies showed that Cu clearance by the choroid plexus in FeD animals was significantly greater than control (p<0.05). Taken together, our results demonstrate that both the BBB and BCB contribute to maintain a stable Cu homeostasis in the brain and CSF. Cu appears to enter the brain primarily via the BBB and is subsequently removed from the CSF by the BCB. FeD has a more profound effect on brain Cu levels than FeO. FeD increases Cu transport at the brain barriers and prompts Cu overload in the CNS. The BCB plays a key role in removing the excess Cu from the CSF.

Expression of orexin A and its receptor 1 in the choroid plexuses from buffalo brain.

The hypothalamic peptide orexin A, deriving from the proteolytic cleavage of the precursor molecule prepro-orexin, has a wide range of physiological effects including the regulation of feeding behaviour, neuroendocrine functions, sleep-wake cycle, and energy homeostasis. Lowered excretion of orexin A into the cerebrospinal fluid (CSF) plays a pathological role in animal and human narcolepsy. Altered levels of orexin A into the CSF have been also found in numerous disorders of the central nervous system, including Parkinson's and Huntington's disease, dementia, and depressive disorders. While the localization of orexin A and its receptor 1, OX(1), has been elicited in many regions of the mammalian brain and in peripheral organs, there are no information on the expression of the neuropeptide and its receptor 1 in the choroid plexuses (CPs) producing the CSF. In this study, we investigated the expression of orexin A and OX(1) in the CPs from the brain of an adult mammalian species, Bubalis bubalis, by immunogold-labelling in scanning electron microscopy. Both orexin A and OX(1) immuno-reactivity appeared to be widely distributed on the surface of choroid epithelium. Interestingly, a marked orexin A labelling was detected in the areas surrounding the CP blood capillaries. The expression of prepro-orexin and OX(1) mRNA transcripts of 200 and 300 bp, respectively, was assessed in the CPs by reverse-transcription polymerase chain reaction, while Western blotting analysis confirmed the presence of these two proteins in the tissue. Our findings provide the first evidence for orexin A and OX(1) expression in the CPs from mammalian brain, and suggest that the levels of orexin A into the CSF are probably regulated by CP activity.