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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The subcellular distribution of chromosome 6-encoded dystrophin-related protein in the brain.

Chromosome 6-encoded dystrophin-related-protein (DRP) shows significant structural similarities to dystrophin at the carboxyl terminus, though the two proteins are encoded on different chromosomes. Both DRP and dystrophin are expressed in muscle and brain and show some similarity in their subcellular localization. For example, in skeletal muscle both are expressed at neuromuscular and myotendinous junctions. However, while dystrophin is absent or severely reduced in Duchenne/Becker muscular dystrophy, DRP continues to be expressed. Within the brain, dystrophin is enriched at the postsynaptic regions of specific subsets of neurons, while the distribution of DRP is yet to be described. In this study we demonstrate a distinct though highly specific pattern of distribution of DRP in the brain. DRP is enriched in the choroid plexus, pia mater, intracerebral vasculature, and ependymal lining. Within the parenchyma proper, DRP is located at the inner plasma face of astrocytic foot processes at the abluminal aspect of the blood-brain barrier. The distribution of DRP is conserved across a large evolutionary distance, from mammals to elasmobranchs, suggesting that DRP may play a role in the maintenance of regional specializations in the brain.

Induction of somatostatin immunoreactivity in cultured ciliary ganglion neurons by activin in choroid cell-conditioned medium.

We have previously shown that the expression of somatostatin-like immunoreactivity in cultured ciliary ganglion neurons is stimulated by a macromolecule found in choroid cell-conditioned medium (ChCM). Here, we present the following evidence that this somatostatin-stimulating activity (SSA) is activin: human recombinant activin induces somatostatin-like immunoreactivity in CG neurons; ChCM induces hemoglobin synthesis in K562 cells, a biological activity characteristic of activin; activin A-specific antibodies recognize a protein in ChCM; cultured choroid cells contain activin RNA; and SSA is inhibited by follistatin, a specific activin-binding protein. Thus, activin is likely to be a neurodifferentiation factor for CG neurons in vivo.

Lipocalin 2 is a choroid plexus acute-phase protein.

Lipocalin 2 (LCN2) is able to sequester iron-loaded bacterial siderophores and, therefore, is known to participate in the mammalian innate immune response. Of notice, LCN2 was shown to display bacteriostatic effects both in in vitro and in vivo. To reach the brain, bacteria must cross the blood-brain or the choroid plexus (CP)/cerebrospinal fluid (CSF) barriers. Additionally, as the CP is responsible for the production of most of the CSF, responses of the CP mediate signaling into the brain. We show here that in conditions of peripheral inflammation, LCN2 behaves as an acute phase protein in the CP. As early as 1 h after lipopolysaccharide peripheral administration, Lcn2 mRNA levels are upregulated, returning to basal levels after 72 h. Increased LCN2 protein is observed in choroidal epithelia and in endothelial cells of blood vessels in the brain parenchyma. Higher levels of LCN2 are also present in the CSF. These observations suggest that expression of LCN2 at the CP/CSF barrier might be bacteriostatic in the brain, avoiding bacteria dissemination within the CSF into the brain parenchyma. This study shows that the LCN2 is produced by the CP as a component of the innate immune response that protects the central nervous system from infection.

The fodrin-ankyrin cytoskeleton of choroid plexus preferentially colocalizes with apical Na+K(+)-ATPase rather than with basolateral anion exchanger AE2.

A unique feature of the choroid plexus as a single-layer epithelium is its localization of Na+K(+)-ATPase at its apical (lumenal) surface. In contrast, a band 3 (AE1)-related anion exchanger protein has been localized to the basolateral surface of the choroid plexus. Both Na+K(+)-ATPase and AE1 in other tissues have been shown to bind via ankyrin to the spectrin-actin-based membrane cytoskeleton. Since linkage of integral membrane proteins to the membrane cytoskeleton is important for their restriction to specialized domains of the cell surface, we investigated the polarity of the choroid plexus membrane cytoskeleton. We developed isoform-specific antibodies to confirm the identity of choroid plexus band 3-related polypeptide as AE2. We demonstrated that ankyrin, fodrin/spectrin, actin, myosin, and alpha-actinin are predominantly apical in choroid plexus and preferentially colocalize with apical Na+K(+)-ATPase rather than with basolateral anion exchanger AE2. Colchicine administration did not alter the polarity of apical cytoskeletal and transport proteins or basolateral AE2 in choroid plexus, suggesting that biosynthetic targeting of these proteins is not microtubule dependent. In choroid plexus papilloma, Na+K(+)-ATPase and AE2 were decreased in amount and failed to preserve their polarized distributions.

Morphometric and some immunohistochemical characteristics of human choroids plexus stroma and psammoma bodies.

Psammoma bodies (PBs) are one of many choroids plexus aging changes. The aim of our research was to perform the quantification of PBs' presence in human choroids plexus stroma, as well as to evaluate the characteristics of choroids plexus stroma in cases in which PBs were present. Afterwards, the observations of the histochemical analysis would be confirmed by immunohistochemical analysis. Choroid plexuses of 30 cadavers were used for the histochemical and, choroids plexuses of 15 cadavers in which PBs' presence was confirmed during the histochemical analysis, were used as material for the immunohistochemical analysis. Light microscopy, histochemical, immunohistochemical, and morphometric method were applied during the study. Classification of the cases was performed by cluster analysis. We observed increase of choroids plexus PBs' presence during the aging process. But this increase is not linear. Their presence is the largest in the second cluster that is younger than the third and older than the first. Nuclear morphometric parameters of the stroma in these cases showed that the cellular composition in this cluster is different than in other two and, that contain larger number of lymphoid cells. Immunohistochemical analysis showed PBs' positive reaction on vimentin, CD45R0, and LCA markers, while in their vicinity, as well as inside them, numerous T-cells were observed. So, the presence of CD45R0 and LCA-positive T cells, PBs' positive reaction on the same markers, indirectly connect these cells with PBs' formation process.

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.

Immunohistochemical expression of prion protein (PrPC) in the human forebrain during development.

The cellular prion protein (PrPC) is a ubiquitous protein whose expression in the adult brain occurs mainly in synapses. We used monoclonal antibodies to study fetal and perinatal PrPC expression in the human forebrain. Double immunofluorescence and confocal microscopy with GFAP, Iba1, MAP2, doublecortin, synaptophysin, and GAP-43 were used to localize PrPC. PrPC immunoreactivity was observed in axonal tracts and fascicles from the 11th week to the end of gestation. Synapses expressed PrPC at increasing levels throughout synaptogenesis. At midgestation, a few PrPC-labeled neurons were detected in the cortical anlage and numerous ameboid and intermediate microglial cells were PrPC-positive. In contrast, at the end of gestation, microglial PrPC expression decreased to almost nothing, whereas neuronal PrPC expression increased, most notably in ischemic areas. In adults, PrPC immunoreactivity was restricted to the synaptic neuropil of the gray matter. At all ages, choroid plexus, ependymal, and endothelial cells were labeled, whereas astrocytes were only occasionally immunoreactive. In conclusion, the early expression of PrPC in the axonal field may suggest a specific role for this molecule in axonal growth during development. Moreover, PrPC may play a role in early microglial cell development.

Circulating hormones and hypothalamic energy balance: regulatory gene expression in the Lou/C and Wistar rats.

To ascertain the mechanisms underlying low caloric intake and low body weight in the Lou/C rat, the circulating hormone levels and gene expression of hypothalamic peptides and receptors important in energy balance and the induction of suppressor of cytokine signalling 3 (SOCS3) gene expression in response to leptin challenge were compared with Wistar rats. Plasma leptin levels were lower in the Lou/C rat, as were levels of rat corticosterone, TSH and T4 but not T3. Ghrelin levels were higher in the Lou/C rat. Total leptin receptor (Ob-R) and the long form of the leptin receptor (Ob-Rb) gene expression were lower in the arcuate (ARC) and ventromedial nuclei (VMN) in Lou/C rat. Ghrelin receptor expression in the ARC and VMN was lower in Lou/C than in Wistar rats. However, agouti gene-related peptide (AgRP) and neuropeptide Y (NPY) gene expression were higher in the Lou/C rat. There was no difference in the level of cocaine- and amphetamine-regulated transcript gene expression in the ARC, but both were higher in the paraventricular nuclei of the Lou/C breed. There was no difference in Ob-R gene expression in, or [(125)I]leptin binding to, the choroid plexus. SOCS3 mRNA induction in response to leptin was lower in the Lou/C rat. This study reveals that the comparatively low plasma leptin, TSH and T4 levels, and high ghrelin levels together with high levels of AgRP and NPY gene expression seen in the Lou/C rat are indicative of a strong drive to eat and decreased energy expenditure, which are in direct opposition to the comparatively low body weight and adiposity of this rat strain.

Reduced cerebrospinal fluid production and intracranial pressure in mice lacking choroid plexus water channel Aquaporin-1.

Aquaporin-1 (AQP1) is a water channel expressed strongly at the ventricular-facing surface of choroid plexus epithelium. We developed novel methods to compare water permeability in isolated choroid plexus of wild-type vs. AQP1 null mice, as well as intracranial pressure (ICP) and cerebrospinal fluid (CSF) production and absorption. Osmotically induced water transport was rapid in choroid plexus from wild-type mice and reduced by fivefold by AQP1 deletion. AQP1 deletion did not affect choroid plexus size or structure. By stereotaxic puncture of the lateral ventricle with a microneedle, ICP was 9.5 +/- 1.4 cm H2O in wild-type mice and 4.2 +/- 0.4 cm H2O in AQP1 null mice. CSF production, an isosmolar fluid secretion process, was measured by a dye dilution method involving fluid collections using a second microneedle introduced into the cisterna magna. CSF production in wild-type mice was (in microl min(-1)) 0.37 +/- 0.04 (control), 0.16 +/- 0.03 (acetazolamide-treated), and 1.14 +/- 0.15 (forskolin-treated), and reduced by approximately 25% in AQP1 null mice. Pressure-dependent CSF outflow, measured from steady-state ICP at different ventricular infusion rates, was not affected by AQP1 deletion. In a model of focal brain injury, AQP1 null mice had remarkably reduced ICP and improved survival compared with wild-type mice. The reduced ICP and CSF production in AQP1 null mice provides direct functional evidence for the involvement of AQP1 in CSF dynamics, suggesting AQP1 inhibition as a novel option for therapy of elevated ICP.