The senses of the choroid plexus.

The composition of cerebrospinal and brain interstitial fluids is ensured by barriers between the blood and the brain parenchyma (the blood-brain barrier) and between the blood and the cerebrospinal fluid (the blood-cerebrospinal fluid barrier). Barrier function results from the combination of tight junctions between cells that impair solute flux via the paracellular pathway, cell membrane transporters that enable selective transcellular solute passage, and intracellular metabolizing enzymes that transform molecules in transit. Collectively, they comprise a chemical surveillance system, essential to protect the brain from toxicants, microorganisms, and other harmful compounds. Conversely, this chemical surveillance system compromises the brain delivery of many pharmacologic agents against brain cancer and brain metastasis, neurodegenerative diseases, and brain infections. Despite their importance, the mechanisms underlying the regulation of the components of this chemical surveillance system in response to alterations in the composition of blood and brain fluids are still poorly understood. We propose that odorant receptors, vomeronasal receptors and taste receptors, recently identified at brain barriers might be upstream components of this surveillance system. These chemosensory receptors are strategically placed to monitor the composition of blood, cerebrospinal and brain interstitial fluids. Upon ligand-binding, they may deploy the action of transporters and detoxifying enzymes or other unprecedented functions in brain barrier cells, to cope with alterations in the composition of blood and brain cerebrospinal and interstitial fluids, working as guardians of the central nervous system.

Sex Hormone Decline and Amyloid ß Synthesis, Transport and Clearance in the Brain.

Sex hormones (SH) are essential regulators of the central nervous system. The decline in SH levels along with ageing may contribute to compromised neuroprotection and set the grounds for neurodegeneration and cognitive impairments. In Alzheimer's disease, besides other pathological features, there is an imbalance between amyloid ß (Aß) production and clearance, leading to its accumulation in the brain of older subjects. Aß accumulation is a primary cause for brain inflammation and degeneration, as well as concomitant cognitive decline. There is mounting evidence that SH modulate Aß production, transport and clearance. Importantly, SH regulate most of the molecules involved in the amyloidogenic pathway, their transport across brain barriers for elimination, and their degradation in the brain interstitial fluid. This review brings together data on the regulation of Aß production, metabolism, degradation and clearance by SH.

Sex Hormones Protect Against Amyloid-ß Induced Oxidative Stress in the Choroid Plexus Cell Line Z310.

The choroid plexus (CP) epithelium is a unique structure in the brain that forms an interface between the peripheral blood on the basal side and the cerebrospinal fluid (CSF) on the apical side. It is a relevant source of many polypeptides secreted to the CSF with neuroprotective functions and also participates in the elimination and detoxification of brain metabolites, such as ß-amyloid (Aß) removal from the CSF through transporter-mediated influx. The CP is also a target tissue for sex hormones (SHs) that have recognised neuroprotective effects against a variety of insults, including Aß toxicity and oxidative stress in the central nervous system. The present study aimed to understand how SHs modulate Aß-induced oxidative stress in a CP cell line (Z310 cell line) by analysing the effects of Aß1-42 on oxidative stress, mitochondrial function and apoptosis, as well as by assessing how 17ß-oestradiol (E2 ) and 5α-dihydrotestosterone (DHT) modulated these effects and the cellular uptake of Aß1-42 by CP cells. Our findings show that E2 and DHT treatment reduce Aß1-42 -induced oxidative stress and the internalisation of Aß1-42 by CP epithelial cells, highlighting the importance of considering the background of SHs and therefore sex-related differences in Aß metabolism and clearance by CP cells.

"Tasting" the cerebrospinal fluid: Another function of the choroid plexus?

The choroid plexus (CP) located in brain ventricles, by forming the interface between the blood and the cerebrospinal fluid (CSF) is in a privileged position to monitor the composition of these body fluids. Yet, the mechanisms involved in this surveillance system remain to be identified. The taste transduction pathway senses some types of molecules, thereby evaluating the chemical content of fluids, not only in the oral cavity but also in other tissues throughout the body, such as some cell types of the airways, the gastrointestinal tract, testis and skin. Therefore, we hypothesized that the taste transduction pathway could also be operating in the CP to assess the composition of the CSF. We found transcripts for some taste receptors (Tas1r1, Tas1r2, Tas1r3, Tas2r109 and Tas2r144) and for downstream signaling molecules (α-Gustducin, Plcß2, ItpR3 and TrpM5) that encode this pathway, and confirmed the expression of the corresponding proteins in Wistar rat CP explants and in the CP epithelial cells (CPEC). The functionality of the T2R receptor expressed in CP cells was assessed by calcium imaging, of CPEC stimulated with the bitter compound D-Salicin, which elicited a rise in the intracellular Ca(2+). This effect was diminished in the presence of the bitter receptor blocker Probenecid. In summary, we described the expression of the taste-related components involved in the transduction signaling cascade in CP. Taken together, our results suggest that the taste transduction pathway in CPEC makes use of T2R receptors in the chemical surveillance of the CSF composition, in particular to sense bitter noxious compounds.

Sex-Related Differences in Rat Choroid Plexus and Cerebrospinal Fluid: A cDNA Microarray and Proteomic Analysis.

The choroid plexus (CP) epithelium is a unique structure in the brain that forms an interface between the peripheral blood and the cerebrospinal fluid (CSF), which is mostly produced by the CP itself. Because the CP transcriptome is regulated by the sex hormone background, the present study compared gene/protein expression profiles in the CP and CSF from male and female rats aiming to better understand sex-related differences in CP functions and brain physiology. We used data previously obtained by cDNA microarrays to compare the CP transcriptome between male and female rats, and complemented these data with the proteomic analysis of the CSF of castrated and sham-operated males and females. Microarray analysis showed that 17 128 and 17 002 genes are expressed in the male and female CP, which allowed the functional annotation of 141 and 134 pathways, respectively. Among the most expressed genes, canonical pathways associated with mitochondrial dysfunctions and oxidative phosphorylation were the most prominent, whereas the most relevant molecular and cellular functions annotated were protein synthesis, cellular growth and proliferation, cell death and survival, molecular transport, and protein trafficking. No significant differences were found between males and females regarding these pathways. Seminal functions of the CP differentially regulated between sexes were circadian rhythm signalling, as well as several canonical pathways related to stem cell differentiation, metabolism and the barrier function of the CP. The proteomic analysis identified five down-regulated proteins in the CSF samples from male rats compared to females and seven proteins exhibiting marked variation in the CSF of gonadectomised males compared to sham animals, whereas no differences were found between sham and ovariectomised females. These data clearly show sex-related differences in CP gene expression and CSF protein composition that may impact upon neurological diseases.

Gender associated circadian oscillations of the clock genes in rat choroid plexus.

It is well-documented that circadian rhythms are controlled by the circadian master clock of the mammalian brain, located in the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN clockwork is a cell autonomous mechanism consisting of a series of interlocked transcriptional/post-translational feedback loops. In turn, the SCN controls the seasonal rhythmicity of various biological processes, in particular the secretion pattern of hormones. Although the effects of gonadal hormones on circadian rhythmicity are clearly established, how the SCN integrates and regulates these hormonal stimuli remains unknown. We have previously found that clock genes are expressed in the choroid plexus (CP). Therefore, we compared the circadian expression of these genes in female and male rat CP. We show that there is a 24-h rhythm in the expression of Per2 and Cry2 in males and females. Bmal1 and Per1 expression also varied along the day, but only in females. Bmal1, Clock and Per1 mRNA did not show any significant differences in the CP of males. Moreover, data from cultured CP cells collected at different timepoints revealed significant circadian rhythms in mRNA abundance of Bmal1, Clock and Per2. In conclusion, our data show that the rat CP expresses all canonical clock genes and that their circadian expression differs between genders suggesting that hormones can regulate circadian rhythmicity in CP.

17beta-estradiol induces transthyretin expression in murine choroid plexus via an oestrogen receptor dependent pathway.

Oestrogen protects against AD by multiple mechanisms, including the enhancement of Abeta clearance. Transthyretin (TTR) is a homotetrameric protein mainly synthesized by the liver and choroid plexus (CP) of the brain that sequesters the amyloid beta (Abeta) peptide. In this study we examined the effects of 17beta-estradiol (E2) on TTR protein and mRNA levels, in primary cultures of rat CP epithelial cells (CPEC) by Western blot and Real Time PCR, respectively. Moreover, the localization of oestrogen receptors alpha (ERalpha) and beta (ERbeta) in response to E2 treatment was analysed by confocal microscopy in these cells. The expression of TTR, ERalpha and ERbeta was also compared in the CP of castrated female mice treated with E2 to vehicle-treated animals by Real Time PCR. TTR concentration in the CSF of all these animals was measured by radioimmunoassay. E2 treatment induced TTR transcription and increased TTR protein content in CPEC. Pre-treatment with ICI 182,780 (ICI) abrogated E2-induced TTR expression suggesting that, TTR is up-regulated via an ER-dependent pathway. Confocal microscopy demonstrated extranuclear ERalpha and ERbeta localization in untreated CPEC. Upon E2 treatment, translocation of ERalpha to the nucleus occurred, while ERbeta remained in the cytosol. These data was concurrent with the up-regulation of TTR expression detected in the CP of castrated female mice subjected to E2 treatment. Our results highlight the importance of E2 on the regulation of TTR, which may participate in the oestrogen-induced decrease in Abeta levels and deposition described in the literature.

5Alpha-dihydrotestosterone up-regulates transthyretin levels in mice and rat choroid plexus via an androgen receptor independent pathway.

Transthyretin (TTR) is a 55 kDa plasma homotetrameric protein mainly synthesized in the liver and choroid plexuses (CPs) of the brain that, functions as a carrier for thyroxin and retinol binding protein. It sequesters amyloid beta (Abeta) peptide, and TTR levels in the cerebrospinal fluid (CSF) appear to be inversely correlated with Alzheimer's disease (AD) onset and progression. Androgen deprivation increases plasma Abeta levels, which indicate that androgens may reduce the levels of soluble Abeta, the peptide widely implicated in the initiation of AD pathogenesis; however, the underlying mechanisms are still poorly understood. In this study we examined the effects of 5alpha-dihydrotestosterone (DHT) on TTR protein and mRNA levels, in primary cultures of rat CPs epithelial cells (CPEC) by Western blot, and real time PCR, respectively. Moreover, TTR concentrations were measured in the CSF of castrated wild-type, and transgenic mice expressing human TTR subjected to DHT treatment, by radioimmunoassay and ELISA, respectively. TTR mRNA expression was also compared in the CPs, of the animals from each experimental group by real time PCR. DHT treatment increased TTR protein levels in CPEC, and induced TTR transcription in these cells. The combination of flutamide with DHT in the treatment of CPEC did not abrogate DHT-induced TTR levels, suggesting that TTR is up-regulated via an androgen receptor independent pathway. In the CPs of both mice strains, DHT also increased TTR mRNA levels, but no significant differences in TTR protein levels were detected in the CSF of these animals. These findings open a wide range of possibilities for future studies on Abeta deposition and cognitive function, in response to androgen induction of TTR in animal models of AD.

Transthyretin is up-regulated by sex hormones in mice liver.

Misfolding and aggregation of mutated and wild-type transthyretin (TTR) can cause familial amyloid polyneuropathy (FAP) and senile systemic amyloidosis (SSA), respectively. In some populations, FAP onset seems to occur on average 2-11 years earlier in men than in women, and SSA appears to be a disease of elderly men. Most (95-100%) SSA patients described in the literature are men, suggesting that amyloid deposition in these patients may be sex hormone related. On the basis of gender-related differences in FAP onset, and on the almost exclusivity of SSA in elder men, we hypothesize that, sex hormones may increase TTR synthesis by the liver, and therefore, may contribute to amyloid deposition. In order to test this hypothesis, castrated female and male mice were implanted with alzet mini-osmotic pumps, delivering 17beta-estradiol (E2) or 5alpha-dihydrotestosterone (DHT), or vehicle only, for 1 week. Sham operated animals were also included in the experiment. After hormonal stimulation, mice were euthanized under anaesthesia, and liver and sera were collected. The expression of TTR in liver, and the levels of TTR in sera in response to E2 and DHT were analysed by Real Time PCR and radioimmunoassay, respectively. Data analysis showed that, both hormones induced TTR transcription, which was concurrent with a consistent increase in the circulating levels of the protein. Taken together, all these data provide an indication that sex hormone stimulation may constitute a risk factor for SSA.

Characterization of oligoadenylate synthetase-1 expression in rat mammary gland and prostate: effects of 17beta-estradiol on the regulation of OAS1g in both tissues.

OAS1 belongs to a protein family of interferon-induced enzymes characterized by their ability to catalyze the synthesis of 2'-5'-linked oligomers of adenosine from ATP (2-5A). 2-5A bind to the latent Ribonuclease L (RNase L), which subsequently dimerizes into the active form, acquiring the capacity of cleaving cellular and viral mRNA. Several studies indicate that OAS1 is an important inducer of apoptosis in human cancer cells and that it may be regulated by 17beta-estradiol (E(2)). The aim of this study was to characterize OAS1 gene expression in rat mammary gland and prostate, and to analyze its regulation by E(2) in both tissues. It is demonstrated that OAS1g is the most abundant OAS1 gene expressed in both tissues, and that OAS1 protein is present in the nucleus of rat mammary gland and prostate epithelial cells. In addition, it is shown by Real Time PCR that OAS1g is up-regulated by E(2) in rat mammary gland, but down-regulated in prostate, suggesting that the OAS1g gene may be related to estrogen dependent pathways in rat mammary gland and prostate physiology.

Regulation of transthyretin by thyroid hormones in fish.

Transthyretin (TTR) is a thyroid hormone-binding protein (THBP) which in its tetrameric form transports thyroid hormones (THs), thyroxine (T(4)) and triiodothyronine (T(3)) in the blood of vertebrates. The principal site of production of TTR is the liver but in the sea bream TTR mRNA is also present in the heart, intestine and brain. The regulation of TTR is unstudied in fish and the normal circulating level of this THBP is unknown. The aim of the present study was to establish factors which regulate TTR production in fish. As a first step a number of tools were generated; sea bream recombinant TTR (sbrTTR) and specific sbrTTR antisera which were used to establish an ELISA (enzyme-linked immunosorbent assay) for measuring TTR plasma levels. Subsequently, an experiment was conducted to determine the influence of THs on TTR production. Circulating physiological levels of TTR in sea bream determined by ELISA are approximately 3.8microgml(-1). Administration of T(3) and T(4) to sea bream significantly increased (p<0.001 and p<0.005, respectively) the concentration of circulating TTR ( approximately or = 11.5microgml(-1)) in relation to control fish, but did not change gene transcription in the liver. Methimazol (MMI) an antithyroid agent, failed to significantly reduce circulating THs below control levels but significantly increased (p<0.005) plasma TTR levels (approximately or = 10.8microgml(-1)) and decreased (p<0.05) transcription in the liver. Future studies will aim to elucidate in more detail these regulatory pathways.

Isolation of a novel aquaglyceroporin from a marine teleost (Sparus auratus): function and tissue distribution.

The aquaporins (formerly called the major intrinsic protein family) are transmembrane channel proteins. The family includes the CHIP group, which are functionally characterised as water channels and the GLP group, which are specialised for glycerol transport. The present study reports the identification and characterisation of a novel GLP family member in a teleost fish, the sea bream Sparus auratus. A sea bream aquaporin (sbAQP) cDNA of 1047 bp and encoding a protein of 298 amino acids was isolated from a kidney cDNA library. Functional characterization of the sbAQP using a Xenopus oocyte assay revealed that the isolated cDNA stimulated osmotic water permeability in a mercury-sensitive manner and also stimulated urea and glycerol uptake. Northern blotting demonstrated that sbAQP was expressed at high levels in the posterior region of the gut, where two transcripts were identified (1.6 kb and 2 kb), and in kidney, where a single transcript was present (2 kb). In situ hybridisation studies with a sbAQP riboprobe revealed its presence in the lamina propria and smooth muscle layer of the posterior region of the gut and in epithelial cells of some kidney tubules. sbAQP was also present in putative chloride cells of the gill. Phylogenetic analysis of sbAQP, including putative GLP genes from Fugu rubripes, revealed that it did not group with any of the previously isolated vertebrate GLPs and instead formed a separate group, suggesting that it may be a novel GLP member.

Developmental ontogeny of prolactin and prolactin receptor in the sea bream (Sparus aurata).

The expression of PRL and its receptor (PRLR) were characterised during sea bream embryonic and larval development, by semi-quantitative and quantitative RT-PCR, respectively, until 46 days post-hatch (DPH). Immunocytochemistry with antisera specific for sea bream PRLR was carried out with larval sections from hatching up to 46 DPH. A single transcript of PRL (1.35 Kb) and PRLR (2.8 Kb) identical to the transcripts previously characterised in adult tissue, are present in sea bream embryos and larvae. PRL expression is first detectable at neurula and in all samples collected thereafter. The lowest levels of PRL mRNA are detected in sea bream embryos up until neurula when expression starts to increase. The maximal levels of PRL expression were detected at 24 DPH. PRLR transcripts first appear at 12h post-fertilisation (0.002 rho mol/microg total larvae RNA) (blastula) and increase significantly during gastrulation (0.245 rho mol/microg total larvae RNA) reaching a maximum at 2 DPH (0.281 rho mol/microg total larvae RNA). After hatching a significant reduction in PRLR expression is observed which reaches a minimum at 4 DPH (0.103 rho mol/microg total larvae RNA), gradually increasing thereafter. Immunocytochemistry revealed the presence of PRLR in early post-hatching stages of larvae in tissues derived from all three germ layers.