Mouse Choroid Plexus Research Articles

Assessment of common housekeeping genes as reference for gene expression studies using RT-qPCR in mouse choroid plexus

Choroid plexus (ChP), a vascularized secretory epithelium located in all brain ventricles, plays critical roles in development, homeostasis and brain repair. Reverse transcription quantitative real-time PCR (RT-qPCR) is a popular and useful technique for measuring gene expression changes and also widely used in ChP studies. However, the reliability of RT-qPCR data is strongly dependent on the choice of reference genes, which are supposed to be stable across all samples. In this study, we validated the expression of 12 well established housekeeping genes in ChP in 2 independent experimental paradigms by using popular stability testing algorithms: BestKeeper, DeltaCq, geNorm and NormFinder. Rer1 and Rpl13a were identified as the most stable genes throughout mouse ChP development, while Hprt1 and Rpl27 were the most stable genes across conditions in a mouse sensory deprivation experiment. In addition, Rpl13a, Rpl27 and Tbp were mutually among the top five most stable genes in both experiments. Normalisation of Ttr and Otx2 expression levels using different housekeeping gene combinations demonstrated the profound effect of reference gene choice on target gene expression. Our study emphasized the importance of validating and selecting stable housekeeping genes under specific experimental conditions.

Multiple Na,K-ATPase Subunits Colocalize in the Brush Border of Mouse Choroid Plexus Epithelial Cells.

(1) Background: The unusual accumulation of Na,K-ATPase complexes in the brush border membrane of choroid plexus epithelial cells have intrigued researchers for decades. However, the full range of the expressed Na,K-ATPase subunits and their relation to the microvillus cytoskeleton remains unknown. (2) Methods: RT-PCR analysis, co-immunoprecipitation, native PAGE, mass spectrometry, and differential centrifugation were combined with high-resolution immunofluorescence histochemistry, proximity ligase assays, and stimulated emission depletion (STED) microscopy on mouse choroid plexus cells or tissues in order to resolve these issues. (3) Results: The choroid plexus epithelium expresses Na,K-ATPase subunits α1, α2, β1, β2, β3, and phospholemman. The α1, α2, β1, and β2, subunits are all localized to the brush border membrane, where they appear to form a complex. The ATPase complexes may stabilize in the brush border membrane via anchoring to microvillar actin indirectly through ankyrin-3 or directly via other co-precipitated proteins. Aquaporin 1 (AQP1) may form part of the proposed multi-protein complexes in contrast to another membrane protein, the Na-K-2Cl cotransporter 1 (NKCC1). NKCC1 expression seems necessary for full brush border membrane accumulation of the Na,K-ATPase in the choroid plexus. (4) Conclusion: A multitude of Na,K-ATPase subunits form molecular complexes in the choroid plexus brush border, which may bind to the cytoskeleton by various alternative actin binding proteins.

Sodium/glucose cotransporter 2 is expressed in choroid plexus epithelial cells and ependymal cells in human and mouse brains.

Diabetes mellitus (DM) is now recognized as one of the risk factors for Alzheimer's disease (AD), and the disease‐modifying effects of anti‐diabetic drugs on AD have recently been attracting great attention. Sodium/glucose cotransporter 2 (SGLT2) inhibitors are a new class of anti‐diabetic drugs targeting the SGLT2/solute carrier family 5 member 2 (SLC5A2) protein, which is known to localize exclusively in the brush border membrane of early proximal tubules in the kidney. However, recent data suggest that it is also expressed in other tissues. In the present study, we investigated the expression of SGLT2/SLC5A2 in human and mouse brains. Immunohistochemical staining of paraffin sections from autopsied human brains and C3H/He mouse brains revealed granular cytoplasmic immunoreactivity in choroid plexus epithelial cells and ependymal cells. Immunoblot analysis of the membrane fraction of mouse choroid plexus showed distinct immunoreactive bands at 70 and 26 kDa. Band patterns around 70 kDa in the membrane fraction of the choroid plexus were different from those in the kidney. Reverse transcription‐polymerase chain reaction analysis confirmed the expression of Slc5a2 mRNA in the mouse choroid plexus. Our results provide in vivo evidence that SGLT2/SLC5A2 is expressed in cells facing the cerebrospinal fluid, in addition to early proximal tubular epithelial cells. These findings suggest that SGLT2 inhibitors may have another site of action in the brain. The effects of SGLT2 inhibitors on brain function and AD progression merit further investigation to develop better treatment options for DM patients.

Proliferation of endothelial cells in the choroid plexus of normal and hydrocephalic mice

The choroid plexus (CP), located at the walls of the brain ventricles, produces and secretes cerebrospinal fluid (CSF). Hydrocephalus is a neurological disorder in which the CP abnormally secretes excess amounts of CSF into the ventricles. There is currently no information on the vascular dynamics of the CP in adult brains under normal and hydrocephalic conditions. In the present study, we reported the continuous proliferation of endothelial cells in the CP of normal mice, which depended on vascular endothelial cell growth factor (VEGF). The proliferation of endothelial cells increased in mice with intraventricular hemorrhage, which was attenuated by a pretreatment with the toll-like receptor 4 (TLR4) inhibitor VIPER. Moreover, the intracerebroventricular infusion of the TLR4 agonist, lipopolysaccharide, increased endothelial cell proliferation in the CP and induced ventriculomegaly. The present results provide insights into the importance of the TLR4-initiated and VEGF-dependent proliferation of endothelial cells in the pathogenesis of hydrocephalus.

Are There Any Sex-Depended Differences in Water Transporting Proteins of Choroid Plexus in Mice?

Are There Any Sex-Depended Differences in Water Transporting Proteins of Choroid Plexus in Mice?

Mechanism of the neuroprotective effect of injecting brain cells on ST36 in an animal model of Parkinson’s disease

In Parkinson’s disease, the dopaminergic neurons of the brain are destroyed. Dopamine is an important neurotransmitter that acts on the basal ganglia of the brain, allowing precise body movement. In the early stages of Parkinson’s disease, levodopa appears to alleviate clinical symptoms; however, during long-term use, motor complications occur. There is no clear treatment or remedy for Parkinson’s disease; therefore, the development of novel therapies is urgently required. In the present study, mouse choroid plexus cells were transplanted into ST36 in a mouse model of Parkinson’s disease to determine whether the motor function could be restored. Pole tests showed changes in motor dysfunction in the mice. The athletic ability of the mice was significantly lowered after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) injection and significantly increased after choroidal neuron cell treatment. Injection of di-alkyl indocarbocyanine (DiI) (as a trace substance) confirmed that the choroid plexus cells injected into acupuncture point ST36 were transferred to the brain. In the Parkinson’s disease model, choroid plexus cell injection into ST36 inhibited the decrease in tyrosine hydroxylase (TH) expression and decreased the activation of inflammatory factors mitochondrial cytochrome C oxidase (COX2) and inducible NO synthase (iNOS). Apoptosis factors Cytochrome C and BCL2 associated X, apoptosis regulator (BAX) levels were decreased and B-Cell CLL/Lymphoma 2 (BCL2) levels were increased. Taken together, these results suggest that the injection of choroid plexus cell at ST36 had neuroprotective effects in the Parkinson’s disease mouse model. The results suggest new possibilities for the treatment of Parkinson's disease.

Ncbe (slc4a10) is a major Na+ loader in the mouse choroid plexus epithelium

Ncbe (<i>slc4a10</i>) is a major Na⁺ loader in the mouse choroid plexus epithelium

Prolactin regulation of insulin‐like growth factor 2 gene expression in the adult mouse choroid plexus

There is clear evidence for carrier-mediated transport of prolactin into the brain, and it has been widely assumed that prolactin receptors (PRLRs) in the choroid plexus (ChP) might mediate this transport. Using PRLR knockout mice, we recently showed that PRLRs in ChP are not required for prolactin transport into the brain. Hence, the function of PRLR in the ChP remains unknown. PRLR expression is increased in the ChP during lactation, suggesting a possible role in adaptive function of prolactin at this time. To gain insight into prolactin function in the ChP, we have utilized RNA sequencing and NanoString techniques to characterize transcriptional changes in response to differing levels of prolactin at diestrus, during pregnancy, and in lactation. We have observed opposing transcriptional effects of prolactin on the ChP in different physiologic states, being primarily inhibitory during diestrus but stimulatory in lactation. Insulin-like growth factor 2 (Igf2), a highly expressing transcript found in the ChP, showed a 6-fold increase at lactation that returned to baseline on suppression of prolactin levels. These results indicate that Igf2 may be an important downstream mediator of prolactin-induced signaling in the ChP.-Phillipps, H. R., Rand, C. J., Brown, R. S. E., Kokay, I. C., Stanton, J.-A., Grattan, D. R. Prolactin regulation of insulin-like growth factor 2 gene expression in the adult mouse choroid plexus.

A new genetically engineered mouse model of choroid plexus carcinoma

Choroid plexus carcinomas (CPCs) are highly malignant brain tumours predominantly found in children and associated to poor prognosis. Improved therapy for these cancers would benefit from the generation of animal models. Here we have created a novel mouse CPC model by expressing a stabilised form of c-Myc (MycT58A) and inactivating Trp53 in the choroid plexus of newborn mice. This induced aberrant proliferation of choroid plexus epithelial cells, leading to aggressive tumour development and death within 150 days. Choroid plexus tumours occurred with a complete penetrance in all brain ventricles, with prevalence in the lateral and fourth ventricles. Histological and cellular analysis indicated that these tumours were CPCs resembling their human counterparts. Comparison of gene expression profiles of CPCs and non-neoplastic tissues revealed profound alterations in cell cycle regulation and DNA damage responses, suggesting that dysregulation of cell division and DNA checkpoint pathways may represent key vulnerabilities. This novel animal model of CPC provides an invaluable tool to elucidate the mechanism of CPC formation and to develop successful therapies against this devastating paediatric cancer.

Application of Europium-Doped Very Small Iron Oxide Nanoparticles to Visualize Neuroinflammation with MRI and Fluorescence Microscopy

Our recent studies demonstrated that electrostatically stabilized very small superparamagnetic iron oxide particles (VSOPs) are promising MRI probes for detecting various pathological aspects of autoimmunity in the central nervous system (CNS). However, investigation of the precise tissue and cellular distribution of VSOP has been technically limited due to the need to use iron detection methods for VSOP visualization. Therefore, we assessed here the utility of europium (Eu)-doped VSOP as an MRI tool for in vivo investigations in the animal model experimental autoimmune encephalomyelitis (EAE), and as a tool to investigate histopathological processes in the CNS using fluorescence microscopy.We demonstrated that Eu-VSOP display the same properties as VSOP in terms of revealing inflammation-mediated changes by binding to brain endothelium in vitro, and in terms of visualizing brain lesions in EAE in vivo. MRI examinations with Eu-VSOP confirm that at peak disease particles accumulated inside the choroid plexus, and in cerebellar and meningeal lesions. Importantly, Eu-VSOP-based MRI showed for the first time in a longitudinal setup that particles were absent from the choroid plexus in mice during remission of EAE, but accumulated again during subsequent relapse. Within the choroid plexus, Eu-VSOP were associated both with monocytes/macrophages present in the plexus stroma, and associated with epithelial cells.Using Eu-VSOP, we demonstrated for the first time the involvement of the choroid plexus in relapses. Thus, Eu-VSOP have the potential to reveal various aspects of choroid plexus involvement in neuroinflammation, including monocyte recruitment from the blood and alterations of the choroid plexus epithelium.

CD8+ T cells are essential for the effects of enriched environment on hippocampus-dependent behavior, hippocampal neurogenesis and synaptic plasticity

Enriched environment (EE) induces plasticity changes in the brain. Recently, CD4+ T cells have been shown to be involved in brain plasticity processes. Here, we show that CD8+ T cells are required for EE-induced brain plasticity in mice, as revealed by measurements of hippocampal volume, neurogenesis in the DG of the hippocampus, spinogenesis and glutamatergic synaptic function in the CA of the hippocampus. As a consequence, EE-induced behavioral benefits depend, at least in part, on CD8+ T cells. In addition, we show that spleen CD8+ T cells from mice housed in standard environment (SE) and EE have different properties in terms of 1) TNFα release after in vitro CD3/CD28 or PMA/Iono stimulation 2) in vitro proliferation properties 3) CD8+ CD44+ CD62Llow and CD62Lhi T cells repartition 4) transcriptomic signature as revealed by RNA sequencing. CD8+ T cells purified from the choroid plexus of SE and EE mice also exhibit different transcriptomic profiles as highlighted by single-cell mRNA sequencing. We show that CD8+ T cells are essential mediators of beneficial EE effects on brain plasticity and cognition. Additionally, we propose that EE differentially primes CD8+ T cells leading to behavioral improvement.

The choroid plexus is a key cerebral invasion route for T cells after stroke.

Neuroinflammation contributes substantially to stroke pathophysiology. Cerebral invasion of peripheral leukocytes-particularly T cells-has been shown to be a key event promoting inflammatory tissue damage after stroke. While previous research has focused on the vascular invasion of T cells into the ischemic brain, the choroid plexus (ChP) as an alternative cerebral T-cell invasion route after stroke has not been investigated. We here report specific accumulation of T cells in the peri-infarct cortex and detection of T cells as the predominant population in the ipsilateral ChP in mice as well as in human post-stroke autopsy samples. T-cell migration from the ChP to the peri-infarct cortex was confirmed by in vivo cell tracking of photoactivated T cells. In turn, significantly less T cells invaded the ischemic brain after photothrombotic lesion of the ipsilateral ChP and in a stroke model encompassing ChP ischemia. We detected a gradient of CCR2 ligands as the potential driving force and characterized the neuroanatomical pathway for the intracerebral migration. In summary, our study demonstrates that the ChP is a key invasion route for post-stroke cerebral T-cell invasion and describes a CCR2-ligand gradient between cortex and ChP as the potential driving mechanism for this invasion route.

Gender-specific expression of ATP-binding cassette (Abc) transporters and cytoprotective genes in mouse choroid plexus.

The choroid plexus (CP) and blood-brain barrier (BBB) control the movement of several drugs and endogenous compounds between the brain and systemic circulation. The multidrug resistance associated protein (Mrp) efflux transporters form part of these barriers. Several Mrp transporters are positively regulated by the transcription factor nuclear factor erythroid-2-related factor (Nrf2) in liver. The Mrps, Nrf2 and Nrf2-dependent genes are cytoprotective and our aim was to examine basal gender differences in expression of Mrp transporters, Nrf2 and Nrf2-dependent genes (Nqo1 and Ho-1) in the brain-barriers. Previous studies have shown higher expression of Mrp1, Mrp2 and Mrp4 in female mouse liver and kidney. We hypothesized that similar renal/hepatic gender-specific patterns are present in the brain-barrier epithelia interfaces. qPCR and immunoblot analyses showed that Mrp4, Ho-1 and Nqo1 expression was higher in female CP. Mrp1, Mrp2 and Nrf2 expression in the CP had no gender pattern. Female Mrp1, Mrp2 and Mrp4 mouse brain expressions in remaining brain areas, excluding CP, were higher than male. Functional analysis of Mrp4 in CP revealed active accumulation of the Mrp4 model substrate fluo-cAMP. WT female CP had 10-fold higher accumulation in the vascular spaces than males and 60% higher than Mrp4-/- females. Probenecid blocked all transport. Methotrexate did as well except in Mrp4-/- females where it had no effect, suggesting compensatory induction of transport occurred in Mrp4-/-. Collectively, our findings indicate significant gender differences in expression of Mrp transporters and cytoprotective genes in the CP and BBB.

Toll-like receptors in the brain of mice following infection with Acanthamoeba spp.

The Toll-like receptors (TLRs) of the innate immune system play an important role in the recognition of pathogens such as bacteria, viruses, fungi, and parasites. In this study, we examined the changes in the level of expression of TLR2 and TLR4 mRNA and protein in the brains of mice infected with Acanthamoeba spp. The Acanthamoeba strains were isolated from a patient with Acanthamoeba keratitis (AK) (Ac55) and Malta Lake (Ac43). In the brain isolated from mice at 2 days post-infection (dpi) with Acanthamoeba strains Ac55 and Ac43, mRNAs for TLR2 and TLR4 were significantly more strongly expressed in comparison with the uninfected mice. In Acanthamoeba-infected mice, TLR2 and TLR4 expression was detected in neurons, glial cells, and endothelial cells within the neocortex. These receptors showed more intense expression in ependymocytes of the choroid plexus of infected mice at 2 dpi. Increased levels of TLR2 and TLR4 mRNA expression in infected mice suggest the involvement of these TLRs in the recognition of Acanthamoeba spp. pathogen-associated molecular patterns (PAMPs).

Abstract 350: Visualizing alectinib distribution in mice brain by using MALDI mass spectrometry imaging technique

Abstract [Introduction] In the development of anticancer drug, measurement of drug concentration in target tissue has been thought to be crucial for predicting its efficacy and safety. However, measuring homogenized tissue by using quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS) completely loss the spatial information of the compound in a target tissue. Mass spectrometry imaging (MSI) has been recently applied as innovative tools to detect pharmaceutical distributions in heterogeneous targets; however, it still needs to be improved in quantitative capability or reproducibility. [Purpose] We propose quantitative approach for MSI (qMSI) that administere matrix-assisted laser desorption ionization (MALDI)-MSI, concurrently with LC-MS/MS by using serial sections. We applied qMSI for visualizing spatial distribution of alectinib, novel ALK inhibitor, in mouse brain and examined the effect of MDR1 (p-glycoprotein) on the pharmacokinetic profiles in FVB and Mdr1a/b knockout mice. [Method] Pharmacokinetic study of alectinib was conducted in FVB and Mdr1a/b KO mice. Plasma, cerebrospinal fluid (CSF) and brain tissue were collected at 1, 1.5, 2, and 4 hours after a single oral administration dose of 4 and 20 mg/kg. The concentration of alectinib in plasma, CSF and brain tissue sections was measured by LC-MS/MS. The distribution of alectinib in mice brain tissue section was visualized by qMSI that we established. [Results] Developing qMSI showed the brain distribution of alectinib in the range from 1.2 to 952.6 pg/mm2 per each section, and the results made with qMSI were in excellent agreement with the complementary measurements by using laser-micro dissection to verify the exposure level. While plasma alectinib concentrations had no difference between both mice, the diffuse alectinib distributions in brain were observed in Mdr1a/b knockout compared with that in FVB mice. The dominant localization of alectinib in choroid plexus and brain blood vessels of FVB mice was observed by 20 μm resolution MSI analysis. [Conclusion] Our qMSI technique is expected to contribute to the development of anticancer drugs and translational research by visualizing drug distribution in target organs and tumors. Citation Format: Hiroaki Aikawa, Mitsuhiro Hayashi, Shoraku Ryu, Makiko Yamashita, Naoto Ohtsuka, Masanobu Nishidate, Akinobu Hamada. Visualizing alectinib distribution in mice brain by using MALDI mass spectrometry imaging technique. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 350.

Expression of NKCC1 and Aquaporins 4, 7 and 9 in Mouse Choroid Plexus and Ependymal Cells

Choroid plexus epithelial cells (CPECs) secrete cerebrospinal fluid (CSF) through poorly understood mechanisms involving Aquaporins (AQPs) and various solute transport proteins like the Na+/K+ pump and the Na+‐K+‐2Cl− cotransporter 1 (NKCC1). Net water movement across CPECs occurs from the basolateral membrane through the apical membrane. However, with the exception of AQP1 in the apical membrane, the molecular pathways for transepithelial water fluxes are either unknown, or their expression is controversial (Damkier et al. 2013, Physiol Rev 93, 1847). Because of their relatively high transcript expression (Cha et al, 2014, FASEB Journal 28 .1 Suppl 1182.3) the present work focuses on the cellular and subcellular localization of AQPs 4, 7 and 9 proteins in mouse CPECs and ependymal cells (EPCs), using immunolabeling with validated antibodies and confocal immuno‐fluorescence. Evidence for AQP4 expression in CPECs is controversial. The present results demonstrate that: 1) AQP 4 is expressed neither in CPECs nor in ependymal cells, but in radial glial endfeet surrounding the basal aspect of EPCs, where it is co‐expressed with NKCC1; the latter was also expressed in the apical membrane of both CPECs and EPCs; 2) AQP 7 expression was found to be restricted to the apical membrane of CPECs and EPCs. 3) AQP9 was expressed neither in CPECs nor in EPCs. The pathways for water influx across the basolateral membrane of CPECs remain to be elucidated. The pattern of expression of NKCC1 and AQP4 suggests a role of these proteins in K+ uptake from the CSF, both in CPECs and EPCs, thus regulating CSF and interstitial brain fluid [K+]. The expression of AQP7 in the apical membrane of CPECs suggests that this protein could work together with AQP1 in CSF secretion.Support or Funding Information(Supported by Wright State University Foundation FOAP 551462)

Proteomic analysis of mouse choroid plexus cell line ECPC-4 treated with lipid A.

Choroid plexus (CP) epithelial cells have multiple functions in the cerebral ventricles, including cerebrospinal fluid (CSF) production and forming part of the blood-CSF barrier. They are also responsible for producing inflammatory mediators involved in meningitis. The present study aimed to elucidate the functions of the CP epithelial cells during CNS inflammation. We analyzed the proteome and phosphoproteome in lipid A-treated ECPC-4 mouse CP cells by gel electrophoresis and mass spectrometry. Levels of 10 proteins and seven phosphoproteins were significantly altered by lipid A in time-dependent manners, including V-type proton ATPase subunit B (ATP6V), protein 40 kD, elongation factor-1δ, coatomer subunit ε (COPE), vimentin (isoform CRA a), purine nucleoside phosphorylase, eukaryotic initiation factor-4F splicing variant, put. β-actin, peroxiredoxin-6 isoform 1, and immunoglobulin heavy chain variable region. These proteins could be classified as having cytoskeleton/intermediate filament, protein-folding, signal-transduction, cell-growth, metabolism, and redox-regulation functions. The identified phosphoproteins were HSP 84, γ-actin, HSP 70 cognate, vimentin, tubulin β-4B chain, protein disulfide-isomerase A6 precursor, and heterogenous nuclear ribonucleoprotein, which could be classified as having cytoskeleton/intermediate filament, protein-folding, and metabolism functions. These results indicate that lipid A can change the levels of proteins and phosphoproteins in ECPC-4 cells, suggesting that the identified proteins and phosphoproteins may play important roles in inflammation of the CP.

Modeling immune functions of the mouse blood-cerebrospinal fluid barrier in vitro: primary rather than immortalized mouse choroid plexus epithelial cells are suited to study immune cell migration across this brain barrier.

BackgroundThe blood–cerebrospinal fluid barrier (BCSFB) established by the choroid plexus (CP) epithelium has been recognized as a potential entry site of immune cells into the central nervous system during immunosurveillance and neuroinflammation. The location of the choroid plexus impedes in vivo analysis of immune cell trafficking across the BCSFB. Thus, research on cellular and molecular mechanisms of immune cell migration across the BCSFB is largely limited to in vitro models. In addition to forming contact-inhibited epithelial monolayers that express adhesion molecules, the optimal in vitro model must establish a tight permeability barrier as this influences immune cell diapedesis.MethodsWe compared cell line models of the mouse BCSFB derived from the Immortomouse® and the ECPC4 line to primary mouse choroid plexus epithelial cell (pmCPEC) cultures for their ability to establish differentiated and tight in vitro models of the BCSFB.ResultsWe found that inducible cell line models established from the Immortomouse® or the ECPC4 tumor cell line did not express characteristic epithelial proteins such as cytokeratin and E-cadherin and failed to reproducibly establish contact-inhibited epithelial monolayers that formed a tight permeability barrier. In contrast, cultures of highly-purified pmCPECs expressed cytokeratin and displayed mature BCSFB characteristic junctional complexes as visualized by the junctional localization of E-cadherin, β-catenin and claudins-1, -2, -3 and -11. pmCPECs formed a tight barrier with low permeability and high electrical resistance. When grown in inverted filter cultures, pmCPECs were suitable to study T cell migration from the basolateral to the apical side of the BCSFB, thus correctly modelling in vivo migration of immune cells from the blood to the CSF.ConclusionsOur study excludes inducible and tumor cell line mouse models as suitable to study immune functions of the BCSFB in vitro. Rather, we introduce here an in vitro inverted filter model of the primary mouse BCSFB suited to study the cellular and molecular mechanisms mediating immune cell migration across the BCSFB during immunosurveillance and neuroinflammation.

Lipid A-activated inducible nitric oxide synthase expression via nuclear factor-κB in mouse choroid plexus cells

Choroid plexus (CP) which is responsible for the inflammatory mediators including nitric oxide (NO) are thought to play a crucial role in the process of bacterial meningitis. The present study investigated the mechanisms regulating inducible nitric oxide synthase (iNOS) expression in the choroid plexus epithelium (CPe) in mice. Initially, the expression of iNOS in mouse CPe was strengthened by intracerebroventriclar (i.c.v.) administration of lipid A, which is part of a Gram-negative bacterial endotoxin located at one end of the lipopolysaccharide (LPS) molecule. Next, the expression of iNOS in the CP epithelial cell line ECPC-4 cells was increased from 24 to 48h after lipid A treatment, although mRNA and proteins of toll-like receptor (TLR)-2 and -4 expressed in ECPC-4 cells were not changed by lipid A. The expression of total nuclear factor κB (NFκB), an inflammatory transcriptional factor, in ECPC-4 cells was not changed for 72h after lipid A treatment, while cytoplasmic NFκB was decreased and nuclear NFκB was increased from 1 to 2h. In addition, the phosphorylation of inhibitor κB (IκB) was peaked at 10min, and the level of IκB was attenuated from 10 to 45min after lipid A treatment. Moreover, the RNA interference (RNAi) of NFκB suppressed the expression of iNOS induced by lipid A. We demonstrated that lipid A-induced iNOS expression in ECPC-4 cells was mainly regulated by the activation of NFκB–IκB intracellular signaling pathway. Thus, we propose that the CPe plays a pivotal role in innate immunity responses of the brain, that is, the signal pathway TLRs on the CPe following inflammatory stimulation such as meningitis is activated, leading to iNOS expression through NFκB.

162. Crossing the Blood-Cerebrospinal Fluid Barrier in the Mouse Choroid Plexus With an Engineered Receptor/Ligand System

Crossing the blood-brain and the blood-cerebrospinal fluid barriers are one of the fundamental challenges in the development of new therapeutic molecules for brain disorders because they prevent entry of most drugs from the blood into the brain. However, some large molecules, like the protein transferrin, cross these barriers using a specific receptor that transports them into the brain. Mimicking nature, we engineered a receptor/ligand system to cross the brain barrier by combining the human transferrin receptor with the cohesin domain from Clostridium thermocellum, and we tested it in the choroid plexus of the mouse brain with a dockerin ligand. By expressing our receptor in choroidal ependymocytes, which are part of the blood-cerebrospinal fluid barrier, we found that our systemically administrated ligand was able to bind to the receptor and accumulate in ependymocytes, where some of the ligand was transported from the blood side to the brain side.