Choroid Plexus Epithelial Cell Line Research Articles

Highly restrictive and directional penetration of the blood cerebral spinal fluid barrier by JCPyV.

The human polyomavirus JCPyV is an opportunistic pathogen that infects greater than 60% of the world's population. The virus establishes a persistent and asymptomatic infection in the urogenital system but can cause a fatal demyelinating disease in immunosuppressed or immunomodulated patients following invasion of the CNS. The mechanisms responsible for JCPyV invasion into CNS tissues are not known but direct invasion from the blood to the cerebral spinal fluid via the choroid plexus has been hypothesized. To study the potential of the choroid plexus as a site of neuroinvasion, we used an adult human choroid plexus epithelial cell line to model the blood-cerebrospinal fluid (B-CSF) barrier in a transwell system. We found that these cells formed a highly restrictive barrier to virus penetration either as free virus or as virus associated with extracellular vesicles (EVJC+). The restriction was not absolute and small amounts of virus or EVJC+ penetrated and were able to establish foci of infection in primary astrocytes. Disruption of the barrier with capsaicin did not increase virus or EVJC+ penetration leading us to hypothesize that virus and EVJC+ were highly cell-associated and crossed the barrier by an active process. An inhibitor of macropinocytosis increased virus penetration from the basolateral (blood side) to the apical side (CSF side). In contrast, inhibitors of clathrin and raft dependent transcytosis reduced virus transport from the basolateral to the apical side of the barrier. None of the drugs inhibited apical to basolateral transport suggesting directionality. Pretreatment with cyclosporin A, an inhibitor of P-gp, MRP2 and BCRP multidrug resistance transporters, restored viral penetration in cells treated with raft and clathrin dependent transcytosis inhibitors. Because choroid plexus epithelial cells are known to be susceptible to JCPyV infection both in vitro and in vivo we also examined the release of infectious virus from the barrier. We found that virus was preferentially released from the cells into the apical (CSF) chamber. These data show clearly that there are two mechanisms of penetration, direct transcytosis which is capable of seeding the CSF with small amounts of virus, and infection followed by directional release of infectious virions into the CSF compartment.

The role of Nrf2 in the alteration of tight junction protein expression in choroid plexus epithelial cells created by lanthanum-activated MMP9

Objective: To investigate the effect of nuclear factor erythroid 2-related factor 2 (Nrf2) in the alteration of tight junction protein expression in choroid plexus epithelial cells created by lanthanum-activated matrix metalloproteinase 9 (MMP9) . Methods: In October 2020, immortalized rat choroid plexus epithelial cell line (Z310) cells were used as the blood-cerebrospinal fluid barrier in vitro, and were divided into control group and 0.125, 0.25, 0.5 mmol/L lanthanum chloride (LaCl(3)) treatment group. After treating Z310 cells with different concentrations of LaCl(3) for 24 hours, the morphological changes of Z310 cells were observed under inverted microscope, the protein expression levels of MMP9, occludin and zonula occludens-1 (ZO-1) were observed by cellular immunofluorescence method, and the protein expression levels of MMP9, tissue inhibitors of metalloproteinase1 (TIMP1) , occludin, ZO-1 and Nrf2 were detected by Western blotting. The level of reactive oxygen species (ROS) in cells was detected by flow cytometry. Results: Compared with the control group, Z310 cells in the LaCl(3) treatment group were smaller in size, with fewer intercellular junctions, and more dead cells and cell fragments. The expression level of MMP9 protein in cells treated with 0.25 and 0.5 mmol/L LaCl(3) was significantly higher than that in the control group (P<0.05) , and the expression level of TIMP1 and tight junction proteins occudin and ZO-1 was significantly lower than that in the control group (P<0.05) . Compared with the control group, the ROS production level in the 0.25, 0.5 mmol/L LaCl(3) treatment group was significantly increased (P<0.05) , and the Nrf2 protein expression level in the 0.125, 0.25, 0.5 mmol/L LaCl(3) treatment group was significantly decreased (P<0.05) . Conclusion: Lanthanum may increase the level of ROS in cells by down regulating the expression of Nrf2, thus activating MMP9 to reduce the expression level of intercellular tight junction proteins occludin and ZO-1.

Characterization of TRPV4-mediated signaling pathways in an optimized human choroid plexus epithelial cell line.

The objectives of these studies were twofold: 1) to characterize the human choroid plexus papilloma (HIBCPP) cell line as a model of the blood-cerebrospinal fluid barrier (BCSFB) via morphology, tightness, and polarization of transporters in choroid plexus epithelia (CPe), and 2) to utilize Ussing-style electrophysiology to elucidate signaling pathways associated with the activation of the transient receptor potential vanilloid 4 (TRPV4) channel involved in cerebrospinal fluid (CSF) secretion. RT-PCR was implemented to determine gene expression of cell fate markers, junctional complex proteins, and transporters of interest. Scanning electron microscopy and confocal three-dimensional renderings of cultures grown on permeable supports were utilized to delineate the morphology of the brush border, junctional complexes, and polarization of key transporters. Electrophysiology was used to understand and explore TRPV4-mediated signaling in the HIBCPP cell line, considering both short-circuit current (Isc) and conductance responses. HIBCPP cells grown under optimized culture conditions exhibited minimal multilayering, developed an intermediate resistance monolayer, retained differentiation properties, and expressed, and correctly localized, junctional proteins and native transporters. We found that activation of TRPV4 resulted in a robust, multiphasic change in electrogenic ion flux and increase in conductance accompanied by substantial fluid secretion. This response appears to be modulated by a number of different effectors, implicating phospholipase C (PLC), protein kinase C (PKC), and phosphoinositide 3-kinase (PI3K) in TRPV4-mediated ion flux. The HIBCPP cell line is a representative model of the human BCSFB, which can be utilized for studies of transporter function, intracellular signaling, and regulation of CSF production.

TRIP6 functions in brain ciliogenesis

TRIP6, a member of the ZYXIN-family of LIM domain proteins, is a focal adhesion component. Trip6 deletion in the mouse, reported here, reveals a function in the brain: ependymal and choroid plexus epithelial cells are carrying, unexpectedly, fewer and shorter cilia, are poorly differentiated, and the mice develop hydrocephalus. TRIP6 carries numerous protein interaction domains and its functions require homodimerization. Indeed, TRIP6 disruption in vitro (in a choroid plexus epithelial cell line), via RNAi or inhibition of its homodimerization, confirms its function in ciliogenesis. Using super-resolution microscopy, we demonstrate TRIP6 localization at the pericentriolar material and along the ciliary axoneme. The requirement for homodimerization which doubles its interaction sites, its punctate localization along the axoneme, and its co-localization with other cilia components suggest a scaffold/co-transporter function for TRIP6 in cilia. Thus, this work uncovers an essential role of a LIM-domain protein assembly factor in mammalian ciliogenesis.

Stimulated Transepithelial Ion Flux in a Choroid Plexus Epithelial Cell Line

Production and maintenance of the cerebral spinal fluid (CSF) is crucial for brain health and normal function. Alterations in normal homeostatic balance can occur in response a variety of pathological etiologies including traumatic brain injury, stroke, infection and cancerous lesions. Overproduction or blockage of CSF flow can also lead to hydrocephalus in both the pediatric population and adults. These alterations are remarkably difficult to treat and, in many cases, surgery is the only option. The choroid plexus, an epithelial cell lined tuft of capillaries that project into the cerebral ventricles, is responsible for a substantial portion of the CSF production. The capillaries of the CP are relatively leaky while the epithelial cells surrounding this vasculature form the blood‐CSF barrier and control the amount and composition of the CSF. A stable porcine choroid plexus epithelial cell line (PCP‐R) with a high transepithelial resistance (TEER) has been developed. We used Ussing style electrophysiology to characterize stimulated transepithelial ion transport measured as short circuit current (SCC) in confluent PCP‐R cells. In response to forskolin, a compound known to activate adenylyl cyclase and increase intracellular cAMP, there is a sustained increase in transepithelial ion flux that is consistent with an increase in cation absorption and/or anion secretion. This stimulated transport is partially inhibited by 1 mM BaCl2, used as a pan inhibitor of K+ channels. It is also partially inhibited by 10 mM T16Ainh‐A01, an inhibitor of the Ca2+ activated Cl− channel TMEM16a and by 50 nM GLY H101, an inhibitor of the cAMP‐stimulated Cl− channel CFTR. Neither the stimulatory nor inhibitory responses were accompanied by a substantial change in TEER. An agonist of the transient receptor potential vanilloid 4 (TRPV4) cation channel, GSK1016790, elicits a prolonged ion transport response that is the opposite of the forskolin stimulated response. The TRPV4 agonist elicits an increase in cation secretion and/or anion absorption. Interestingly, this response to the agonist is accompanied by a reversible decrease in TEER (&gt;50%). The effect of the agonist on both SCC and TEER can be blocked by pretreatment with HC067047, a TRPV4 antagonist. If added after agonist stimulation, the TRPV4 antagonist can also fully reverse the agonist stimulated ion flux and can substantially restore the agonist stimulated decrease in TEER. Studies such as these are important for characterizing the factors that may disturb the normal CSF production and lead to an accumulation of CSF that can, over time, cause cellular loss and brain injury.Support or Funding InformationThis research was supported by an Indiana University Collaborative Research Grant fund of the Office of the Vice President for Research, and by a grant from the Vice Chancellor's Office, IUPUI.

Loss of TP53 expression in immortalized choroid plexus epithelial cells results in increased resistance to anticancer agents

Choroid plexus carcinomas are malignant brain tumors predominantly arising in young children. Because a prognostic role of p53 alterations has been demonstrated, further research into potential underlying mechanisms is essential. Our objective was, therefore, to investigate the role of p53 in the growth-inhibitory potential of a variety of anticancer agents in the rodent choroid plexus epithelial cell line Z310. Furthermore, association of p53 alterations with proliferative activity (Ki67/MIB1) in choroid plexus carcinoma samples (N = 20) was examined by use of immunohistochemistry. Silencing of TP53 expression did not significantly alter metabolic activity in Z310 cells and p53 protein expression status was not associated with increased proliferative activity in choroid plexus carcinomas. However, the growth-inhibitory activity of vincristine, doxorubicin, carboplatin, etoposide, and temozolomide was significantly impaired by silencing of TP53. In conclusion, these results indicate a potential predictive role of p53 in choroid plexus carcinomas. Alterations of p53 should be taken into account when evaluating the effect of anticancer agents in future clinical trials.

Characterization of immortalized choroid plexus epithelial cell lines for studies of transport processes across the blood-cerebrospinal fluid barrier

BackgroundTwo rodent choroid plexus (CP) epithelial cell lines, Z310 and TR-CSFB, were compared with primary rat CP epithelial cells and intact CP tissue with respect to transport protein expression, function and tight junction (TJ) formation.MethodsFor expression profiles of transporters and TJ proteins, qPCR and western blot analysis were used. Uptake assays were performed to study the functional activity of transporters and TJ formation was measured by trans-epithelial electrical resistance (TEER) and visualized by electron microscopy.ResultsThe expression of known ATP-binding cassette (Abc) transporter and solute carrier (Slc) genes in CP was confirmed by qPCR. Primary cells and cell lines showed similar, but overall lower expression of Abc transporters and absent Slc expression when compared to intact tissue. Consistent with this Mrp1, Mrp4 and P-gp protein levels were higher in intact CP compared to cell lines. Functionality of P-gp and Mrp1 was confirmed by Calcein-AM and CMFDA uptake assays and studies using [3H]bis-POM-PMEA as a substrate indicated Mrp4 function. Cell lines showed low or absent TJ protein expression. After treatment of cell lines with corticosteroids, RNA expression of claudin1, 2 and 11 and occludin was elevated, as well as claudin1 and occludin protein expression. TJ formation was further investigated by freeze-fracture electron microscopy and only rarely observed. Increases in TJ particles with steroid treatment were not accompanied by an increase in transepithelial electrical resistance (TEER).ConclusionTaken together, immortalized cell lines may be a tool to study transport processes mediated by P-gp, Mrp1 or Mrp4, but overall expression of transport proteins and TJ formation do not reflect the situation in intact CP tissue.

LPS enhance the expression of nitric oxide synthases in the choroid plexus epithelial cell line ECPC-4 cells

LPS enhance the expression of nitric oxide synthases in the choroid plexus epithelial cell line ECPC-4 cells

Platelet-Derived Growth Factor Receptor Expression and Activation in Choroid Plexus Tumors

Choroid plexus tumors are intraventricular neoplasms predominantly affecting young children. In contrast to choroid plexus papillomas, choroid plexus carcinomas progress frequently, necessitating the development of adjuvant treatment concepts. Platelet derived growth factor (PDGF) signaling has been shown to support growth in a variety of tumors. The finding of PDGF receptor expression in choroid plexus tumors prompted us to elucidate PDGF receptor activation state using a novel method, in situ proximity ligation assay, on formalin-fixed, paraffin-embedded, archival samples of 19 choroid plexus tumors. As assessed by in situ proximity ligation assay, the proportion of phosphorylated PDGF receptor alpha was low in choroid plexus papillomas and choroid plexus carcinomas, whereas phosphorylated PDGF receptor beta was found to be significantly higher in choroid plexus carcinomas. In the immortalized choroid plexus epithelial cell line Z310 expressing PDGF receptor beta, PDGF-BB exhibited a time- and dose-dependent proliferative response, which was significantly attenuated by imatinib (gleevec). In conclusion, our findings suggest that PDGF receptor beta is functionally involved in the biology of choroid plexus tumors and may represent a molecular target for therapy. In addition, this study demonstrates the feasibility and usefulness of in situ proximity ligation assay for monitoring receptor tyrosine kinase activation in formalin-fixed, paraffin-embedded, archival tissues.

TWIST-1 is overexpressed in neoplastic choroid plexus epithelial cells and promotes proliferation and invasion.

The pathogenesis of choroid plexus papillomas, intraventricular papillary neoplasms most often occurring sporadically in children and young adults, remains poorly understood. To identify pathways operative in the development of choroid plexus papillomas, gene expression profiles obtained from laser-microdissected human choroid plexus papilloma cells (n = 7) were compared with that of normal choroid plexus epithelial cells laser microdissected from autopsy tissue (n = 8). On DNA microarray data analysis, 53 probe sets were differentially expressed in choroid plexus papilloma tumor cells (>7-fold). Up-regulation of TWIST1, WIF1, TRPM3, BCLAF1, and AJAP1, as well as down-regulation of IL6ST was confirmed using quantitative reverse transcription-PCR. Knockdown of Twist1 gene expression in the rat choroid plexus epithelial cell line Z310 significantly reduced proliferation as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and cell invasion in a Matrigel assay, whereas cell migration was not affected. Screening for expressional changes of cancer-related genes upon Twist1 knockdown revealed up-regulation of Cdkn1a, Cflar, and Serpinb2 and down-regulation of Figf. To conclude, using gene expression profiling, several genes differentially expressed in human choroid plexus papillomas could be identified. Among those, TWIST1 is highly expressed in choroid plexus papillomas and promotes proliferation and invasion.

Expression and possible role of creatine transporter in the brain and at the blood‐cerebrospinal fluid barrier as a transporting protein of guanidinoacetate, an endogenous convulsant

Little is known about the cerebral distribution and clearance of guanidinoacetate (GAA), the accumulation of which induces convulsions. The purpose of the present study was to identify creatine transporter (CRT)-mediated GAA transport and to clarify its cerebral expression and role in GAA efflux transport at the blood-cerebrospinal fluid barrier (BCSFB). CRT mediated GAA transport with a K(m) value of 269 microM/412 microM which was approximately 10-fold greater than that of CRT for creatine. There was wide and distinct cerebral expression of CRT and localization of CRT on the brush-border membrane of choroid plexus epithelial cells. The in vivo elimination clearance of GAA from the CSF was 13-fold greater than that of d-mannitol reflecting bulk flow of the CSF. This process was partially inhibited by creatine. The characteristics of GAA uptake by isolated choroid plexus and an immortalized rat choroid plexus epithelial cell line (TR-CSFB cells) used as an in vitro model of BCSFB are partially consistent with those of CRT. These results suggest that CRT plays a role in the cerebral distribution of GAA and GAA uptake by the choroid plexus. However, in the presence of endogenous creatine in the CSF, CRT may make only a limited contribution to the GAA efflux transport at the BCSFB.

Expression of junctional proteins in choroid plexus epithelial cell lines: a comparative study

BackgroundThere is an increasing interest in using choroid plexus (CP) epithelial cell lines to study the properties of the blood-cerebrospinal fluid barrier (BCSFB). Currently, there are three major CP-derived cell lines available. Z310 and TR-CSFB3, two immortalized cell lines carrying the simian virus 40 large T-antigen gene, were derived from rat CP epithelium, whereas the CPC-2 cell line was derived from human CP carcinoma. Although these cell lines have previously been used in various functional studies, the expression of adherens junction (AJ) and tight junction (TJ) proteins in these epithelial cells has not been systematically studied. Accordingly, in the present study, we sought to characterize the expression of these junctional proteins in these three cell lines.MethodsThe cells were grown in six-well cell culture plates. Reverse-transcriptase polymerase chain reaction, Western blotting, and immunocytochemistry were used to characterize the expression of AJ and TJ proteins in the CP cell lines.ResultsZ310 and TR-CSFB3 cells expressed a TJ protein, occludin, and its cytosolic binding partner, zonula occludens 1, as well as an AJ protein, E-cadherin, and β-catenin, a cytoplasmic protein that interacts with E-cadherin. However, the expression of occludin and E-cadherin in TR-CSFB3 cells at both the mRNA and protein level was weaker than that found in Z301 cells. The immunocytochemical analysis also demonstrated that the staining pattern for these junctional proteins in TR-CSFB3 cells was discontinuous and the staining intensity was weaker than that observed in Z310 cells. The message for claudin 1 and claudin 2 was expressed at low levels in TR-CSFB3 cells and these cells were weakly immunopositive for claudin 1. In comparison, the message for these TJ proteins could not be detected in Z310 cells. CPC-2 cells expressed occludin, which was localized to areas of cell-cell contact, but the staining pattern for this TJ protein was found to be variable and irregular. Although CPC-2 cells expressed mRNA for claudin 1, claudin 2, and claudin 11, only claudin 1 was expressed at the protein level and it was localized to the nuclei rather than to areas of cell-cell contact. An AJ protein, E-cadherin, was also found to be mislocalized in CPC-2 cells, even though its cytosolic binding partner, β-catenin, was restricted to areas of cell-cell contact, as in normal CP.ConclusionThe three CP cell lines analyzed in this study vary considerably with regard to the expression of AJ and TJ proteins, which is likely reflected by different barrier properties of these in vitro models of BCSFB.

細胞治療とＤＤＳ 条件的不死化脳関門細胞株を用いた脳へのＤＤＳ研究の新展開

Elucidating the molecular mechanism of the brain harrier transport is a key step toward successful drug delivery to the brain. In this review, we focused on the usefulness and the limitations of the newly developed in vitro model. To overcome several limitations of classical in vitro model, conditionally immortalized mouse (TM-BBB) and rat (TR-BBB) brain capillary endothelial cell lines and a choroid plexus epithelial cell line (TR-CSFB) were established from the temperature sensitive SU 40 large T antigen gene transgenic animals. mRNA and protein expression of GLUT1 in TR-BBB was 100 folds greater than that of primary culture, Several known transporter genes were identified for TR-BBB or TM-BBB, including organic anion transporter 3 (OAT 3) and organic anion transporting polypeptide 2 (oatp 2). Employing TM-BBB, new brain barrier functions have been clarified such as creatine transporter (CRT) and GABA transporter (GAT 2/BGT-1). TR-BBB has been applied for the study of expressional regulation of transporters at the BBB, e. g., L-proline and glycine transporter (ATA 2) and taurine transporter (TAUT). Moreover, one of the most important subjects for the in vitro BBB model is to clarify the expression and regulation of the tight junction proteins at the BBB. Co-culture systems among TR-BBB, conditionally immortalized astrocyte (TR-AST) and pericyte (TR-PCT) cell lines were developed and revealed that the expression of occludin, a tight-junction component, was induced by soluble factors secreted from TR-AST and TR-PCT. For the brain drug delivery research, the new in vitro model will provide rational and efficient strategies.

Characterization of the amino acid transport of new immortalized choroid plexus epithelial cell lines: a novel in vitro system for investigating transport functions at the blood-cerebrospinal fluid barrier.

To establish and characterize a choroid plexus epithelial cell line (TR-CSFB) from a new type of transgenic rat harboring the temperature-sensitive simian virus 40 (ts SV 40) large T-antigen gene (Tg rat). Choroid plexus epithelial cells were isolated from the Tg rat and cultured on a collagen-coated dish at 37 degrees C during the first period of 3 days. Cells were subsequently cultured at 33 degrees C to activate large T-antigen. At the third passage, cells were cloned by colony formation and isolated from other cells using a penicillin cup. Five immortalized cell lines of choroid plexus epithelial cells (TR-CSFB 1 approximately 5) were obtained from two Tg rats. These cell lines had a polygonal cell morphology, expressed the typical choroid plexus epithelial cell marker, transthyretin, and possessed Na+, K+-ATPase on their apical side. TR-CSFBs cells expressed a large T-antigen and grew well at 33 degrees C with a doubling-time of 35 approximately 40 hr. [3H]-L-Proline uptake by TR-CSFB cells took place in an Na+-dependent, ouabain-sensitive, energy-dependent, and concentration-dependent manner. It was also inhibited by alpha-methylaminoisobutylic acid, suggesting that system A for amino acids operates in TR-CSFB cells. When [3H]-L-proline uptake was measured using the Transwell device, the L-proline uptake rate following application to the apical side was five-fold greater than that following application to the basal side. In addition, both Na+-dependent and Na+-independent L-glutamic acid (L-Glu) uptake processes were present in TR-CSFB cells. Immortalized choroid plexus epithelial cell lines were successfully established from Tg rats and have the properties of choroid plexus epithelial cells, and amino acid transport activity was observed in vivo.

Conditionally immortalized cell lines as a new in vitro model for the study of barrier functions.

Conditionally immortalized brain and retinal capillary endothelial and choroid plexus epithelial cell lines were established from a transgenic rat (Tg rat) and mouse (Tg mouse) harboring the temperature-sensitive simian virus 40 (ts SV 40) large T-antigen. These cell lines exhibit temperature-sensitive cell growth due to the expression of ts SV 40 large T-antigen. Mouse brain (TM-BBB) and rat brain (TR-BBB) and rat retinal (TR-iBRB) capillary endothelial cell lines appear to have a spindle-fiber shaped morphology and exhibit the typical endothelial markers, such as von Willebrand factor and acetylated low-density lipoprotein uptake. These cell lines express in vivo influx and efflux transporters, such as P-glycoprotein (P-gp) and GLUT1, which is capable of 3-O-methyl-D-glucose transport. TM-BBB cells are able to undergo efflux transport of cyclosporin A, which is a substrate for P-gp transport activity. They may also express oatp2 and exhibit dehydroepiandrosterone sulfate and digoxin uptake activity. TR-BBB cells express the mRNA of multidrug resistance associated protein 1 (MRP1) and a large neutral amino acid transporter, which consists of LAT1 and 4F2hc. TR-iBRB cells exhibit pH-dependent L-lactic acid transport activity and express the mRNA of monocarboxylate transporter (MCT) 1 and 2. The choroid plexus epithelial cell line (TR-CSFB) has polygonal cell morphology, expresses the typical choroid plexus epithelial cell marker, transthyretin, and has Na+, K+-ATPase located on the apical side. TR-CSFB cells also exhibit amino acid transport activity which has been observed in vivo. These barrier cell lines established from the Tg rat and Tg mouse have in vivo transport functions and are good in vitro models for drug transport to the brain and retina and as a screen for drugs which might be capable of delivery to the brain and retina.

Expression of neuropeptide processing enzymes and neurosecretory proteins in ependyma and choroid plexus epithelium

Recent studies suggest that brain ependyma and choroid plexus produce neuropeptide processing enzymes. To facilitate the understanding of these cells and their ability to produce biologically active peptides, we developed cultures of defined cell type. Ependymal cells were characterized by morphological criteria, and choroid plexus epithelial cell lines were characterized by the presence of the mRNA for IGF-II and transthyretin, a thyroxine binding protein produced in liver and choroid plexus. The ependymal cells and the choroid plexus epithelial cell lines were then examined for the presence of mRNAs for various neuropeptide processing enzymes. Northern blot analysis revealed high levels of furin, car☐ypeptidase E, and peptidyl glycine α-amidating monooxygenase mRNAs, with levels in ependymal cells comparable to those in brain or pituitary. Car☐ypeptidase E activity was detected in medium from cultured ependymal cells; this activity was identified as car☐ypeptidase E based on the acidic pH optimum and sensitivity to various inhibitors. The mRNAs for other neuropeptide processing enzymes, such as prohormone convertases 1 and 2, were not detected on Northern blots of RNA from ependyma or choroid plexus epithelium. Since ependyma and choroid plexus epithelium express a subset of processing enzymes, we suggest that these cells have the capacity to produce biologically active peptides. Initial screening by reverse transcriptase-polymerase chain reaction assays has demonstrated the presence of mRNA for the neurosecretory proteins chromogranin B and secretogranin II in both ependyma and choroid plexus epithelium.

Immunofluorescent cytometry and electron microscopic immunolocalization of insulin-like growth factor (IGF)-II receptors in infant rat choroid plexus

The choroid plexus contains receptors for insulin-like growth factor (IGF)-II, it synthesizes IGF-II, and it secretes soluble IGF binding proteins with high affinity for IGF-II. Although the actions of IGFs in the choroid plexus are unknown, IGFs may participate in CNS growth and differentiation, CNS injury/repair mechanisms, regulation of satiety, and growth hormone secretion. Because the choroid plexus is a heterogeneous tissue containing several cell types, to study the role of IGF-II in the choroid plexus we need to know precisely which cells contain IGF-II receptors. Therefore, we studied the distribution of IGF-II receptors in infant rat choroid plexus by electron microscopic immunohistochemistry and immunofluorescent cytometry of dispersed choroid plexus cells using a well-characterized, highly specific rabbit anti-rat IGF-II receptor antibody. These studies show that IGF-II receptors are detectable in large quantities in both choroid plexus epithelial cells and in the fenestrated capillary endothelial cells. Results were confirmed by immunofluorescent cell sorting using the anti-IGF-II receptor antiserum. Other cell types in the choroid plexus contained lower levels of immunoreactivity, including red blood cells. Physiological studies of IGF-II effects on intact choroid plexus may, therefore, reflect effects on both of these cell populations. These data provide a compelling rationale for the development of choroid plexus epithelial cell lines or pure culture techniques to study the physiology of IGF-II in individual cell types.