Adult Choroid Plexus Research Articles

ETMR-24. SOX2 INFLUENCES IDENTITY OF GLIA-LIKE PROGENITORS AND TUMORIGENESIS IN CHOROID PLEXUS THROUGH LIM HOMEODOMAIN TRANSCRIPTION FACTORS

Abstract Choroid plexus (CP) tumors encompass a spectrum of rare brain neoplasms ranging from benign papilloma (CPP) to aggressive carcinoma (CPC) that collectively affect the pediatric population. Poor outcomes for high-grade lesions and side effects of current treatment paradigms underscore a critical need to develop safer and more efficacious therapies guided by biological understanding of these tumors. We developed mouse models from common molecular defects found in human disease. These models exploit sustained NOTCH signaling activation in the hindbrain upper rhombic lip to generate CPP and CPC. To delineate cellular composition of CP tumors, we conducted snRNA-seq in murine CP tumors. Similar to adult CP, cells in NOTCH-driven CPP partitioned into clusters identified as epithelial, mesenchymal, endothelial, immune, neuronal, and glial cells. Though tumor cells are confined to epithelial compartment based on inferCNV analysis of snRNA-seq data, they are derived from glia-like progenitors. Tumor cells are in a distinct differentiation state characterized by a lack of epithelial maturation, and aberrant expression of markers of neural progenitor/stem-like cells including SRY-Box Transcription Factor 2 (SOX2). Consistently, SOX2 inactivation decreased tumor cell proliferation and blocked NOTCH-driven CP tumor formation. ChIP-seq studies revealed SOX2 binding to promoters of LIM homeodomain transcription factors Lmx1a and Lmx1b. Enforced SOX2 expression activated the expression Lmx1a and Lmx1b, whereas Sox2 loss reduced their expressions in tumor cells. Importantly, knockdown of Lmx1a or Lmx1b decreased tumor cell proliferation, whereas overexpression of Lmx1a/b rescued proliferation of tumor cells subjected to SOX2 suppression. Contextualizing these results in CP development, SOX2 critically maintains progenitor cell identity through regulating Lmx1a/b expression in the rhombic lip. Finally, analysis of human CP tumor samples revealed abnormal expressions of SOX2 and LMX1A. Together, these results indicate that oncogenic signaling subverts SOX2 transcriptional programs that specify normal progenitor identity and lock tumor cells in unique differentiation state to facilitate CP tumor growth.

Primary adult choroid plexus carcinomas: a single-center experience with a systematic review.

Primary adult choroid plexus carcinomas (PACPCs) are extremely rare brain tumors. The existing literature primarily comprises case reports, which limits our understanding of this uncommon disease. This study aims to describe the clinical characteristics and prognosis of PACPCs, as well as to identify optimal treatment strategies. We conducted a comprehensive analysis of clinical data from 7 patients with PACPCs who underwent surgical treatment at the Department of Neurosurgery, Beijing Tiantan Hospital, between March 2011 and March 2023. Additionally, a thorough search of the PubMed database was performed using the keywords "choroid plexus carcinoma" or "choroid plexus carcinomas" within the time frame of August 1975 to April 2023, which yielded a total of 28 identified cases. Subsequently, we evaluated risk factors for progression-free survival (PFS) and overall survival (OS) based on the pooled cases. The pooled cohort, consisting of 7 cases from our institution and 28 cases from the literature, included 20 males and 15 females with a mean age of 44.3 ± 14.7 years (range: 21-73 years). Gross-total resection (GTR) and non-GTR were achieved in 22 (62.9%) and 13 (37.1%) patients, respectively. Radiotherapy and chemotherapy were administered to 29 (90.6%) and 13 (40.6%) patients, respectively. After a mean follow-up of 21.0 ± 26.7 months (range: 2-132 months), 18 patients were alive, and 11 patients had died. The multivariate Cox regression model demonstrated that non-GTR (HR 5.262, 95% CI 1.350-20.516, p=0.017) was a negative prognostic factor for OS. However, we did not find any risk factors for PFS. Complete surgical resection should be considered as the primary treatment approach for this rare disease. Chemotherapy and radiotherapy appear to have limited effectiveness in treating this condition. Further research with large cohorts is needed to validate our conclusions.

Efflux transporters in rat placenta and developing brain: transcriptomic and functional response to paracetamol

Adenosine triphosphate binding cassette (ABC) transporters transfer lipid-soluble molecules across cellular interfaces either directly or after enzymatic metabolism. RNAseq analysis identified transcripts for ABC transporters and enzymes in rat E19, P5 and adult brain and choroid plexus and E19 placenta. Their functional capacity to efflux small molecules was studied by quantitative analysis of paracetamol (acetaminophen) and its metabolites using liquid scintillation counting, autoradiography and ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). Animals were treated acutely (30 min) and chronically (5 days, twice daily) with paracetamol (15 mg/kg) to investigate ability of brain and placenta barriers to regulate ABC transport functionality during extended treatment. Results indicated that transcripts of many efflux-associated ABC transporters were higher in adult brain and choroid plexus than at earlier ages. Chronic treatment upregulated certain transcripts only in adult brain and altered concentrations of paracetamol metabolites in circulation of pregnant dams. Combination of changes to metabolites and transport system transcripts may explain observed changes in paracetamol entry into adult and fetal brains. Analysis of lower paracetamol dosing (3.75 mg/kg) indicated dose-dependent changes in paracetamol metabolism. Transcripts of ABC transporters and enzymes at key barriers responsible for molecular transport into the developing brain showed alterations in paracetamol pharmacokinetics in pregnancy following different treatment regimens.

OTX2 Homeoprotein Functions in Adult Choroid Plexus.

The choroid plexus is an important blood barrier that secretes cerebrospinal fluid, which essential for embryonic brain development and adult brain homeostasis. The OTX2 homeoprotein is a transcription factor that is critical for choroid plexus development and remains highly expressed in adult choroid plexus. Through RNA sequencing analyses of constitutive and conditional knockdown adult mouse models, we reveal putative functional roles for OTX2 in adult choroid plexus function, including cell signaling and adhesion, and show that OTX2 regulates the expression of factors that are secreted into the cerebrospinal fluid, notably transthyretin. We also show that Otx2 expression impacts choroid plexus immune and stress responses, and affects splicing, leading to changes in the mRNA isoforms of proteins that are implicated in the oxidative stress response and DNA repair. Through mass spectrometry analysis of OTX2 protein partners in the choroid plexus, and in known non-cell-autonomous target regions, such as the visual cortex and subventricular zone, we identify putative targets that are involved in cell adhesion, chromatin structure, and RNA processing. Thus, OTX2 retains important roles for regulating choroid plexus function and brain homeostasis throughout life.

The genetic landscape of choroid plexus tumors in children and adults.

Choroid plexus tumors (CPTs) are intraventricular brain tumors predominantly arising in children but also affecting adults. In most cases, driver mutations have not been identified, although there are reports of frequent chromosome-wide copy-number alterations and TP53 mutations, especially in choroid plexus carcinomas (CPCs). DNA methylation profiling and RNA-sequencing was performed in a series of 47 CPTs. Samples comprised 35 choroid plexus papillomas (CPPs), 6 atypical choroid plexus papillomas (aCPPs) and 6 CPCs plus three recurrences thereof. Targeted TP53 and TERT promotor sequencing was performed in all samples. Whole exome sequencing (WES) and linked-read whole genome sequencing (WGS) was performed in 25 and 4 samples, respectively. Tumors comprised the molecular subgroups "pediatric A" (N=11), "pediatric B" (N=12) and "adult" (N=27). Copy-number alterations mainly represented whole-chromosomal alterations with subgroup-specific enrichments (gains of Chr1, 2 and 21q in "pediatric B" and gains of Chr5 and 9 and loss of Chr21q in "adult"). RNA sequencing yielded a novel CCDC47-PRKCA fusion transcript in one adult choroid plexus papilloma patient with aggressive clinical course; an underlying Chr17 inversion was demonstrated by linked-read WGS. WES and targeted sequencing showed TP53 mutations in 7/47 CPTs (15%), five of which were children. On the contrary, TERT promoter mutations were encountered in 7/28 adult patients (25%) and associated with shorter progression-free survival (log-rank test, p=0.015). Pediatric CPTs lack recurrent driver alterations except for TP53, whereas CPTs in adults show TERT promoter mutations or a novel CCDC47-PRKCA gene fusion, being associated with a more unfavorable clinical course.

Claudin-1, -2 and -3 Are Selectively Expressed in the Epithelia of the Choroid Plexus of the Mouse from Early Development and into Adulthood While Claudin-5 is Restricted to Endothelial Cells.

A primary function of epithelial and endothelial monolayers is the formation of barriers that separate tissues into functional compartments. Tight junctions (TJs) seal the intercellular space between the single cells of a monolayer. TJs thus contribute importantly to the homeostasis of the cerebrospinal fluid as they help in maintaining the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (CSF). The composition of TJs differs by its localization as well as the stage of development according to its respective function. Claudin-3 is typically present in the epithelia and has been claimed to be a constituent of the BBB. It is, however, notoriously difficult to demonstrate its expression in endothelial cells of the brain vasculature at the morphological level by means of immunohistochemical techniques. Using an improved fixation strategy (4% paraformaldehyde at pH 11, in the presence of EDTA) and the sensitive alkaline phosphatase as a detection system, we show that claudin-3 is present in mouse epithelia from embryonic day 14 onwards. In brain, it is restricted to the anlage of choroid plexus in the ventricles, together with claudin-1 and -2. In adult mice, it is clearly delineating the epithelium of the choroid plexus in the lateral and fourth ventricles. In contrast, in cerebral blood vessels claudin-3 as well as claudin-1 and -2 are absent in cerebral blood vessels during all developmental stages up to adulthood. Rather, the BBB is characterized by the presence of claudin-5, ZO-1 and occludin. Thus, in mice claudin-3 is an important constituent of TJ in the embryonic and in the adult choroid plexus.

Spatial and temporal expression of immunoglobulin superfamily member 1 in the rat.

Loss-of-function mutations in the immunoglobulin superfamily member 1 (IGSF1) gene cause an X-linked syndrome of central hypothyroidism, macroorchidism, variable prolactin and GH deficiency, delayed pubertal testosterone rise, and obesity. To understand the pathophysiology of this syndrome, knowledge on IGSF1's place in normal development is imperative. Therefore, we investigated spatial and temporal protein and mRNA expression of IGSF1 in rats using immunohistochemistry, real-time quantitative PCR (qPCR), and in situ hybridization. We observed high levels of IGSF1 expression in the brain, specifically the embryonic and adult choroid plexus and hypothalamus (principally in glial cells), and in the pituitary gland (PIT1-lineage of GH, TSH, and PRL-producing cells). IGSF1 is also expressed in the embryonic and adult zona glomerulosa of the adrenal gland, islets of Langerhans of the pancreas, and costameres of the heart and skeletal muscle. IGSF1 is highly expressed in fetal liver, but is absent shortly after birth. In the adult testis, IGSF1 is present in Sertoli cells (epithelial stages XIII-VI), and elongating spermatids (stages X-XII). Specificity of protein expression was corroborated with Igsf1 mRNA expression in all tissues. The expression patterns of IGSF1 in the pituitary gland and testis are consistent with the pituitary hormone deficiencies and macroorchidism observed in patients with IGSF1 deficiency. The expression in the brain, adrenal gland, pancreas, liver, and muscle suggest IGSF1's function in endocrine physiology might be more extensive than previously considered.

The neural milieu of the developing choroid plexus: neural stem cells, neurons and innervation.

The choroid plexus produces cerebrospinal fluid and plays an important role in brain homeostasis both pre and postnatally. In vitro studies have suggested that cells from adult choroid plexus have stem/progenitor cell-like properties. Our initial aim was to investigate whether such a cell population is present in vivo during development of the choroid plexus, focusing mainly on the chick choroid plexus. Cells expressing neural markers were indeed present in the choroid plexus of chick and also those of rodent and human embryos, both within their epithelium and mesenchyme. ß3-tubulin-positive cells with neuronal morphology could be detected as early as at E8 in chick choroid plexus and their morphological complexity increased with development. Whole mount immunochemistry demonstrated the presence of neurons throughout choroid plexus development and they appeared to be mainly catecholaminergic, as indicated by tyrosine-hydroxylase reactivity. The presence of cells co-labeling for BrdU and the neuroblast marker, doublecortin, in organotypic choroid plexus cultures supported the hypothesis that neurogenesis can occur from neural precursors within the developing choroid plexus. Furthermore, we found that extrinsic innervation is present in the developing choroid plexus, unlike previously suggested. Altogether, our data are consistent with the presence of neural progenitors within the choroid plexus, suggest that at least some of the choroid plexus neurons are born locally, and show for the first time that choroid plexus innervation occurs prenatally. Hence, we propose the existence of a complex neural regulatory network within the developing choroid plexus that may play a crucial role in modulating its function during development as well as throughout life.

Clinical outcome of adult choroid plexus tumors: retrospective analysis of a single institute.

Choroid plexus tumors are rare brain tumors with clinical features that vary according to the histological grade. We reviewed the treatment outcomes of 15 adult patients with choroid plexus tumors, focusing on surgical outcomes and current therapeutic strategies. Patient demographic and clinical characteristics, operative findings, adjuvant therapies, disease progression and survival rates were reviewed. The median age at diagnosis was 33.7 ± 10 years (19-59 years) for patients with choroid plexus tumors. Postoperative chemotherapy was given to 26.7 % of patients, and 13.3 % of patients received radiotherapy. The Ki-67 labeling index and mitotic index increased at higher histological grades. All of the choroid plexus papilloma and atypical choroid plexus papilloma patients have survived. The overall survival rate of patients with choroid plexus carcinoma was 50 % in the first year, but none of the patients survived to the second year. Five patients underwent permanent cerebrospinal fluid diversion surgery because of hydrocephalus or subdural effusion. Choroid plexus papilloma and atypical choroid plexus papilloma patients can be treated with complete surgical resection. Choroid plexus carcinoma has a poor prognosis, and aggressive multi-modal treatments are generally needed for treatment. Chemotherapy and radiotherapy are important adjuvant therapies for choroid plexus carcinoma. If hydrocephalus and/or subdural effusion occur, permanent cerebrospinal fluid (CSF) diversion should be added to the therapeutic strategy.

The relative patient benefit of gross total resection in adult choroid plexus papillomas

Choroid plexus papillomas are rare neuroepithelial tumors found mainly in children. Although well studied in the pediatric population, there is a paucity of literature focusing specifically on adults. We sought to assess the relative advantage of gross total resection (GTR) and further characterize the natural history of this disease in adults. A comprehensive PubMed search was performed to identify adults who underwent surgical resection for choroid plexus papillomas with clearly reported age, tumor location, and extent of resection. Kaplan–Meier analysis was used to assess progression-free survival (PFS) and overall survival (OS). Multivariate analysis was performed using Cox proportional hazards models. A total of 193 patients were identified with a mean age of 39.9±1.1years. GTR was achieved in 72% of patients with subtotal resection (STR) in 28%. GTR was associated with a significant increase in both PFS (p=0.015) and OS (p=0.004) compared to STR. In a multivariate Cox proportional hazards model we found that only GTR was associated with recurrence (hazard ratio [HR]=0.47, 95% confidence interval [CI] 0.25–0.90), while both age (HR=1.03, 95% CI 1.00–1.05) and GTR (HR=0.36, 95% CI 0.17–0.78) were associated with OS. Interestingly, our observed recurrence and death rates were higher than those in previously published studies. These findings demonstrate the benefit of GTR for the treatment of choroid plexus papillomas in adults. Our analysis suggests that these lesions are not as indolent as previously thought and while GTR is preferred, it is not always curative.

Recurrent Adult Choroid Plexus Carcinoma Treated with High-Dose Chemotherapy and Syngeneic Stem Cell (Bone Marrow) Transplant

Choroid plexus carcinomas (CPCs) are rare epithelial central nervous system tumors. CPC occurs mainly in infants and young children, comprising ≈ 1 to 4% of all pediatric brain neoplasms. There is very limited information available regarding tumor biology and CPC treatment due to its rarity. There have been various case reports and meta-analyses of reported cases with CPC. Surgical resection is often challenging but remains a well-established treatment option. Chemotherapy is often reserved for recurrent or refractory cases, but the goal of treatment is usually palliative. We present a case of recurrent, adult CPC with disseminated leptomeningeal involvement treated with salvage chemotherapy including high-dose ifosfamide, carboplatin, and etoposide; once a remission was achieved, this response was consolidated with a syngeneic stem cell (bone marrow) transplant after a preparative regimen of high-dose chemotherapy with carboplatin, etoposide, and thiotepa. Although the patient tolerated the transplant well and remained disease-free for 12 months, she subsequently succumbed to relapsed disease 18 months posttransplant. We believe that this is the first report of using syngeneic stem cell transplant in CPC to consolidate a remission achieved by salvage chemotherapy.

A molecular characterization of the choroid plexus and stress-induced gene regulation

The role of the choroid plexus (CP) in brain homeostasis is being increasingly recognized and recent studies suggest that the CP has a more important role in physiological and pathological brain functions than currently appreciated. To obtain additional insight on the CP function, we performed a proteomics and transcriptomics characterization employing a combination of high resolution tandem mass spectrometry and gene expression analyses in normal rodent brain. Using multiple protein fractionation approaches, we identified 1400 CP proteins in adult CP. Microarray-based comparison of CP gene expression with the kidney, cortex and hippocampus showed significant overlap between the CP and the kidney. CP gene profiles were validated by in situ hybridization analysis of several target genes including klotho, CLIC 6, OATP 14 and Ezrin. Immunohistochemical analyses were performed for CP and enpendyma detection of several target proteins including cytokeratin, Rab7, klotho, tissue inhibitor of metalloprotease 1 (TIMP1), MMP9 and glial fibrillary acidic protein (GFAP). The molecular functions associated with various proteins of the CP proteome indicate that it is a blood–cerebrospinal fluid (CSF) barrier that exhibits high levels of metabolic activity. We also analyzed the gene expression changes induced by stress, an exacerbating factor for many illnesses, particularly mood disorders. Chronic stress altered the expression of several genes, downregulating 5HT2C, glucocorticoid receptor and the cilia genes IFT88 and smoothened while upregulating 5HT2A, BDNF, TNFα and IL-1b. The data presented here attach additional significance to the emerging importance of CP function in brain health and CNS disease states.

The path from the choroid plexus to the subventricular zone: go with the flow!

In adult mammals, under physiological conditions, neurogenesis, the process of generating new functional neurons from precursor cells, occurs mainly in two brain areas: the subgranular zone in the dentate gyrus of the hippocampus, and the subventricular zone (SVZ) lining the walls of the brain lateral ventricles. Taking into account the location of the SVZ and the cytoarchitecture of this periventricular neural progenitor cell niche, namely the fact that the slow dividing primary progenitor cells (type B cells) of the SVZ extend an apical primary cilium toward the brain ventricular space which is filled with cerebrospinal fluid (CSF), it becomes likely that the composition of the CSF can modulate both self-renewal, proliferation and differentiation of SVZ neural stem cells. The major site of CSF synthesis is the choroid plexus (CP); quite surprisingly, however, it is still largely unknown the contribution of molecules specifically secreted by the adult CP as modulators of the SVZ adult neurogenesis. This is even more relevant in light of recent evidence showing the ability of the CP to adapt its transcriptome and secretome to various physiologic and pathologic stimuli. By giving particular emphasizes to growth factors and axonal guidance molecules we will illustrate how CP-born molecules might play an important role in the SVZ niche cell population dynamics.

ECRG-4 expression in normal and neoplastic choroid plexus

The choroid plexus is a major site of gene expression of esophageal cancer-related gene (ECRG)-4 during development, suggesting that its gene product may be involved in cerebrospinal fluid (CSF) homeostasis. Yet, ECRG-4 is also a novel candidate tumor suppressor gene whose expression is downregulated and is inversely associated with a worse prognosis in several different cancers. Reduced expression of ECRG-4 has been demonstrated in most tumors, including colorectal carcinoma and malignant glioma, to be mediated by hypermethylation of its promoter. Materials and methods In this study, samples of normal human choroid plexus (both fetal and adult) and choroid plexus neoplasms (WHO grade I papilloma, grade II atypical papilloma, and grade III carcinoma) were stained with antibodies that we generated to augurin, the gene product of ECRG4. DNA was then extracted from the tissue, treated with bisulfite, and subjected to PCR using a 217-base pair region encompassing the ECRG-4 promotor to detect methylation. Results Both fetal and adult human choroid plexus cells demonstrated a robust positive immunostaining at the apical surface that is consistent with our prior results in human, rat, and mouse brains. In contrast, there was a near-complete absence of immunostaining in all of the choroid plexus neoplasms examined. The choroid plexus carcinoma demonstrated significant methylation of the ECRG-4 promotor region. Conclusions Taken together, these data suggest that ECRG-4 is downregulated in neoplasms of the choroid plexus just as has been observed in other central nervous system (CNS) and non-CNS cancers. This is likely due to hypermethylation of the ECRG-4 promotor, as shown in the choroid plexus carcinoma. Further analysis is underway to determine the (1) physiologic and (2) pathophysiologic consequences of ECRG-4 over- and under- expression in the choroid plexus on CSF formation, function, and composition.

The expression of twisted gastrulation in postnatal mouse brain and functional implications

Twisted gastrulation (TWSG1), an extracellular regulator of bone morphogenetic protein (BMP) signaling, is critical for embryonic brain development. Mice deficient in TWSG1 have abnormal forebrain development manifesting as holoprosencephaly. The expression and potential roles of TWSG1 in postnatal brain development are less well understood. We show that Twsg1 is expressed in the adult mouse brain in the choroid plexus (CP), hippocampus, and other regions, with the strongest expression observed in CP. TWSG1 was also detected in a human fetal brain at mid-gestation, with highest levels in the epithelium of CP. Bmp1, Bmp2, Bmp4–Bmp7 as well as BmprIA and BmprII, but not BmprIB, were expressed in CP. BMP antagonists Chordin (Chrd) and Noggin were not detected in CP, however Chrd-like 1 and brain-specific Chrd-like (Brorin) were expressed. Electrophysiological study of synaptic plasticity revealed normal paired-pulse facilitation and long-term potentiation in the CA1 region of hippocampus in Twsg1 −/− mice. Among the homozygous mutants that survive beyond the first 2 weeks, the prevalence of hydrocephalus was 4.3%, compared to 1.5% in a wild type colony ( P =0.0133) between 3 and 10 weeks of life. We detected a high level of BMP signaling in CP in wild type adult mice that was 17-fold higher than in the hippocampus ( P =0.005). In contrast, transforming growth factor beta (TGFβ) signaling was predominant in the hippocampus. Both BMP signaling and the expression of BMP downstream targets Msx1 and Msx2 were reduced in CP in Twsg1 −/− mice. In summary, we show that Twsg1 is expressed in the adult mouse and human fetal CP. We also show that BMP is a branch of TGFβ superfamily that is dominant in CP. This presents an interesting avenue for future research in light of the novel roles of CP in neural progenitor differentiation and neuronal repair, especially since TWSG1 appears to be the main regulator of BMP present in CP.

Dynamic expression of Dab2 in the mouse embryonic central nervous system

BackgroundDab2, one of two mammalian orthologs of Drosophila Disabled, has been shown to be involved in cell positioning and formation of visceral endoderm during mouse embryogenesis, but its role in neuronal development is not yet fully understood. In this report, we have examined the localization of the Dab2 protein in the mouse embryonic central nervous system (CNS) at different developmental stages.ResultsDab2 protein was transiently expressed in rhombomeres 5 and 6 of the developing hindbrain between E8.5 and E11.5, and in the floor plate of the neural tube from E9.5 to E12.5, following which it was no longer detectable within these regions. Dab2 protein was also identified within circumventricular organs including the choroid plexus, subcommissural organ and pineal gland during their early development. While Dab2 was still strongly expressed in the adult choroid plexus, immunoreactivity within the subcommissural organ and pineal gland was lost after birth. In addition, Dab2 was transiently expressed within a subpopulation of Iba1-positive mononuclear phagocytes (including presumed microglial progenitors) within the neural tube from E10.0 and was lost by E14.5. Dab2 was separately localized to Iba1 positive cells from E9.5 and subsequently to F4/80 positive cells (mature macrophage/myeloid-derived dendritic cells) positioned outside the neural tube from E12.5 onwards, implicating Dab2 expression in early cells of the mononuclear phagocyte lineage. Dab2 did not co-localize with the pan-neuronal marker PGP9.5 at any developmental stage, suggesting that Dab2 positive cells in the developing CNS are unlikely to be differentiating neurons.ConclusionThis is the first study to demonstrate the dynamic spatiotemporal expression of Dab2 protein within the CNS during development.

VEGF and TGF-β are required for the maintenance of the choroid plexus and ependyma

Although the role of vascular endothelial growth factor (VEGF) in developmental and pathological angiogenesis is well established, its function in the adult is less clear. Similarly, although transforming growth factor (TGF) β is involved in angiogenesis, presumably by mediating capillary (endothelial cell [EC]) stability, its involvement in quiescent vasculature is virtually uninvestigated. Given the neurological findings in patients treated with VEGF-neutralizing therapy (bevacizumab) and in patients with severe preeclampsia, which is mediated by soluble VEGF receptor 1/soluble Fms-like tyrosine kinase receptor 1 and soluble endoglin, a TGF-β signaling inhibitor, we investigated the roles of VEGF and TGF-β in choroid plexus (CP) integrity and function in adult mice. Receptors for VEGF and TGF-β were detected in adult CP, as well as on ependymal cells. Inhibition of VEGF led to decreased CP vascular perfusion, which was associated with fibrin deposition. Simultaneous blockade of VEGF and TGF-β resulted in the loss of fenestrae on CP vasculature and thickening of the otherwise attenuated capillary endothelium, as well as the disappearance of ependymal cell microvilli and the development of periventricular edema. These results provide compelling evidence that both VEGF and TGF-β are involved in the regulation of EC stability, ependymal cell function, and periventricular permeability.

Use of a new polyclonal antibody to study the distribution and glycosylation of the sodium-coupled bicarbonate transporter NCBE in rodent brain

NCBE (SLC4A10) is a member of the SLC4 family of bicarbonate transporters, several of which play important roles in intracellular-pH regulation and transepithelial HCO 3 − transport. Here we characterize a new antibody that was generated in rabbit against a fusion protein consisting of maltose-binding protein and the first 135 amino acids (aa) of the N-terminus of human NCBE. Western blotting—both of purified peptides representing the initial ∼120 aa of the transporters and of full-length transporters expressed in Xenopus oocytes—demonstrated that the antibody is specific for NCBE versus the two most closely related proteins, NDCBE (SLC4A8) and NBCn1 (SLC4A7). Western blotting of tissue in four regions of adult mouse brain indicates that NCBE is expressed most abundantly in cerebral cortex (CX), cerebellum (CB) and hippocampus (HC), and less so in subcortex (SCX). NCBE protein was present in CX, CB, and HC microdissected to avoid choroid plexus. Immunocytochemistry shows that NCBE is present at the basolateral membrane of embryonic day 18 (E18) fetal and adult choroid plexus. NCBE protein is present by Western blot and immunocytochemistry in cultured and freshly dissociated HC neurons but not astrocytes. By Western blot, nearly all NCBE in mouse and rat brain is highly N-glycosylated (∼150 kDa). PNGase F reduces the molecular weight (MW) of natural NCBE in mouse brain or human NCBE expressed in oocytes to approximately the predicted MW of the unglycosylated protein. In oocytes, mutating any one of the three consensus N-glycosylation sites reduces glycosylation of the other two, and the triple mutant exhibits negligible functional expression.

Chromosomal Imbalances in Choroid Plexus Tumors

We studied 49 choroid plexus tumors by comparative genomic hybridization. Chromosomal imbalances were found in 32 of 34 choroid plexus papillomas and 15 of 15 choroid plexus carcinomas. Choroid plexus papillomas frequently showed +7q (65%), +5q (62%), +7p (59%), +5p (56%), +9p (50%), +9q (41%), +12p, +12q (38%), and +8q (35%) as well as -10q (56%), -10p, and -22q (47%); choroid plexus carcinomas mainly showed +12p, +12q, +20p (60%), +1, +4q, +20q (53%), +4p (47%), +8q, +14q (40%), +7q, +9p, +21 (33%) as well as -22q (73%), -5q (40%), -5p, and -18q (33%). Several chromosomal imbalance differences could be found that were characteristic for a tumor entity or age group. In choroid plexus papillomas +5q, +6q, +7q, +9q, +15q, +18q, and -21q were significantly more common whereas choroid plexus carcinomas were characterized by +1, +4q, +10, +14q, +20q, +21q, -5q, -9p, -11, -15q, and -18q. Among choroid plexus papillomas, children more often showed +8q, +14q, +12, and +20q; adults mainly presented with +5q, +6q, +15q, +18q, and -22q. Although the number of aberrations overall as well as of gains and losses on their own bore no significance on survival among choroid plexus tumors, a significantly longer survival among patients with choroid plexus carcinomas was associated with +9p and -10q. Our results show that aberrations differ between choroid plexus papillomas and choroid plexus carcinomas as well as between pediatric and adult choroid plexus papillomas, supporting the notion of different genetic pathways. Furthermore, gain of 9p and loss of 10q seem to be correlated with a more favorable prognosis in choroid plexus carcinomas.

Immunolocalization of NAIP in the human brain and spinal cord.

The neuronal apoptosis inhibitory protein (NAIP) is known to have anti-apoptotic functions, and its gene is often mutated in severe cases of spinal muscular atrophy (SMA), a disease characterized by motor neuron degeneration. In this study, we examined the distribution of the endogenous NAIP protein in normal human spinal cord and brain tissue by using a polyclonal antibody against NAIP. Immunohistochemical staining demonstrated that NAIP is strongly expressed in anterior horn and motor cortex neurons of normal brains, and it is not altered in the remaining motor neurons of patients with amyotrophic lateral sclerosis (ALS). NAIP is also located in human fetal neurons and in adult choroid plexus cells. These results suggest that the anti apoptotic molecule NAIP may be important in motor neurons, but it specifically does not appear to be altered in ALS.