System-N transport plays an important role in l-glutamine uptake into isolated rat choroid plexus but its role in the transport of another System-N substrate, l-histidine, has yet to be determined. Similarly, the possible effects on System-N mediated l-histidine transport of changes in pH and extracellular l-glutamine, such as occur in cerebral ischemia and hepatic encephalopathy, have yet to be examined. In the absence of competing amino acids, l-[3H]histidine uptake in isolated rat choroid plexus was mediated by both Na+-independent and Na+-dependent transport. The former was inhibited by 2-amino-2-norbornane carboxlic acid, indicating System-L transport, while the latter appears System-N mediated as it was inhibited by three System-N substrates but not substrates for System-A and -ASC. The Na+-dependent uptake had a Km of 0.2 mM and a Vmax of 1.4 nmol/mg/min. It accounted for 30% of l-histidine uptake in the presence of physiological concentrations of amino acids. Reductions in pH markedly inhibited Na+-dependent but not Na+-independent transport indicating that, as in liver but not neurons, System-N mediated transport at the choroid plexus is pH sensitive. Increases in l-glutamine concentration in the pathophysiological range reduced l-histidine uptake via both System-L and -N.

Although fibroblast growth factor (Fgf) signalling plays crucial roles in several developing and mature tissues, little information is currently available on expression of Fgf2 during early choroid plexus development and whether Fgf2 directly affects the behaviour of the choroid plexus epithelium (CPe). The purpose of this study was to investigate expression of Fgf2 in rodent and human developing CPe and possible function of Fgf2, using in vitro models. The application of Fgf2 to brain in vivo can affect the whole tissue, making it difficult to assess specific responses of the CPe.

Wet AMD (wAMD) is associated with cellular senescence. However, senescent cell-targeted therapies for wAMD have rarely been comprehensively studied. This study aimed to explore the therapeutic effects of senolytic agents on choroidal neovascularization (CNV).

Telocytes (TCs) are a recently identified type of interstitial cells present in a wide variety of organs in humans and mammals (www.telocytes.com). They are characterized by a small cell body, but extremely long cell processes - telopodes (Tp), and a specific phenotype. TCs establish close contacts with blood capillaries, nerve fibers and stem cells. We report here identification of TCs by electron microscopy and immunofluorescence in rat meninges and choroid plexus/subventricular zone, in the vicinity of putative stem cells. The presence of TCs in brain areas involved in adult neurogenesis might indicate that they have a role in modulation of neural stem cell fate.

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.

Emmetropization is an active and visually guided process that involves the retina, choroid and sclera, and results in compensatory changes in eye growth. This guided growth is the result of visual cues and possibly mechanical interactions being translated into growth signals via molecular events from the retina into the choroid and sclera, through the choroidal scleral transition zone. If mechanical interactions were a part of the choroid-sclera signaling transduction cascade, specific morphological arrangements should be detectable in this region at the ultrastructural level. The goal of this study was to investigate the ultrastructural features of the choroidal scleral transition zone by comparing avian, non-human primate and human eyes, with the goal to confirm whether specific mechanical structures are present. Choroidal and scleral tissue from chicken, marmoset, and human eyes were imaged using transmission electron microscopy to document the choroid-sclera transition zone. In chicken eyes, fibroblast lamellae bordered the scleral matrix and formed thin end elongated processes that were undercut by scleral collagen fibrils. These processes back-looped into the scleral matrix, and displayed small club-like membrane protrusions. Differences in these arrangements in mature vs young chickens were not detected. The club-like membrane protrusions identified in chickens were rare in marmoset eyes, which instead exhibited two types of collagen fibrils discriminated by size, and were absent in the human eyes investigated. In marmoset and human eyes, elastic components were detected in the transition zone that were absent in chickens. In summary, cellular/membrane specializations indicating a mechanical interaction at the choroid-sclera transition zone were not detected in chicken, non-human primate or human eyes. If mechanotransduction is necessary for scleral growth, matrix integrity or development, alternative structural arrangements might be required.

Immune system alterations have been implicated in various dopamine-related disorders, such as schizophrenia, bipolar disorder, and attention-deficit/hyperactivity disorder (ADHD). How immunity might be influenced by dopaminergic dysfunction and impact on clinically-relevant behaviors is still uncertain. We performed a peripheral and cerebral immunophenotyping in mice bearing dopaminergic alteration produced by genetic liability (hypofunction of the dopamine transporter DAT) and psychostimulant (amphetamine) administration. We found that DAT hypofunction influences immune tolerance by increasing functional Tregs and adrenomedullin levels in the thymus and spleen, while reducing microglia activation and infiltration of brain monocyte-derived macrophages (mo-MΦ). Remarkably, both DAT hypofunction and amphetamine treatment are associated with a weaker activation of the choroid plexus (CP) gateway. Conversely, amphetamine reactivated the CP in the setting of DAT hypofunction, paralleling its paradoxical ADHD-relevant behavioral effects. These findings add new knowledge on dopaminergic immunopharmacology and support the immunomodulation of CP functionality as a promising therapeutic strategy for neurodevelopmental and psychiatric disorders.

We report a previously undescribed inflammatory lesion consisting of deposition of activated complement (C3d and C9neo) in association with major histocompatibility complex type II (MHC2)-positive activated microglia in choroid plexus villi exhibiting classical fibrous thickening of the pericapillary filtration membrane. The proportion of villi affected ranged from 5% to 90% in 56 adult subjects with diseases of the CNS and 11 subjects with no preexisting disease of the CNS. In 3 of the 4 children studied, 2% or less of examined villi showed stromal thickening, complement deposition, and the presence of MHC2-positive microglia; in adults, the proportion of villi affected increased with age. Other features of the lesion included loss of capillaries and failure by macrophages to clear extracellular particulate electron-dense material by clathrin-mediated phagocytosis. This choroid plexus lesion may relate pathogenetically to age-related macular degeneration and to Alzheimer disease, 2 other conditions with no known risk factors other than increasing age. All 3 conditions are characterized by the presence of damaged capillaries, inflammatory extracellular aggregates of mixed molecular composition and defective clearance of the deposits by macrophages.

Tumor-associated hydrocephalus (TAH) is a common and lethal complication of brain metastases. Although other factors beyond mechanical obstructions have been suggested, the exact mechanisms are unknown. Using single-nucleus RNA sequencing and spatial transcriptomics, we find that a distinct population of mast cells locate in the choroid plexus and dramatically increase during TAH. Genetic fate tracing and intracranial mast-cell-specific tryptase knockout showed that choroid plexus mast cells (CPMCs) disrupt cilia of choroid plexus epithelia via the tryptase-PAR2-FoxJ1 pathway and consequently increase cerebrospinal fluid production. Mast cells are also found in the human choroid plexus. Levels of tryptase in cerebrospinal fluid are closely associated with clinical severity of TAH. BMS-262084, an inhibitor of tryptase, can cross the blood-brain barrier, inhibit TAH in vivo, and alleviate mast-cell-induced damage of epithelial cilia in a human pluripotent stem-cell-derived choroid plexus organoid model. Collectively, we uncover the function of CPMCs and provide an attractive therapy for TAH.

While there are murine and rat choroid plexus epithelial cell cultures, a translationally relevant model for choroid plexus activation and function is still lacking. The rhesus macaque is the gold standard for modeling viral infection and activation of CNS, including HIV-associated neurocognitive disorders. We have developed a rhesus macaque choroid plexus epithelial cell culture model which we believe to be suitable for studies of inflammation associated with viral infection of the CNS. Epithelial morphology and function were assessed using vimentin, phalloidin, the tight junction protein zonula-occludens-1 (ZO-1), and focal adhesion kinase (FAK). Choroid plexus epithelial cell type was confirmed using immunofluorescence with two proteins highly expressed in the choroid plexus: transthyretin and α-klotho. Finally, barrier properties of the model were monitored using pro- and anti-inflammatory mediators (TNF-α, the TLR2 agonist PamCys3K, and dexamethasone). When pro-inflammatory TNF-α was added to the xCelligence wells, there was a decrease in barrier function, which decreased in a step-wise fashion with each additional administration. This barrier function was repaired upon addition of the steroid dexamethasone. The TLR2 agonist PAM3CysK increased barrier functions in TNF-α treated wells. We have presented a model of the blood-CSF barrier that will allow study into pro- and anti-inflammatory conditions in the brain, while simultaneously measuring real time changes to epithelial cells.

The choroid plexus (CP) is a highly vascularized structure containing endothelial and epithelial cells located in the ventricular system of the central nervous system (CNS). The role of the fenestrated CP endothelium is under-researched and requires the generation of an immortalized CP endothelial cell line with preserved features. Transduction of primary human CP endothelial cells (HCPEnC) with the human telomerase reverse transcriptase (hTERT) resulted in immortalized HCPEnC (iHCPEnC), which grew as monolayer with contact inhibition, formed capillary-like tubes in Matrigel, and showed no colony growth in soft agar. iHCPEnC expressed pan-endothelial markers and presented characteristic plasmalemma vesicle-associated protein-containing structures. Cultivation of iHCPEnC and human epithelial CP papilloma (HIBCPP) cells on opposite sides of cell culture filter inserts generated an in vitro model with a consistently enhanced barrier function specifically by iHCPEnC. Overall, iHCPEnC present a tool that will contribute to the understanding of CP organ functions, especially endothelial-epithelial interplay.

The choroid plexus (CP) consists of specialized ependymal cells and underlying blood vessels and stroma producing the bulk of the cerebrospinal fluid (CSF). CP epithelial cells are considered the site of the internal blood-cerebrospinal fluid barrier, show epithelial characteristics (basal lamina, tight junctions), and express aquaporin-1 (AQP1) apically. In this study, we analyzed the expression of aquaporins in the human CP using immunofluorescence and qPCR. As previously reported, AQP1 was expressed apically in CP epithelial cells. Surprisingly, and previously unknown, many cells in the CP epithelium were also positive for aquaporin-4 (AQP4), normally restricted to ventricle-lining ependymal cells and astrocytes in the brain. Expression of AQP1 and AQP4 was found in the CP of all eight body donors investigated (3 males, 5 females; age 74-91). These results were confirmed by qPCR, and by electron microscopy detecting orthogonal arrays of particles. To find out whether AQP4 expression correlated with the expression pattern of relevant transport-related proteins we also investigated expression of NKCC1, and Na/K-ATPase. Immunostaining with NKCC1 was similar to AQP1 and revealed no particular pattern related to AQP4. Co-staining of AQP4 and Na/K-ATPase indicated a trend for an inverse correlation of their expression. We hypothesized that AQP4 expression in the CP was caused by age-related changes. To address this, we investigated mouse brains from young (2 months), adult (12 months) and old (30 months) mice. We found a significant increase of AQP4 on the mRNA level in old mice compared to young and adult animals. Taken together, we provide evidence for AQP4 expression in the CP of the aging brain which likely contributes to the water flow through the CP epithelium and CSF production. In two alternative hypotheses, we discuss this as a beneficial compensatory, or a detrimental mechanism influencing the previously observed CSF changes during aging.

The choroid plexus, located in brain ventricles, has received surprisingly little attention in clinical neuroscience. In morphometric brain analysis, we serendipitously found a 21% increase in choroid plexus volume in 12 patients suffering from complex regional pain syndrome (CRPS) compared with age- and gender-matched healthy subjects. No enlargement was observed in a group of 8 patients suffering from chronic pain of other etiologies. Our findings suggest involvement of the choroid plexus in the pathogenesis of CRPS. Since the choroid plexus can mediate interaction between peripheral and brain inflammation, our findings pinpoint the choroid plexus as an important target for future research of central pain mechanisms.

Brain plasticity is often restricted to critical periods in early life. Here, we show that a key regulator of this process in the visual cortex, Otx2 homeoprotein, is synthesized and secreted globally from the choroid plexus. Consequently, Otx2 is maintained in selected GABA cells unexpectedly throughout the mature forebrain. Genetic disruption of choroid-expressed Otx2 impacts these distant circuits and in the primary visual cortex reopens binocular plasticity to restore vision in amblyopic mice. The potential to regulate adult cortical plasticity through the choroid plexus underscores the importance of this structure in brain physiology and offers therapeutic approaches to recovery from a broad range of neurodevelopmental disorders.

Although the gene expression profile of several tissues in humans and in rodent animal models has been explored, analysis of the complete choroid plexus (CP) transcriptome is still lacking. A better characterization of the CP transcriptome can provide key insights into its functions as one of the barriers that separate the brain from the periphery and in the production of cerebrospinal fluid.

The choroid plexus (CP) has a key role in maintaining brain homeostasis by producing cerebrospinal fluid (CSF), by mediating transport of nutrients and removing metabolic products from the central nervous system and by responding to peripheral inflammatory signals. Although abnormal markers of immune response and inflammation are apparent in individuals with schizophrenia, the CP of these individuals has not been characterized. We therefore sequenced mRNA from the CP from two independent collections of individuals with schizophrenia and unaffected controls. Genes related to immune function and inflammation were upregulated in both collections. In addition, a co-expression module related to immune/inflammation response that was generated by combining mRNA-Seq data from both collections was significantly associated with disease status. The immune/inflammation-related co-expression module was positively correlated with levels of C-reactive protein (CRP), cortisol and several immune modulator proteins in the serum of the same individuals and was also positively correlated with CRP, cortisol and pro-inflammatory cytokines in the frontal cortex of the same individuals. In addition, we found a substantial number of nodes (genes) that were common to our schizophrenia-associated immune/inflammation module from the pooled data and a module we generated from lippopolysaccharides-treated mouse model data. These results suggest that the CP of individuals with schizophrenia are responding to signals from the periphery by upregulating immune/inflammation-related genes to protect the brain and maintain the homeostasis but nevertheless fails to completely prevent immune/inflammation related changes in the brain.

To study the choroid in uncomplicated pregnant women using advanced optical coherence tomography (OCT) imaging and analysis.

1. Using conventional microelectrode techniques, membrane potentials were recorded from smooth muscle cells of guinea-pig choroidal arterioles. 2. Transmural stimulation initiated excitatory junction potentials (EJPs) which were abolished by either guanethidine or alpha,beta-methylene-ATP but not by phentolamine, indicating that they resulted from activation of purinoceptors. 3. Trains of stimuli evoked EJPs which were followed by a slow depolarization, an inhibitory junction potential (IJP) or a biphasic membrane potential change which consisted of an IJP and a subsequent slow depolarization. 4. Slow depolarizations were abolished by either phentolamine or guanethidine, indicating that they resulted from activation of alpha-adrenoceptors. 5. IJPs were abolished by atropine but not by guanethidine, and were reduced by 50 % by apamin with the residual response being abolished by charybdotoxin, indicating that they resulted from the activation of muscarinic receptors which open two sets of Ca2+-activated K+ channels. 6. Most responses were followed by slow hyperpolarizations. These were almost abolished by L-nitroarginine, an effect which was partly overcome by L-arginine, and were abolished by glibenclamide, indicating that they resulted from the release of NO and activation of ATP-sensitive K+ channels. 7. Immunohistochemical analysis showed that arterioles were densely innervated by adrenergic nerve fibres. A population of fibres, likely to be cholinergic, was also identified. Furthermore, populations of nerve fibres immunoreactive to antibodies against either nitric oxide synthase (NOS) or substance P (SP) were also identified. 8. These findings indicate that choroidal arterioles of the guinea-pig are innervated by at least three different populations of nerves, adrenergic nerves which evoke excitatory responses, cholinergic nerves which evoke inhibitory responses and a population of nerves which cause the release of NO.

The choroid plexus, an epithelial-based structure localized in the brain ventricle, is the major component of the blood-cerebrospinal fluid barrier. The choroid plexus produces the cerebrospinal fluid and regulates the components of the cerebrospinal fluid. Abnormal choroid plexus function is associated with neurodegenerative diseases, tumor formation in the choroid plexus epithelium, and hydrocephaly. In this study, we used zebrafish (Danio rerio) as a model system to understand the genetic components of choroid plexus development. We generated an enhancer trap line, Et(cp:EGFP) (sj2), that expresses enhanced green fluorescent protein (EGFP) in the choroid plexus epithelium. Using immunohistochemistry and fluorescent tracers, we demonstrated that the zebrafish choroid plexus possesses brain barrier properties such as tight junctions and transporter activity. Thus, we have established zebrafish as a functionally relevant model to study choroid plexus development. Using an unbiased approach, we performed a forward genetic dissection of the choroid plexus to identify genes essential for its formation and function. Using Et(cp:EGFP) (sj2), we isolated 10 recessive mutant lines with choroid plexus abnormalities, which were grouped into five classes based on GFP intensity, epithelial localization, and overall choroid plexus morphology. We also mapped the mutation for two mutant lines to chromosomes 4 and 21, respectively. The mutants generated in this study can be used to elucidate specific genes and signaling pathways essential for choroid plexus development, function, and/or maintenance and will provide important insights into how these genetic mutations contribute to disease.

Investigation of the choroid plexus in schizophrenia has seen growing interest due to its role in the interaction between neuroinflammation and brain dysfunction. Most previous studies included treated and long-term ill patients, while antipsychotics and illness course might both affect the choroid plexus. Here, we recruited first-episode antipsychotic-naïve schizophrenia patients, performed high-resolution structural brain scan and manually extracted choroid plexus volume. Choroid plexus volume was compared between patients and healthy controls after controlling for age, sex and intracranial volume. Age and sex effects were examined on choroid plexus volume in patient and healthy control groups respectively. In patients, we also examined the correlation of choroid plexus volume with volume measures of cortical and subcortical gray matter, white matter, lateral ventricular as well as symptom severity and cognitive function. Schizophrenia patients showed significantly enlarged choroid plexus volume compared with healthy controls. Choroid plexus volume was positively correlated with age in only patient group and we found significantly larger choroid plexus volumes in males than females in both patient and healthy control groups, while the sex effects did not differ between groups. Choroid plexus volume was only found correlated with lateral ventricular volume among the brain volume measures. No significant correlation between choroid plexus volume and clinical ratings or cognitive performance was observed. Without potential confounding effects of pharmacotherapy or illness course, our findings indicated the enlargement of choroid plexus in schizophrenia might be an enduring trait for schizophrenia.