Choroid plexus defects in Down syndrome brain organoids enhance neurotropism of SARS-CoV-2.

Why individuals with Down syndrome (DS) are more susceptible to SARS-CoV-2-induced neuropathology remains elusive. Choroid plexus (ChP) plays critical roles in barrier function and immune response modulation and expresses the ACE2 receptor and the chromosome 21-encoded TMPRSS2 protease, suggesting its substantial role in establishing SARS-CoV-2 infection in the brain. To explore this, we established brain organoids from DS and isogenic euploid iPSC that consist of a core of functional cortical neurons surrounded by a functional ChP-like epithelium (ChPCOs). DS-ChPCOs recapitulated abnormal DS cortical development and revealed defects in ciliogenesis and epithelial cell polarity in ChP-like epithelium. We then demonstrated that the ChP-like epithelium facilitates infection and replication of SARS-CoV-2 in cortical neurons and that this is increased in DS. Inhibiting TMPRSS2 and furin activity reduced viral replication in DS-ChPCOs to euploid levels. This model enables dissection of the role of ChP in neurotropic virus infection and euploid forebrain development and permits screening of therapeutics for SARS-CoV-2-induced neuropathogenesis.

The circadian clock in the choroid plexus drives rhythms in multiple cellular processes under the control of the suprachiasmatic nucleus.

Choroid plexus (ChP), the brain structure primarily responsible for cerebrospinal fluid production, contains a robust circadian clock, whose role remains to be elucidated. The aim of our study was to [1] identify rhythmically controlled cellular processes in the mouse ChP and [2] assess the role and nature of signals derived from the master clock in the suprachiasmatic nuclei (SCN) that control ChP rhythms. To accomplish this goal, we used various mouse models (WT, mPer2Luc, ChP-specific Bmal1 knockout) and combined multiple experimental approaches, including surgical lesion of the SCN (SCNx), time-resolved transcriptomics, and single cell luminescence microscopy. In ChP of control (Ctrl) mice collected every 4 h over 2 circadian cycles in darkness, we found that the ChP clock regulates many processes, including the cerebrospinal fluid circadian secretome, precisely times endoplasmic reticulum stress response, and controls genes involved in neurodegenerative diseases (Alzheimer's disease, Huntington's disease, and frontotemporal dementia). In ChP of SCNx mice, the rhythmicity detected in vivo and ex vivo was severely dampened to a comparable extent as in mice with ChP-specific Bmal1 knockout, and the dampened cellular rhythms were restored by daily injections of dexamethasone in mice. Our data demonstrate that the ChP clock controls tissue-specific gene expression and is strongly dependent on the presence of a functional connection with the SCN. The results may contribute to the search for a novel link between ChP clock disruption and impaired brain health.

c-fos induction in the choroid plexus, tanycytes and pars tuberalis is an early indicator of spontaneous arousal from torpor in a deep hibernator.

Hibernation is an extreme state of seasonal energy conservation, reducing metabolic rate to as little as 1% of the active state. During the hibernation season, many species of hibernating mammals cycle repeatedly between the active (aroused) and hibernating (torpid) states (T-A cycling), using brown adipose tissue (BAT) to drive cyclical rewarming. The regulatory mechanisms controlling this process remain undefined but are presumed to involve thermoregulatory centres in the hypothalamus. Here, we used the golden hamster (Mesocricetus auratus), and high-resolution monitoring of BAT, core body temperature and ventilation rate, to sample at precisely defined phases of the T-A cycle. Using c-fos as a marker of cellular activity, we show that although the dorsomedial hypothalamus is active during torpor entry, neither it nor the pre-optic area shows any significant changes during the earliest stages of spontaneous arousal. Contrastingly, in three non-neuronal sites previously linked to control of metabolic physiology over seasonal and daily time scales - the choroid plexus, pars tuberalis and third ventricle tanycytes - peak c-fos expression is seen at arousal initiation. We suggest that through their sensitivity to factors in the blood or cerebrospinal fluid, these sites may mediate metabolic feedback-based initiation of the spontaneous arousal process.

Circadian ABCG2 Expression Influences the Brain Uptake of Donepezil across the Blood-Cerebrospinal Fluid Barrier.

Donepezil (DNPZ) is a cholinesterase inhibitor used for the management of Alzheimer's disease (AD) and is dependent on membrane transporters such as ABCG2 to actively cross brain barriers and reach its target site of action in the brain. Located in the brain ventricles, the choroid plexus (CP) forms an interface between the cerebrospinal fluid (CSF) and the bloodstream, known as the blood-CSF barrier (BCSFB). Historically, the BCSFB has received little attention as a potential pathway for drug delivery to the central nervous system (CNS). Nonetheless, this barrier is presently viewed as a dynamic transport interface that limits the traffic of molecules into and out of the CNS through the presence of membrane transporters, with parallel activity with the BBB. The localization and expression of drug transporters in brain barriers represent a huge obstacle for drug delivery to the brain and a major challenge for the development of therapeutic approaches to CNS disorders. The widespread interest in understanding how circadian clocks modulate many processes that define drug delivery in order to predict the variability in drug safety and efficacy is the next bridge to improve effective treatment. In this context, this study aims at characterizing the circadian expression of ABCG2 and DNPZ circadian transport profile using an in vitro model of the BCSFB. We found that ABCG2 displays a circadian pattern and DNPZ is transported in a circadian way across this barrier. This study will strongly impact on the capacity to modulate the BCSFB in order to control the penetration of DNPZ into the brain and improve therapeutic strategies for the treatment of AD according to the time of the day.

The Pattern of GH Action in the Mouse Brain.

GH acts in numerous organs expressing the GH receptor (GHR), including the brain. However, the mechanisms behind the brain's permeability to GH and how this hormone accesses different brain regions remain unclear. It is well-known that an acute GH administration induces phosphorylation of the signal transducer and activator of transcription 5 (pSTAT5) in the mouse brain. Thus, the pattern of pSTAT5 immunoreactive cells was analyzed at different time points after IP or intracerebroventricular GH injections. After a systemic GH injection, the first cells expressing pSTAT5 were those near circumventricular organs, such as arcuate nucleus neurons adjacent to the median eminence. Both systemic and central GH injections induced a medial-to-lateral pattern of pSTAT5 immunoreactivity over time because GH-responsive cells were initially observed in periventricular areas and were progressively detected in lateral brain structures. Very few choroid plexus cells exhibited GH-induced pSTAT5. Additionally, Ghr mRNA was poorly expressed in the mouse choroid plexus. In contrast, some tanycytes lining the floor of the third ventricle expressed Ghr mRNA and exhibited GH-induced pSTAT5. The transport of radiolabeled GH into the hypothalamus did not differ between wild-type and dwarf Ghr knockout mice, indicating that GH transport into the mouse brain is GHR independent. Also, single-photon emission computed tomography confirmed that radiolabeled GH rapidly reaches the ventral part of the tuberal hypothalamus. In conclusion, our study provides novel and valuable information about the pattern and mechanisms behind GH transport into the mouse brain.

Single-cell analysis of mesenchymal cells in permeable neural vasculature reveals novel diverse subpopulations of fibroblasts.

BACKGROUND: In the choroid plexus and pituitary gland, vasculature is known to have a permeable, fenestrated phenotype which allows for the free passage of molecules in contrast to the blood brain barrier observed in the rest of the CNS. The endothelium of these compartments, along with secretory, neural-lineage cells (choroid epithelium and pituitary endocrine cells) have been studied in detail, but less attention has been given to the perivascular mesenchymal cells of these compartments. METHODS: The Hic1CreERT2 Rosa26LSL-TdTomato mouse model was used in conjunction with a PdgfraH2B-EGFP mouse model to examine mesenchymal cells, which can be subdivided into Pdgfra+ fibroblasts and Pdgfra- pericytes within the choroid plexus (CP) and pituitary gland (PG), by histological, immunofluorescence staining and single-cell RNA-sequencing analyses. RESULTS: We found that both CP and PG possess substantial populations of distinct Hic1+ mesenchymal cells, including an abundance of Pdgfra+ fibroblasts. Within the pituitary, we identified distinct subpopulations of Hic1+ fibroblasts in the glandular anterior pituitary and the neurosecretory posterior pituitary. We also identified multiple distinct markers of CP, PG, and the meningeal mesenchymal compartment, including alkaline phosphatase, indole-n-methyltransferase and CD34. CONCLUSIONS: Novel, distinct subpopulations of mesenchymal cells can be found in permeable vascular interfaces, including the CP, PG, and meninges, and make distinct contributions to both organs through the production of structural proteins, enzymes, transporters, and trophic molecules.

Olfactory Receptor OR2K2 Expression in Human Choroid Plexus as a Potential Marker in Early Sporadic Alzheimer's Disease.

Epithelial cells comprising the choroid plexus (CP) form a crucial barrier between the blood and the cerebrospinal fluid, thereby assuming a central position in brain homeostasis and signaling. Mounting evidence suggests that the impairment of CP function may be a significant contributor to Alzheimer's disease (AD) pathogenesis. CP function relies on the expression of specific receptors, and the potential involvement of olfactory receptors (ORs) and taste receptors (TASRs) in chemical surveillance within the CP is being investigated. Previous studies have implicated ORs and TASRs in neurodegenerative disorders like AD, although the direct evidence of their expression in the human CP remains to be established. In this study, we conducted a transcriptomic analysis encompassing eleven ORs and TASRs in the CP, comparing samples from healthy age-matched controls to those from patients with AD spanning Braak stages I to VI. Among these receptors, a striking finding emerged-OR2K2 exhibited robust expression, with a statistically significant upregulation noted at Braak stage I. Surprisingly, at the protein level, OR2K2 showed a significant decrease in both Braak stage I and VI. Additionally, we identified CP epithelial cells as the source of OR2K2 expression, where it colocalized with autophagy markers LC3 and p62. We postulate that OR2K2 could be subjected to degradation by autophagy in the early stages of AD, triggering a compensatory mechanism that leads to increased OR2K2 mRNA transcription. This study uncovers a potential role for OR2K2 in AD pathogenesis, offering a novel perspective on the intricate dynamics at play in this neurodegenerative disorder.

Cerebrospinal fluid pH regulation.

The cerebrospinal fluid (CSF) fills the brain ventricles and the subarachnoid space surrounding the brain and spinal cord. The fluid compartment of the brain ventricles communicates with the interstitial fluid of the brain across the ependyma. In comparison to blood, the CSF contains very little protein to buffer acid-base challenges. Nevertheless, the CSF responds efficiently to changes in systemic pH by mechanisms that are dependent on the CO2/HCO3- buffer system. This is evident from early studies showing that the CSF secretion is sensitive to inhibitors of acid/base transporters and carbonic anhydrase. The CSF is primarily generated by the choroid plexus, which is a well-vascularized structure arising from the pial lining of the brain ventricles. The epithelial cells of the choroid plexus host a range of acid/base transporters, many of which participate in CSF secretion and most likely contribute to the transport of acid/base equivalents into the ventricles. This review describes the current understanding of the molecular mechanisms in choroid plexus acid/base regulation and the possible role in CSF pH regulation.

Choroid plexus and its relations with age-related diseases.

The choroid plexus is composed of epithelial cells situated on the basal layer. The tight junctions between adjacent choroid plexus epithelial cells establish the blood-cerebrospinal fluid barrier. This barrier, in conjunction with the blood-brain barrier, is crucial for the homeostasis of the brain microenvironment. The choroid plexus epithelium secretes cerebrospinal fluid, growth factors, neuropeptides, and lipids into the ventricles and also serves as a gateway for immune cells to enter the brain. The pathophysiology of aging and neurodegenerative diseases remains largely enigmatic, with an increasing body of research linking the choroid plexus to the etiology of these age-related disorders. In this review, we summarize the known relationship between the choroid plexus epithelium and age-related diseases, aiming to provide new therapeutic clues for these disorders.

A Review of Cerebrospinal Fluid Circulation and the Pathogenesis of Congenital Hydrocephalus.

The brain's ventricles are filled with a colorless fluid known as cerebrospinal fluid (CSF). When there is an excessive accumulation of CSF in the ventricles, it can result in high intracranial pressure, ventricular enlargement, and compression of the surrounding brain tissue, leading to potential damage. This condition is referred to as hydrocephalus. Hydrocephalus is classified into two categories: congenital and acquired. Congenital hydrocephalus (CH) poses significant challenges for affected children and their families, particularly in resource-poor countries. Recognizing the psychological and economic impacts is crucial for developing interventions and support systems that can help alleviate the distress and burden faced by these families. As our understanding of CSF production and circulation improves, we are gaining clearer insights into the causes of CH. In this article, we will summarize the current knowledge regarding CSF circulation pathways and the underlying causes of CH. The main causes of CH include abnormalities in the FoxJ1 pathway of ventricular cilia, dysfunctions in the choroid plexus transporter Na+-K+-2Cl- contransporter isoform 1, developmental abnormalities in the cerebral cortex, and structural abnormalities within the brain. Understanding the causes of CH is indeed crucial for advancing research and developing effective treatment strategies. In this review, we will summarize the findings from existing studies on the causes of CH and propose potential research directions to further our understanding of this condition.

Multi-omics analyses of choroid plexus carcinoma cell lines reveal potential targetable pathways and alterations.

PURPOSE: Choroid plexus carcinomas (CPCs) are extremely rare brain tumors and carry a dismal prognosis. Treatment options are limited and there is an urgent need to develop models to further research. In the present study, we established two CPC cell lines and performed multi-omics analyses. These cell lines serve as valuable models to propose new treatments in these rare but deadly brain tumors. METHODS: Multi-omic profiling including, (i) methylation array (EPIC 850 K), (ii) whole genome sequencing (WGS), (iii) CANCERPLEX cancer genome panel testing, (iv) RNA sequencing (RNA-seq), and (v) proteomics analyses were performed in CCHE-45 and NGT131 cell lines. RESULTS: Both cell lines were classified as methylation class B. Both harbored pathogenic TP53 point mutations; CCHE-45 additionally displayed TP53 loss. Furthermore, alterations of the NOTCH and WNT pathways were also detected in both cell lines. Two protein-coding gene fusions, BZW2-URGCP, and CTTNBP2-ERBB4, mutations of two oncodrivers, GBP-4 and KRTAP-12-2, and several copy number alterations were observed in CCHE-45, but not NGT131. Transcriptome and proteome analysis identified shared and unique signatures, suggesting that variability in choroid plexus carcinoma tumors may exist. The discovered difference's importance and implications highlight the possible diversity of choroid plexus carcinoma and call for additional research to fully understand disease pathogenesis. CONCLUSION: Multi-omics analyses revealed that the two choroid plexus carcinoma cell lines shared TP53 mutations and other common pathway alterations and activation of NOTCH and WNT pathways. Noticeable differences were also observed. These cell lines can serve as valuable models to propose new treatments in these rare but deadly brain tumors.

The gut-brain axis: Correlation of choroid plexus volume and permeability with inflammatory biomarkers in Crohn's disease.

BACKGROUND: The dysregulation of the gut-brain axis in chronic inflammatory bowel diseases can cause neuro-psychological disturbances, but the underlying mechanisms are still not fully understood. The choroid plexus (CP) maintains brain homeostasis and nourishment through the secretion and clearance of cerebrospinal fluid. Recent research has demonstrated the existence of a CP vascular barrier in mice which is modulated during intestinal inflammation. This study investigates possible correlations between CP modifications and inflammatory activity in patients with Crohn's disease (CD). METHODS: In this prospective study, 17 patients with CD underwent concomitant abdominal and brain 3 T MRI. The volume and permeability of CP were compared with levels of C-reactive protein (CRP), fecal calprotectin (FC), sMARIA and SES-CD scores. RESULTS: The CP volume was negatively correlated with CRP levels (R = -0.643, p-value = 0.024) and FC (R = -0.571, p-value = 0.050). DCE metrics normalized by CP volume were positively correlated with CRP (K-trans: R = 0.587, p-value = 0.045; Vp: R = 0.706, p-value = 0.010; T1: R = 0.699, p-value = 0.011), and FC (Vp: R = 0.606, p-value = 0.037). CONCLUSIONS: Inflammatory activity in patients with CD is associated with changes in CP volume and permeability, thus supporting the hypothesis that intestinal inflammation could affect the brain through the modulation of CP vascular barrier also in humans.

Circadian clock in choroid plexus is resistant to immune challenge but dampens in response to chronodisruption.

The choroid plexus (ChP) in the brain ventricles has a major influence on brain homeostasis. In this study, we aimed to determine whether the circadian clock located in ChP is affected by chronodisruption caused by misalignment with the external light/dark cycle and/or inflammation. Adult mPer2Luc mice were maintained in the LD12:12 cycle or exposed to one of two models of chronic chronodisruption - constant light for 22-25 weeks (cLL) or 6-hour phase advances of the LD12:12 cycle repeated weekly for 12 weeks (cLD-shifts). Locomotor activity was monitored before the 4th ventricle ChP and suprachiasmatic nuclei (SCN) explants were recorded in real time for PER2-driven population and single-cell bioluminescence rhythms. In addition, plasma immune marker concentrations and gene expression in ChP, prefrontal cortex, hippocampus and cerebellum were analyzed. cLL dampened the SCN clock but did not shorten the inactivity interval (sleep). cLD-shifts had no effect on the SCN clock, but transiently affected sleep duration and fragmentation. Both chronodisruption protocols dampened the ChP clock. Although immune markers were elevated in plasma and hippocampus, levels in ChP were unaffected, and unlike the liver clock, the ChP clock was resistant to lipopolysaccharide treatment. Importantly, both chronodisruption protocols reduced glucocorticoid signaling in ChP. The data demonstrate the high resistance of the ChP clock to inflammation, highlighting its role in protecting the brain from neuroinflammation, and on the other hand its high sensitivity to chronodisruption. Our results provide a novel link between human lifestyle-induced chronodisruption and the impairment of ChP-dependent brain homeostasis.

Choroid plexus volume is increased in mood disorders and associates with circulating inflammatory cytokines.

Depressed patients exhibit altered levels of immune-inflammatory markers both in the peripheral blood and in the cerebrospinal fluid (CSF) and inflammatory processes have been widely implicated in the pathophysiology of mood disorders. The Choroid Plexus (ChP), located at the base of each of the four brain ventricles, regulates the exchange of substances between the blood and CSF and several evidence supported a key role for ChP as a neuro-immunological interface between the brain and circulating immune cells. Given the role of ChP as a regulatory gate between periphery, CSF spaces and the brain, we compared ChP volumes in patients with bipolar disorder (BP) or major depressive disorder (MDD) and healthy controls, exploring their association with history of illness and levels of circulating cytokines. Plasma levels of inflammatory markers and MRI scans were acquired for 73 MDD, 79 BD and 72 age- and sex-matched healthy controls (HC). Patients with either BD or MDD had higher ChP volumes than HC. With increasing age, the bilateral ChP volume was larger in patients, an effect driven by the duration of illness; while only minor effects were observed in HC. Right ChP volumes were proportional to higher levels of circulating cytokines in the clinical groups, including IFN-γ, IL-13 and IL-17. Specific effects in the two diagnostic groups were observed when considering the left ChP, with positive association with IL-1ra, IL-13, IL-17, and CCL3 in BD, and negative associations with IL-2, IL-4, IL-1ra, and IFN-γ in MDD. These results suggest that ChP could represent a reliable and easy-to-assess biomarker to evaluate the brain effects of inflammatory status in mood disorders, contributing to personalized diagnosis and tailored treatment strategies.

DPA714 PET Imaging Shows That Inflammation of the Choroid Plexus Is Active in Chronic-Phase Intracerebral Hemorrhage.

PURPOSE: Our aims were to investigate the presence of choroid plexus (CP) inflammation in chronic-phase intracerebral hemorrhage (ICH) patients and to characterize any inflammatory cells in the CP. PATIENTS AND METHODS: An in vivo 18 F-DPA714 PET study was undertaken in 22 chronic-phase ICH patients who were admitted to the First Affiliated Hospital of Fujian Medical University or Tianjin Medical University General Hospital from April 2017 to June 2020. Ten control participants with nonhemorrhagic central nervous system diseases were included. Choroid plexus 18 F-DPA714 uptake was calculated as the average SUVR. To aid the interpretation of the 18 F-DPA714 uptake results at the CP level, Cy5-DPA714 in vivo imaging and immunofluorescence staining were used to show the presence of CP inflammation in an ICH mouse model during the chronic phase (14 weeks after ICH). Then immunofluorescence staining against translocator protein and other specific biomarkers was used to characterize the cells present in the inflamed CP of ICH mice in the chronic phase. RESULTS: PET imaging showed that CP DPA714 SUVRs in chronic-phase ICH patients were higher than in controls (mean CP SUVR ± SD; ICH group: 1.05 ± 0.35; control group: 0.81 ± 0.21; P = 0.006). Immunofluorescence staining of the CP in ICH model mice identified a population of CD45 + immune cells, peripheral monocyte-derived CD14 + cells, CD68 + phagocytes, and CD11b + resident microglia/macrophages expressing translocator protein, possibly contributing to the increased 18 F-DPA714 uptake. CONCLUSIONS: Our study shows that CP DPA714 uptake in chronic-phase ICH patients was higher than that of participants with nonhemorrhagic central nervous system diseases, which means that CP inflammation is still active in chronic-phase ICH patients.

Choroid plexus mast cells drive tumor-associated hydrocephalus.

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.

Cholesterol 24-hydroxylase at the choroid plexus contributes to brain immune homeostasis.

The choroid plexus (CP) plays a key role in remotely controlling brain function in health, aging, and disease. Here, we report that CP epithelial cells express the brain-specific cholesterol 24-hydroxylase (CYP46A1) and that its levels are decreased under different mouse and human brain conditions, including amyloidosis, aging, and SARS-CoV-2 infection. Using primary mouse CP cell cultures, we demonstrate that the enzymatic product of CYP46A1, 24(S)-hydroxycholesterol, downregulates inflammatory transcriptomic signatures within the CP, found here to be elevated across multiple neurological conditions. In vitro, the pro-inflammatory cytokine tumor necrosis factor α (TNF-α) downregulates CYP46A1 expression, while overexpression of CYP46A1 or its pharmacological activation in mouse CP organ cultures increases resilience to TNF-α. In vivo, overexpression of CYP46A1 in the CP in transgenic mice with amyloidosis is associated with better cognitive performance and decreased brain inflammation. Our findings suggest that CYP46A1 expression in the CP impacts the role of this niche as a guardian of brain immune homeostasis.

Cycle-dependent sex differences in expression of membrane proteins involved in cerebrospinal fluid secretion at rat choroid plexus.

BACKGROUND: Female sex is a known risk factor of brain disorders with raised intracranial pressure (ICP) and sex hormones have been suggested to alter cerebrospinal fluid (CSF) dynamics, thus impairing ICP regulation in CSF disorders such as idiopathic intracranial hypertension (IIH). The choroid plexus (CP) is the tissue producing CSF and it has been hypothesized that altered hormonal composition could affect the activity of transporters involved in CSF secretion, thus affecting ICP. Therefore, we aimed to investigate if expression of various transporters involved in CSF secretion at CP were different between males and females and between females in different estrous cycle states. Steroid levels in serum was also investigated. METHODS: Female and male rats were used to determine sex-differences in the genes encoding for the transporters Aqp1 and 4, NKCC1, NBCe2, NCBE; carbonic anhydrase enzymes II and III (CA), subunits of the Na+/K+-ATPase including Atp1a1, Atp1b1 and Fxyd1 at CP. The estrous cycle stage metestrus (MET) and estrous (ES) were determined before euthanasia. Serum and CP were collected and subjected to RT-qPCR analysis and western blots. Serum was used to measure steroid levels using liquid chromatography tandem mass spectrometry (LC-MS/MS). RESULTS: Significant differences in gene expression and steroid levels between males and ES females were found, while no differences were found between male and MET females. During ES, expression of Aqp1 was lower (p < 0.01) and NKCC1 was higher in females compared to males. CAII was lower while CAIII was higher in ES females (p < 0.0001). Gene expression of Atp1a1 was lower in ES compared to male (p = 0.0008). Several of these choroidal genes were also significantly different in MET compared to females in ES. Differences in gene expression during the estrus cycle were correlated to serum level of steroid hormones. Protein expression of AQP1 (p = 0.008) and CAII (p = 0.035) was reduced in ES females compared to males. CONCLUSIONS: This study demonstrates for the first time that expression at CP is sex-dependent and markedly affected by the estrous cycle in female rats. Further, expression was related to hormone levels in serum. This opens a completely new avenue for steroid regulation of the expression of CSF transporters and the close link to the understanding of CSF disorders such as IIH.

SARS-CoV-2 infects epithelial cells of the blood-cerebrospinal fluid barrier rather than endothelial cells or pericytes of the blood-brain barrier.

BACKGROUND: As a consequence of SARS-CoV-2 infection various neurocognitive and neuropsychiatric symptoms can appear, which may persist for several months post infection. However, cell type-specific routes of brain infection and underlying mechanisms resulting in neuroglial dysfunction are not well understood. METHODS: Here, we investigated the susceptibility of cells constituting the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB) of the choroid plexus (ChP) to SARS-CoV-2 infection using human induced pluripotent stem cell (hiPSC)-derived cellular models and a ChP papilloma-derived epithelial cell line as well as ChP tissue from COVID-19 patients, respectively. RESULTS: We noted a differential infectibility of hiPSC-derived brain microvascular endothelial cells (BMECs) depending on the differentiation method. Extended endothelial culture method (EECM)-BMECs characterized by a complete set of endothelial markers, good barrier properties and a mature immune phenotype were refractory to SARS-CoV-2 infection and did not exhibit an activated phenotype after prolonged SARS-CoV-2 inoculation. In contrast, defined medium method (DMM)-BMECs, characterized by a mixed endothelial and epithelial phenotype and excellent barrier properties were productively infected by SARS-CoV-2 in an ACE2-dependent manner. hiPSC-derived brain pericyte-like cells (BPLCs) lacking ACE2 expression were not susceptible to SARS-CoV-2 infection. Furthermore, the human choroid plexus papilloma-derived epithelial cell line HIBCPP, modeling the BCSFB was productively infected by SARS-CoV-2 preferentially from the basolateral side, facing the blood compartment. Assessment of ChP tissue from COVID-19 patients by RNA in situ hybridization revealed SARS-CoV-2 transcripts in ChP epithelial and ChP stromal cells. CONCLUSIONS: Our study shows that the BCSFB of the ChP rather than the BBB is susceptible to direct SARS-CoV-2 infection. Thus, neuropsychiatric symptoms because of COVID-19 may rather be associated with dysfunction of the BCSFB than the BBB. Future studies should consider a role of the ChP in underlying neuropsychiatric symptoms following SARS-CoV-2 infection.

In vitro investigation of the effect of proinflammatory cytokines on mouse choroid plexus membrane transporters Ncbe and NKCC1.

Intraventricular hemorrhage is a potentially life-threatening condition. Approximately 20% of patients develop posthemorrhagic hydrocephalus with increased ventricular volume and intracranial pressure. Hydrocephalus develops partially due to increased secretion of cerebrospinal fluid by the choroid plexus. During hemorrhage a multitude of factors are released into the cerebrospinal fluid. Many of these have been implicated in the hypersecretion. In this study, we have investigated the isolated effect of inflammatory components, on the abundance of two membrane transporters involved in cerebrospinal fluid secretion by the choroid plexus: the Na+-dependent Cl-/HCO3- exchanger, Ncbe, and the Na+, K+, 2Cl- cotransporter, NKCC1. We have established a primary choroid plexus epithelial cell culture from 1 to 7 days old mouse pups. Seven days after seeding, the cells formed a monolayer. The cells were treated with either tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL-1ß), or interleukin 6 (IL-6) to mimic inflammation. The data show that treatment with TNFα, and IL-1ß only transiently increased NKCC1 abundance whereas the effect on Ncbe abundance was a transient decrease. IL-6 however significantly increased NKCC1 (242%), the phosphorylated NKCC1 (147%), as well as pSPAK (406%) abundance, but had no effect on Ncbe. This study suggests that the inflammatory pathway involved in hypersecretion primarily is mediated by activation of basolateral receptors in the choroid plexus, mainly facilitated by IL-6. This study highlights the complexity of the pathophysiological circumstances occurring during intraventricular hemorrhage.