Immune response and pathogen invasion at the choroid plexus in the onset of cerebral toxoplasmosis

Background Toxoplasma gondii (T. gondii) is a highly successful parasite being able to cross all biological barriers of the body, finally reaching the central nervous system (CNS). Previous studies have highlighted the critical involvement of the blood–brain barrier (BBB) during T. gondii invasion and development of subsequent neuroinflammation. Still, the potential contribution of the choroid plexus (CP), the main structure forming the blood–cerebrospinal fluid (CSF) barrier (BCSFB) have not been addressed. Methods To investigate T. gondii invasion at the onset of neuroinflammation, the CP and brain microvessels (BMV) were isolated and analyzed for parasite burden. Additionally, immuno-stained brain sections and three-dimensional whole mount preparations were evaluated for parasite localization and morphological alterations. Activation of choroidal and brain endothelial cells were characterized by flow cytometry. To evaluate the impact of early immune responses on CP and BMV, expression levels of inflammatory mediators, tight junctions (TJ) and matrix metalloproteinases (MMPs) were quantified. Additionally, FITC-dextran was applied to determine infection-related changes in BCSFB permeability. Finally, the response of primary CP epithelial cells to T. gondii parasites was tested in vitro. Results Here we revealed that endothelial cells in the CP are initially infected by T. gondii, and become activated prior to BBB endothelial cells indicated by MHCII upregulation. Additionally, CP elicited early local immune response with upregulation of IFN-γ, TNF, IL-6, host-defence factors as well as swift expression of CXCL9 chemokine, when compared to the BMV. Consequently, we uncovered distinct TJ disturbances of claudins, associated with upregulation of MMP-8 and MMP-13 expression in infected CP in vivo, which was confirmed by in vitro infection of primary CP epithelial cells. Notably, we detected early barrier damage and functional loss by increased BCSFB permeability to FITC-dextran in vivo, which was extended over the infection course. Conclusions Altogether, our data reveal a close interaction between T. gondii infection at the CP and the impairment of the BCSFB function indicating that infection-related neuroinflammation is initiated in the CP.

The Wilms Tumor Gene wt1a Contributes to Blood-Cerebrospinal Fluid Barrier Function in Zebrafish

The Wilms tumor suppressor gene Wt1 encodes a zinc finger transcription factor, which is highly conserved among vertebrates. It is a key regulator of urogenital development and homeostasis but also plays a role in other organs including the spleen and the heart. More recently additional functions for Wt1 in the mammalian central nervous system have been described. In contrast to mammals, bony fish possess two paralogous Wt1 genes, namely wt1a and wt1b. By performing detailed in situ hybridization analyses during zebrafish development, we discovered new expression domains for wt1a in the dorsal hindbrain, the caudal medulla and the spinal cord. Marker analysis identified wt1a expressing cells of the dorsal hindbrain as ependymal cells of the choroid plexus in the myelencephalic ventricle. The choroid plexus acts as a blood-cerebrospinal fluid barrier and thus is crucial for brain homeostasis. By employing wt1a mutant larvae and a dye accumulation assay with fluorescent tracers we demonstrate that Wt1a is required for proper choroid plexus formation and function. Thus, Wt1a contributes to the barrier properties of the choroid plexus in zebrafish, revealing an unexpected role for Wt1 in the zebrafish brain.

Final results of the Choroid Plexus Tumor study CPT-SIOP-2000

Introduction Standards for chemotherapy against choroid plexus tumors (CPT) have not yet been established. Methods CPT-SIOP-2000 (NCT00500890) was an international registry for all CPT nesting a chemotherapy randomization for high-risk CPT with Carboplatin/Etoposide/Vincristine (CarbEV) versus Cyclophosphamide/Etoposide/Vincristine (CycEV). Patients older than three years were recommended to receive irradiation: focal fields for non-metastatic CPC, incompletely resected atypical choroid plexus papilloma (APP) or metastatic choroid plexus papilloma (CPP); craniospinal fields for metastatic CPC/APP and non-responsive CPC. High risk was defined as choroid plexus carcinoma (CPC), incompletely resected APP, and all metastatic CPT. From 2000 until 2010, 158 CPT patients from 23 countries were enrolled. Results For randomized CPC, the 5/10 year progression free survival (PFS) of patients on CarbEV (n = 20) were 62%/47%, respectively, compared to 27%/18%, on CycEV (n = 15), (intention-to-treat, HR 2.6, p = 0.032). Within the registry, histological grading was the most influential prognostic factor: for CPP (n = 55) the 5/10 year overall survival (OS) and the event free survival (EFS) probabilities were 100%/97% and 92%/92%, respectively; for APP (n = 49) 96%/96% and 76%/76%, respectively; and for CPC (n = 54) 65%/51% and 41%/39%, respectively. Without irradiation, 12 out of 33 patients with CPC younger than three years were alive for a median of 8.52 years. Extent of surgery and metastases were not independent prognosticators. Conclusions Chemotherapy for Choroid Plexus Carcinoma is feasible and effective. CarbEV is superior to CycEV. A subset of CPC can be cured without irradiation.

Widespread choroid plexus contamination in sampling and profiling of brain tissue

The choroid plexus, a tissue responsible for producing cerebrospinal fluid, is found predominantly in the lateral and fourth ventricles of the brain. This highly vascularized and ciliated tissue is made up of specialized epithelial cells and capillary networks surrounded by connective tissue. Given the complex structure of the choroid plexus, this can potentially result in contamination during routine tissue dissection. Bulk and single-cell RNA sequencing studies, as well as genome-wide in situ hybridization experiments (Allen Brain Atlas), have identified several canonical markers of choroid plexus such as Ttr, Folr1, and Prlr. We used the Ttr gene as a marker to query the Gene Expression Omnibus database for transcriptome studies of brain tissue and identified at least some level of likely choroid contamination in numerous studies that could have potentially confounded data analysis and interpretation. We also analyzed transcriptomic datasets from human samples from Allen Brain Atlas and the Genotype-Tissue Expression (GTEx) database and found abundant choroid contamination, with regions in closer proximity to choroid more likely to be impacted such as hippocampus, cervical spinal cord, substantia nigra, hypothalamus, and amygdala. In addition, analysis of both the Allen Brain Atlas and GTEx datasets for differentially expressed genes between likely “high contamination” and “low contamination” groups revealed a clear enrichment of choroid plexus marker genes and gene ontology pathways characteristic of these ciliated choroid cells. Inclusion of these contaminated samples could result in biological misinterpretation or simply add to the statistical noise and mask true effects. We cannot assert that Ttr or other genes/proteins queried in targeted assays are artifacts from choroid contamination as some of these differentials may be due to true biological effects. However, for studies that have an unequal distribution of choroid contamination among groups, investigators may wish to remove contaminated samples from analyses or incorporate choroid marker gene expression into their statistical modeling. In addition, we suggest that a simple RT-qPCR or western blot for choroid markers would mitigate unintended choroid contamination for any experiment, but particularly for samples intended for more costly omic profiling. This study highlights an unexpected problem for neuroscientists, but it is also quite possible that unintended contamination of adjacent structures occurs during dissections for other tissues but has not been widely recognized.

Management of Choroid Plexus Papillomas

Introduction: Choroid plexus papillomas are rare neuroepithelial tumors found primarily in children. It represents less than 1% of all central nervous system tumors. Materials and methods: A retrospective study including 14 patients with choroid plexus papilloma tumors were performed at the Neurosurgery Department in Ait IDDIR Health Hospital Establishment between January 2010 and December 2017. In each case, diagnosis was made clinically and confirmed radiologically and histo-pathologically. All patients were operated. Results and discussion: The mean age was 26 years (ranged 3 months –48 years) .In our department, we grouped together 14 cases of choroid plexus papilloma tumors. For mortality we had one case who died during surgery, survival rate for 04 years is 100% .We had not recurrence during the study period.All patients had intracranial hypertension (HIC) without neurological deficit and benefited from brain CT, MRI and an Angiography. The location of the tumor was: Lateral ventricle, Fourth ventricle, Third ventricle. All patients underwent surgical excision with or without ventriculo-peritoneal shunt. Conclusion: Choroid plexus papillomas are rare neuroepithelial tumors, typically considered benign lesions, derived from the choroid plexus and appear like cauliflower.

Role of SPAK-NKCC1 Signaling Cascade in the Choroid Plexus Blood-CSF Barrier Damage After Stroke

Background: The mechanisms underlying dysfunction of choroid plexus (ChP) blood-cerebrospinal fluid (CSF) barrier and lymphocyte invasion in neuroinflammatory responses to stroke are not well understood. In this study, we investigated whether stroke damaged the blood-CSF barrier integrity due to dysregulation of major ChP ion transport system Na+-K+-Cl- cotransporter (NKCC1) and regulatory Ste20-related proline-alanine-rich kinase (SPAK). Methods: Sham or ischemic stroke was induced in C57Bl/6J mice. Changes of the SPAK-NKCC1 complex and tight junction proteins (TJs) in the ChP were quantified by immunofluorescence staining and immunoblotting. Immune cell infiltration in the ChP was assessed by flow cytometry and immunostaining. Cultured ChP epithelium cells (CPECs) and cortical neurons were used to evaluate H2O2-mediated oxidative stress in stimulating the SPAK-NKCC1 complex and cellular damage. In vivo or in vitro pharmacological blockade of the ChP SPAK-NKCC1 cascade with SPAK inhibitor ZT-1a or NKCC1 inhibitor bumetanide were examined. Results: Ischemic stroke stimulated activation of the CPECs apical membrane SPAK-NKCC1 complex, NF-κB, and MMP9, which was associated with loss of the blood-CSF barrier integrity and increased immune cell infiltration into the ChP. Oxidative stress directly activated SPAK-NKCC1 pathway and resulted in apoptosis, neurodegeneration, and NKCC1-mediated ion influx. Pharmacological blockade of the SPAK-NKCC1 pathway protected the ChP barrier integrity, attenuated ChP immune cell infiltration or neuronal death. Conclusion: Stroke-induced pathological stimulation of the SPAK-NKCC1 cascade caused CPECs damage and disruption of TJs at the blood-CSF barrier. The ChP SPAK-NKCC1 complex emerged as a therapeutic target for attenuating ChP dysfunction and lymphocyte invasion after stroke.

Techniques for visualizing fibroblast-vessel interactions in the developing and adult CNS

Fibroblasts are found associated with blood vessels in various locations across the CNS: in the meninges, the choroid plexus, and in the parenchyma within perivascular spaces. CNS fibroblasts have been characterized using transcriptional profiling and a Col1a1-GFP mouse line used to identify CNS fibroblasts in vivo. However, current methods for visualizing CNS fibroblasts are lacking and, in particular, prevent adequate assessment of fibroblast-vessel interactions. Here, we describe methods for whole mount visualization of meningeal and choroid plexus fibroblasts, and optical tissue clearing methods for visualization of parenchymal vessel-associated fibroblasts. Importantly, these techniques can be combined with immunohistochemistry methods for labeling different cell types in the meninges and blood vasculature as well as EdU-based cell proliferation assays. These methods are ideal for visualization of vessel-fibroblast interactions in these CNS structures and provide significant improvement over traditional sectioning and staining methods. We expect these methods will advance studies of CNS fibroblast development and functions in homeostasis, injury, and disease.