an-advanced-co-culture-model-human-blood-cerebrospinal-fluid-barrier

An Advanced Co-Culture Model of the Human Blood-Cerebrospinal Fluid Barrier for Separate Analysis of Choroid Plexus Epithelium and Endothelium

The highly perfused choroid plexus (CP), located in the ventricular system of the brain, consists of several structural components, including a cuboid ...

Heidelberg University

Single-molecule localization microscopy (SMLM) enables imaging of biological structures in the nanometre range. Long measurement times are the consequence of﻿ this kind of microscopy due to the need of acquiring thousands of images. We built a setup that automatically detects target structures using confocal microscopy and images them with SMLM. Utilizing the Konstanz Information Miner (KNIME), we were able to connect a confocal microscope with an SMLM unit for targeted screening. In this process, we developed KNIME plugins to communicate with the microscope components and combined them to a workflow. Thus, measuring biological nanometre-sized structures in a sufficient number to get statistical significance becomes feasible. For proof of principle HIV-1 assembly complexes in HeLa cells ﻿﻿derived from transfection of replication deficient viral construct﻿ were imaged by a fully automated screen.

Fully Automated Targeted Confocal and Single-Molecule Localization Microscopy.

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  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.

Immortalized human choroid plexus endothelial cells enable an advanced endothelial-epithelial two-cell type  model of the choroid plexus.

Choroid plexus, located in brain ventricles, is the site of blood-cerebrospinal fluid barrier that contains endothelial cells and an epithelial monolayer separated by stroma. We established a two-cell-type model of the human choroid plexus consisting of immortalized endothelial cells (iHCPEnC) and epithelial papilloma (HIBCPP) cells grown on opposite sides of filter supports. In this protocol, we describe the preparation of this model, the measurement of transepithelial electrical resistance (TEER), and immunofluorescence imaging-based analysis to determine the barrier function. For complete details on the use and execution of this protocol, please refer to Muranyi et al. (2022).

Protocol for the setup and use of a human choroid plexus endothelial-epithelial two-cell-type  model.

Endothelial cells play a major part in the regulation of vascular permeability and angiogenesis. According to their duty to fit the needs of the underlying tissue, endothelial cells developed different subtypes with specific endothelial microdomains as caveolae, fenestrae and transendothelial channels which regulate nutrient exchange, leukocyte migration, and permeability. These microdomains can exhibit diaphragms that are formed by the endothelial cell-specific protein plasmalemma vesicle-associated protein (PLVAP), the only known protein component of these diaphragms. Several studies displayed an involvement of PLVAP in diseases as cancer, traumatic spinal cord injury, acute ischemic brain disease, transplant glomerulopathy, Norrie disease and diabetic retinopathy. Besides an upregulation of PLVAP expression within these diseases, pro-angiogenic or pro-inflammatory responses were observed. On the other hand, loss of PLVAP in knockout mice leads to premature mortality due to disrupted homeostasis. Generally, PLVAP is considered as a major factor influencing the permeability of endothelial cells and, finally, to be involved in the regulation of vascular permeability. Following these observations, PLVAP is debated as a novel therapeutic target with respect to the different vascular beds and tissues. In this review, we highlight the structure and functions of PLVAP in different endothelial types in health and disease.

The role of PLVAP in endothelial cells.

The Gram-negative bacterium , which causes meningitis in humans, has been demonstrated to manipulate or alter host signalling pathways during infection of the central nervous system (CNS). However, these complex signalling networks are not completely understood. We investigate the phosphoproteome of an  model of the blood-cerebrospinal fluid barrier (BCSFB) based on human epithelial choroid plexus (CP) papilloma (HIBCPP) cells during infection with the  serogroup B strain MC58 in presence and absence of the bacterial capsule. Interestingly, our data demonstrates a stronger impact on the phosphoproteome of the cells by the capsule-deficient mutant of MC58. Using enrichment analyses, potential pathways, molecular processes, biological processes, cellular components and kinases were determined to be regulated as a consequence of  infection of the BCSFB. Our data highlight a variety of protein regulations that are altered during infection of CP epithelial cells with N. meningitidis, with the regulation of several pathways and molecular events only being detected after infection with the capsule-deficient mutant. Mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD038560.

The phosphoproteome of choroid plexus epithelial cells following infection with Neisseria meningitidis.

[This corrects the article DOI: 10.3389/fcimb.2023.1113528.].

Corrigendum: The phosphoproteome of choroid plexus epithelial cells following infection with Neisseria meningitidis.

The human choroid plexus (CP) is the location of the blood-cerebrospinal fluid (CSF) barrier (BCSFB). Whereas the epithelial cells of the CP mainly contribute to the formation of the BCSFB, the vessels of the CP are built by fenestrated endothelial cells. Still, the CP endothelium can contribute to barrier function. By ectopic expression of human telomerase reverse transcriptase (hTERT) in primary human CP endothelial cells (HCPEnCs), we recently generated and characterized immortalized HCPEnCs (iHCPEnCs). Here, we compared primary cells of the sixth passage (HCPEnCs p6) with a lower (p20) and a higher passage (p50) of iHCPEnCs by transcriptome analysis. A high concordance of HCPEnCs and both passages of iHCPEnCs was observed, as only small proportions of the transcripts examined were significantly altered. Differentially expressed genes (DEGs) were identified and assigned to potentially affected biological processes by gene set enrichment analysis (GSEA). Various components of the endothelial barrier-relevant Wnt signaling were detected in HCPEnCs and iHCPEnCs. Functional analysis of HCPEnCs and iHCPEnCs showed equal marginal activation of Wnt signaling, supporting the downregulation of β-catenin (CTNNB) signaling in CP endothelial cells, and a contribution to the barrier function by the CP endothelium was retained until passage 100 (p100) of iHCPEnCs. Overall, our data support the suitability of iHCPEnCs as an in vitro model of the CP endothelium over extended passages.

Transcriptome and Functional Comparison of Primary and Immortalized Endothelial Cells of the Human Choroid Plexus at the Blood-Cerebrospinal Fluid Barrier.

Efficient delivery of therapeutics to the central nervous system (CNS) is one of the major challenges in treating neurological diseases due to brain barriers, which prevent entry of almost all potential therapeutic agents into the CNS. Targeting receptors that induce receptor-mediated transcytosis (RMT) across brain barriers has long been heralded as a potential solution to this problem, but this approach has yet to deliver clinical improvements for patients. Here, we set out to identify and characterize bivalent antibodies against the transferrin receptor 1 (TfR) as mediators of RMT. We identified the antibody YU904-F06 (hereafter referred to as F06) that showed efficient transcytosis as a bivalent IgG in two independent in vitro models of brain barriers. Despite its high affinity at extracellular pH levels, we determined that F06's binding to TfR was greatly reduced at lower pH levels expected during endocytic acidification. We postulated, with the support of a validated predictive mathematical model of RMT, that the pH-sensitivity of F06 allowed it to overcome the lysosomal degradation that has been previously reported for high affinity bivalent binders of TfR. Finally, we demonstrated that F06 could mediate the transcytosis of scFvs that target TREM2 or EGFRvIII as potential therapeutic cargos. In conclusion, we present a proof-of-concept antibody and rationale for the design of high affinity bivalent anti-TfR antibodies that effectively induce RMT by exploiting pH-sensitivity in binding.

Article	Year
Fully Automated Targeted Confocal and Single-Molecule Localization Microscopy. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 28924665	2017
Immortalized human choroid plexus endothelial cells enable an advanced endothelial-epithelial two-cell type model of the choroid plexus. iScience\| PubMed ID: 35633941	2022
Protocol for the setup and use of a human choroid plexus endothelial-epithelial two-cell-type model. STAR protocols\| PubMed ID: 36386888	2022
The role of PLVAP in endothelial cells. Cell and tissue research\| PubMed ID: 36781482	2023
The phosphoproteome of choroid plexus epithelial cells following infection with Neisseria meningitidis. Frontiers in cellular and infection microbiology\| PubMed ID: 37065199	2023
Corrigendum: The phosphoproteome of choroid plexus epithelial cells following infection with Neisseria meningitidis. Frontiers in cellular and infection microbiology\| PubMed ID: 37637459	2023
Transcriptome and Functional Comparison of Primary and Immortalized Endothelial Cells of the Human Choroid Plexus at the Blood-Cerebrospinal Fluid Barrier. International journal of molecular sciences\| PubMed ID: 40004242	2025
A pH-sensitive binding modality allows successful transferrin receptor-mediated transcytosis of a bivalent antibody across brain barriers. mAbs\| PubMed ID: 40983601	2025

Walter Muranyi

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