Though congenital hydrocephalus is heritable, it has been linked only to eight genes, one of which is MPDZ. Humans and mice that carry a truncated version of MPDZ incur severe hydrocephalus resulting in acute morbidity and lethality. We show by magnetic resonance imaging that contrast medium penetrates into the brain ventricles of mice carrying a Mpdz loss‐of‐function mutation, whereas none is detected in the ventricles of normal mice, implying that the permeability of the choroid plexus epithelial cell monolayer is abnormally high. Comparative proteomic analysis of the cerebrospinal fluid of normal and hydrocephalic mice revealed up to a 53‐fold increase in protein concentration, suggesting that transcytosis through the choroid plexus epithelial cells of Mpdz KO mice is substantially higher than in normal mice. These conclusions are supported by ultrastructural evidence, and by immunohistochemistry and cytology data. Our results provide a straightforward and concise explanation for the pathophysiology of Mpdz‐linked hydrocephalus.

Clustering of synthetic biotinylated K1 domain from HGF/SF by streptavidin is sufficient to generate a potent MET agonist.

No abstract

Background The pathways that control protein transport across the blood–brain barrier (BBB) remain poorly characterized. Despite great advances in recapitulating the human BBB in vitro, current models are not suitable for systematic analysis of the molecular mechanisms of antibody transport. The gaps in our mechanistic understanding of antibody transcytosis hinder new therapeutic delivery strategy development. Methods We applied a novel bioengineering approach to generate human BBB organoids by the self-assembly of astrocytes, pericytes and brain endothelial cells with unprecedented throughput and reproducibility using micro patterned hydrogels. We designed a semi-automated and scalable imaging assay to measure receptor-mediated transcytosis of antibodies. Finally, we developed a workflow to use CRISPR/Cas9 gene editing in BBB organoid arrays to knock out regulators of endocytosis specifically in brain endothelial cells in order to dissect the molecular mechanisms of receptor-mediated transcytosis. Results BBB organoid arrays allowed the simultaneous growth of more than 3000 homogenous organoids per individual experiment in a highly reproducible manner. BBB organoid arrays showed low permeability to macromolecules and prevented transport of human non-targeting antibodies. In contrast, a monovalent antibody targeting the human transferrin receptor underwent dose- and time-dependent transcytosis in organoids. Using CRISPR/Cas9 gene editing in BBB organoid arrays, we showed that clathrin, but not caveolin, is required for transferrin receptor-dependent transcytosis. Conclusions Human BBB organoid arrays are a robust high-throughput platform that can be used to discover new mechanisms of receptor-mediated antibody transcytosis. The implementation of this platform during early stages of drug discovery can accelerate the development of new brain delivery technologies.

Though congenital hydrocephalus is heritable, it has been linked only to eight genes, one of which is MPDZ. Humans and mice that carry a truncated version of MPDZ incur severe hydrocephalus resulting in acute morbidity and lethality. We show by magnetic resonance imaging that contrast-medium penetrates into the brain ventricles of mice carrying a Mpdz loss-of-function mutation, whereas none is detected in the ventricles of normal mice, implying that the permeability of the choroid plexus epithelial cell monolayer is abnormally high. Comparative proteomic analysis of the cerebrospinal fluid of normal and hydrocephalic mice revealed up to a 53-fold increase in protein concentration, suggesting that transcytosis through the choroid plexus epithelial cells of Mpdz KO mice is substantially higher than in normal mice. These conclusions are supported by ultrastructural evidence, and by immunohistochemistry and cytology data. Our results provide a straightforward and concise explanation for the pathophysiology of Mpdz-linked hydrocephalus.Keywords: hydrocephalus/choroid plexus/magnetic resonance imaging/cerebrospinal fluid/proteomics Hamazaki et al., 2002;Jeansonne et al., 2003;Lanaspa et al., 2008;Poliak et al., 2002). The abundance of MPDZ in the central nervous system is highest in the choroid plexus (CP) (Sitek et al., 2003), a network of capillaries walled by fenestrated endothelial cells, surrounded by a monolayer of cuboidal epithelial cells (Maxwell and Pease, 1956). The CP is the principal source of the CSF (Lun et al., 2015;Spector et al., 2015).We used a mouse model (Milner et al., 2015) similar to that of Feldner et al. to test the differences between the permeability of the CP of Mpdz +/+ and Mpdz -/mice, and between the composition of their CSF, using approaches that have not been employed before to these ends. Based on our findings and detailed observations of the ultrastructure of the CP, we propose a new pathophysiological mechanism to explain the formation of hydrocephalus in the Mpdz LOF mouse model. The same mechanism could conceivably account for severe congenital hydrocephalus in humans carrying LOF variants of MPDZ. RESULTS Mpdz -/mice harbor severe congenital hydrocephalusOut of a total of 112 mice bred by crossing heterozygous Mpdz mice, approximately 9 percent (10 mice) were homozygous for a gene-trap-induced mutation G510Vfs*19 (Milner et al., 2015). Consequently, the exons coding for PDZ domains 4-13 were truncated, likely resulting in nonsense-mediated mRNA decay. Mpdz -/pups were indistinguishable from their littermates at birth, but their heads started to bulge and form a domed forehead as early as P4, becoming gradually more pronounced (Fig. 1A). This is a malformation indicative of hydrocephalus. The life-span of Mpdz -/mice did not exceed 3 weeks, and by P18-P21 they were approximately 35 percent lighter than their wild type littermates (Fig. 1B). To substantiate the presence of hydrocephalus in the brains of Mpdz -/mice, and to distinguish between metabolically-active and inert, possibl...

Hepatocyte growth factor/scatter factor (HGF/SF) and its cognate receptor MET play several essential roles in embryogenesis and regeneration in postnatal life of epithelial organs such as the liver, kidney, lung, and pancreas, prompting a strong interest in harnessing HGF/SF-MET signalling for regeneration of epithelial organs after acute or chronic damage. The limited stability and tissue diffusion of native HGF/SF, however, which reflect the tightly controlled, local mechanism of action of the morphogen, have led to a major search of HGF/SF mimics for therapy. In this work, we describe the rational design, production, and characterization of K1K1, a novel minimal MET agonist consisting of two copies of the kringle 1 domain of HGF/SF in tandem orientation. K1K1 is highly stable and displays biological activities equivalent or superior to native HGF/SF in a variety of in vitro assay systems and in a mouse model of liver disease. These data suggest that this engineered ligand may find wide applications in acute and chronic diseases of the liver and other epithelial organs dependent of MET activation.

Degenerative diseases of major internal epithelial organs such as liver, lung and kidney account for more than one third of mortality worldwide. The huge demand for drugs able to limit epithelial tissue degradation and eventually restore its functionality, place mimics of the hepatocyte growth factor/scatter factor (HGF/SF), the physiological ligand for the MET receptor tyrosine kinase, at the forefront of potential drug candidates. HGF/SF is a growth and motility factor with essential physiological roles in development and regeneration of epithelial organs. Unfortunately, HGF/SF itself is unsuitable for therapy because naturally the factor acts only locally as a morphogen and chemoattractant and has poor in vivo distribution and shelf life profile. We have therefore designed, produced, solved the crystal structure and characterized the biochemical and biological properties of K1K1, a new engineered fragment of HGF/SF for applications in tissue/organ regeneration. K1K1, a covalent dimer of the first kringle domain of HGF/SF, is recombinantly produced in bacterial cells, shows superior stability at physiological pH and ionic strength and is a potent receptor agonist as demonstrated in a wide range of biological assays with cells in culture and initial in vivo studies. K1K1 has broad potential in regenerative medicine with diseases such as acute liver failure, non-alcoholic steatohepatitis, chronic obstructive pulmonary disease and acute kidney injury.

The molecular mechanisms governing the formation of lymphatic vasculature are not yet well understood. Pannexins are transmembrane proteins that form channels which allow for diffusion of ions and small molecules (<1 kDa) between the extracellular space and the cytosol. The expression and function of pannexins in blood vessels have been studied in the last few decades. Meanwhile, no studies have been conducted to evaluate the role of pannexins during human lymphatic vessel formation. Here we show, using primary human dermal lymphatic endothelial cells (HDLECs), pharmacological tools (probenecid, Brilliant Blue FCF, mimetic peptides [10Panx]) and siRNA-mediated knockdown that Pannexin-1 is necessary for capillary tube formation on Matrigel and for VEGF-C-induced invasion. These results newly identify Pannexin-1 as a protein highly expressed in HDLECs and its requirement during in vitro lymphangiogenesis.