Les interfaces sang-cerveau comme la barrière hémato-encéphalique (BHE), les plexus choroïdes (PC) ou les organes circumventriculaires (OCV), constituent des barrières physiologiques nécessaires au fonctionnement du système nerveux central. Ces barrières sont à la fois « physiques », constituées de jonctions serrées, et « enzymatiques ». Longtemps considérées comme immatures chez le fœtus, ces barrières sont en réalité présentes précocement au cours du développement. Leurs caractéristiques et leurs propriétés restent peu connues chez l'homme. Nos travaux montrent que les PC expriment, précocement au cours du développement, des protéines de jonction serrée, les claudines (CLDN) 1, 2 et 3 chez le rat et chez l'homme. Cette expression est dynamique au cours du développement avec une apparition progressive de la CLDN2 pouvant avoir un lien avec la sécrétion du liquide céphalo-rachidien. Les CLDN 1 et 3 sont identifiées chez le fœtus humain au niveau de l'organe sous-commissural (OSC), un des OCV. La CLDN5 est exprimée précocement au niveau de la BHE chez le rat et chez l'homme et son expression est altérée dans un modèle d'excitotoxicité néonatale. Nos travaux montrent également que l'analyse du profil des CLDN est utile en pathologie tumorale notamment dans la compréhension et le diagnostic de tumeurs développées à partir des PC ou de l'OSC. Enfin, diverses enzymes antioxydantes et de détoxification dont l'époxyde hydrolase microsomale sont exprimées à 22 semaines d'aménorrhée principalement au niveau des PC du fœtus humain. Ces données suggèrent des capacités de détoxification des PC, d'installation précoce au cours du développement chez l'homme / Blood-brain interfaces including blood-brain barrier (BBB), choroid plexuses (CP) or circumventricular organs (CVO) are physiological barriers required for brain homeostasis. These barriers are “physical”, with tight junctions, and “enzymatic”. Though long considered immature in fetuses, these barriers are present from an early stage of development. Their characteristics and their properties are largely unknown in humans. Our work demonstrates that CP express tight junction-associated proteins claudins (CLDN) 1, 2, and 3 at early stages of development in rat and human. This expression is dynamic during development as shown by the progressive increase of CLDN2 immunopositivity that may follow increase in cerebrospinal fluid secretion. CLDN 1 and 3 are identified in human fetal subcommissural organ (SCO), one of the CVO. CLDN5 is early expressed in rat and human BBB and its expression is disrupted by excitotoxic injury. Our work also shows that CLDN immunohistochemical profile is useful in tumoral pathology, notably to better understand and diagnose tumors arising from CP or the SCO. Finally, various antioxidant and detoxifying enzymes such as the microsomal epoxide hydrolase are expressed at 22 weeks of gestation in the human fetus, mainly in CP. These results suggest a high detoxifying capacity for the CP during development in humans

Abstract Collagen XVIII is a multidomain basement membrane proteoglycan with three tissue-specific isoforms. Endostatin, the C-terminal part of collagen XVIII, has antiangiogenic properties, while the frizzled-like domain of the longest isoform is suggested to be capable of inhibiting the Wnt/β-catenin signaling network. This study utilized several genetically modified mouse lines and electron microscopy to achieve new information on the biological role of collagen XVIII, its different isoforms, and the frizzled domain. Lack of collagen XVIII was found to affect the integrity of basement membranes of various tissues, leading to an abnormally loosened network structure. In the choroid plexus, the change in the basement membrane ultrastructure caused alterations in the production of the cerebrospinal fluid and predisposed to the development of hydrocephalus. In the kidney, broadening of the proximal tubular basement membrane was shown to be due specifically to the lack of the short isoform, while the lack of the two longer isoforms led to podocyte foot process effacement. Moreover, lack of collagen XVIII was found to cause softening of the kidney glomeruli and the levels of serum creatinine were elevated in the mutant animals, indicating altered kidney function. The hair follicle cycle was used as a model to study the possible role of the frizzled domain of collagen XVIII in the Wnt/β-catenin signaling cascade. The longer collagen XVIII isoforms were shown to be expressed in the basement membrane facing the dermal papilla and in the hair follicle bulge, containing the follicular stem cells. Lack of the long isoforms led to abnormalities in the progression of the first hair cycle, and the phenotype could be rescued via transgenic delivery of the frizzled domain of the longest isoform, suggesting its involvement in the regulation of the Wnt/β-catening signaling network during the cyclic growth of the hair. / Tiivistelmä Kollageeni XVIII on useista toiminnallisista osista koostuva tyvikalvojen proteoglykaani, jolla on kolme eri kudoksissa esiintyvää isomuotoa. Sen C-terminaalisella endostatiini-osalla on verisuonten kasvua estäviä vaikutuksia, kun taas pisimmän isomuodon frizzled-osan uskotaan estävän Wnt/β-kateniini signalointireitin toimintaa. Tässä tutkimuksessa saatiin uutta tietoa kollageeni XVIII:n, sen eri isomuotojen sekä frizzled-osan biologisesta merkityksestä useiden geenimuunneltujen hiirimallien sekä elektronimikroskopian avulla. Kollageeni XVIII:n puutoksen todettiin vaikuttavan tyvikalvojen rakenteen eheyteen useissa eri kudoksisssa, johtaen epänormaalisti löyhtyvään verkkorakenteeseen. Suonipunoksessa tämä tyvikalvon hienorakenteen muutos vaikutti aivo-selkäydinnesteen tuottumiseen ja altisti vesipään kehittymiselle. Munuaisessa proksimaalisen munuaistiehyen tyvikalvon levenemisen osoitettiin johtuvan lyhyen isomuodon puutoksesta, kun taas kahden pidemmän isomuodon puuttuminen aiheutti podosyyttien jalkalisäkkeiden leviämistä. Lisäksi kollageeni XVIII:n puuttumisen osoitettiin johtavan hiirimallien munuaiskerästen pehmenemiseen sekä veren kreatiniinitason kohoamiseen, viitaten munuaistoiminnan häiriöihin. Karvatuppien syklistä kasvua käytettiin mallina tutkittaessa kollageeni XVIII:n frizzled-osan mahdollisia vaikutuksia Wnt/β-kateniini signalointireittiin. Pidempien kollageeni XVIII isomuotojen osoitettiin tuottuvan karvanystyn tyvikalvossa sekä karvatupin kantasolut sisältävällä pullistuma-alueella. Pitkien isomuotojen puuttuminen johti karvojen ensimmäisen kasvukierron epänormaaliin etenemiseen. Tämä voitiin estää siirtogeenisen frizzled-osan avulla, mikä viittasi sen osallisuuteen Wnt/β-kateniini signalointireitin säätelyyn karvan syklisen kasvun aikana.

Streptococcus suis (S. suis) is an important opportunistic pathogen, which can cause septicemia and meningitis in pigs and humans. Previous in vivo observations in S. suisinfected pigs revealed lesions at the choroid plexus (CP). In vitro experiments with primary porcine CP epithelial cells (PCPEC) and human CP epithelial papilloma (HIBCPP) cells demonstrated that S. suis can invade and traverse the CP epithelium, and that the CP contributes to the inflammatory response via cytokine expression. Here, next generation sequencing (RNA-seq) was used to compare global transcriptome profiles of PCPEC and HIBCPP cells challenged with S. suis serotype (ST) 2 infected in vitro, and of pigs infected in vivo. Identified differentially expressed genes (DEGs) were, amongst others, involved in inflammatory responses and hypoxia. The RNA-seq data were validated via quantitative PCR of selected DEGs. Employing Gene Set Enrichment Analysis (GSEA), 18, 28, and 21 enriched hallmark gene sets (GSs) were identified for infected HIBCPP cells, PCPEC, and in the CP of pigs suffering from S. suis ST2 meningitis, respectively, of which eight GSs overlapped between the three different sample sets. The majority of these GSs are involved in cellular signaling and pathways, immune response, and development, including inflammatory response and hypoxia. In contrast, suppressed GSs observed during in vitro and in vivo S. suis ST2 infections included those, which were involved in cellular proliferation and metabolic processes. This study suggests that similar cellular processes occur in infected human and porcine CP epithelial cells, especially in terms of inflammatory response.

T cells infiltrate the degenerating brain and influence central nervous system (CNS) inflammation and neuronal health. In mice, the choroid plexus and the meninges have been implicated in regulating T cell entry and egress from the CNS, respectively. Further, antigen presenting cells in the mouse meninges present CNS-derived antigens to T cells and may represent a method for the peripheral immune system to sense and respond to CNS immune triggers. However, whether these processes occur in the human choroid plexus and meninges has not been comprehensively studied. Further, the antigens towards which T cells in the degenerating human brain and its borders respond remain unknown. Therefore, I implemented a multi-omics approach using fresh postmortem tissue from patients diagnosed with amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), Parkinson’s disease (PD), and non-neurodegenerative controls to identify not only the T cell-associated changes that occur in the degenerating human CNS and surrounding tissues but also identified a library of putative antigen targets for disease-associated T cell populations. Specifically, using single cell RNA and TCR sequencing information from paired postmortem choroid plexus, leptomeninges, and brain I lineage traced T cells using their TCR information and found that T cell access to leptomeninges and brain is likely limited and controlled by anti-inflammatory macrophage activity at the blood/CSF barrier (BCSFB). Once past the BCSFB, I present evidence that T cells access the CNS where they interact with MHC expressed by microglia. T cells also accumulate in the leptomeninges where they become tissue resident memory T cells. These tissue resident memory T cells likely serve as a reservoir for a rapid antigen-driven immune response to future CNS inflammatory insults. Finally, by performing immunopeptidomics to identify peptides presented by MHC in the same patients’ CNS and border tissues, I identified a library of putative antigenic triggers that may drive high levels of T cell clonal expansion in the brain and surrounding tissues. Altogether, this thesis serves as a resource for understanding the trajectory of T cells as they travel into the degenerating human brain and as a foundation for the development of antigen-specific precision medicines to treat neurodegeneration.

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<p>Hydrocephalus is a neurological disorder characterised by the pathological accumulation of cerebrospinal fluid (CSF) within the brain ventricles. Surgical interventions, including shunt placement, remain the gold standard treatment option for this life-threatening condition, despite these often requiring further revision surgeries. Unfortunately, there is currently no effective, pharmaceutical therapeutic agent available for the treatment of hydrocephalus. CSF is primarily produced by the choroid plexus (CP), a specialized, branched structure found in the ventricles of the brain. The CP comprises a high resistance epithelial monolayer surrounding a fenestrated capillary network, forming the blood-CSF barrier (BCSFB). The choroid plexus epithelium (CPe) critically modulates CSF production by regulating ion and water transport from the blood into the intraventricular space. This process is thought to be controlled by a host of intracellular mediators, as well as transporter proteins present on either the apical or basolateral membrane of the CPe. Though many of these proteins have been identified in the native tissue, exactly how they interact and modulate signal cascades to mediate CSF secretion remains less clear.</p> <p><br></p> <p>Transient potential receptor vanilloid 4 (TRPV4) is a non-selective cation channel that can be activated by a range of stimuli and is expressed in the CP. TRPV4 has been implicated in the regulation of CSF production through stimulating ion flux across the CPe. In a continuous CP cell line, activation of TRPV4, through the addition of a TRPV4 specific agonist GSK1016790A, stimulated a change in net transepithelial ion flux and increase in conductance. In order to develop a pharmaceutical therapeutic for the treatment of hydrocephalus, we must first understand the mechanism of CSF secretion in health and disease. Therefore, a representative <em>in vitro</em> model is critical to elucidate the signaling pathways orchestrating CSF production in the CP.</p> <p><br></p> <p>This research aims to characterize an <em>in vitro</em> culture model that can be utilized to study both the BCSFB and CSF production, to investigate and identify additional transporters, ion channels and intracellular mediators involved in TRPV4-mediated signaling in the CPe, primarily through a technique called Ussing-style electrophysiology which considers electrogenic ion flux across a monolayer. These studies implicated several potential modulators, specifically phospholipase C (PLC), phosphoinositide 3-kinase (PI3K), protein kinase C (PKC), intermediate conductance K+ channel (IK), transmembrane member 16A (TMEM16A), cystic fibrosis transmembrane conductance regulator (CFTR) and protein kinase A (PKA), in TRPV4-mediated ion flux.</p>