Thesis (Ph. D.)--Columbia University, 1961. / Includes abstract. Part 2 contains musical transcriptions. Discography: pt. 1, p. 472. Includes bibliographical references (pt. 1, p. 363-471).

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Claudin Tight Junction Proteine sind wichtige Komponenten für den parazellulären Ionentransport in Epithelien. In der Niere sind Claudine an der Ionenresorption im Rahmen der Urinbildung beteiligt. In meiner Arbeit wurde die renale Funktion von zwei Vertretern dieser Familie, Claudin-16 und Claudin-10, untersucht. Durch gerichtetes Gentargeting wurden murine Defizienzlinien für Cldn16 und Cldn10 generiert und hinsichtlich ihres Phänotyps charakterisiert, der Fokus der Untersuchungen wurde auf die Nierenfunktion gelegt. Claudin-16-Fehlfunktion wurde mit der herediären Nierenerkrankung FHHNC assoziiert. FHHNC ist charakterisiert durch renalem Salzverlust und die Ausbildung einer Nephrokalzinose, welche schließlich zu terminalem Nierenversagen führt. Claudin-16-defiziente Mäuse weisen ähnliche Elektrolytimbalanzen wie humane FHHNC-Patienten auf, zeigen jedoch kompensatorische Mechanismen auf physiologischer, endokrinologischer und Transkriptions-Ebene, welche eine Progression der Krankheit verhindern. Das Mausmodell eignet sich somit für Studien zum renalen Salzverlust und zur Analyse der zugrunde liegenden molekularen Mechanismen. Zusätzlich konnten zwei putative Transporter gefunden werden, welche als interessante Kandidaten im renalen Magnesiumtransport weiteren Analysen unterzogen werden. Claudin-10 ist ubiquitär in Epithelien exprimiert, seine physiologische Rolle jedoch bis dato nur unzureichend geklärt. Claudin-10-defiziente Mäuse versterben wenige Stunden nach der Geburt. Untersuchungen an pulmonalem und renalem Gewebe konnten bislang keine Ursache für die Mortalität aufzeigen. Urinanalysen deuten jedoch auf eine Imbalanz der Magnesiumhomöostase hin. Durch konditionelle Knockout-Strategien soll im Folgenden die Claudin-10-Defizienz in einzelnen Geweben, etwa der Niere, untersucht werden. Auf diese Weise soll die Aufarbeitung des Phänotyps komplettiert und die Ursache der Letalität gefunden werden. / Claudin tight junction proteins are essential components in the regulation of paracellular fluxes through epithelial layers. In the kidney, claudins contribute to the resorption of ions in urine formation. This thesis highlights the physiological role of two renally expressed family members, claudin-16 and claudin-10. Via conditional gene targeting, murine Cldn16 and Cldn10 deficiency strains have been generated and evaluated with respect to their phenotype, with a focus on the renal function. Claudin-16 has been attributed to the resorption of bivalent ions in the thick ascending limb of Henle’s loop. Protein malfunction in humans goes in hand with FHHNC, a genetic disorder characterized by renal loss of bivalent ions, finally leading to nephrocalcinosis and end stage renal disease. Claudin-16 deficient mice display similar electrolyte disorders as human patients, but also resemble compensatory mechanisms on physiological, endocrinological and transcriptional levels to prevent the progression of the disease state. Hence, Cldn16 knockout mice serve as an adequate model to study renal salt wasting as well as counterregulatory mechanisms which highlights molecular pathways underlying renal salt wasting. Additionally, we could identify putative transport proteins which are attractive candidates for transcellular magnesium transport in the kidney. Claudin-10 shows a wide distribution in various tissues, its physiological role, however, is still under evaluation. Mice lacking claudin-10 display a lethal phenotype shortly after birth. Investigations of renal and pulmonary histology has not yet revealed the cause for the mortality under claudin-10-deficiency. However, urine analysis suggests an imbalance in the magnesium homeostasis in these animals. By subsequent conditional gene knockout approaches, this model will serve as a basis for tissue specific claudin-10 ablation and thereby enables investigation into the contribution of claudin-10 to distinct organ functions.

Currently, there is a growing body of research characterizing the blood-brain barrier (BBB) under normal physiological conditions; however, little is known about BBB regulation under pathophysiological conditions, such as inflammatory pain. This dissertation elucidates peripheral inflammatory pain effects on the BBB both functionally in terms of permeability and structurally via tight junction (TJ) protein expression and regulation.Inflammation was produced by subcutaneous injection of formalin, lambda-carrageenan, or complete Freund's adjuvant (CFA) into the right hind paw of rats. In situ perfusion and Western blot analyses were performed to assess BBB integrity after inflammatory insult. In situ brain perfusion determined that peripheral inflammation significantly increased the uptake of a membrane impermeant marker, sucrose into the cerebral hemispheres in all inflammatory models. Subsequently, a 0-168h time course study of lambda-carrageenan-induced inflammatory pain elicited a biphasic increase in BBB permeability of sucrose with the first phase occurring from 1-6h and the second phase occuring at 48h. Lambda-carrageenan-induced inflammatory pain also increased brain uptake of a commonly used analgesic, codeine at the same time-points. This is the first known observation that peripheral inflammation results in greater analgesic drug uptake to the brain. This uptake also correlated with its antinociceptive profile over a 168h time course. This suggests the presence of inflammatory pain may be an important consideration in therapeutic drug dosing, potential adverse effects and/or neurotoxicity.Western blot analyses showed altered TJ protein expression during peripheral inflammation. Occludin significantly decreased in the lambda-carrageenan- and CFA-treated groups. ZO-1 expression was significantly increased in all pain models. Claudin-1 protein expression was present at the BBB and remained unchanged during inflammation. Actin expression was significantly increased in the lambda-carrageenan- and CFA-treated groups. Over a 72h time period with lambda-carrageenan-induced inflammatory pain, altered TJ protein expression of occludin and ZO-1 correlated with permeability changes in BBB function. This is the first report of peripheral inflammation inducing alterations in TJs and increasing permeability of the BBB. This dissertation demonstrates that changes in the structure of TJs leading to alterations in the BBB may have important clinical ramifications concerning central nervous system homeostasis and therapeutic drug delivery.

Central nervous system (CNS) myelination is important for proper nervous system function in vertebrates. In demyelinating diseases such as multiple sclerosis, autoimmune-mediated myelin destruction results in neurological impairment; and although remyelination does occur spontaneously, it is poorly understood and insufficient in humans. Zebrafish (Danio rerio) are known to harbour tremendous regenerative capacity of various CNS tissues; however, there is presently only little knowledge of their myelin repair efficiency. An experimental model of myelin injury in zebrafish would permit study of the mechanisms involved in successful remyelination and could potentially guide the development of novel therapeutic agents for mammalian remyelination. This doctoral thesis describes the characterisation of the novel myelin protein Claudin k in zebrafish, demonstrates the establishment of adult zebrafish as an experimental model for CNS de- and remyelination and explores some mechanisms underlying myelin repair. A variety of myelin markers have previously been investigated in zebrafish, including myelin basic protein and myelin protein zero. However, the use of these is limited by either late developmental expression or presence in compact myelin only. Claudin k is a novel tight junction protein specific to zebrafish CNS and PNS, which can be observed early in development and throughout nervous system regeneration. Utilising specific antibodies and a novel transgenic zebrafish line, in which the claudin k promoter drives the expression of green fluorescent protein in myelinating cells, the studies herein characterise the expression of Claudin k, demonstrate the fidelity of the transgenic construct, and investigate the relationship of Claudin k with established myelin and CNS inflammation markers. Data demonstrate that Claudin k expression closely resembles expression patterns of the endogenous gene, and as such provides a key tool for examining CNS myelination in zebrafish. For the study of de- and remyelination in the zebrafish, the experiments herein describe the use of lysophosphatidylcholine (LPC), a detergent-like myelin toxin, which is used widely in rodent models to demyelinate axons. Its application to the adult zebrafish optic nerve induced focal demyelinating lesions, critically without detectable axonal injury, and permitted the study of time course and efficiency of remyelination. Myelin in the lesion area was reduced as detected by both immunohistochemistry and electron microscopy at 8 days post lesion (dpl), and return of the markers to control levels suggested regeneration by 28 dpl. In addition microglial activation was observed along the optic pathway, which also returned to levels compared to unlesioned control by 28 dpl. In young zebrafish (aged 4-6 months), the myelin thickness of remyelinated fibres showed no difference to the pre-lesion state, which is different to mammals, where the myelin thickness is reduced. However, in old fish (aged 18+ months), remyelinated fibres presented with thinner myelin, suggesting that the regenerative capacity of zebrafish declines with age. While the zebrafish as an experimental system has tremendous benefits, such as potential for drug screens using the transparent larvae, capacity for transgenesis and live imaging, experimental models in zebrafish potentially bear several limitations, in particular their distant relationship to humans. To determine whether zebrafish remyelination involves homologous signalling mechanisms to mammals, demyelinated zebrafish optic nerves were treated with human recombinant Semaphorin 3A, an axonal guidance molecule which is well known to inhibit oligodendrocyte precursor cell (OPC) recruitment and remyelination in mammals. Results demonstrated fewer oligodendroglial cells at 14 dpl and less myelinated fibres at 28 dpl in the optic nerve lesion area compared to control treated animals, supporting the hypothesis that zebrafish remyelination may indeed respond to human signalling molecules. Taken together, the findings in this doctoral thesis suggest that this new experimental zebrafish-based model of CNS remyelination can be added to the suite of current models to better understand the remyelination process and that some signalling mechanisms observed in mammals around myelination and OPC recruitment are likely conserved in the zebrafish. In addition, it could potentially be used to discover novel therapeutic targets that promote myelination in injury.

The lungs represent one of the earliest interfaces for pathogens and noxious stimuli to interact with the body. As such, careful maintenance of the permeability barrier is vital in providing homeostasis within the lung. Essential to maintaining this barrier is the tight junction, which primarily acts as a paracellular seal and regulator of ionic transport, but also contributes to establishing cell polarity, cell-to-cell integrity, and regulating cell proliferation and differentiation. The loss of these tight junctions has been documented to result in alterations in inflammation, and ultimately the development of many respiratory disorders such as COPD, Asthma, ARDS, and pulmonary fibrosis. One critical contributor that creates this permeability barrier is the tight junctional protein Claudin. While studies have begun to elucidate the various functions and roles of various Claudins, our understanding is still limited. To initially investigate these proteins, we looked at both temporal and spatial expression patterns for family members during development. A consistent pattern was demonstrated in mRNA expression for the majority of Claudin members. In general, Claudin expression underwent rapid increase during time periods that correlate with the pseudoglanduar/canalicular periods. One notable exception was Claudin 6 (Cldn6), which demonstrated decreasing levels of mRNA expression throughout gestation. We also sought to understand expression dynamics during the addition of maternal secondhand smoke (SHS) which resulted in an almost universal decrease in Claudin proteins. To more fully explore expression mechanisms that affect Claudin-6 (Cldn6), we exposed pulmonary alveolar type II (A549) cells to cigarette smoke extract (CSE) and found that it transcriptionally regulated Cldn6 expression. Using a luciferase reporter, we determined that transcription was negatively regulated at multiple promoter response elements by CSE, and transcription was equally hindered by hypoxic conditions. These findings identified Cldn6 as a potential target of SHS and other respiratory irritants such as diesel particulate matter (DPM). We next sought to assess whether an increase in Cldn6 was sufficient to provide a protective advantage against harmful exogenous exposure. To test this, we utilized a doxycycline induced Cldn6 over-expressing mouse, and subjected it to SHS for 30 days to stimulate an inflammatory state. Our findings demonstrated that Cldn6 transgenic animals have decreased inflammation as evidence by decreased total cell infiltration into the airways, decreased polymorphonuclocyte (PMNs) extravasation, total protein in bronchoalveolar lavage fluid (BALF), and decreased cytokine secretion. Anti-inflammatory advantages were also discovered during experiments involving acute exposure to DPM. In both cases, while stimulation of transgenic mice with SHS or DPM diminished Cldn6 expression, anti-inflammatory evidence emerged suggesting that genetic up-regulation of Cldn6 likely causes the recruitment of other tight junctional components during an organism's response to environmental assault.

The Sertoli cell tight junction (TJ) of the seminiferous epithelium is important for the developmental process of spermatogenesis as it separates germ cells in the seminiferous tubules from the general circulation in the testicular interstitium. Absence of the TJ leads to spermatogenic arrest and infertility. TJs form at puberty as circulating gonadotrophins luteinising hormone/testosterone and follicle stimulating hormone increase. Several studies have demonstrated hormonal regulation of the two major TJ proteins, claudin-11 and occludin, and also of TJ function in vitro and in vivo. Men with low levels of circulating gonadotrophins exhibit an immature and dysfunctional TJ phenotype, which is reversed upon the exogenous application of gonadotrophins. This thesis hypothesises that claudin-11 and occludin are the major contributors to TJ function, and that gonadotrophins regulate TJ function and structure via these two proteins in several species including humans. This PhD was divided into four separate studies to address these hypotheses. The first study selectively silenced the genetic expression of claudin-11 and occludin with small interfering RNA (siRNA) in cultured immature rat Sertoli cells to determine their contribution to Sertoli cell TJ function in vitro. siRNA treatment against either protein significantly (p less than 0.01) reduced TJ function by ~50% as assessed by transepithelial electrical resistance. Immunocytochemistry displayed marked reductions in the localisation of these proteins to the TJ after siRNA treatment. It was concluded that both proteins significantly contributed to TJ function in vitro. The second and third studies then aimed to study hormonal regulation of the TJ in vivo. Weekly injections of the gonadotrophin releasing hormone antagonist acyline were used to suppress circulating gonadotrophins and spermatogenesis in adult rats. Acyline treatment disrupted i) the localisation of occludin to the TJ and ii) TJ function as shown by permeability to a biotin tracer, which was impermeable to TJs in controls. Short-term hormone replacement partially restored the effects of gonadotrophin suppression. It was concluded that gonadotrophins regulate the maintenance of the TJ in rats in vivo. The third study used the hypogonadal (hpg) mouse, which is a naturally occurring model of gonadotrophin deficiency with inactive spermatogenesis. Claudin-11 in hpg mice was not localised at the TJs, and these were dysfunctional as shown by permeability to biotin. Following hormone treatment, TJs were structurally and functionally competent, demonstrating that gonadotrophins also regulate the formation of TJs in vivo. The fourth study subsequently analysed TJs in gonadotrophin suppressed men, and it was found that claudin-11 staining was reduced from continuous bands in control men, to punctate staining in gonadotrophin-suppressed men, demonstrating that gonadotrophins also regulate the localisation of claudin-11 to the TJ in men in vivo. In summary, it is concluded that the Sertoli cell TJ is hormonally regulated, and that the major contributors to TJ function in vivo and in vitro are claudin-11 and occludin. It is hypothesised that the reduction of claudin-11 localisation to the TJ in men may also result in a loss of human Sertoli cell TJ function, suggesting that the TJ may be a potential target of hormonal contraception in men.

Claudin-5 is a transmembrane cell junction protein that is a component of tight junctions in endothelial cell layers. We have previously shown that claudin-5 also localizes to lateral membranes of murine cardiomyocytes at their junction with the extracellular matrix. Claudin-5 levels are specifically reduced in myocytes from a mouse model of muscular dystrophy with cardiomyopathy. To establish whether claudin-5 is similarly specifically reduced in human cardiomyopathy, we compared the levels of claudin-5 with other cell junction proteins in 62 cardiomyopathic end-stage explant samples. We show that claudin-5 levels are reduced in at least 60% of patient samples compared with non-failing controls. Importantly, claudin-5 reductions can be independent of connexin-43, a gap junction protein previously reported to be reduced in failing heart samples. Other cell junction proteins including α-catenin, β-catenin, γ-catenin, desmoplakin, and N-cadherin are reduced in only a small number of failing samples and only in combination with reduced claudin-5 or connexin-43 levels. We also show that reduced claudin-5 levels can be present independently from dystrophin alterations, which are known to be capable of causing and resulting from cardiomyopathy. These data are the first to show alterations of a tight junction protein in human cardiomyopathy samples and suggest that claudin-5 may participate in novel mechanisms in the pathway to end-stage heart failure.

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The tight junctions (TJ), which are located in the apical region between epithelial and endothelial cells, regulate the paracellular diffusion of ions and small molecules and play an important role in maintaining cell polarity, cell-cell integrity, and permeability. In the lung, epithelial cells are attached by TJ structures. They provide a permeable barrier and cell communication. The loss of barrier integrity, which is maintained by the expression of claudins (Cldn), results in cellular permibilization and leads to paracellular diffusion of solutes and harmful molecules. There are 27 known Cldn homologous members in mice and human. Cldn6 is mostly expressed in embryonic stem cells and associated with the programing of epithelial cells during embryo development and lung morphogenesis. In order to test the hypothesis that Cldn6 expression affects lung morphogenesis, we analyzed the expression pattern of Cldn6 during lung ontogenesis to examine cell-specific expression pattern of Cldn6 during each embryonic period in the mouse lung. Also, we assessed transcriptional regulators and control mechanisms that precisely influence Cldn6 expression in pulmonary cells. We discovered that Cldn6 is an important tight junctional component expressed by pulmonary epithelium during lung organogenesis. We found that normal down-regulation of Cldn6 as development proceeds influences differentiation associated with the transition between the embryonic to the alveolar stage. Conditional gain-of-function and loss-of-function experiments in animal models prove to be the most beneficial tool in deciphering the impact of Cldn in organ formation and maintenance. We generated a conditional transgenic mouse that provides the opportunity to genetically up-regulate Cldn6 in distal lung. Our transgenic mouse showed a delay in lung development and down-regulation of transcriptional factors. Cldn6 is both temporally and spatially controlled in the developing lung and its regulation is maintained by critical transcriptional control networks managed by TTF-1. In lung diseases, altered Cldn expression leads to diseases such as COPD, asthma, and ARDS. The tight junctional proteins are differentially regulated by tobacco smoke exposure and Cldn6 is potentially involved as neighboring epithelial cells respond to tobacco smoke. We exposed adult mice to controlled doses of second hand smoke during four days and A-549 cells to 10% CSE for 6 hours. We discovered that mice lungs respond by down-regulating Cldn6 basal levels and impair barrier function. These results reveal that midgestational up-regulation of Cldn6 and its marked down-regulation as development proceeds illustrate the notion that Cldn6 function is important during early programming stages of lung morphogenesis.