Schizophrenia is a neurological disorder with a complex psychopathology, which is far from fully elucidated. In the patients with this disorder, changes on anatomical, cellular, and neurotransmitter level have been found. The aim of this work is to elucidate the function of specific ionotropic glutamate receptors in NG2 glia in the hippocampus of a mouse model of schizophrenia. For this purpose, a mouse model of schizophrenia was generated and validated using immunohistochemistry and behavioural testing. Mice with NG2 glia labelled by a fluorescent protein with a calcium indicator also in NG2 glia were used to observe the activity of glutamate channels and the properties of the extracellular space in these mice. Changes were found in the schizophrenic animals when compared to control animals in the numbers of hippocampal oligodendrocyte lineage cells, in prepulse inhibition and in both volume fraction and tortuosity of the extracellular space in hippocampus. Moreover, the percentage of cells responding to glutamate receptor agonists in NG2 glia in hippocampus also differed significantly between the schizophrenic and the control animals. In conclusion, it can be said that we were able to observe significant changes in the mouse model of schizophrenia that we generated in comparison to control...

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18183号 / 医博第3903号 / 新制||医||1004(附属図書館) / 31041 / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邉 大, 教授 髙橋 良輔, 教授 髙橋 淳, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Objective: Aortic valve (AV) leaflets rely on a precise extracellular matrix (ECM) microarchitecture for appropriate biomechanical performance. The ECM structure is maintained by valvular interstitial cells (VICs), which reside within the leaflets. The presence of pigment produced by a melanocytic population of VICs in mice with dark coats has been generally regarded as a nuisance, as it interferes with histological analysis of the AV leaflets. However, our previous studies have shown that the presence of pigment correlates with increased mechanical stiffness within the leaflets as measured by nanoindentation analyses. In the current study, we seek to better characterize the phenotype of understudied melanocytic VICs, explore the role of these VICs in ECM patterning, and assess the presence of these VICs in human aortic valve tissues. Approach and Results: Immunofluorescence and immunohistochemistry revealed that melanocytes within murine AV leaflets express phenotypic markers of either neuronal or glial cells. These VIC subpopulations exhibited regional patterns that corresponded to the distribution of elastin and glycosaminoglycan ECM proteins, respectively. VICs with neuronal and glial phenotypes were also found in human AV leaflets and showed ECM associations similar to those observed in murine leaflets. A subset of VICs within human AV leaflets also expressed dopachrome tautomerase, a common melanocyte marker. A spontaneous mouse mutant with no aortic valve pigmentation lacked elastic fibers and had reduced elastin gene expression within AV leaflets. A hyperpigmented transgenic mouse exhibited increased AV leaflet elastic fibers and elastin gene expression. Conclusions: Melanocytic VIC subpopulations appear critical for appropriate elastogenesis in mouse AVs, providing new insight into the regulation of AV ECM homeostasis. The identification of a similar VIC population in human AVs suggests conservation across species.

Carotid body (CB) receptor type I cells transduce blood-borne chemical stimuli into electrical signals and release the excitatory neurotransmitter ATP onto afferent terminals that project to the breathing centre located in the brainstem. Within the CB, type I cells are ensheathed by glial-like processes of type II cells. Recently, it was hypothesized that type II cells have a paracrine function in CB chemotransduction by acting as an ATP amplifier and enhancing chemoexcitation (Zhang et al. 2012). Given this recent development, the primary goal of this thesis was to further elucidate the paracrine function of type II cells and characterize the signalling mechanisms involved in type I and type II cell interactions. Ratiometric calcium imaging was used to investigate type II cell sensitivity to two prominent CB neuromodulators, angiotensin II (ANG II) and 5-HT, in rat CB cultures. Both ANG II and 5-HT elicited large rises in intracellular Ca<sup>2+<sup> that were present in the absence of extracellular Ca<sup>2+<sup> and were inhibited by intracellular store depletion agents. ANG II and 5-HT acted on their respective G-protein coupled receptors, AT<sub>1<sub> receptor and 5-HT<sub>2A<sub> receptor, to initiate these Ca<sup>2+<sup> responses presumably via a PLC-IP<sub>3<sub> mediated mechanism. Interestingly, these Ca<sup>2+<sup> responses were required to activate pannexin-1 channels (Panx-1), a channel that has been previously shown to be a conduit for ATP in type II cells (Zhang et al. 2012). We were also interested in determining whether type II cells were capable of indirectly responding to a chemostimulus such that the stimulus would elicit neurosecretion from type I cells and result in a secondary Ca<sup>2+<sup> responses in type II cells. Isohydric hypercapnia and a depolarizing stimulus (30 mM KCl saline) were capable of eliciting indirect Ca<sup>2+<sup> responses in type II cells. These secondary Ca<sup>2+<sup> responses in type II cells were partially inhibited by suramin, a purinergic P2Y2 receptor antagonist, suggesting that ATP was the predominant neurotransmitter responsible for type I to type II crosstalk. Similarly, a selective agonist for type II cells, UTP, evoked indirect Ca<sup>2+<sup> responses in nearby type I cells. Type II to type I cell communication was dependent on Panx-1 channels since the secondary Ca<sup>2+<sup> responses in type I cells were inhibited by the Panx-1 blocker, carbenoxolone (5 µM). UTP-evoked indirect Ca<sup>2+<sup> in type I cells were partially inhibited by adenosine A<sub>2<sub> receptor antagonists suggesting that the neuromodulator, adenosine, governs cross-talk between type II and type I cells. This study elucidates the importance of purinergic signalling in the bi-directional cross-talk between receptor type I cells and glial-like type II cells. / Thesis / Master of Science (MSc)

The immune response is a complex series of cell-mediated reactions by which an organism combats infection, responds to injury, external stresses, or disease. In both Drosophila melanogaster and vertebrates, aging is associated with progressive declines in physiological functions as well as susceptibility to stress and disease. Naturally, the immune activity is increased with age, yet the efficacy of this response is reduced with age. Conversely, when the immune activation is artificially-induced by Lipopolysaccharide, aging is accelerated. Like aging, neurodegeneration is also associated with increased immune activity. The Blood-Brain barrier (BBB) is a physical barrier with highly selective permeability that isolates the brain from the rest of the body. This barrier is essential for ion homeostasis, and exclusion or efflux of exogenous chemicals. The exclusion properties of the Dm BBB are facilitated by paracellular septate junctions of subperineural glia (SPG), which prevent diffusion into or out of the brain. Using the GAL/UAS system in Drosophila, we found that activation of a glial-specific immune response in either immunodeficiency (IMD) or Toll pathways led to reductions in lifespan and age-dependent negative geotaxis. These reductions were also correlated with an early sensitivity towards oxidative and thermal stresses. Furthermore, we found that a SPG-specific immune response of the Toll pathway or disruption of the paracellular BBB itself was sufficient to show the same reductions as pan-glial activation. In short, we found that flies with CNS-specific immune activation showed an inability to cope with long-term and acute forms of stress, and that SPG-specific Toll Activation was sufficient to show these effects. This implicates chronic immune response as a negative factor during aging, neurodegenerative disease, and brain homeostasis. / Thesis / Master of Science (MSc)

The demise of retinal ganglion cells (RGCs) is characteristic of diseases of the retina such as glaucoma and diabetic or ischemic retinopathies. Pigment epithelium-derived factor (PEDF) is a multifunctional secreted protein that mediates neuroprotection and inhibition of angiogenesis in the retina. We have studied expression and regulation of two of several receptors for PEDF, patatin-like phospholipase 2 gene product/PEDF-R and laminin receptor (LR), in serum-starved RGC under normoxia and hypoxia and investigated their involvement in the survival of retinal neuronal cells. We show that PEDF-R and LR are co-expressed in RGC and R28 retinal precursor cells. Expression of both receptors was enhanced in the presence of complex secretions from retinal glial (Müller) cells and upregulated by VEGF and under hypoxic conditions. PEDF-R- and LR-knocked-down cells demonstrated a markedly attenuated expression of anti-apoptotic Bcl-2 family members (Bcl-2, Bcl-xL) and neuroprotective mediators (PEDF, VEGF, BDNF) suggesting that both PEDF-R and LR mediate pro-survival effects of PEDF on RGC. While this study does not provide evidence for a differential survival-promoting influence of either PEDF-R or LR, it nevertheless highlights the importance of both PEDF receptors for the viability of retinal neurons.

Steigende Zahlen von Patienten mit neurodegenerativen Erkrankungen sind ein überzeugender Grund, das menschliche Gehirn und seinen fortschreitenden Verfall zu untersuchen, wobei aber viele essenzielle biochemische Funktionen bisher noch nicht vollends geklärt sind. In vitro Forschung zur Hirnfunktion auf geeigneten Plattformen ist ein vielversprechender Weg, diese Lücke zu schließen. Eigenschaften der brain-machine Grenzfläche müssen erforscht werden, um neue Biomaterialien effektiv für lab-on-a-chip Anwendungen wie bspw. Multielektrodenarrays (MEAs) einzusetzen. Diese brain-on-a-chip Anwendungen können dazu dienen, die Zahl der Tierexperimente zu reduzieren, damit Forschung zu beschleunigen und Kosten zu senken. In dieser Hinsicht erfordert die Miniaturisierung von MEAs für eine detailliertere Messung von neuronalen Funktionen die Entwicklung von neuen Biomaterialien mit vorteilhaften elektrischen Eigenschaften. Die Wechselwirkung dieser Biomaterialien mit Zellen muss untersucht werden, um gute Zelladhäsion, Proliferation und elektrische Kopplung zu gewährleisten. Die vorliegende Arbeit dient der Charakterisierung der Wechselwirkung von humanen neuronalen Zellen und Gliazellen (neuronenartige SH-SY5Y und gliaartige U-87 MG Zellen) mit dem Elektrodenmaterial Titannitrid mit nanokolumnarer Oberfläche (TiN nano) und dessen Vorteile bezüglich elektrischer und bioaktiver Eigenschaften im Vergleich mit Gold (Au) und Indiumzinnoxid (ITO), welche derzeit für MEAs und Neuroelektroden verwendet werden. Das Ziel der Arbeit ist die Implementierung neuer aus der theoretischen Physik, Mathematik und Computerwissenschaft entlehnten Techniken, um eine bildbasierte Methode zu entwickeln, die auf minimalen Experimenten beruht und trotzdem wichtige Hinweise zur Biokompatibiliät eines Materials liefert. Das schließt die Analyse von Zellnetzwerken, Zellverteilung, Adhäsion und elektrochemischer Eigenschaften in mono- und co-Kultur ein. Dazu werden Autokorrelation, selbstlernende Algorithmen und die Analyse nächstgelegener Nachbarn eingesetzt, um einen Weg von klassischen biochemischen Assays weg zu einem rechnerischen Ansatz zu finden. Die Ergebnisse zeigen eine Überlegenheit von Tin nano als potenzielles Biomaterial für lab-on-a-chip Anwendungen und in vivo neuraler Stimulation. Die präsentierte bildbasierte Analysemethode für die Untersuchung von Zellverteilungen erweist sich als wertvolles Werkzeug für die Bewertung von Biokompatibilität. Sie ist universell einsetzbar für verschiedene Zelltypen und quantifiziert die Wechselwirkung von Zellen mit Biomaterialien. / Rising numbers of patients with neurodegenerative diseases are a compelling reason to study the human brain and its progressive deterioration but many essential biochemical functions are still under investigation. Conducting research on brain function in vitro with suitable platforms is a promising solution to close these gaps. Characteristics of the brain-machine interface need to be investigated to effectively employ new biomaterials for lab-on-a-chip devices, such as multielectrode arrays (MEAs) for example. These brain-on-a-chip devices will potentially reduce the number of conducted animal experiments and therewith accelerate future research and reduce costs. In this context, miniaturization of MEAs for more detailed measurements of neuronal function calls for new biomaterials with advantageous electrical characteristics. The interaction of these biomaterials with cells needs to be investigated to ensure good cell adhesion, proliferation, and electrical coupling. This thesis aims to study and characterize the interaction of human neuronal and glial cells (neuron-like SH-SY5Y and glia-like U-87 MG cells) with the electrode material titanium nitride with nanocolumnar surface topography (TiN nano) and its advantages in terms of electric and bioactive properties compared to gold (Au) and indium tin oxide (ITO) which are currently employed for MEAs and neuroelectrodes. The overall goal of this study is the implementation of new techniques drawn from theoretical physics, mathematics, and computer science to establish an image-based method that relies on minimal experimental effort but nevertheless yields important evidence of biocompatibility of the material. Analysis includes the investigation of cellular networks, cell distribution, adhesion, and electrochemical properties in mono- and co-culture experiments. To this end, autocorrelation function, self-learning algorithms, and nearest neighbor analysis are deployed to move away from classical biochemical assays toward a more computational approach. Results show the superiority of TiN nano as a potential biomaterial employed for lab-on-a-chip designs as well as for in vivo neural stimulation. The proposed image-based analysis method for the investigation of cellular distribution turns out to be a valuable tool for the assessment of biocompatibility. It is universally applicable to cell types other than neuronal and quantifies the interaction of cells with biomaterials.

In contrast to mammals, adult zebrafish undergo successful neural regeneration following spinal cord injury (SCI). Radial glia (RG) lining the zebrafish central canal undergo injury-induced proliferation and subsequent neuronal differentiation to replace damaged cells and restore motor function. However, the molecular mechanisms that underlie these processes remain elusive. Here, we demonstrate that signaling through the evolutionarily conserved purinergic P2X7 receptor is involved. Within the zebrafish spinal cord, P2X7 receptors have widespread distribution with specific localization to neurons and radial glia. At the protein level, the predominant P2X7 receptor isoforms in zebrafish did not include the full-length variant expressed throughout the murine central nervous system, but two truncated splice variants. In response to SCI, protein expression of the 50 kDa isoform became downregulated at 7 dpi and returned to basal levels of expression at 14 and 21 dpi when compared to naïve controls. Meanwhile, expression of the 37 kDa isoform did not change following injury. Pharmacological activation of P2X7 following SCI resulted in a greater number of proliferating cells around the central canal by 7 dpi, while P2X7 inhibition appeared to have no effect. At 14 dpi, these treatments did not have a significant effect on the number of neurons within the injured spinal cord. This data indicates that P2X7 receptor activation is sufficient to induce cellular proliferation, but not a necessary mediator of either proliferation or neurogenesis following SCI in adult zebrafish. Our findings suggest that unlike in humans, P2X7 signaling may not play a maladaptive role following SCI in adult zebrafish. / Thesis / Master of Science (MSc) / Spinal cord injury in mammals causes widespread neuronal cell death and paralysis. In comparison, zebrafish regenerate damaged neurons and restore motor function. Radial glial cells within the zebrafish spinal cord maintain stem-cell properties. Following injury, these cells divide and replace motor neurons. Since mammals have similar cell-types within the spinal cord, understanding the molecular cues driving this adaptive response is of great interest. Here, we examined the evolutionarily conserved purinergic signaling system and found that the expression of the P2X7 receptor varies significantly from mammals and promotes radial glia division following injury.

No description available.