Zugl.: Leipzig, Univ., Diss. A. Wurm, 2007

No description available.

Glutamate is a major stress-sensitive excitatory input to the noradrenergic locus coeruleus (LC) and pathology of both glutamatergic and noradrenergic systems is strongly implicated in major depressive disorder (MDD). Glutamatergic innervation of the LC originates in the frontal cortex and in local brainstem nuclei. Stress increases the release of glutamate in the LC which results in activation of noradrenergic LC neurons. Previous research has demonstrated abnormalities in the levels of glutamate receptor proteins and gene expressions in the postmortem LC of subjects with MDD as compared to normal control subjects providing further evidence for disruption of glutamate-norepinephrine communication in MDD. A principal mechanism for the termination of the action of neuronally-released glutamate is by reuptake via excitatory amino acid transporters (EAAT) located on glia, cells which express both EAAT1 and EAAT2. Here, the potential contribution of glia to glutamate pathology in the LC in MDD was studied by measuring gene expression of EAAT1 and EAAT2 in astrocytes captured from the immediate region of the postmortem LC from 6 pairs of subjects with MDD and psychiatrically normal control subjects. MDD and control subjects were carefully matched for age, RIN value (RNA integrity number), gender, cigarette smoking or non-smoking, and brain tissue pH. Laser capture microdissection was used to capture astrocytes from tissue sections labeled with a modified rapid GFAP-immunostaining, and gene expression levels were analyzed by quantitative PCR. Three reference genes were used as internal controls and the quality of the capture of astrocytes was confirmed by examining the gene expression of cell-type specific markers, including markers for noradrenergic neurons and oligodendrocytes. The gene expression of EAATs was significantly lower (EAAT1, -60%; p<0.001; EAAT2, -25%, P<0.01) in LC astrocytes from MDD subjects as compared to normal control subjects. To determine the regional specificity of these findings, gene expression levels of EAAT1 and EAAT2 were measured in homogenates of both gray and white matter from Brodmann’s area 10 of the cortex. No differences in EAAT gene expression in these cortical tissues were observed comparing MDD to control subjects. These findings indicate that disrupted glial transport of glutamate may contribute to altered glutamatergic transmission in the noradrenergic LC in MDD.

Autism spectrum disorder (ASD) is considered a disease of neuronal dysfunction based on pathological alterations in axon thickness and neuronal disorganization. Recent findings suggest non-neuronal cells may also play a role in ASD brain pathology. White matter areas in the ASD brain display enlargement paired with a reduction in structural integrity. These structural abnormalities are likely associated with dysfunction of the most prevalent cell types present in white matter, astrocytes and oligodendrocytes. In fact, myelin abnormalities and structural changes of reactive astrocytes have been reported in ASD. The goal of the present study was to further investigate glia pathology in the white matter of the ASD brain. The primary brain area of interest was the anterior cingulate cortex (BA24) because this brain region mediates social interactive behavior, disruption of which is a core behavioral feature of ASD. Furthermore, a reduction in the structural integrity of white matter in BA24 has been observed in ASD. Postmortem brain tissues were obtained from highly characterized ASD and developmentally normal control donors. Quantitative Western blotting was used to measure glial fibrillary acidic protein (GFAP) and myelin oligodendrocyte glycoprotein (MOG) produced by astrocytes and oligodendrocytes, respectively. A significant elevation in levels of GFAP-immunoreactivity (p=0.005) in BA24 white matter was observed in ASD as compared to control donors. In contrast, levels of MOG-immunoreactivity in BA24 white matter were similar when comparing ASD to control donors. Levels of both GFAP and MOG in the BA24 gray matter from the same subjects were similar comparing the two groups of donors. Measurement of GFAP gene expression in BA24 white matter did not reveal any difference between ASD and control donors. To further examine the regional specificity of the observed glial pathology, we analyzed GFAP and MOG protein expression in the anterior prefrontal cortex (BA10) white matter. Levels of GFAP- and MOG-immunoreactivities were unchanged in BA10 white matter comparing ASD to control donors. These findings demonstrate that astrocytic pathology is both tissue-specific (white matter) and regionally selective (BA24) in ASD. Elevation of GFAP protein in BA24 white matter implies an activation of astrocytes possibly as a result of a yet undefined cellular insult. Research is needed to investigate the molecular pathways that underlie this astrocyte reaction and such research may yield important clues regarding the etiology of ASD.

Poster exhibited at GPSC Student Showcase, February 24th, 2016, University of Arizona. Recipient of the 2016 Katheryne B. Willock Library Research Award. / The nervous system is composed of two types of cells: neurons and glia. In neuronal circuits, neurons communicate through synapses and glia play a crucial modulatory role. To modulate chemical reuptake, glia send processes close to synapses and many glia directly appose or ensheathe a synapse. This structural motif is one of the elements often included in describing a vertebrate tripartite synapse, which includes a bidirectional functional neuron-glia relationship. The exact nature of this neuron-glia communication is not well understood. In the invertebrate fruit fly, we have also found that particular neurons and glia also have a bidirectional functional relationship. This allows us to ask new questions about glial morphology. Throughout multiple images, I identified particular neuronal synapses and surrounding glia. After creating a 3D reconstruction, I measured the distance between a particular neuronal synapse and its closest glial process. Interestingly, the neuronal synapses were not directly apposed or ensheathed by glia, and the distance to the closest glial process varied one-hundred-fold. With variable distance, functional communication is consistently present. These findings provide important insight into invertebrate neuron-glia communication, and offer new avenues to investigate the structural neuron-glia relationships that are required for reciprocal signaling between the two cell classes.

<p>The blood-brain barrier regulates the transport into the brain and protects the central nerve system (CNS) from toxics substances. However some areas of the brain, called circumventricular organs (CVO), lack the blood-brain barrier. One of these is area postrema (AP), which is located in the brainstem immediately adjacent to the nucleus tractus solitarius (NTS). These two areas together regulate autonomic behaviours such as food intake, and also make up the vomiting center.</p><p>The hormones leptin and ghrelin, which regulate food intake, are too big to pass the blood-brain barrier, but have receptors in NTS.</p><p>In this study we used immunohistochemistry to obtain a detailed map of the different components of the blood-brain barrier in AP and NTS.</p><p>The results suggest that there is a barrier that prevents diffusion of substances from AP into NTS. However, there seems to be some vessels in NTS that have a weaker or no barrier characteristics. These vessels could provide an entrance for peripheral substances to neurons in NTS.</p>

Olfactory ensheathing cells (OECs), the glia of the olfactory nerve, are promising candidates for patient-specific cell-mediated repair of both peripheral nerves and the spinal cord. The recent discovery that OECs originate from the neural crest, rather than the olfactory epithelium as previously thought, potentially means that homogeneous populations of OECs for repair could be expanded in culture from neural crest stem cells persisting in the patient's own skin and hair follicles. The first step towards this long-term goal is to understand the molecular mechanisms underlying neural crest differentiation into OECs, as opposed to Schwann cells (the glia of all other peripheral nerves), which are less effective in spinal cord repair. To identify transcription factors and signalling pathways that might be involved in OEC versus Schwann cell differentiation, I took an unbiased transcriptome profiling approach. Taking advantage of Sox10 expression throughout both OEC and Schwann cell development, I used laser-capture microdissection on cryosections of mouse embryos carrying a Sox10:H2BVenus transgene, to isolate OEC subpopulations (olfactory mucosal OECs, from the olfactory nerve, and olfactory nerve layer OECs, from the olfactory nerve layer surrounding the olfactory bulb) at different stages of development, and Schwann cells from trigeminal nerve branches on the same sections, for RNA-seq and cross-wise comparison of transcriptomes. Validation of candidate genes by in situ hybridisation revealed some contamination with adjacent cells from mesenchyme, olfactory epithelium or olfactory bulb, but also identified the expression in developing OECs of various genes previously reported to be expressed in adult OECs, and of over 20 genes previously unknown in OECs. Some of these genes are expressed by OECs but not Schwann cells; some are expressed by olfactory nerve layer OECs but not olfactory mucosal OECs, while some are expressed by olfactory mucosal OECs and Schwann cells but not olfactory nerve layer OECs. For a subset of the genes, I was also able to analyse OEC differentiation in mouse mutants. I also collected transcriptome data from neural crest-derived cells that persist on the olfactory nerve in Sox10-null embryos (in which neural crest-derived cells colonise the olfactory nerve, but normal OEC differentiation is disrupted). Comparison with wild-type OEC transcriptome data from the same embryonic stage identified genes whose expression is likely either downregulated or up-regulated in the absence of Sox10, supporting a role in normal OEC differentiation. Overall, these various transcriptomic comparisons (between OECs at different developmental stages, different OEC subpopulations, OECs versus Schwann cells, and OECs versus Sox10-null neural crest-derived cells on the olfactory nerve) have identified multiple transcription factor and signalling pathway genes, amongst others, that are expressed during OEC development in vivo (including some specific to different OEC subpopulations) and that may be important for OEC differentiation. Furthermore, some of these genes are not expressed by embryonic Schwann cells. This work provides a foundation for understanding how to promote OEC rather than Schwann cell differentiation from neural crest stem cells in culture, with the potential for clinical application in the future.

The enteric nervous system (ENS) innervates the gastrointestinal (GI) tract and controls central aspects of GI physiology including contractility of the intestinal musculature, glandular secretion and intestinal blood flow. The ENS is composed of neurons that conduct electrical signals and of enteric glial cells (EGCs). EGCs resemble central nervous system (CNS) astrocytes in their morphology and in the expression of shared markers such as the intermediate filament protein glial fibrillary acidic protein (GFAP). They are strategically located at the interface of ENS neurons and their effector cells to modulate intestinal motility, epithelial barrier stability and inflammatory processes. The specific contributions of EGCs to the maintenance of intestinal homeostasis are subject of current research. From a clinical point of view EGC involvement in pathophysiological processes such as intestinal inflammation is highly relevant. Like CNS astrocytes ECGs can acquire a reactive, tissue-protective phenotype in response to intestinal injury. In patients with chronic inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis, alterations in the EGC network are well known, particularly a differential expression of GFAP, which is a hallmark of reactive gliosis in the CNS. With increasing recognition of the role of EGCs in intestinal health and disease comes the need to study the glial population in its complexity. The overall aim of this thesis was to comprehensively study EGCs with focus on the reactive GFAP-expressing subpopulation under inflammatory conditions in vivo and in vitro. In a first step, a novel in vivo rat model of acute systemic inflammation mimicking sepsis was employed to investigate rapidly occuring responses of EGCs to inflammation. This study revealed that within a short time frame of a few hours, EGCs responded to the inflammation with an upregulation of Gfap gene expression. This inflammation-induced upregulation was confined to the myenteric plexus and varied in intensity along the intestinal rostro-caudal axis. This highly responsive myenteric GFAP-expressing EGC population was further characterized in vivo andin vitro using a transgenic mouse model (hGFAP-eGFP mice). Primary purified murine GFAP-EGC cultures in vitro were established and it was assessed how the transcriptomic and proteomic profiles of these cells change upon inflammatory stimulation. Here, myenteric GFAP-EGCs were found to undergo a shift in gene expression profile that predominantly affects expression of genes associated with inflammatory responses. Further, a secretion of inflammatory mediators was validated on protein level. The GFAP+ subpopulation is hence an active participant in inflammatory pathophysiology. In an acute murine IBD model in vivo, GFAP-EGCs were found to express components of the major histocompatibility complex (MHC) class II in inflamed tissue, which also indicates a crosstalk of EGCs with the innate and the adaptive lamina propria immune system in acute inflammation. Taken together, this work advances our knowledge on EGC (patho-)physiology by identifying and characterizing an EGC subpopulation rapidly responsive to inflammation. This study further provides the transcriptomic profile of this population in vivo and in vitro, which can be used to identify targets for therapeutic intervention. Due to the modulating influence of EGCs on the intestinal microenvironment, the study further underlines the importance of integrating EGCs into in vitro test systems that aim to model intestinal tissues in vitro and presents an outlook on a potential strategy. / Das enterische Nervensystem (ENS) innerviert den gastrointestinalen Trakt und kontrolliert zentrale Aspekte der gastrointetinalen Physiologie, wie die Kontraktilität der intestinalen Muskulatur, Sekretion und den intestinalen Blutfluss. Das ENS setzt sich aus elektrisch leitenden Neuronen und enterischen Gliazellen (EGZ) zusammen. EGZ ähneln Astrozyten des zentralen Nervensystems (ZNS) hinsichtlich ihrer Morphologie und der Expression gemeinsamer Marker wie dem Intermediärfilament Saures Gliafaserprotein (GFAP von engl. glial fibrillary acidic protein). EGZ sind strategisch an der Kontaktstelle zwischen ENS-Neuronen und deren Effektorzellen positioniert, um die intestinale Motilität, die epitheliale Barrierestabilität sowie inflammatorischen Prozesse zu modulieren. Die spezifische Beteiligung der EGZ an der Aufrechterhaltung der Darmhomöostase wird gegenwärtig erforscht. Aus klinischer Sicht ist die Beteiligung von EGZ an pathophysiologischen Prozessen wie der intestinalen Entzündung besonders relevant. Wie ZNS-Astrozyten können EGZ bei intestinalen Schädigungen einen reaktiven, gewebe-protektiven Phänotyp annehmen. Bei Patienten mit chronisch-entzündlichen Darmerkrankungen (IBD, von engl. inflammatory bowel disease) wie Morbus Crohn und Colitis ulcerosa sind Veränderungen im EGZ-Netzwerk bekannt, besonders eine veränderte Expression von GFAP, welches ein prominentes Kennzeichen der reaktiven Gliose im ZNS ist. Nachdem sich die Bedeutung der EGZ im gesunden und kranken Darm zunehmend herausgestellt hat, muss ein stärkerer Fokus auf die Erforschung der glialen Population gelegt werden. Die Zielsetzung dieser Arbeit war die umfassende Untersuchung der EGZ mit Fokus auf die reaktive GFAP-exprimierende Population unter entzündlichen Bedingungen in vivo und in vitro}. In einem ersten Schritt wurde ein neuartiges in vivo-Rattenmodell einer akuten systemischen Entzündung verwendet, um die schnell stattfindenden Veränderungen der EGZ unter entzündlichen Bedingungen zu untersuchen. Diese Studie ergab, dass innerhalb von wenigen Stunden EGZ mit einer Hochregulation der Gfap-Genexpression auf die Entzündung reagieren. Diese entzündungsinduzierte Hochregulation war lokal auf den myenterischen Plexus begrenzt und entlang der rostro-kaudalen Achse des Darms unterschiedlich stark ausgeprägt. Die responsive, GFAP-exprimierende myenterische EGZ-Population wurde daraufhin in vivo und in vitro charakterisiert unter Zuhilfenahme eines transgenen Mausmodells (hGFAP-eGFP-exprimierende Mäuse). Primäre, aufgereinigte GFAP-EGZ-Zellkulturen wurden etabliert und dahingehend untersucht, wie sich das transkriptomische und proteomische Profil der Population unter entzündlichen Bedingungen verändert. Hierbei wurde reproduzierbar eine Verschiebung des transkriptomischen Profils myenterischer GFAP-exprimierender EGZ gefunden. Die davon betroffenen Gene sind vorwiegend mit Immunantworten assoziiert. Weiterhin wurde die Sekretion solcher Immunmediatoren auf Proteinebene validiert. Die GFAP+ Subpopulation ist somit ein aktiver Modulator entzündlicher pathophysiologischer Prozesse. In einem akuten IBD-Mausmodell konnte weiterhin gezeigt werden, dass GFAP-EGZ verstärkt Komponenten des Haupthistokompatibilitätskomplex (MHC) Klasse II im entzündeten Gewebe exprimieren. Dies weist auf eine direkt Interaktion der EGZ mit dem Immunsystem in der Lamina propria hin. Insgesamt konnte mit dieser Arbeit das Wissen über die (Patho-)Physiologie von EGZ erweitert werden, indem eine schnell responsive EGZ-Subpopulation identifizert und charakterisiert wurde. Weiterhin wurde im Rahmen dieser Arbeit das gesamte Transkriptomprofil der GFAP-Subpopulation in vivo und in vitro veröffentlicht, welches für weitere Studien zur Identifikation möglicher therapeutischer Anwendungen genutzt werden kann. Aufgrund des modulierenden Einflusses der EGZ auf die Darmphysiologie betont diese Studie die Notwendigkeit EGZs in in-vitro-Gewebemodelle des Darms zu integrieren und präsentiert einen Ausblick auf eine mögliche Strategie.

The blood-brain barrier regulates the transport into the brain and protects the central nerve system (CNS) from toxics substances. However some areas of the brain, called circumventricular organs (CVO), lack the blood-brain barrier. One of these is area postrema (AP), which is located in the brainstem immediately adjacent to the nucleus tractus solitarius (NTS). These two areas together regulate autonomic behaviours such as food intake, and also make up the vomiting center. The hormones leptin and ghrelin, which regulate food intake, are too big to pass the blood-brain barrier, but have receptors in NTS. In this study we used immunohistochemistry to obtain a detailed map of the different components of the blood-brain barrier in AP and NTS. The results suggest that there is a barrier that prevents diffusion of substances from AP into NTS. However, there seems to be some vessels in NTS that have a weaker or no barrier characteristics. These vessels could provide an entrance for peripheral substances to neurons in NTS.

The olfactory system exhibits remarkable regenerative ability in it’s neuronal population. The success of continuous neurogenesis is thought to be due, at least in part, to its unique glia – olfactory ensheathing cells (OECs). OECs bear characteristics of both peripheral and central glia, and serve to ensheath, guide and promote growth of olfactory receptor neurons (ORNs) throughout both development and adult life. Brain lipid binding protein (BLBP) is most highly expressed by radial glia during embryonic development. It is largely down-regulated in the adult CNS, but BLBP expression is retained in the adult by special subpopulations of glia, including OECs. BLBP expression is induced in radial glia via Notch signaling, but it is not known if these same mechanisms regulate BLBP expression in the adult CNS. Axonal-glial signaling is a dynamic process whereby closely apposed neuronal and glial cells regulate the growth, maintenance and plasticity of one another through direct cell-cell signaling. Delta/Notch-like EGF-related receptor (DNER) is a transmembrane protein expressed by Purkinje cells which has been implicated in the regulation of BLBP in Bergmann glia during cerebellum development through Notch1 deltex-dependent non-canonical signaling. We have found that DNER is expressed in more mature ORNs, and other exclusive subpopulations of cells within the CNS. OECs in close apposition with DNER-expressing ORNs in vivo appear to maintain the highest BLBP expression found in the nervous system through development and adulthood. Immunofluorescence shows that this close relationship between BLBP expressing cells and DNER expressing cells also appears to be retained in specialized areas such as the hippocampus, retina and spinal cord, throughout mouse CNS development as well as in the mature system. Removing DNER or axonal input in vivo decreases the robustness of OEC BLBP expression, and the number of cells in OEC culture expressing BLBP decreases rapidly with time. OEC co-culture with a DNER expressing monolayer increases the number of OECs in vitro which express BLBP, providing evidence for the regulation of BLBP expression in OECs by DNER expression in apposing ORNs.