<p>Evidence from experimental models of epilepsy support that prolonged seizures (status epilepticus, SE) promote pathological hippocampal synaptodendritic remodeling which contributes to the development of seizures and cognitive decline. One potential mechanism underlying the SE-induced sequelae is microgliosis. </p> <p>Evidence from models of experimental epilepsy supports a significant spatiotemporal correlation between SE-induced decreases in the microtubule associated protein 2 (Map2) loss and microgliosis in the hippocampus. In addition, pharmacological suppression of microgliosis after SE with the drug rapamycin attenuated the losses of Map2 and the dendritic ion channels Kv.4.2 and HCN1 in the hippocampus. This microglia suppression paralleled a recovery of the SE-induced recognition and spatial memory deficits. Based on these studies, we hypothesized that the inhibition of microgliosis during epileptogenesis will attenuate the SE-induced hippocampal dendritic and cognitive pathology. To further investigate the role of microgliosis in the SE-induced dendritic pathology, we tested the efficacy of a more selective inhibitor <a>of the survival and proliferation of </a>microglia, PLX3397, using the pilocarpine model of SE and acquired epilepsy. PLX3397 binds to colony stimulating factor 1 receptor (CSF1R) on microglia and inhibits the downstream signaling responsible for survival and proliferation of these cells. </p> <p>To test this hypothesis, we induced SE in male rats with pilocarpine (280-300mg/kg) <a>while and controls (Ctrl) received saline. </a>Rats were randomly assigned to a diet of either chow alone (vehicle; Veh) or chow with PLX3397 (50mg/kg) for 20 days post-SE. At two weeks post-SE, rats were subjected to novel object recognition (NOR) and Barnes maze (BM) to evaluate hippocampal-dependent recognition memory, and spatial learning and memory, respectively. Following the behavioral assessments, rats were sacrificed for brain analysis at 20 days post-SE. We used histological analysis to determine the amount of microgliosis with IBA1 and dendritic stability with Map2. We used western blotting to measure the protein levels of molecules involved in the crosstalk between microglia and astrocytes: GFAP, IL-6, C3, and iC3b. We also measured the protein levels of the dendritic ion channels Kv4.2 and HCN1, and the synaptic marker PSD95.</p> <p>NOR showed that the Ctrl+Veh and Ctrl+PLX3397 groups spent significantly more time exploring the novel object (<i>p</i> < .05), while the SE+Veh and SE+PLX3397 did not. Similar results were observed in the BM test, Ctrl+Veh and Ctrl+PLX3397 groups had a faster latency to find the target compared to the SE+Veh and SE+PLX3397 groups (<i>p</i> < .05). These data suggest that recognition and spatial memory deficits induced by SE were not attenuated by treatment with PLX3397. We found that the PLX3397 treatment significantly decreased microgliosis in Ctrl+PLX3397 rats compared to Ctrl+Veh rats (<i>p</i> < .05). As expected, we found a significant increase in the number of microglial cells in hippocampi of SE+Veh rats compared to Ctrl+Veh rats (<i>p</i> < .05). Interestingly, in the PLX3397-treated SE group, we observed two distinctive groups which we categorized as responders and non-responders when compared to the SE+Veh group. The SE+PLX responders had significantly decreased microgliosis compared to the SE+Veh group (<i>p</i> < .05). The SE+PLX non-responders had higher levels of microgliosis compared to the SE+Veh group (<i>p</i> < .05). We found levels of GFAP were increased in the SE+Veh group compared to the Ctrl+Veh group (<i>p</i> < .05). Treatment with PLX3397 in the SE group reduced these levels compared to the vehicle treated SE group (<i>p</i> < .05). We also found increases in C3 and iC3b following the induction of SE compared to Ctrl+Veh group (<i>p</i> < .05), and these levels remained similar in the SE+PLX3397 group compare to the SE+Veh group (<i>p</i> > .05). There was a reduction in Map2 immunoreactivity as well as the protein levels of Kv4.2 and PSD95 in the SE+Veh group compared to the Ctrl+Veh group (<i>p</i> < .05). We found that treatment with PLX3397 recovered the SE-induced loss of Map2 labeled dendrites compared to SE+Veh group (<i>p</i> < .05). However, treatment with PLX3397 did not recover the SE-induced reduction Kv4.2 and PSD95 (<i>p</i> > .05). <a>In parallel, we found that a group of SE+PLX3397 animals did not have reduced microgliosis compared to the SE+Veh group (<i>p </i>< .05), and therefore was categorized as a non-responder group. </a></p> Our findings are the first to show that blocking CSF1R signaling with PLX3397 suppressed microgliosis in the hippocampus, partially recovered the SE-induced decline of Map2 immunoreactivity in the hippocampal CA1 region but had no effect in the recognition or spatial memory deficits. These data suggest that while hippocampal microgliosis may play a role in the disruption of dendritic structural stability in the hippocampus it does not seem to critically contribute to the memory decline that occurs during epileptogenesis.

Adult neurogenesis, is a lifelong process by which relatively few cells are added into two restricted regions of the brain. Integration of the cells into the existing neuronal circulatory, with the unique properties involved in the maturation of these cells, is possibly critical to the acquisition and retrieval of new memories. With the chronological aging of the organism a process of cellular senescence occurs throughout the body; a portion of which is independent of primary alterations to the stem cells; instead, it appears to be dependent on the environment where the cells reside, and is in part regulated by inflammation. Microglia, the resident immune cells in the brain, are neuroprotective but chronic activation of the microglia, such as the chronic activation that occurs with advanced age, can promote neurotoxic inflammation. However, it not clear if the aged-related increase in neuroinflammation is at least partly responsible for the aged related decrease in neurogenesis. To address the involvement in neuroinflammation in regulating neurogenesis we used 3 different potential therapeutically relevant manipulations. The first was a targeted approach directed at disrupting the synthesis of Interleukin-1beta (IL-1B), which is a proinflammatory cytokine that is consistently found elevated in the aged brain. The second was a cell therapy approach in which human umbilical cord blood cells were injected into the systemic circulation. The final approach was directed at a chemokine system, fractalkine/CX3CR1, which has been shown as an important paracrine signal, from neurons that regulates the activation state of microglia. While the three approaches used to manipulate, aging-rodent model system were different, a consistent finding was reached in all three studies. In the aged brain, microglia which are the predominate produces of IL-1B, negatively regulate neurogenesis. When IL-1B is decreased or microglia activation is decreased, neurogenesis can be partially restored in the aged brain. The results of these studies, demonstrate a key role for microglia in regulating the neurogenic neiche, which are amendable to therapeutic manipulations.

In der vorliegenden Arbeit wurde die Funktion des G-Protein-gekoppelten Rezeptors 34 (GPR34) in Mikroglia untersucht. Dieser Rezeptor weist eine hohe Expression auf Gliazellen auf, jedoch ist über dessen Aufgabe innerhalb dieser Zellpopulation bisher nichts bekannt. In bisherigen Arbeiten wurde dem GPR34 eine Rolle in der Immunantwort zugeschrieben. Knock-out (ko)-Mäuse, welche mit Cryptococcus neoformans infiziert wurden, zeigten im Vergleich zum infizierten Wildtyp (wt) eine deutlich höhere Pathogenlast in verschiedenen Geweben u.a. im Gehirn, was für eine inadäquate Immunantwort spricht. In dieser Arbeit konnte mittels morphologischer Studien gezeigt werden, dass eine GPR34- Defizienz zu einer veränderten Gestalt der Mikroglia im Cortex sowie der Retina führt. Mikrogliazellen aus ko-Mäusen sind kleiner und deutlich weniger ramifiziert. Mit Hilfe von Transkriptomanalysen wurde eine große Vielfalt an unterschiedlich exprimierten Genen zwischen ko- und wt-Tieren identifiziert. Hierunter befanden sich Gene, die die Motilität, aber auch die Phagozytose der Mikroglia beeinflussen. Um den Einfluss der GPR34-Defizienz auf diese Vorgänge zu untersuchen, wurden zahlreiche funktionelle Untersuchungen an murinen Mikrogliazellen durchgeführt. Mittels basalen Motilitätsstudien aber auch unter Stimulation durch Laserläsion und Läsion des entorhinalen Cortex konnten keine Unterschiede in der Beweglichkeit von Mikroglia aufgedeckt werden. Jedoch zeigten ko- Mikrogliazellen des Cortex und der Retina eine deutlich geringere Phagozytoseaktivität. Dies ist ein möglicher Erklärungsansatz für die beschriebene erhöhte Pathogenlast in den GPR34- defizienten Tieren. Da die Phagozytoseaktivität von Mikroglia in neurodegenerativen Erkrankungen wie Multipler Sklerose oder der Alzheimer´schen Demenz eine bedeutende Rolle spielt, sollte zukünftig die Relevanz des GPR34 bei diesen Erkrankungen untersucht werden.

Bei der vorliegenden Arbeit handelt es sich um eine experimentell durchgeführte Charakterisierung von CD11c-positiven Zellen des Gehirns im direkten Vergleich zu CD11c-positiven Zellen aus Lunge, Leber und Milz. Mittels Konfokal- und Fluoreszenzmikroskopie wurde die Existenz von intraparenchymalen Zellen nachgewiesen, welche den Zellmarker für Dendritische Zellen CD11c exprimieren. Durch die Etablierung einer einheitlichen Isolierungsmethode von CD11c-positiven mononukleären Zellen aus dem Gehirn, Milz, Lunge und Leber, war es uns möglich, diese mittels Durchflusszytometrie, auf die Expression wichtiger Marker für mononukleäre Zellen zu untersuchen und phänotypisch miteinander zu vergleichen. Durch diese Zellanalysen zeigten wir, dass CD11c-positive Zellen des Gehirns sowohl aufgrund ihrer spezifischen CD45-Expression, als auch durch die Expression von CD11b einen Mikrogliaphänotyp aufwiesen. Dabei konnten wir beobachten, dass CD11c-positive Zellen aus dem Gehirn einzigartig in der Eigenschaft ihrer geringen Major Histocompatibility Complex (MHC)-II-Expression sind. Mit Hilfe einer transgenen Mauslinie, welche unter dem Promotor von MHC-II das grün-fluoreszierende Protein (GFP) exprimiert, konnten wir nachweisen, dass Mikroglia selbst in der Umgebung von MHC-II-positiven Zellen, kultiviert auf Schnittkulturen der Milz, ihre MHC-II-Negativität behalten. Im Vergleich dazu adaptierten sich MHC-II-positive Splenozyten, kultiviert auf Schnittkulturen vom Hippocampus, an die neue Umgebung und verringerten die Expression von MHC-II. Unsere Daten lassen also die Schlussfolgerung zu, dass sich CD11c-positive Mikroglia hinsichtlich ihrer Expression von MHC-II intrinsisch von CD11c-positiven Zellen anderer Organe unterscheiden. Ebenso scheinen auch lokale Faktoren im Gehirn dazu beizutragen, die Expression von MHC-II unter physiologischen Bedingungen wirkungsvoll zu unterdrücken.

Microglia, the brain’s resident immune cells, exhibit constitutive expression of the ionized calcium-binding adaptor molecule 1 (IBA1). They are long-lived cells that exhibit a senescent morphology (dystrophy) with aging. It has been reported that dystrophy of IBA1-positive microglia is exacerbated in obese humans. Furthermore, we detected another microglial abnormality, which is the loss of IBA1 immunoreactivity that can create large areas in the brain seemingly devoid of all microglial cells. Here, we systematically compared microglial appearance in human hippocampi derived from obese individuals compared to controls by morphological and spatial analysis. In both groups, areas that were negative for IBA1 contained P2YR12 and glutathione-peroxidase 1 (GPX)-positive microglia. The number and extent of IBA1-negative regions was increased in obese cases. Since some cases of non-obese individuals also exhibited loss of IBA-1 immunoreactivity, we searched for possible confounders and found that hepatic dysfunction strongly impacts the distribution of microglial cells: a spatial analysis of scanned IBA1-stained sections increased Mean Empty Space distances (p=0.016) and IBA1-negative areas (p=0.090) which were independent of the cause of liver dysfunction, but also from aging, were detected. Thus, we report on a novel type of microglia pathological change, i.e. localized loss of IBA1 that is linked, at least in part, to obesity and hepatic dysfunction.:1 Introduction . . . . .. . . . . . . . .. . . . . . . . . . .. 2 1.1 Formation and morphology. .. . . . . . . . . . 2 1.2 Microglia and aging . . . . . . . . . . . . . . . . . 3 1.3 Microglia and obesity . . . . . . . .. . . . .. . . .5 1.4 Hippocampus . . . . . . . . . . . . . . . . . .. . . . 6 1.5 IBA1 . . . . . . . . . . . . . . . . . . . . . . . . .. . . . 7 1.6 Microglial markers . . . . . . . . . . . . .. . . . . 9 1.7 Hepatic dysfunction. . . . . . . . . . . . . . . . .10 1.8 Hepatic encephalopathy . . . . . . . . . . . . .10 1.9 Microglia in hepatic encephalopathy . . . 11 2 Aims and outlines of the dissertation . . . . .13 3 Manuscript of publication . . . . . . . . . . .. . . 15 4 Abstract . . . . . . . . . . .. . . . . . . . . . .. . . . . .25 5 Bibliography. . . . . . . . . . .. . . . . . . . . . .. . . 29 6 Appendix . . . . . . . . . . .. . . . . . . . . . .. . . . . 38 6.1 Supplemental Material .. . . . . . . . . . . . .. 38 7 Darstellung des eigenen Beitrags . . . . . . . 41 8 Erklärung über die eigenständige Abfassung der Arbeit . . . . . . . . . . .. . . . . .. . 42 10 Publications . . . . . . . . . . .. . . . . . . . . . . . .45 11 Acknowledgements. . . . . . . . . . .. . . . . . . 46

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid-β peptide (Aβ) in the brain and intraneuronal hyperphosphorylated tau. Microglia in the brain adopt M1 (pro-inflammatory) or M2 (anti-inflammatory) phenotypes similar to peripheral monocytes. M1 microglia negatively affect neurogenesis and have reduced phagocytic capabilities whereas M2 microglia can enhance neurogenesis and support phagocytosis. Cluster of Differentiation-200 (CD200) is an anti-inflammatory glycoprotein physiologically expressed on neurons and lymphocytes, and its receptors (CD200R1 and CD200R3) are expressed on glia. Both AD patients and mouse models of AD show an age-related or Aβ-induced reduction in neural CD200 that may contribute to M1-skewing of microglia in AD. We hypothesize that CD200 skews microglia to an M2 phenotype, and that genetic over-expression of CD200 in transgenic mice expressing the Swedish familial AD mutation of human β-amyloid precursor protein (APP mice) can restore neurogenesis and enhance Aβ clearance in the hippocampus. In this study, we constructed a tetracycline-controlled transactivator-inducible adeno-associated virus serotype 2/1 expressing full-length CD200 (AAV2/1-CD200) or green fluorescent protein (AAV2/1- GFP). These were bilaterally injected into the hippocampi at 6 months of age, and mice were sacrificed at 12 months of age. AAV2/1-GFP-injected APP mice showed a reduction in number of proliferating neural stem cells (NSCs) by 65.0% and differentiating NSCs by 70.5% in the dentate gyrus compared to wild-type controls. AAV2/1-CD200 restored these neurogenic deficits to those of wild-type mouse levels. AAV2/1-CD200 reduced diffuse Aβ plaques in the hippocampal region by 65.5% compared to AAV2/1-GFP-injected APP mice, but did not alter thioflavin-S-positive compact plaques as measured by protein and immunohistochemical assays. In vitro studies demonstrated that CD200-stimulated microglia co-cultured in transwells increased differentiation and complexity of neural stem cells. CD200 also directly enhanced Aβ phagocytosis by microglia. CD200 enhanced expression of the adaptor protein TYRO protein tyrosine kinase binding protein (TYROBP), suggesting this may be the mechanism by which CD200 enhances phagocytosis of Aβ. Overall, the data presented here indicate that CD200 is a plausible therapeutic agent in patients with AD to enhance neural differentiation and microglial-mediated clearance of Aβ.

L’hypertension artérielle chronique représente le premier facteur de risque de l’AVC ischémique, mais elle en est aussi le principal facteur aggravant. Les mécanismes à l’origine du risque ischémique lié à l’hypertension ne sont pas encore entièrement compris. Plusieurs études ont montré l’existence d’une forte composante inflammatoire délétère impliquée dans la physiopathologie de l’hypertension. Au niveau cérébral, la présence d’une intense réactivité microgliale hypothalamique, participant à l’aggravation de la pathologie a été observé. Dans le travail présenté ici, nous nous sommes intéressés à l’impact de l’hypertension artérielle sur l’état inflammatoire du cortex cérébral, une région particulièrement touchée lors d’un AVC ischémique chez le patient. Nous avons tiré parti de la souche transgénique de souris CX3CR1GFP/+ pour l’imagerie de la dynamique microgliale, principal acteur de l’immunité cérébrale. Par une analyse in vivo réalisée en microscopie biphotonique, nous avons montré que l’hypertension induite par infusion chronique d’angiotensine-II altère la morphologie de la microglie, mais surtout sa capacité de surveillance du parenchyme cérébral et sa capacité cicatricielle. Nous avons aussi montré que ce type d’hypertension endommage la structure et la fonctionnalité des vaisseaux corticaux. L’ensemble de ces résultats pourrait expliquer, au moins en partie, la sensibilisation du cerveau aux lésions ischémiques par l’hypertension artérielle, avant même la survenue de l’AVC. / Chronic high blood pressure is ischemic stroke’s leading risk factor, but it is also its main aggravating factor. The mechanisms underlying hypertension-induced ischemic brain lesion exacerbation are not yet fully understood. Several studies highlighted the existence of a strong inflammatory component in the pathophysiology of hypertension. In the brain, the presence of intense hypothalamic microglial reactivity, contributing to the pathology worsening has been shown. In this work, we focused on the impact of high blood pressure on the inflammatory state of the cerebral cortex, a region particularly affected by ischemic stroke in the patient. We took advantage of the CX3CR1GFP/+ mice transgenic strain for imaging microglia dynamics. By using in vivo two-photon microscopy, we have shown that hypertension induced by chronic infusion of angiotensin-II alters the microglia morphology, especially its parenchymal surveilling activity and its cicatricial capacity. We have also shown that this type of hypertension disrupts the structure and the functioning of cortical vessels. All of these results can explain, at least in part, the brain sensitization to ischemic lesions under arterial hypertension, before the onset of stroke.

Microglia are the resident macrophage in the central nervous system (CNS) that actively survey their environment and participate in shaping neuronal circuits. Among the transcription factors necessary for microglia development, interferon regulatory factor 8 (IRF8) is a known risk gene for multiple sclerosis and lupus and it has recently been shown to be downregulated in schizophrenia. These studies suggest that lack of microglial IRF8 can subsequently impact neuronal function in disease, but the mechanisms underlying these effects remain unknown. While most studies have focused on IRF8-dependent regulation of immune cell function, little is known about how it impacts neural circuits. To interrogate the impact of disrupted microglial IRF8 signaling on brain circuits, I first show by RNAseq that several genes known to regulate neuronal function are dysregulated basally in Irf8-/- brains. I then found that these molecular changes are reflected in heightened neural excitability and a profound increase in susceptibility to chemically-induced lethal seizures in Irf8-/- mice. Importantly, I also show that developmental synaptic pruning, a key function for microglia, proceeds normally in Irf8-/-mice. Finally, I identified that these IRF8-dependent effects on circuits are due to elevated TNF-α in the CNS as genetic or acute pharmacological blockade of TNF-α in the Irf8-/- CNS rescued the seizure phenotype. These results provide important insights into the consequences of IRF8 signaling and TNF-α on neural circuits. The next steps are to use cell-specific genetic approaches to manipulate this signaling, which I have further developed over the course of this project.

Das Chemokin SDF-1 spielt eine wichtige Rolle bei der Hämatopoese, bei Immunreaktionen sowie bei der Entwicklung des Herzens, der Extremitätenmuskulatur und des zentralen und peripheren Nervensystems. Lange Zeit galt CXCR4 als der einzige Chemokinrezeptor für SDF-1, bis vor wenigen Jahren CXCR7 als ein alternativer Rezeptor für SDF-1 identifiziert wurde. Da alle Zelltypen des zentralen Nervensystems (ZNS) sensitiv für SDF-1 sind, sollte in dieser Arbeit die Funktion der beiden Rezeptoren in primärer Mikroglia und primären Astrozyten untersucht werden. Bisher konnte CXCR7 nur als Scavenger-Rezeptor für SDF-1 oder als atypischer Chemokinrezeptor nachgewiesen werden. Die Untersuchungen ergaben einen mitogenen und chemotaktischen Effekt von SDF-1 auf primäre Mikroglia, wobei sowohl CXCR4 als auch CXCR7 für das SDF-1-Signalverhalten essentiell sind. Nach Aktivierung von Mikroglia in vitro und in vivo wurden beide Rezeptoren verstärkt expremiert. In primären Astrozyten ergab sich ein ligandenabhängiges Signalverhalten von CXCR7. So führte die Bindung von SDF-1 an CXCR7 zu einer Aktivierung von G-Proteinen, während die Kopplung von interferon-inducible T cell alpha chemoattractant (I-TAC), als zweiten Liganden von CXCR7, eine Signalweiterleitung über ß-Arrestin2 zur Folge hatte. Zudem konnte die G-Protein-gekoppelte Rezeptorkinase (Grk)2 als ein positiver Regulator des SDF-1-CXCR7-Signalverhaltens in Astrozyten identifiziert werden.

Microglia are the resident innate immune cells that are critical for innate and adaptive immune responses within the CNS. They recognize and are activated by pathogen-associated molecular patterns (PAMPs) present on the surface of pathogens. β-glucans, the major PAMP present within fungal cell walls, are recognized by Dectin-1, which mediates numerous intracellular events invoked by β-glucans in various immune cells. Previously, we showed that Dectin-1 mediates phagocytosis of β-glucan and subsequent superoxide production in microglia. Here, we report that the guanine nucleotide exchange factor Vav1 as well as phosphoinositide-3 kinase (PI3K) are downstream mediators of what is now recognized as the Dectin-1 signaling pathway. Both Vav1 and PI3K are activated upon stimulation of microglia with β-glucans, and the two proteins are required for phagocytosis of the glucan particles and for subsequent superoxide production. We also show that Vav1 functions upstream of PI3K and is required for activation of PI3K. Together, our results provide an important insight into the mechanistic aspects of microglial activation in response to β-glucans.