Mikroglia sind residente Makrophagen-artige Zellen des Zentralnervensystems (ZNS), die das Gewebe kontinuierlich auf Anzeichen homöostatischer Störungen überwachen. Als die wesentlichen immunkompetenten Effektorzellen im Hirnparenchym exprimieren sie eine Vielzahl von Rezeptoren für pathogen-assoziierte molekulare Strukturmuster (pathogen-associated molecular patterns, PAMPs). Zu diesen Rezeptoren zählt der Toll-like receptor (TLR) 4, der nicht nur Reaktionen der Mikroglia auf bakterielle Infektionen, sondern auch auf Gewebe Schädigungen ermöglicht. Stimulation des TLR4 mit bakteriellem Lipopolysaccharid (LPS) und endogenen schädigung-sassoziierten molekularen Strukturen (damage-associated molecular patterns, DAMPs), die durch Gewebebeeinträchtigung freigesetzt werden, löst sowohl TRIF- als auch MyD88-abhängige Signalkaskaden aus. Die damit induzierte Freisetzung von Zytokinen und Chemokinen rekrutiert und instruiert periphere Immunzellen für eine Protektion und unterstützende Geweberegeneration des ZNS. Wir zeigen hier, dass der TLR4-Korezeptor CD14 ein essenzieller gate keeper für die Generierung von Immunantworten im ZNS ist, die durch LPS oder E. coli-Verabreichung, aber auch durch mechanisches Trauma und ischämischen Schlaganfall ausgelöst werden. In gewissem Gegensatz zu extraneuralen Makrophagen nutzen Mikroglia CD14 zur Erlangung einer extremen Sensitivität gegenüber sehr geringen LPS-Mengen. Gleichzeitig schützt CD14 Mikroglia vor überschießenden Reaktionen auf hohe LPS-Dosen und verhindert dabei insbesondere die exzessive Produktion von CXCL1, eines chemoattraktiven Signals für neutrophile Granulozyten. Entsprechend unterstützt CD14 die ZNS-Rekrutierung von Monozyten und Neutrophilen durch niedrige LPS-Dosen, während es die verstärkte Einwanderung von Neutrophilen durch hohe Dosen von LPS oder E. coli verhindert. Als eine besonders wichtige Funktion beschreiben wir dabei die absolute CD14-Abhängigkeit DAMP-ausgelöster und TLR4-vermittelter Immunreaktionen. CD14-Defizienz (unter cd14-/--Bedingungen) oder CD14 Blockade (durch Antikörper) löschen mikrogliale Reaktionen, die durch Plasma-Fibronektin (als repräsentatives DAMP-Molekül) ausgelöst werden können, komplett aus und beeinträchtigen die Leukozyten-Infiltration nach ZNS-Trauma. Bei einer ischämischen ZNS-Schädigung weisen cd14-/--Mäuse im Gehirn nicht nur weniger Monozyten auf, sondern gleichzeitig ein vergrößertes Infarktvolumen. Wir konnten für Interferon (IFN) b eine Schlüsselfunktion in der CD14-vermittelten Eindämmung der CXCL1-Synthese darstellen, die auf eine negative CD14/TLR4-TRIF-IFNβ-INAR1-Jak- Rückkopplung für MyD88-getriebene Chemokine schließen lässt. Obwohl CD14 somit TLR4-vermittelte Reaktionen auf infektiöse und nicht-infektiöse Agenzien orchestriert, wird seine Expression durch verschiedene TLR-Liganden und Zytokine reguliert. Letztlich unterliegen damit CD14-kontrollierte Funktionen selbst einer komplexen Kontrolle durch ZNS-residente und eingewanderte periphere Zellen. Diese Regulationen können über die Einbeziehung oder den Ausschluss der Kapazitäten des TLR4-Komplexes für eine Schadenserkennung während der ZNS-Reaktionen in unterschiedlichsten pathologischen Szenarien entscheiden.

The blood-brain barrier (BBB) is located at the level of the cerebral microcapillaries, and functions to maintain environmental homeostasis by allowing the neurons access to the required nutrients and enabling the exchange of metabolic waste. BBB dysfunction has been observed in a number of pathophysiologic statres including peripheral inflammatory pain (Huber et al., 2001b). Using the lamda-carrageenan inflammatory pain (CIP) model, we observed alterations in the tight junction (TJ) proteins paralleled by an increase in BBB permeability to [14C] sucrose. The mechanisms by which these perturbations occurred remain to be elucidated. In the current study, we investigate the central mechanism for the BBB perturbations under CIP. It is our hypothesis that the modulations of the BBB under CIP, are mediated via a central signaling pathway. First, to investigate the involvement of neuronal input from pain activity on alterations in BBB, we developed a method for inhibiting the nociceptive input from the paw. Using a perineural injection of 0.75% bupivacaine into the right hind leg prior to CIP, we were able inhibit development thermal hyperalgesia induced by CIP, as tested by infrared heat stimulus, without effecting edema formation 1 h post CIP. Upon inhibition of nociception under CIP, there was an attenuation of both the changes in permeability and the changes in tight junction protein expression, with both returning to control levels. Next, we investigated intercellular adhesion molecule-1 (ICAM-1), a key signaling protein at the BBB, which in the presence of proinflammatory mediators, increases in expression leading to the activation of signaling pathways as well as morphological changes. We found a region specific increase in ICAM-1 mRNA and protein expression following CIP which directly correlated with increased expression of activated microglia. Finally, we investigated the influence activated microglia had on BBB permeability. Using an 0.150 mg intrathecal bolus injection of minocycline, a potent inhibitor of microglia activation (Klein and Cunha, 1995), we were able to inhibit the increased expression of activated microglia, and saw an attenuation of permeability to control levels. These findings suggest CIP induced BBB disruption is localized and has a central-mediated component independent of peripheral influence.

Microglia, immune cells of the central nervous system, activate in response to pathophysiological stimuli. One of their reactive phenotypes is to migrate to site of injury where they could have either beneficial or detrimental effects. However, little is known regarding the mechanisms underlying microglial migration and how they traverse the unique extracellular environment in brain tissue to reach their destination. Our laboratory first discovered that microglia express structures called podosomes, which can adhere to as well as degrade extracellular matrix. In this study, I further characterize microglial podosomes, and show that they associate with Iba1, Orai1 and calmodulin, proteins not yet observed in podosomes of other cell types. I also present evidence that podosome formation depends on Ca2+ and its entry through store-operated Ca2+ channels. The findings in this thesis contribute to a better understanding of podosome dynamics and their probable roles in microglia migration.

Microglia, immune cells of the central nervous system, activate in response to pathophysiological stimuli. One of their reactive phenotypes is to migrate to site of injury where they could have either beneficial or detrimental effects. However, little is known regarding the mechanisms underlying microglial migration and how they traverse the unique extracellular environment in brain tissue to reach their destination. Our laboratory first discovered that microglia express structures called podosomes, which can adhere to as well as degrade extracellular matrix. In this study, I further characterize microglial podosomes, and show that they associate with Iba1, Orai1 and calmodulin, proteins not yet observed in podosomes of other cell types. I also present evidence that podosome formation depends on Ca2+ and its entry through store-operated Ca2+ channels. The findings in this thesis contribute to a better understanding of podosome dynamics and their probable roles in microglia migration.

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.

Accumulating evidence suggests that inflammation plays an important role in the progression ofParkinson's disease (PD). Among many inflammatory factors found in the PD brain, cyclooxygenase(COX), especially the inducible isoform, COX-2, is believed to be the critical enzyme in theinflammatory response. Induction of COX-2 is also found in an experimental model of PD producedby administration of 1-methy-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). To investigate whetherinhibition of COX-2 by valdecoxib or deficiency in COX-2 could prevent dopaminergic neuronaltoxicity and locomotor activity impairment, we injected MPTP into valdecoxib-treated C57BL/6N miceand COX-2 deficient mice, respectively. Both automated total distance and vertical activitymeasurements of the open-field test were significantly reduced in the vehicle-treated mice at two weekspost-MPTP injection. In contrast, valdecoxib treatment significantly attenuated these deficits.Similarly, COX-2 deficiency attenuated MPTP-induced loss of coordination on a rotarod assay.Valdecoxib or deficiency of COX-2 reduced microglial activation while preventing loss of tyrosinehydroxylase (TH)-positive neurons in the substantia nigra pars compacta (SNpc). The total number ofactivated microglia in the SNpc had a strong positive correlation with the level of COX-2 anddopaminergic neurodegeneration. The results of this study indicate that reducing the activity of COX-2can mitigate the progressive loss of dopaminergic neurons as well as the motor deficits caused byMPTP neurotoxicity, possibly by suppressing the activation of microglia in the SNpc.

Prion diseases are curious neurodegenerative diseases characterized by the conversion of a cellular protein, PrPC, into an infectious isoform, PrPSc. One of the earliest hallmarks of disease and concurrent with prion deposition, is the activation of the brain’s principal immune effector cells, microglia. In prion disease, activated microglia synthesize fairly low levels of pro-inflammatory cytokines, presumably to ameliorate the severe pathology that can arise in host tissue as a result of an acute inflammatory response. The specific stimuli and signaling pathways that lead to this modulation of function are as yet unknown. However, the involvement of miRNAs, a recently identified class of regulatory molecules, is likely. Recently, miR-146a was found to be upregulated in the brains of prion infected mice. In addition, its expression was found to be enriched in cells of microglial origin. It was hypothesized that, given the immunomodulatory role ascribed to miR-146a in macrophages, upregulation of miR-146a may function to attenuate the microglial immune response to prion infection. The first objective was to identify inflammatory related miRNAs associated with prion disease in microglia. Using Taqman Low Density Arrays, allowing for the detection of hundreds of miRNAs at once, the miRNAs of microglia treated with inflammatory agonists were profiled. The miRNA profile of activated microglia was found to be similar to that of macrophages. Furthermore, among the miRNAs profiled, miR-146a and miR-155 were the most highly induced and persistently expressed over 24 hours. The second objective was to investigate miR-146a induction. Therefore, microglia were treated with various agonists and miR-146a expression was determined using Taqman miR-146a assays. Although treatment with a PrP-mimic did not induce miR-146a expression, stimulation of TLRs 1, 2, 4, and 5, resulted in significant over-expression similar to what has been described previously. Moreover, in contrast to the rapid and transient induction of inflammatory mediators, miR-146a follows alternate kinetics functioning to prolong the dampening of the innate immune response following activation via TLR4 and TLR2. By employing a functional proteomic strategy, the third objective was to identify miR-146a regulated proteins. First, miR-146a expression was manipulated using miR-146a mimics and miR-146a inhibitors. Secondly, the functional model was validated by confirming decreased expression of IL6 by ELISA in miR-146a over-expressing microglia cells. Lastly, using Tandem Mass Tag labels to discriminate between treatment group (miR-146a mimic and TLR2 agonist) and control group (scrambled-miR and TLR2 agonist), the effect of miR-146a on the proteome was determined. In total, 172 proteins were identified as being miR-146a regulated and gene ontology assignment resulted in an over-representation of proteins involved in cellular dynamics capable of altering the activation state of microglia. After filtering for bioinformatically predicted targets and those implicated in a similar genomic study, it was decided to further investigate proteins ARF6, RhoA and NOS2 based on their role in modulating the phagocytic potential of microglia. The final objective was to validate miR-146a putative direct targets identified from the proteomics analysis. Luciferase expression of the 3’UTR of targets upon transfection with miR-146a were determined. Based on luciferase analysis, NOS2 appears to be directly targeted by miR-146a and this was also confirmed by western blot. While production of NOS2 by microglia under an acute activation state serves to support and protect CNS homeostasis, chronic expression of this factor can lead to neurotoxicity. Therefore, miR-146a appears to have an overarching role in altering microglial activation during prion disease thus protecting neurons from bystander damage. Taken together, these results suggest that miR-146a could play an important role in the prion disease process by specifically attenuating the microglial induced immune response. Therefore, manipulation of miR-146a may represent a novel therapeutic strategy. Furthermore, given that miR-146a de-regulation has been observed in other neurodegenerative diseases, these results may well extend beyond the realm of prion disease. Lastly, although practical limitations relating to the sensitivity of the comparative proteomics methodology meant that it alone were not sufficient to identify miRNA targets, an integrated approach that takes into consideration genomic and bioinformatic strategies is most promising.

<p>The interactions between the brain and the immune system are extensive and each has a profound influence on the other. The hippocampus is a brain region that is strongly impacted by the immune system, especially considering its large population of microglia, the resident immune cells of the brain. Cytokines and chemokines, the signaling molecules from immune cells, signal within the central nervous system (CNS) as well, and they are critical in hippocampal function. The relationship between the immune system and the hippocampus may underlie its particular vulnerability to diseases and disorders of the nervous system and the periphery. Conversely, immune signaling within the hippocampus is affected by alterations in hippocampal resilience and flexibility, such that increased hippocampal plasticity reduces vulnerability to immune challenges. The balance between risk and resilience in the hippocampus is modulated by immune signaling, especially by microglia.</p><p> The hippocampus is vulnerable to immune challenges, disease and injury, but it is simultaneously a region capable of profound plasticity and flexibility. The following dissertation experiments were designed to assess the roles of microglia and their signaling molecules, cytokines and chemokines, during normal hippocampal processes, such as learning and memory and response to immune challenge. The first set of experiments examined the effects of a neonatal bacterial infection in rats on hippocampal-dependent learning and memory as well as neuronal and microglial signaling in adulthood. In the first experiment, neonatally infected rats have impaired memory during fear conditioning following an immune challenge in adulthood. The impairment is caused by the exaggerated expression of the pro-inflammatory cytokine, interleukin (IL)-1&#946;, within the hippocampus during learning. Hippocampal microglia are the primary source of IL-1&#946; and the microglia in neonatally infected rats are "primed" by the infection into adulthood. In the second experiment, neonatally infected rats are more accurate on a Morris Water maze task following minimal training in adulthood, but have significantly impaired memory for a reversal platform location. In addition to improved accuracy, they have lower neural activation as measured by Arc protein expression within the dentate gyrus (DG) of the hippocampus. The next set of experiments assessed the effects of increasing hippocampal plasticity on immune signaling within the hippocampus. Following 7 weeks of environmental enrichment (EE), enriched rats had an attenuated pro-inflammatory response within the hippocampus in response to an in vivo peripheral immune challenge. The reduced immune response was specific to a subset of cytokines and chemokines and occurred only within the hippocampus and not adjacent cortical regions. Enrichment increased glial antigen expression within the DG as well. In another group of enriched rats, an ex vivo stimulation of isolated hippocampal microglia from EE rats demonstrated that the reduced microglial reactivity observed in vivo requires influence of other neural cell types on microglia phenotype, such that microglia within the DG of EE rats are smaller than controls. Taken together, these experiments define cellular and molecular mechanisms of hippocampal vulnerability and resilience as a function of interactions between the brain and the immune system.</p> / Dissertation

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 98-112).

Thesis (Ph. D.)--University of Virginia, 2005. / CD-ROM has .tiff and .mov files. Includes bibliographical references (leaves 129-132). Also available online through Digital Dissertations.