NG2-glia, a fourth major glial cell population, were shown to posses wide proliferation and differentiation potential in vitro and in vivo, therefore the aim of this study was to compare the rate of proliferation and differentiation potential of NG2-glia after different types of brain injuries, such as global and focal cerebral ischemia (GCI, FCI) or stab wound (SW), as well as during aging. Moreover, we aimed to determine the role of Sonic hedgehog (Shh) in NG2-glia proliferation/differentiation after FCI. We used transgenic mice, in which tamoxifen triggers the expression of red fluorescent protein (tdTomato) in NG2-glia and cells derived therefrom. Proliferation and differentiation potential of tdTomato+ cells in sham operated animals (controls) and those after injury were determined by immunohistochemistry employing antibodies against proliferating cell nuclear antigen and glial fibrillary acidic protein. FCI was induced by middle cerebral artery occlusion, GCI by carotid occlusion with hypotension and SW by sagittal cortical cut. Shh signaling in vivo was activated or inhibited by Smoothened agonist or Cyclopamine, respectively. Compared to controls, the proliferation rate of tdTomato+ cells was increased after all types of injuries, while it declined in aged mice (15-18- months-old) after...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Magdaléna Svobodová Supervisor: Assoc. Prof. Přemysl Mladěnka, Ph.D. Assoc. Prof. Maria da Glória Correia da Silva Queiroz, Ph.D. Title of diploma thesis: Microglia control adenosine A2A-receptor mediated astrogliosis In the central nervous system, astrocytes and microglia are the main cells coordinating the inflammatory response. During inflammation, dying or temporarily damaged cells release ATP, as a danger-associated signal molecule, that contributes to the induction of astrogliosis and promotes clearance of the debris by immune cells such as microglia. Adenosine that results from ATP metabolism also stimulates astrogliosis. However, the effects of adenosine on astrogliosis may be more complex, since it also modulates microglia phenotype and microglia have been shown to prevent excessive astroglial proliferation mediated by nucleotides. In this context, ATP and adenosine are assumed as relevant signalling molecules in the control of astrogliosis and its modulation by microglia. However, it is still unknown whether and how microglia modulate adenosine-mediated astrogliosis. The present study aims to clarify the impact of microglia in the control of adenosine-induced astrogliosis. Two...

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21015号 / 医博第4361号 / 新制||医||1028(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 高橋 淳, 教授 渡邉 大, 教授 宮本 享 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Primary cilia are sensory organelles that are important for initiating cell division in the brain, especially through sonic hedgehog (Shh) signaling. Several lines of evidence suggest that the mitogenic effect of Shh requires primary cilia. Proliferation initiated by Shh signaling plays key roles in brain development, in neurogenesis in the adult hippocampus, and in the generation of glial cells in response to cortical injury. In spite of the likely involvement of cilia in these events, little is known about their characteristics. Centrosomes, which are associated with primary cilia, also have multiple influences on the cell cycle, and they are important in assembling microtubules for the maintenance of the cell’s cytoskeleton and cilia. The cilia of terminally differentiated neurons have been previously examined with respect to length, incidence, and receptors present. However, almost nothing is known about primary cilia in stem cells, progenitors, or differentiated glial cells. Moreover, it is not known how the properties of cilia and centrosomes may vary with cell cycle or proliferative potential, in brain or other tissues. This dissertation focuses first on neurogenesis in the hippocampal subgranular zone (SGZ). The SGZ is one of the few brain regions in mammals that gives rise to a substantial number of new neurons throughout adulthood. The neuron lineage contains a progression of identifiable precursor cell types with different proliferation rates. This present study found that primary cilia were present in every cell type in the neuronal lineage in SGZ. Cilium length and incidence were positively correlated among these cell types. Ciliary levels of adenylyl cyclase type III (ACIII) levels relative to ADP-ribosylation factor-like protein 13b (Arl13b) was higher in neurons than in precursor cells and glia, and also changed with the cell cycle. G-protein coupled receptors, SstR3, MCHR1, and Gpr161 receptors were only found in neuronal cilia. The levels and distribution of three centrosomal proteins, γ-tubulin, pericentrin and cenexin in neurons was different from the distributions in precursors and glia. The second focus of study is glial responses to injury in the neocortex, which has been widely studied as an injury model. This study found that in the normal adult somatosensory cortex, primary cilia were present in astrocytes and polydendrocytes but not in microglia. Following injury, the incidence of primary cilia decreased in astrocytes. Also, a new cell type expressing GFAP, NG2 and Olig2 was seen 3 days following injury, but was not present in normal mice. The characteristics of primary cilia and centrosome described here suggest that in stem cells and progenitors their characteristics may be well suited for proliferation, whereas in neurons, the cilia and centrosomes are important for other sensory functions.

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The analysis of Gene Regulatory Networks (GRNs) is essential to understanding the complete process of embryo development. Elucidating every gene regulatory circuit from maternal regulatory inputs all the way to the activation of differentiation gene batteries is an important step in increasing our understanding of developmental biology. In this work I study the cis-regulatory architecture of a pigment cell differentiation gene, polyketide synthase (SpPks) in the sea urchin Strongylocentrotus purpuratus. SpPks encodes an enzyme that is responsible for the biosynthesis of the sea urchin pigment echinochrome in larval pigment cells. The analysis of the promoter of a differentiation gene will lead to identifying the direct upstream regulators and ultimately to elucidating the structure of the upstream gene regulatory network, which is mostly uncharacterized. From previous studies the transcription factors SpGcm and SpGatae are predicted to be positive regulators of SpPks. Here, I identify a minimal 1kb promoter region containing putative DNA-binding sites for both GCM and GATAE that is able to recapitulate the expression of SpPks. I further show by mutagenesis that a putative DNA-binding site for GCM located 1,179 base pairs upstream of the start of transcription is a direct target for the positive cis-regulation of SpPks. Quantitative analysis of the transcriptional regulatory function of the GCM-mutagenized construct suggests that GCM is not necessary for the start of SpPks transcription but is required for its maintenance. Several GATA E binding sites have been identified within the minimal promoter for SpPks by means of consensus sequence. My analysis suggests that GATA E may be a direct positive regulator and could potentially be required for the onset of transcription of SpPks, though further experimentation will be necessary to characterize the exact regulatory function of GATA E.

Purpose: The purpose of this study was to investigate the role of the vasculature and immune system in models of glaucoma. Vascular changes have been implicated in glaucoma. As well there is mounting evidence that the immune system plays a role in the disease. It is my hypothesis that the vasculature and immune system play a role in the retinal response to injury in models of glaucoma. Methods: Immunohistochemistry, in vivo retinal imaging (Bright field, fluorescent, optical coherence tomography), Slit2 injections and Evan’s blue labeling were used to investigate vascular and immune changes associated with retinal ganglion cell death after optic nerve crush up to 28 days after injury. Histology, immunohistochemistry, and intravascular labeling were utilized to investigate the role of the vascular degeneration and the systemic immune response to elevated intraocular pressure in 8-16 week old AP-2β Neural Crest Cell Knockout (AP-2β NCC KO) mice. Results: The vascular and immune responses to optic nerve crush were not found to play a significant role in the response to retinal ganglion cell death. Conversely the role of vascular degeneration and immune cell recruitment to the retinas of AP-2β NCC KO mice demonstrated that these factors played a significant role in the retinal response to injury. Conclusion: The vasculature and immune system play a varied role in the response to retinal injury and neurodegeneration depending on the model being studied. The vascular and immune changes were of minimal significance in acute optic nerve crush injury. On the other hand, the chronic injury associated with elevated intraocular pressure in AP-2β NCC KO mice was associated with significant vascular degeneration and systemic immune cell infiltration. / Thesis / Doctor of Philosophy (PhD)