The role of adenosine as an endogenous neuromodulator is well established, but the mechanism(s) mediating the extensive modulatory and regulatory actions of adenosine have not yet been fully elucidated. In fact, although adenosine, through activation of adenosine A1 and A2A receptors, has been demonstrated as neuroprotective or neurodegenerative, respectively, little is known about the mechanism by which adenosine mediates these actions. In the hippocampus, essential physiological processes rely on adenosine signaling, including regulation of long-term potentiation (LTP) and long-term depression (LTD). Neuromodulation by adenosine is dominantly inhibitory in the hippocampus, mediated by the abundant and high-affinity adenosine A1 receptor. In ischemia and hypoxia, A1 receptor activation induces rapid synaptic depression which is mediated by multiple signaling pathways including the induction of excitatory AMPA glutamate receptor internalization, which inhibits synaptic transmission in the hippocampus. Considerable effort has been devoted to investigating the role of adenosine in ischemic stroke, due to the fact that in cerebral ischemia or hypoxia, extracellular levels of adenosine increase dramatically. This thesis explores the functional consequences of adenosine signaling in hypoxia and ischemia, which mediate GluA1 AMPA receptor subunit internalization. Three major serine/threonine protein phosphatases (PPs), PP1, PP2A, and PP2B are investigated and shown to mediate A1 receptor-mediated GluA1 internalization in hypoxic conditions in the rat hippocampus. Further experiments demonstrate the role of adenosine A2A receptors in potentiating hippocampal synaptic transmission in reperfusion by increasing GluA1 surface expression through increased phosphorylation of regulatory C-terminal phosphorylation sites of GluA1. The mechanism of extracellular adenosine regulation by equilibrative nucleoside transporters (ENTs) and casein kinase 2 (CK2) are examined and shown to interact in hypoxia/reperfusion experiments on hippocampal slices. Finally, using a pial vessel disruption (PVD) permanent focal cortical ischemia stroke model, experiments demonstrate increased adenosine tone in the hippocampus, which mediates increased adenosine-induced synaptic depression. CK2 inhibition was also neuroprotective after 20min hypoxia. This shows that adenosine tone is increased in the hippocampus after a small cortical stroke, implying a potential global effect of focal ischemia. Together, these studies further reveal the paramount role of adenosine as a neuromodulator in the hippocampus during neuronal insults, furthering our understanding of the mechanism of neuronal death in hypoxic and ischemic conditions.The role of adenosine as an endogenous neuromodulator is well established, but the mechanism(s) mediating the extensive modulatory and regulatory actions of adenosine have not yet been fully elucidated. In fact, although adenosine, through activation of adenosine A1 and A2A receptors, has been demonstrated as neuroprotective or neurodegenerative, respectively, little is known about the mechanism by which adenosine mediates these actions. In the hippocampus, essential physiological processes rely on adenosine signaling, including regulation of long-term potentiation (LTP) and long-term depression (LTD). Neuromodulation by adenosine is dominantly inhibitory in the hippocampus, mediated by the abundant and high-affinity adenosine A1 receptor. In ischemia and hypoxia, A1 receptor activation induces rapid synaptic depression which is mediated by multiple signaling pathways including the induction of excitatory AMPA glutamate receptor internalization, which inhibits synaptic transmission in the hippocampus. Considerable effort has been devoted to investigating the role of adenosine in ischemic stroke, due to the fact that in cerebral ischemia or hypoxia, extracellular levels of adenosine increase dramatically. This thesis explores the functional consequences of adenosine signaling in hypoxia and ischemia, which mediate GluA1 AMPA receptor subunit internalization. Three major serine/threonine protein phosphatases (PPs), PP1, PP2A, and PP2B are investigated and shown to mediate A1 receptor-mediated GluA1 internalization in hypoxic conditions in the rat hippocampus. Further experiments demonstrate the role of adenosine A2A receptors in potentiating hippocampal synaptic transmission in reperfusion by increasing GluA1 surface expression through increased phosphorylation of regulatory C-terminal phosphorylation sites of GluA1. The mechanism of extracellular adenosine regulation by equilibrative nucleoside transporters (ENTs) and casein kinase 2 (CK2) are examined and shown to interact in hypoxia/reperfusion experiments on hippocampal slices. Finally, using a pial vessel disruption (PVD) permanent focal cortical ischemia stroke model, experiments demonstrate increased adenosine tone in the hippocampus, which mediates increased adenosine-induced synaptic depression. CK2 inhibition was also neuroprotective after 20min hypoxia. This shows that adenosine tone is increased in the hippocampus after a small cortical stroke, implying a potential global effect of focal ischemia. Together, these studies further reveal the paramount role of adenosine as a neuromodulator in the hippocampus during neuronal insults, furthering our understanding of the mechanism of neuronal death in hypoxic and ischemic conditions.

Thesis (M.S.)--West Virginia University, 2008. / Title from document title page. Document formatted into pages; contains ix, 128 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 118-127).

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Thesis (M.S.)--West Virginia University, 2005. / Title from document title page. Document formatted into pages; contains vi, 90 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 79-90).

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2007. / On t.p. "2̳+̳" is superscript. Includes bibliographical references (leaves 137-155). Also available in electronic version.

Emerging evidence indicates that the dysregulation of protein ubiquitination plays a crucial role in aging-associated diseases. Smad-dependent canonical BMP signaling pathway is indispensable for osteoblastic bone formation, which could be disrupted by the ubiquitination and subsequent proteasomal degradation of Smad1/5, the key molecules for BMP signaling transduction. However, whether the dysregulation of Smad1/5 ubiquitination and disrupted BMP signaling pathway are responsible for the age-related bone formation reduction is still underexplored. Casein kinase-2 interacting protein-1 (CKIP-1), also known as Pleckstrin homology domain-containing family O member 1 (PLEKHO1), is a previously identified ubiquitination-related molecule that could specifically target the linker region between the WW domains of Smurf1 to promote the ubiquitination of Smad1/5. Here, we found an age-related increase in the expression of CKIP-1 in bone specimens from either fractured patients or aging rodents, which was associated with the age-related reduction in Smad-dependent BMP signaling and bone formation. By genetic approach, we demonstrated that loss of Ckip-1 in osteoblasts could promote the Smad-dependent BMP signaling and alleviated the age-related bone formation reduction. In addition, osteoblast-specific Smad1 overexpression had beneficial effect on bone formation during aging, which could be counteracted after overexpressing Ckip-1 within osteoblasts. By pharmacological approach, we showed that osteoblast-targeted CKIP-1 siRNA treatment could enhance Smad-dependent BMP signaling and promote bone formation in aging rodents. Taken together, it suggests that the increased CKIP-1 could suppress Smad-dependent BMP signaling to inhibit bone formation during aging, indicating the translational potential of targeting CKIP-1 in osteoblast as a novel bone anabolic strategy for reversing established osteoporosis during aging.

CK2α is a serine-threonine kinase that is involved in a large number of biological processes, including embryonic development, cancer, and cell proliferation. Recently, it has been found that mutations in CK2α results in a developmental condition known as Okur-Chung neurodevelopmental syndrome (OCNDS). This disorder commonly results in intellectual disability, congenital heart defects (CHDs), gross motor delay, and facial abnormalities. CK2α inhibition has so far primarily been studied in mice, through methods such as knockout, gene floxing, and CRISPR/Cas9 mutations. In this thesis, we provide a proof of principle that chemical inhibition of CK2 in Xenopus laevis embryos can induce a phenocopy similar to the heart phenotype of the CK2α knockout mouse model, and demonstrate the potential of Xenopus laevis as an animal model to study molecular mechanisms that may underlie OCNDS. Here we carefully examined whole embryos, sections stained with multiple antibodies, sections stained with hematoxylin and eosin, and assessment of proliferation and apoptosis rates. The phenotypes observed in the Xenopus laevis model were analyzed and compared to both the CK2α knockout mouse model and OCNDS patients. Results found commonalities among facial features, heart deformities, and muscle patterning between the animal models, which overlapped heavily with patient symptoms. Thus, this work has established Xenopus laevis treated with chemical inhibitors as an appropriate animal model for further characterization of the mechanisms that may underlie OCNDS. / 2023-11-18T00:00:00Z

CK2α is a serine-threonine kinase that is involved in a large number of biological processes, including embryonic development, cancer, and cell proliferation. Recently, it has been found that mutations in CK2α results in a developmental condition known as Okur-Chung neurodevelopmental syndrome (OCNDS). This disorder commonly results in intellectual disability, congenital heart defects (CHDs), gross motor delay, and facial abnormalities. CK2α inhibition has so far primarily been studied in mice, through methods such as knockout, gene floxing, and CRISPR/Cas9 mutations. In this thesis, we provide a proof of principle that chemical inhibition of CK2 in Xenopus laevis embryos can induce a phenocopy similar to the heart phenotype of the CK2α knockout mouse model, and demonstrate the potential of Xenopus laevis as an animal model to study molecular mechanisms that may underlie OCNDS. Here we carefully examined whole embryos, sections stained with multiple antibodies, sections stained with hematoxylin and eosin, and assessment of proliferation and apoptosis rates. The phenotypes observed in the Xenopus laevis model were analyzed and compared to both the CK2α knockout mouse model and OCNDS patients. Results found commonalities among facial features, heart deformities, and muscle patterning between the animal models, which overlapped heavily with patient symptoms. Thus, this work has established Xenopus laevis treated with chemical inhibitors as an appropriate animal model for further characterization of the mechanisms that may underlie OCNDS. / 2023-11-18T00:00:00Z

La transcription par l’ARN polymérase II (ARNP II) est un mécanisme promiscuitaire à l’origine d’évènements transcriptionnels hasardeux, qui génèrent continuellement des transcrits aberrants dans la cellule. L’organisation précise du matériel génétique sous forme de chromatine est cruciale pour améliorer la précision de l’ARNP II en orientant sa fonction pour limiter ses erreurs et empêcher cette transcription cryptique. La structure chromatinienne est très dynamique et plusieurs mécanismes moléculaires coopèrent afin de déstabiliser les nucléosomes en amont de l’ARNP II, pour autoriser la lecture de l’ADN, et de les reconstituer dans son sillage, pour maintenir l’organisation chromatinienne du génome. De façon intéressante, une fonction potentielle de la caséine kinase 2 (CK2) dans la dynamique de la chromatine est suggérée dans la littérature. CK2 est une protéine kinase essentielle, conservée chez les eucaryotes, et impliquée dans des processus cellulaires variés. Dans notre étude, nous explorons le rôle de CK2 dans les modulations de la chromatine associées à la transcription. Nous avons démontré que CK2 phosphoryle le chaperon d’histones Spt6, régulant ainsi sa stabilité et sa fonction d’organisateur chromatinien. L’inactivation de cette voie de régulation conduit à l’accumulation considérable de transcrits cryptiques provenant d’initiations opportunistes intragéniques sens et antisens. La phosphorylation de Spt6 par CK2 favorise le recyclage des histones H3/H4 en 3’ des régions codantes et participe ainsi à la conservation de la structure de la chromatine lors de la transcription et à la suppression de la transcription cryptique. Notre étude suggère en outre que les fonctions de CK2 dans la modulation de la chromatine et la précision transcriptionnelle pourraient s’étendre au-delà de la régulation de Spt6, via la modulation de facteurs tels que les complexes PAF ou FACT. Enfin, nous proposons que la suppression de la transcription cryptique par CK2 contribue à optimiser la transcription afin d’améliorer la réponse transcriptionnelle à des stress extérieurs. L’ensemble de notre étude montre que CK2 stimule la précision transcriptionnelle en régulant directement Spt6 et probablement d’autres facteurs impliqués dans le maintien co-transcriptionnel de la chromatine. Ce mécanisme est crucial pour préserver le programme d’expression du génome et favorise la plasticité et l’efficacité de la réponse transcriptionnelle aux signaux de stress, nécessaires à l’adaptation de la cellule à son environnement. / Transcription by RNA polymerase II (RNAPII) is pervasive and aberrant transcripts are permanently generated within cells. Precise and controlled genomic organization in chromatin structure is essential to improve RNAPII accuracy and prevent cryptic transcripts accumulation. Chromatin structure is highly dynamic during transcription, unfolded to give access to DNA and refolded back in the wake of RNAPII to prevent spurious transcription. Multiple mechanisms act together to make this process highly efficient. Casein Kinase 2 (CK2) is a protein kinase ubiquitously present among eukaryotes and implicated in various important cellular processes. Interestingly, a potential function of this kinase in chromatin dynamics through the regulation of chromatin factors has previously been suggested. In this study, we address the role of CK2 in chromatin modulations associated with transcription. We found that CK2 depletion from yeast cells results in an increase of histone turnover in 3’ of transcribed regions and spurious transcription from cryptic promoters. Interestingly, we demonstrate that CK2 modulates directly Spt6 histone chaperone stability and function. This regulation promotes histone recycling during transcription elongation and maintain chromatin organization within coding regions, thereby inhibiting cryptic intragenic and antisense transcription. Our study also suggests that CK2 suppression of spurious transcription extend beyond Spt6 regulation. Indeed, we describe that additional role of CK2 with respect to spurious transcription could be related to its regulation of RNAP II activity through CTD Ser2 phosphorylation. Chromatin regulators such as PAF complex and FACT could also be involved in this regulation process. Finally, we propose that CK2 suppression of spurious transcription is essential for transcriptional optimal and efficient responses to environmental signals. Altogether, our data highlights CK2 signaling pathway as a regulator of transcription accuracy by affecting the essential histone chaperone Spt6, and probably other factors directly involved in the transcriptional process. This mechanism is important to the suppression of cryptic transcription in steady state conditions but also seems to contribute to the fitness of an optimal cellular response to stress signals.

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