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The central nervous system controls many physiological processes including energy metabolism, immune response, reproduction, and development. In turn, hormones synthesized in and secreted by peripheral organs can be transported across the blood-brain barrier to modulate the development of the brain, the formation of new neurons, neural activity, behavior, and the secretion of brain-derived hormones. The central control of bone mass, mediated by the adipocyte-derived hormone leptin, has raised questions of whether the skeleton may signal back to the brain. In recent years, the Karsenty laboratory has uncovered the endocrine role of the bone-derived hormone osteocalcin. Through the use of a vast array of genetic tools, the Karsenty lab has discovered that osteocalcin is a potent regulator of glucose homeostasis, adaptation to exercise, energy metabolism, and male fertility. The multifunctional role of osteocalcin led us to hypothesize that it may act as a molecular means of communication between the skeleton and the brain. We asked whether osteocalcin could regulate brain development during embryogenesis and behavioral functions in adulthood. In addressing these questions, we observed that bone-derived osteocalcin crosses the blood-brain barrier, accumulates in discrete parts of the brain including the hippocampus, and binds to several neuronal populations to favor the synthesis of monoamine neurotransmitters (serotonin, dopamine, and norepinephrine), and to impede the synthesis of the inhibitory neurotransmitter, GABA. Osteocalcin-/- mice have increased anxiety and depression and impaired learning and memory when compared to WT littermates. We also uncovered that the absence of maternal osteocalcin during embryogenesis hinders brain development and causes defects in spatial learning and memory in the adult offspring. Upon characterizing the necessity of osteocalcin for brain development and cognitive function, we investigated whether bone health is a determinant of cognition, and whether osteocalcin may be sufficient to reverse age-related cognitive decline. In addressing the first question, we found that impairment in either bone formation or bone resorption negatively impacts both anxiety and memory. In addressing the second question, we found that osteocalcin is also necessary for the beneficial effect of young blood on cognitive functions. Finally, we observed reduced anxiety and improved memory in aged mice receiving osteocalcin peripherally. This action appears to require an increase in brain-derived neurotrophic factor levels in the hippocampus. Against the backdrop of our progressively aging population, it is important for future studies to determine whether osteocalcin may act therapeutically in humans to treat age-related cognitive decline. Additionally, to identify potential drug targets, it is important to fully characterize the molecular mechanism by which osteocalcin acts on neurons.

Yang, Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 222-244). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Awards and Presentations --- p.ii / Abstract in English --- p.iii / Abstract in Chinese --- p.vii / Table of Contents --- p.x / List of Tables --- p.xv / List of Figures --- p.xvii / List of Abbreviations --- p.xx / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Gliomas --- p.1 / Chapter 1.1.1 --- Oligodendroglial tumors (OTs) --- p.3 / Chapter 1.1.2 --- Glioblastoma multiforme (GBM) --- p.3 / Chapter 1.1.3 --- Molecular pathology of gliomas --- p.4 / Chapter 1.1.3.1 --- Genetic alterations in OTs --- p.4 / Chapter 1.1.3.2 --- Prognostic and predictive factors in OTs --- p.7 / Chapter 1.1.3.3 --- Genetic alterations in GBM --- p.8 / Chapter 1.1.3.4 --- Prognostic and predictive factors in GBM --- p.10 / Chapter 1.2 --- microRNA(miRNA) --- p.13 / Chapter 1.2.1 --- miRNA biogenesis and function --- p.13 / Chapter 1.2.2 --- miRNA involvement in cancer --- p.17 / Chapter 1.2.2.1 --- Dysregulation of miRNAs in human malignancies --- p.17 / Chapter 1.2.2.2 --- Function and potential application of miRNAs --- p.17 / Chapter 1.2.3 --- Role of miRNAs in glioma --- p.19 / Chapter 1.2.3.1 --- miRNAs in OTs --- p.19 / Chapter 1.2.3.2 --- miRNAs in GBM --- p.20 / Chapter 1.3 --- miR-137 --- p.30 / Chapter 1.3.1 --- Biology of miR-137 --- p.30 / Chapter 1.3.2 --- Role of miR-137 in carcinogenesis --- p.33 / Chapter 1.3.2.1 --- Deregulation of miR-137 in cancer --- p.33 / Chapter 1.3.2.2 --- Regulation of miR-137 expression in cancer --- p.33 / Chapter 1.3.2.3 --- Biological functions of miR-137 in cancer --- p.37 / Chapter 1.3.3 --- Role of miR-137 in differentiation and neurogenesis --- p.39 / Chapter CHAPTER 2 --- AIMS OF STUDY --- p.43 / Chapter CHARPTER 3 --- MATERIALS AND METHODS --- p.45 / Chapter 3.1 --- Tumor samples --- p.45 / Chapter 3.2 --- Cell lines and culture conditions --- p.48 / Chapter 3.3 --- Fluorescence in situ hybridization (FISH) --- p.49 / Chapter 3.4 --- Cell transfection --- p.52 / Chapter 3.4.1 --- Transfection of oligonucleotides --- p.52 / Chapter 3.4.1.1 --- Oligonucleotide preparation --- p.52 / Chapter 3.4.1.2 --- Optimization of transfection condition --- p.52 / Chapter 3.4.2 --- Cotransfection of plasmids and miRNA mimic --- p.53 / Chapter 3.4.2.1 --- Optimization of transfection condition --- p.53 / Chapter 3.4.2.2 --- Procedure of transfection --- p.54 / Chapter 3.5 --- Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) --- p.55 / Chapter 3.5.1 --- RNA extraction from frozen tissues and cell lines --- p.55 / Chapter 3.5.2 --- qRT-PCR for miR-137 --- p.56 / Chapter 3.5.3 --- qRT-PCR for CSE1L and ERBB4 transcripts --- p.57 / Chapter 3.6 --- 5-aza-2'-deoxycytidine (5-aza-dC) and Trichostatin A (TSA) treatment --- p.61 / Chapter 3.7 --- Western blotting --- p.62 / Chapter 3.7.1 --- Preparation of cell lysate --- p.62 / Chapter 3.7.2 --- Measurement of protein concentration --- p.62 / Chapter 3.7.3 --- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.63 / Chapter 3.7.4 --- Electroblotting of proteins --- p.67 / Chapter 3.7.5 --- Immunoblotting --- p.67 / Chapter 3.8 --- Dual-luciferase reporter assay --- p.70 / Chapter 3.8.1 --- Construction of reporter plasmids --- p.70 / Chapter 3.8.1.1 --- Experimental outline --- p.70 / Chapter 3.8.1.2 --- PCR Amplification of MREs --- p.70 / Chapter 3.8.1.3 --- TA cloning --- p.71 / Chapter 3.8.1.4 --- Transformation --- p.72 / Chapter 3.8.1.5 --- Blue/white screening and validation of recombinants --- p.72 / Chapter 3.8.1.6 --- Subcloning of 3'UTR fragments into pMIR-reproter vector --- p.73 / Chapter 3.8.2 --- Site-directed mutagenesis --- p.74 / Chapter 3.8.3 --- Plasmid and miRNA mimic cotransfection --- p.76 / Chapter 3.8.4 --- Determination of luciferase activity --- p.76 / Chapter 3.9 --- Functional assays : --- p.79 / Chapter 3.9.1 --- Cell growth and proliferation assay --- p.79 / Chapter 3.9.1.1 --- "3-(4，5-Dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay" --- p.79 / Chapter 3.9.1.2 --- Cell counting --- p.80 / Chapter 3.9.1.3 --- 5-Bromo-2'-deoxyuridine (BrdU) incorporation assay --- p.80 / Chapter 3.9.2 --- Apoptosis assay --- p.82 / Chapter 3.9.3 --- Anchorage-independent growth assay --- p.82 / Chapter 3.9.4 --- Wound healing assay --- p.83 / Chapter 3.9.5 --- Matrigel invasion assay --- p.84 / Chapter 3.9.6 --- Cell differentiation assay --- p.85 / Chapter 3.10 --- Immunohistochemical analysis --- p.86 / Chapter 3.10.1 --- H&E staining --- p.86 / Chapter 3.10.2 --- Detection of Ki-67 expression --- p.87 / Chapter 3.10.3 --- Detection of CSE1L expression --- p.87 / Chapter 3.10.4 --- Scoring methods --- p.88 / Chapter 3.11 --- Bioinformatic analysis --- p.90 / Chapter 3.12 --- Statistical analysis --- p.92 / Chapter CHAPTER 4 --- RESULTS --- p.93 / Chapter 4.1 --- Expression of miR-137 in glioma cells and clinical significance --- p.93 / Chapter 4.1.1 --- Description of 36 OT samples --- p.93 / Chapter 4.1.2 --- miR-137 level in oligodendroglial tumors and glioma cells --- p.102 / Chapter 4.1.3 --- "Association of miR-137 expression with clinicopathological features, lp/19q status and Ki-67 expression" --- p.104 / Chapter 4.2 --- miR-137 levels in glioma cells after demethylation treatment --- p.113 / Chapter 4.3 --- Biological effects of miR-137 overexpression in glioma cells --- p.118 / Chapter 4.3.1 --- Cell growth --- p.118 / Chapter 4.3.1.1 --- Cell viability --- p.118 / Chapter 4.3.1.2 --- Cell number --- p.123 / Chapter 4.3.1.3 --- Cell cycle analysis : --- p.127 / Chapter 4.3.2 --- Anchorage-independent cell growth --- p.130 / Chapter 4.3.3 --- Cell apoptosis --- p.134 / Chapter 4.3.4 --- Cell motility --- p.136 / Chapter 4.3.5 --- Cell differentiation : --- p.142 / Chapter 4.4 --- Identification of miR-137 targets --- p.144 / Chapter 4.4.1 --- In silico prediction of potential miR-137 targets --- p.144 / Chapter 4.4.2 --- Experimental validation of miR-137 targets by dual-luciferase reporter assay --- p.147 / Chapter 4.4.3 --- "Expression of miR-137 candidate targets, CSE1L and ERBB4 in glioma cells" --- p.152 / Chapter 4.4.4 --- Effects of miR-137 on CSE1L transcript and protein levels --- p.154 / Chapter 4.5 --- Expression of CSE1L in OTs --- p.156 / Chapter 4.5.1 --- CSE1L expression in OTs by qRT-PCR and IHC --- p.156 / Chapter 4.5.2 --- Correlation of CSE1L expression with clinicopathological features --- p.165 / Chapter 4.6 --- Effects of CSE1L knockdown in glioma cells --- p.168 / Chapter 4.6.1 --- Cell growth --- p.170 / Chapter 4.6.1.1 --- Cell viability --- p.170 / Chapter 4.6.1.2 --- Cell number --- p.173 / Chapter 4.6.1.3 --- Cell cycle analysis --- p.176 / Chapter 4.6.2 --- Anchorage-independent cell growth --- p.179 / Chapter 4.6.3 --- Cell apoptosis --- p.182 / Chapter 4.6.4 --- Cell motility --- p.184 / Chapter CHAPTER 5 --- DISCUSSION --- p.190 / Chapter 5.1 --- Expression of miR-137 transcript level in OTs and glioma cell lines --- p.190 / Chapter 5.2 --- Association of miR-137 expression with OT clinical and molecular parameters --- p.192 / Chapter 5.3 --- Prognostic significance of clinical features and miR-137 expression in OTs --- p.194 / Chapter 5.4 --- Inactivation mechanisms of miR-137 in glioma --- p.196 / Chapter 5.5 --- Biological effects of miR-137 overexpression in glioma cells --- p.198 / Chapter 5.6 --- CSE1L is a novel miR-137 target in glioma --- p.200 / Chapter 5.7 --- Expression of CSE1L in glioma --- p.203 / Chapter 5.8 --- Intracellular distribution of CSElL in OTs --- p.206 / Chapter 5.9 --- Correlation of CSE1L expression with clinicopathological and molecular features in OTs --- p.208 / Chapter 5.10 --- CSE1L mediates effects of miR-137 in glioma cells --- p.210 / Chapter 5.11 --- Biological roles of CSE1L in glioma cells 226}0Ø. --- p.212 / Chapter 5.11.1 --- CSE1L in glioma cell proliferation --- p.212 / Chapter 5.11.2 --- CSE1L in glioma cell apoptosis --- p.213 / Chapter 5.11.3 --- CSE1L in glioma cell invasion --- p.215 / Chapter CHAPTER 6 --- CONCLUSIONS --- p.216 / Chapter CHAPTER 7 --- FUTURE STUDIES --- p.219 / Chapter 7.1 --- Expression Molecular mechanisms for miR-137 inactivation in glioma --- p.219 / Chapter 7.2 --- Identification of more miR-137 targets in glioma --- p.219 / Chapter 7.3 --- Role of miR-137 and CSE1L in drug-induced apoptosis in glioma --- p.220 / Chapter 7.4 --- Deciphering dysregulated and clinical relevant miRNAs in glioma --- p.220 / Chapter 7.5 --- Effects of miR-137 in vivo and the therapeutic potential in glioma treatment --- p.221 / REFERENCES --- p.222

Thesis (Ph. D.)--University of Wisconsin, 1954. / Typescript (carbon copy). eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves [53]-[60]).

The effects of a range of neuronal excitants were examined on the firing of central neurones of the cerebral cortex, ventrobasal thalamus, dentate gyrus and dorsal and ventral horns of the spinal cords of urethane anaesthetized rats. These responses were pharmacologically characterized on the basis of their susceptibilities to a number of antagonists and from these results, inferences were made concerning probable receptor mechanisms employed by the agonists. Throughout these experiments the technique of iontophoresis was found to be an ideal one for evaluating the effects of agonists and antagonists on single neurones. Neurones in the cortex, thalamus and Renshaw cells of the spinal cord were readily excited by acetylcholine. These responses were elicited also by both nicotinic and muscarinic cholinomimetics. Excitations produced by acetylcholine and acetyl-β-methylcholine were antagonized by atropine and those of acetylcholine and nicotinic agonists were blocked by nicotinic antagonists. The results may be interpreted as revealing a difference between excitatory cholinergic receptors in the rat and in the cat; the nature of these receptors is discussed. to The excitatory responses of ventrobasal thalamic neurones iontophoretically applied amino acids related to glutamate and aspartate could be blocked both by glutamate diethylester and α-aminoadipate. These two antagonists were found to possess different mechanisms of action however, as the ranking orders of susceptibility of the agonists differed for each antagonist. An analysis of these orders led to the proposal that more than one and possibly as many as three different receptors for the excitatory amino acids exist on central neurones. A number of additional compounds were tested for an evaluation of their antagonistic properties against the amino acid induced responses, and these results are discussed in light of possible steric requirements of the receptors. Granule cells of the dentate gyrus were excited by the amino acids and by their synaptic responses to stimulation of perforant path and commissural inputs. A differential effectiveness of glutamate diethylester and α-aminoadipate was suggestive that two distinct excitatory amino acid receptors, both of which appear to be of synaptic significance, coexist on the same neurones. The effects of octopamine were compared with those of catecholamines on neurones of the cortex and dorsal horn of the spinal cord. Both excitation and depression of neuronal firing was observed with octopamine and these responses appeared not to be correlated with those effected by the catecholamines. A further separation of the actions of octopamine and the catecholamines was evident when the amine induced responses were compared in the presence of the antagonists, propranolol and α-flupenthixol. These blocking compounds were effective in attenuating the effects of the catecholamines, but had no effect upon the octopamine induced changes in firing rate. The results suggest that receptors sensitive to octopamine and which appear to be pharmacologically distinct from those previously categorized as catecholamine receptors, may exist on central neurones of the rat. On the basis of the present findings, it was evident that when the technique of iontophoresis is combined with standard neurophysiological methods of identifying central neurones by their responses to synaptic stimulation, valuable information can be obtained concerning the nature of the synaptic transmitters employed by these cells. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Diagnostic Radiology / Doctoral / Doctor of Philosophy

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