Nitric Oxide Mediated Signal Transduction in Networks of Human ...

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Summary The complex mammalian central nervous system (CNS) undergoes sequential but partially overlapping cellular developmental processes that include cell proliferation, migration, differentiation and synaptogenesis. The gaseous messenger nitric oxide (NO) has been implicated to regulate each of these early neuronal developmental processes. However, it is not known whether NO signaling is involved in the development of human CNS. Here, I used human neuronal stem cells as a model of developing nerve cells to investigate the potential role of NO signaling in neuronal migration, differentiation and pre-synaptic vesicle release. In the first part, a model of human neuronal precursor cells was established from embryonic carcinoma stem cells (NT2 cells) as three-dimensional free-floating aggregate culture. Upon retinoic acid treatment the NT2 spherical aggregates generate cells that proliferate, migrate and differentiate into neuronal phenotypes which mimic cellular processes that occur during early development of CNS. Interestingly, the migrating cells and cells inside the spheres respond to exogenous stimulation with NO by synthesizing cyclic guanosine-monophosphate (cGMP), indicating that the NT2 spherical aggregates express functional NO/cGMP signaling. By employing small bioactive enzyme activators and inhibitors of neuronal nitric oxide synthase (nNOS), soluble guanylyl cyclase (sGC) and protein kinase G (PKG), I showed that the NO/cGMP/PKG signaling cascade is a positive regulator of the migration of human neuronal precursor cells. Moreover, antiproliferative effect of NO was observed at relatively higher concentration of a NO donor. A modest but none significant effect of NO/cGMP signaling on neuronal differentiation was also noted. In the second part, pre-synaptic maturation of human NT2 neurons were followed after the cells migrate out of NT2 spherical aggregates and form neuronal networks. Human NT2 neurons express increasing levels of the axonal marker (Tau) and typical pre-synaptic proteins (synapsin and synaptotagmin I) depending on the length of in vitro culture. By employing an antibody directed against the luminal domain of synaptotagmin I and the fluorescent dye (FM1-43), I showed that depolarized mature NT2 neurons display calcium-dependent exo-endocytotic synaptic vesicle recycling. NT2 neurons express nNOS and a functional NO receptor, sGC. Next, I examined whether NO signal transduction modulates synaptic vesicle turnover, a pathway that may contribute to activity dependent synaptic maturation in human model neurons. Two NO donors (sodium nitroprusside, and N-Ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino ethanamine)) and cGMP analog (8-Br-cGMP) enhanced synaptic vesicle turnover while a sGC inhibitor blocked the effect of NO donors. NO donors evoked vesicle exocytosis which was partially blocked by the sGC inhibitor. The activator of adenylyl cyclase, forskolin, and a cAMP analog induced synaptic vesicle recycling and exocytosis via a parallel acting protein kinase A pathway. These data suggest that 31
NO/cyclic nucleotide signaling pathways may facilitate pre-synaptic neurotransmitter release in human brain. Finally, I tested the presence and function of NO/cGMP signaling in fetal human neural progenitor cells (hNPCs). Fetal hNPCs were cultured as three-dimensional neurospheres that proliferate, migrate and differentiate into both neuronal and glial cells. The migrating cells from human neurosphere express functional sGC that synthesize increasing level of cGMP upon activation with NO. Application of enzyme inhibitors of sGC and PKG blocked the migration of cells out of human neurospheres. Inhibition of sGC can be rescued by a membrane permeable analog of cGMP. In gain of function experiments both NO donor and cGMP analog facilitate cell migration suggesting that NO-cGMP signaling positively regulates hNPCs migration. These results provide first experimental evidence for a role of NO/cGMP signal transduction as a regulator of cell migration during early development of the human brain. 32
Page 1: Fehlerhafte Abgabe! Original Artike
Page 4 and 5: Supervisor: Prof. Dr. Gerd Bicker D
Page 6 and 7: Publications: Tegenge, M. A., and B
Page 8 and 9: List of abbreviations AD, Alzheimer
Page 10 and 11: 1. Introduction 1.1. Nitric oxide s
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Page 14 and 15: 1994). In embryonic grasshopper NO
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Page 26 and 27: which were accompanied by different
Page 28 and 29: Breier, J. M., Gassmann, K., Kayser
Page 30 and 31: disturb differentiation of normal h
Page 32 and 33: 23-35. Knipp, S., and Bicker, G. (2
Page 34 and 35: Ogilvie, P., Schilling, K., Billing
Page 36 and 37: Silverman, R. B. (2009). Design of
Page 38 and 39: Yamazaki, M., Chiba, K., Mohri, T.,
Page 42 and 43: Zusammenfassung Das komplexe Zentra
Page 44 and 45: JOURNAL OF NEUROCHEMISTRY | 2009 |
Page 46 and 47: 1830 | M. A. Tegenge and G. Bicker
Page 58 and 59: JOURNAL OF NEUROCHEMISTRY | 2009 |
Page 60 and 61: 1436 | M. A. Tegenge et al. and Bic
Page 62 and 63: 1438 | M. A. Tegenge et al. (a) (b)
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Page 70 and 71: 1446 | M. A. Tegenge et al. Micheva
Page 72 and 73: Abstract Neuronal migration is a ce
Page 74 and 75: (8-Bromoguanosine-3’, 5’-cyclop
Page 76 and 77: Fig. 1 The antibody against nNOS re
Page 78 and 79: Fig. 3 Immunocytochemical character
Page 80 and 81: Fig. 5 NO donor and cell membrane p
Page 82 and 83: NO/cGMP/PKG signaling mediate the m
Page 84 and 85: Bredt, D. S. and Snyder, S. H. (199
Page 86: Declaration I herewith declare that

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