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Modulation of the Progenitor Cell and Homeostatic Capacities of Müller Glia Cells in Retina

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Modulation of the Progenitor Cell and Homeostatic Capacities of Müller Glia Cells in Retina Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1201 Modulation of the Progenitor Cell and Homeostatic Capacities of Müller Glia Cells in Retina Focus on α2-Adrenergic and Endothelin Receptor Signaling Systems MOHAMMAD HARUN-OR-RASHID ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6206 ISBN 978-91-554-9527-5 UPPSALA urn:nbn:se:uu:diva-281569 2016 Dissertation presented at Uppsala University to be publicly examined in B21, BMC, Husagatan 03, Uppsala, Thursday, 19 May 2016 at 09:15 for the degree of Doctor of Philosophy (Faculty of Medicine). The examination will be conducted in English. Faculty examiner: Docent Per Ekström (Lund University). Abstract Harun-Or-Rashid, M. 2016. Modulation of the Progenitor Cell and Homeostatic Capacities of Müller Glia Cells in Retina. Focus on α2-Adrenergic and Endothelin Receptor Signaling Systems. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1201. 73 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-554-9527-5. Müller cells are major glial cells in the retina and have a broad range of functions that are vital for the retinal neurons. During retinal injury gliotic response either leads to Müller cell dedifferentiation and formation of a retinal progenitor or to maintenance of mature Müller cell functions. The overall aim of this thesis was to investigate the intra- and extracellular signaling of Müller cells, to understand how Müller cells communicate during an injury and how their properties can be regulated after injury. Focus has been on the α2-adrenergic receptor (α2-ADR) and endothelin receptor (EDNR)-induced modulation of Müller cell-properties after injury. The results show that α2-ADR stimulation by brimonidine (BMD) triggers Src-kinase mediated ligand-dependent and ligand-independent transactivation of epidermal growth factor receptor (EGFR) in both chicken and human Müller cells. The effects of this transactivation in injured retina attenuate injury-induced activation and dedifferentiation of Müller cells by attenuating injury-induced ERK signaling. The attenuation was concomitant with a synergistic up-regulation of negative ERK- and RTK-feedback regulators during injury. The data suggest that adrenergic stress-signals modulate glial responses during retinal injury and that α2- ADR pharmacology can be used to modulate glial injury-response. We studied the effects of this attenuation of Müller cell dedifferentiation on injured retina from the perspective of neuroprotection. We analyzed retinal ganglion cell (RGC) survival after α2-ADR stimulation of excitotoxically injured chicken retina and our results show that α2-ADR stimulation protects RGCs against the excitotoxic injury. We propose that α2-ADR-induced protection of RGCs in injured retina is due to enhancing the attenuation of the glial injury response and to sustaining mature glial functions. Moreover, we studied endothelin-induced intracellular signaling in Müller cells and our results show that stimulation of EDNRB transactivates EGFR in Müller cells in a similar way as seen after α2-ADR stimulation. These results outline a mechanism of how injury-induced endothelins may modulate the gliotic responses of Müller cells. The results obtained in this thesis are pivotal and provide new insights into glial functions, thereby uncovering possibilities to target Müller cells by designing neuroprotective treatments of retinal degenerative diseases or acute retinal injury. Keywords: Alpha2-adrenergic receptor, Brimonidine, Brn3a, Dedifferentiation, Endothelin, EGFR, ERK1/2, Neuroprotection, NMDA, MIO-M1 human Müller cell, Müller cells, Retina, Retinal ganglion cells, Src-kinase, Transactivation. Mohammad Harun-Or-Rashid, , Department of Neuroscience, Developmental Neuroscience, Box 593, Uppsala University, SE-751 24 Uppsala, Sweden. © Mohammad Harun-Or-Rashid 2016 ISSN 1651-6206 ISBN 978-91-554-9527-5 urn:nbn:se:uu:diva-281569 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-281569) Dedication To my family “Imagination is more important than knowledge” Albert Einstein Cover: Isodensity map of Brn3a+ RGCs on whole-mount post-natal day 4 chicken retinas. (Left) Saline-treated retina, (Middle) N-methyl-D-aspartate-treated retina, (Right) brimonidine pretreated and N-methyl-D-aspartate-treated retina. (Image: Caridad Galindo-Romero) List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Harun-Or-Rashid M, Lindqvist N, and Hallbook F. (2014). Transactivation of EGF Receptors in Chicken Muller Cells by α2A-Adrenergic Receptors Stimulated by Brimonidine. Invest Ophthalmol Vis Sci. 29;55(6):3385-94. II Harun-Or-Rashid M, and Hallbook F. (2016). Alpha2- Adrenergic Agonist Brimonidine Stimulates ERK1/2 and AKT Signaling via Transactivation of EGF Receptors in MIO-M1 Human Müller Cells. (Manuscript). III Harun-Or-Rashid M, Díaz-DelCastillo M, Galindo-Romero C, and Hallböök F. (2015). Alpha2-Adrenergic-Agonist Brimonidine Stimulates Negative Feedback and Attenuates Injury-Induced Phospho-ERK and Dedifferentiation of Chicken Müller Cells. Invest Ophthalmol Vis Sci. 56(10):5933-45. IV Galindo-Romero C., Harun-Or-Rashid M, Jiménez-López M, Vidal-Sanz M, Agudo-Barriuso M and Hallböök F. (2016). Neuroprotection by α2-Adrenergic Receptor Stimulation after Excitotoxic Injury of Chicken Retinal Ganglion Cells: A Population Study. (Submitted to The Journal of Comparative Neurology). V Harun-Or-Rashid M., Konjusha D., Galindo-Romero C., and Hallböök F. (2016). The Endothelin B Receptor Transactivates Epidermal Growth Factor Receptors in Primary Chicken Müller Cells and in MIO-M1 Human Müller Cells. (Under review in Molecular and Cellular Neuroscience). Reprints were made with permission from the respective publishers. Contents Preface ........................................................................................................... 11 Introduction ................................................................................................... 13 Introduction to the retina .......................................................................... 13 Retinal cell-types and function of the retina ........................................ 13 Structural organization of the retina .................................................... 14 Cells commonly affected by retinal diseases and injuries ................... 15 Excitotoxic retinal injury model ............................................................... 18 Mechanisms of cell death ......................................................................... 18 Mechanisms of RGC death .................................................................. 19 Glial cells and their role in the central nervous system (CNS) ................ 20 Müller cells ............................................................................................... 21 Müller cells in neuroprotection ............................................................ 23 Müller cells in regeneration ................................................................. 24 Factors that stimulate Müller cells in the retina ................................... 25 Brimonidine and its effects in the retina ................................................... 27 Alpha2-adrenergic receptors ..................................................................... 27 Endothelins and their effects in the retina ................................................ 29 Endothelin receptors ................................................................................. 29 Epidermal growth factor receptors ........................................................... 30 Epidermal growth factor receptor signaling ........................................ 31 Regulation of epidermal growth factor receptor signaling .................. 35 Aims of the thesis .......................................................................................... 38 The specific aims were: ....................................................................... 38 Results and discussion ................................................................................... 39 Paper I ....................................................................................................... 39 Paper II ..................................................................................................... 40 Paper III .................................................................................................... 41 Paper IV .................................................................................................... 43 Paper V ..................................................................................................... 45 Conclusions and perspectives ........................................................................ 48 Future prospects ............................................................................................ 50 Materials and methods .................................................................................. 52 Animals ..................................................................................................... 52 Intraocular injection .................................................................................. 52 Müller cell cultures ................................................................................... 52 Immunohistochemistry and Cytochemistry .............................................. 54 Microscopy ..............................................................................................
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