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

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.

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 ...... 54 Quantitative Reverse Transcription-PCR ...... 55 Western blot analysis ...... 56 Statistical analysis ...... 56 Acknowledgements ...... 57 References ...... 58

Abbreviations

AC Amacrine cell BP Bipolar cell BMD Brimonidine BDNF Brain-derived neurotrophic factor Brn3a Brain specific homeobox/POU domain protein 3a CASH1 CASH1-basic helix-loop-helix protein Chx10 Chx10-homeobox protein CNTF Ciliary neurotrophic factor CRALBP Cellular retinaldehyde-binding protein DUSP Dual-specificity phosphatase E Embryonic day EDN Endothelin EDNR Endothelin receptor EGFR Epidermal growth factor receptor FGF Fibroblast growth factor Foxn4 Forkhead box N4 GC Ganglion cell layer GDNF Glial cell-derived neurotrophic factor GCL Ganglion cell GPCR G protein-coupled receptor Grb2 Growth factor receptor-bound protein 2 GS Glutamine Synthetase HC Horizontal cell HB-EGF Heparin binding-epidermal growth factor INL Inner nuclear layer IOP Intraocular pressure IPL Inner plexiform layer IR Immunoreactivity JNK c-Jun N-terminal kinase MAPK Mitogen-activated protein kinase MC Müller cell MEK MAP/ERK-kinase MIO-M1 Moorfields/institute of ophthalmology-Müller 1 MIG6 Mitogen-inducing gene 6 MKK MAP-kinase-kinase MKP Mitogen-activated protein kinase phosphatase

MMP Matrix metalloproteinase NFL Nerve fiber layer NGF Nerve growth factor NMDA N-methyl-D-aspartate OLM Outer limiting membrane ONL Outer nuclear layer OPL Outer plexiform layer Pax6 Paired box gene 6 PDK Phosphoinositide-dependent kinase PEC Pigment epithelial cell P-ERK1/2 Phospho-Extracellular signal-regulated kinase PI3K Phosphoinositide 3-kinase PKC Protein kinase C PR Photoreceptor p38 p38 Mitogen-activated protein kinase p75NTR p75 neurotrophin receptor QRT-PCR Quantitative Reverse transcription-PCR Ras Ras-GTPase Raf Raf-kinase RGC Retinal ganglion cell RPE Retinal pigment epithelium Sox2 Sex determining region Y-box containing gene 2 SPRY2 Sprouty RTK signaling antagonist 2 SOS Son of sevenless protein Y Tyrosine α2-ADR Alpha2-adrenergic receptor

Preface

It has been a long time that I have started my journey as a PhD student. Dur- ing this period of time, I have had the great opportunity to be involved in scientific research. It has been exciting, informative and a very challenging experience. I am grateful for all the experiences and knowledge that I have obtained. I started my scientific research in Finn Hallböök’s lab with a side project related with chicken comb development, which was finally resulted as a scientific publication (Boije et al., 2012). In addition to this project, I was involved in several other projects, which also resulted in published arti- cles. I investigated eomesodermin expression in the embryonic chicken comb and characterized duplex comb phenotype (Dorshorst et al., 2015). I also studied association between vascular endothelia growth factor receptor- 2 and VE-cadherin in Shb-knockout lung endothelial cells (Zang et al., 2013). Moreover, I studied the expression of carnitine palmitoyl-CoA transferase-1B in two chicken lines selected for high and low body weight (Ka et al., 2013). Although those projects are not included in my thesis but involvement on those projects expanded my knowledge in research and helped me for critical thinking in my PhD project.

Introduction

Understanding neural regeneration of the retina is fundamental for developing therapeutic approaches in different retinal degenerative diseases including macular degeneration and glaucoma. One of the keys is understanding the activation and signaling of Müller cells, glial cells that have unique regenerative ability, in response to injury. Even though it is both intellectually challenging and attractive, the idea that endogenous neurogenesis in an injured retina is always beneficial can be questioned. If neurogenesis is fa- vored on the expense of glial homeostatic functions during injury or disease, the long-term outcome may be worse than if neurogenesis was restricted. This antagonistic relationship between neurogenic potential and glial homeostasis is explained by the fact that the same cell type has both capacities in the adult retina: the Müller cell. In this context, it is essential to understand which cellular and molecular events lead to degeneration of retina, particularly the cellular mechanisms. Müller cells are regulated by critical cellular signaling pathways including alpha2-adrenergic receptor, epidermal growth factor receptor and endothelin receptor signaling pathways. In my thesis, I use excitotoxic retinal damage to understand these signaling pathways and answer the following questions: (1) Does alpha2-adrenergic receptor signaling transactivate epidermal growth factor receptors in Müller cells? (Paper I and II) (2) Does alpha2-adrenergic receptor signaling stimulate negative feedback and attenuate injury-induced Müller cell dedifferentiation? (Paper III) (3) Does alpha2-adrenergic receptor signaling protect retinal ganglion cells against the excitotoxic injury? (Paper IV) (4) Does injury-induced endothelin-signlaing modulate the Müller cell response by transactivating epidermal growth factor receptors? (Paper V)

Introduction to the retina Retinal cell-types and function of the retina The visual sense, found in nearly all vertebrates and many invertebrates, allows organisms to perceive light reflected off of objects in the environment. The retina, a major neural network of the eye, is fundamental to vision, because it receives, processes, and converts incoming light stimuli into neural signals (figure. 1). There are five different types of neurons and one

13 principal glia cell in the retina, all of which are generated from multi-potent retinal progenitor cells. The first cells to differentiate from the progenitor pool are retinal ganglion cells (RGCs), followed by cone photoreceptor cells, horizontal cells and amacrine cells. The late-born cell types are bipolar cells, rod photoreceptor cells and Müller cells (MCs) (Masland, 2001). Both types of photoreceptor cells capture light rays and convert the incoming photons into neural signals. Cone photoreceptor cells are involved in color and normal lighting vision, whereas rod photoreceptor cells are responsible for vision under dim conditions. The neural signals from photoreceptors undergo processing by all classes of neurons of the retina. Processing may enhance or silence the signals depending on the strength of the visual input and how the various neurons respond to and integrate their signals. The photoreceptors connect to bipolar cells to transmit the neural signals, while the interneuron horizontal cells use inhibitory synapses to modulate the output signals from photoreceptors. The bipolar cells then transmit the signals to retinal ganglion cells, whereas amacrine cells, which act as inhibitory interneurons, modulate the signals from bipolar cells by forming inhibitory synapses to retinal ganglion cells and modulating neurotransmitter release from bipolar cells. The neural signals are transmitted along the axons of retinal ganglion cells, which form the optic nerve. In humans, the signals are partly transmitted to lateral geniculate nucleus (LGN) and then to the primary visual cortex where the signals are processed and read as images (Masland, 2001). The neural signals are also transmitted to other regions of the brain, namely the pretectal nucleus, suprachiasmatic nucleus and superior colliculus, which is associated with balance, pupillary reflex control, regulation of circadian clock, and co- ordination of head and eye movements (Berson et al., 2002).

Structural organization of the retina The vertebrate retina is comprised of two main layered structures, the neural retina, which contains all neurons and glia cells, and the retinal pigment epithelium, which is a monolayer of pigmented cells. The neural retina has a well-organized structure, where different cell types are positioned in distinct laminas. The cell bodies of retinal neurons and glia are organized into three nuclear layers (ganglion cell layer, GCL; inner nuclear layer, INL; outer nuclear layer, ONL), and most of the synapses, axons and dendrites are con- fined to the plexiform layers (inner plexiform layer, IPL; outer plexiform layer (OPL). The axons of the ganglion cells on their way to the optic nerve head are situated on the nerve fiber layer (NFL). The photoreceptor cells, cones and rods form the ONL; horizontal cells, bipolar cells, and amacrine cells are found in the INL; the nuclei of Müller cells are also located in INL but their somata span the entire thickness of the retina; ganglion cells and displaced amacrine cells are located in GCL (Cepko et al., 1996; Masland, 2001).

Figure 1: Schematic representation of vertebrate retina. RPE, retinal pigment epithelium; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer, NFL, nerve fiber layer; PEC, pigment epithelial cell; PRs, photoreceptors; HC, horizontal cell; BP, bipolar cell; MC, Müller cell; AC, amacrine cell; RGC, retinal ganglion cell.

Cells commonly affected by retinal diseases and injuries Various environmental, pathological and genetic risk factors make the retina vulnerable to a variety of diseases and injuries, leading to vision loss and blindness. Retinal diseases and injuries commonly affect the photoreceptors and retinal ganglion cells.

Diseases of photoreceptor cells A number of retinal diseases such as macular degeneration, retinitis pigmentosa, and retinal detachments lead to photoreceptors death in the retina. Macular degeneration, also known as age-related macular degeneration (AMD), is a common cause of blindness in elderly people. It primarily af-

15 fects the macular region of the retina, which contains a high density of cone photoreceptors, and gradually spreads to peripheral region of retina (Thornton et al., 2005). Retinitis pigmentosa leads to progressive degeneration of rod photoreceptor cells due to inherited mutation of rod photoreceptor genes in the retina. It is the leading cause of inherited blindness, and around one in every four thousands individuals experience this disease within their lifetime (Hamel, 2006; Parmeggiani, 2011). Retinal detachment causes the accumulation of fluid between the retina and the retinal pigment epithelium, which causes degeneration of photoreceptors. People with severe myopia, complications from cataracts surgery and diabetic retinopathy are particularly prone to retinal detachment (Haug and Bhisitkul, 2012; Mattioli et al., 2009).

Diseases of retinal ganglion cells RGCs are primarily affected by glaucoma, retinal ischemia and retinal trauma. Glaucoma is an optic neuropathy, which leads to RGC death and one of the most common causes of blindness in worldwide. The underlying cause of glaucoma remains elusive, but elevation of intraocular pressure (IOP), increasing age, and family history of glaucoma are considered to be major risk factors for glaucoma (Leske, 1983; Quigley and Broman, 2006). Retinal ischemia is defined as a pathological condition involving inadequate blood supply to retina. It is thought that retinal ischemia results in thinning of NFL containing the RGC axons, which consequently leads to RGC death. Retinal trauma is characterized as either a blunt force or penetrating injury to the retina, which can be caused by a number of unusual injuries that lead to death of RGCs. (Lu and Zang, 1997).

Retinal injuries Retinal injury is a common cause of post-traumatic vision loss. The outcomes of different retinal injuries are very poor due to retinal cell death, retinal scarring and a failure of functional tissue regeneration (Wong et al., 2000). Generally retinal injuries are categorized into two types: closed globe injury and open globe injury (Blanch et al., 2012). Two common closed globe injuries are ‘Commotio retinae’ and blast injuries. ‘Commotio retinae’ is defined by gray-white opacification of the retina after blunt ocular trauma, which usually settles after few days to months. Vision loss may occur transi- ently or permanently when the macula is affected (Eagling, 1974). Blast injury is another type of closed globe injury, which is usually caused by the detonation of an exclusive blast shockwave that leads to degeneration of RGC axon and RGC apoptosis (Chen et al., 2003; Petras et al., 1997).

Experimental retinal injuries There are several types of open globe retinal injuries that can be induced in animal models including incision retinal injury, optic nerve crush injury,

16 optic nerve transection injury, retinal detachment injury, intravitreal N- methyl-D-aspartate (NMDA) injury, retinal ischemia injury, intraocular pressure (IOP) injury, and retinal light injury (Blanch et al., 2012). Retinal incision injuries can be induced by incision through the sclera, choroid and the retina, which affects the cells of all retinal layers near the incision area (Turner et al., 1986). Optic nerve crush injury can be induced by crushing the optic nerve using a pair of calibrated forceps, leading to RGC death. The severity of injury depends on the length of optic nerve from globe. Proximal injury, about 0.5 mm from the globe, causes more aggres- sive RGC death than distal injury (>8 mm from globe) (Berkelaar et al., 1994). Optic nerve transection injury is done by axotomized the axon of RGC, which leads to RGC death (Berkelaar et al., 1994). Retinal detachment can be induced by sub-retinal injection of balanced salt solution or hyaluron- ic acid with or without vitrectomy and lensectomy, which causes degeneration of photoreceptors, horizontal cells, and RGCs (Fisher et al., 2005; Fon- tainhas and Townes-Anderson, 2011). Intravitreal injection of NMDA leads to dose-dependent loss of RGCs and amacrine cells (Ohta et al., 2008). Retinal ischemia injury causes RGC death, which can be induced by ligation of ophthalmic vessels for a fixed period of time (Lonngren et al., 2006). The severity of injury depends on the period of ligation time. IOP injury leads to RGC death, which is commonly induced by cauterization of the episcleral veins using a laser. The cauterization reduces the liquid outflow from the eye, which consequently increases IOP. IOP can also be increased by injection of hypertonic saline solution or laser scarring of the aqueous outflow pathways (Morrison, 2005). Retinal light injury causes degeneration of photoreceptors, which can be induced by intense bright light or strong monochromatic light. Light-induced retinal injury can occur through three fundamental processes: photochemical, photothermal and photomechanical (Glickman, 2002; Wu et al., 2006). Pho- tochemical injury is the most common cause of light-induced phototoxicity. It occurs when photons are absorbed by a chromophore, leading to the generation of an electronically excited state of that chromophore molecule, which consequently undergoes either chemical transformation or interacts with other molecules, causing chemical changes of both interacting molecules or transferring the excitation energy to other molecules, ultimately damage the tissue (Wu et al., 2006). Photothermal injury occurs when the rate of photon energy deposition by thermal deactivation is quicker than thermal diffusion, which results in rising of temperature of the exposed tissue, leading to thermal damage (Delori et al., 2007). Photomechanical damage occurs when photon energy is deposited quicker than mechanical relaxation of tissue can occur, which results in a generation of thermo elastic pressure wave, and exposed tissue is disrupted by shear forces (Delori et al., 2007).

17 Excitotoxic retinal injury model There are different types of retinal injury models to study RGC death in experimental animals. The excitotoxic retinal injury is a widely used model to study the molecular mechanism of RGC death and its protection by different neuroprotective agents. The injury method that has been used in this thesis to induce RGC death in chicken retina is NMDA-mediated excitotoxicity (paper IV). NMDA is a synthetic glutamate analogue that acts on NMDA-type glutamate receptors and triggers excitotoxicity of neurons by hyperpolarization of its receptors, which leads to a massive ca2+ influx into the cells and consequently stimulates pro-apoptotic signaling cascades (Shen et al., 2006). In the retina, RGCs and amacrine cells preferentially express NMDA-type glutamate receptors. Therefore, NMDA-induced excitotoxicity particularly affects RGCs and amacrine cells. NMDA-induced excitotoxicity is concentration dependent. Twenty four hours after intraocular injection of 4.4 µg NMDA in mouse retina approximately kills half of the cells in the GCL, which comprises both RGC and displaced amacrine cells (Li et al., 1999). In post-natal chicken retina around 50% of RGCs die 7 days after intraocular injection of 10 µg NMDA (paper IV). A single lower dose of NMDA can damage a large number of cells in the GCL and the IPL without affecting the other retinal layers (Goldblum and Mittag, 2002).

Mechanisms of cell death The most common mechanisms of cell death in all multicellular organisms are necrosis and apoptosis. In addition ‘autophagy’ is another form of cell death mechanism, in which cells digest their own organelles and macromol- ecules for energy production during starving and growth factor deprivation (Hotchkiss et al., 2009). Necrosis occurs after tissue damage or injury that leads to swelling of the cell and its organelles and rupturing of plasma membrane, which cause the cell contents to spill out into the extracellular space. Necrosis often leads to inflammation. In contrast, apoptosis is a programmed cell death pathway. The characteristic features of apoptosis are shrinkage of the cell with condensation of the cytoplasm and the nucleus, nuclear frag- mentation and the formation of plasma-membrane blebs (Kroemer et al., 2009). Apoptosis is an evolutionary conserved cell death mechanism that regulates the number of cells during normal development of multicellular organisms. For example, the final RGC number in functional retina is adjust- ed by apoptosis of excess RGCs produced in the immature retina (Galli- Resta and Ensini, 1996). Apoptosis can be induced in absence of survival factors, the presence of either intrinsic or extrinsic death factors, or both (Pettmann and Henderson, 1998).

18 There are two major molecular pathways that have been identified in apoptosis: the death-receptor pathway and the mitochondrial pathway (Hotchkiss et al., 2009). The members of the tumor necrosis factor (TNF) superfamily are associated with the activation of the death-receptor pathway. The members of TNF superfamily bind to TNF-receptors (cell surface death receptors) thereby initiating the death-inducing signaling complex. Accumu- lation of this complex triggers the catalytic activity of caspase 8, which is a central mediator of apoptosis. The members of the BCL2 family regulate the mitochondrial apoptotic pathway. The BCL2 family members control mitochondrial outer membrane permeabilization and can act either as pro- apoptotic or as anti-apoptotic (Chao and Korsmeyer, 1998). The Reactive oxygen species, DNA damage response, unfolded protein response and growth factor deprivation initiate the mitochondrial apoptotic pathway, which consequently leads to increased outer mitochondrial permeability, resulting in the release of pro-apoptotic proteins such as cytochrome c into the cytosol. It is reported that pro-apoptotic protein Bax facilitates the release of cytochrome c into the cytosol (Nomura et al., 1999). Cytochrome c binds to apoptotic protease-activating factor 1(Apaf-1), leading to activation of caspase 9. Thus, activated caspase 8 from the death receptor pathway and caspase 9 from the mitochondrial pathway subsequently stimulate a cascade of caspases including caspase 3 that demolish the cell by cleaving numerous proteins and activating DNases (Hotchkiss et al., 2009; Strasser, 2005).

Mechanisms of RGC death RGC death is the common feature of glaucoma and the cause of RGC death in glaucoma is not fully understood. Several mechanisms have been suggested for RGC death in glaucomatous retina. For example a mechanical theory of increased IOP explains RGC death in glaucomatous retina. Increased IOP leads to optic nerve cupping and excavation, which consequently blocks the retrograde RGC axonal transport from its axonal terminals to the cell bodies of the neurons (Lampert et al., 1968; Minckler et al., 1976). The retrograde transport of neurotrophic factors from the brain is essential for RGC survival. Thus, the shrinkage of RGC axons reduces axonal transport of neurotrophic factors causing RGC death by trophic insufficiency. Experimental glaucomas in rats support the theory that increased IOP reduces the retrograde transport of brain-derived neurotrophic factor (BDNF) and its TrkB receptor (Pease et al., 2000; Quigley et al., 2000). A vascular mechanism in which ischemia or chronic hypoxia has also been proposed to explain RGC death in glaucomatous retina (Flammer, 1994; Osborne et al., 2001; Tezel and Wax, 2004). Recent findings in human and monkey eyes support the vascular mechanism. Immunohistochemical analysis in postmortem human glaucomatous eyes has shown that the oxygen-regulated transcription activator, hypoxia-inducible factor-1 (HIF-1)

19 alpha was up-regulated in the retina and optic nerve head of glaucomatous eyes (Tezel and Wax, 2004). Experimentally induced chronic ischemia in monkey eyes elicits optic nerve damage, which is IOP independent and consequently leads to RGC axon loss and RGC death (Cioffi et al., 2004). The oxidative stress- and reactive oxygen species-induced mitochondrial dysfunction has also been suggested to explain apoptotic RGC loss in glaucomatous retina (Abu-Amero et al., 2006; Tatton et al., 2001). Mitochondrial dysfunction triggers apoptotic cell death via stimulation of the mitochondrial permeability transition pore complex and consequent caspase activation. Mitochondrial dysfunction was shown to have a central role in animals and human glaucoma (Tatton et al., 2001). The inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and nitric oxide have also been suggested to involve in RGC death in human glaucomatous retina (Neufeld et al., 1997; Yuan and Neufeld, 2000). Func- tional studies on experimental glaucoma and cultured RGCs have revealed that RGC death can be reduced by treatment with substances that inhibit TNF-α production (Fuchs et al., 2005; Kitaoka et al., 2006). Lastly, excitotoxicity due to excess amount of glutamate has been suggested as another mechanism for RGC death in glaucoma (Vorwerk et al., 1999). It was found that vitreous humor of glaucomatous human and monkey retina contain increased amount of glutamate (Dreyer et al., 1996). Stimulation of glutamate receptors by excess glutamate leads to excess influx of calcium, which activates calcium dependent enzymes and results in necrotic cell death. A phase III clinical trial has shown that treatment with a selective glutamate antagonist reduces the visual field loss in glaucoma patients (Larsson et al., 1988).

Glial cells and their role in the central nervous system (CNS) Glial cells are also called neuroglia or simply glia. The ‘neuroglia’ is a Greek term means ‘nerve glue’ and was first introduced in the nineteenth century by a German pathologist Rudolf Virchow. During his search for connective tissue in the CNS, Virchow discovered cells, which were not nerve cells, and named them glia. Recently, it is thought that due to this un- attractive name these cells were neglected by most of neuroscientists for more than a century. Glial cells were initially thought to be passive cells that only physically support the neurons. However, over the last 3 decades, neuroscientists revealed that glial cells have crucial contributions during ontogenesis, mature functioning, and pathology of the CNS (Bringmann et al., 2006).

20 Glia can be defined as the cells in the CNS that are not neurons and do not belong to mesenchymal structures such as the blood vessels and the me- ninges. Four main functions have been identified for glial cells: (1) to cover the neurons and hold them in place, (2) to provide nutrients and oxygen to neurons, (3) to shield neurons from each other and (4) to combat pathogens and clear dead neurons (Bringmann et al., 2006). Neuroglia can be divided into two sub-types: macroglia and microglia. Macroglia comprises a wide variety of cell-types arising from the primitive neuroectoderm together with neurons, which includes astrocytes, oligodendrocytes, redial glia (tanycytes and Müller cells), ependymocytes, choroid plexus epithelial cells and pigment epithelial cells (Campbell and Gotz, 2002; Johansson et al., 1999; Torres et al., 2012). Microglia are the primary immune cells of the CNS, which are blood-borne macrophages and during their late ontogenesis invade the brain via blood vessels. Microglia comprise 10-15% of cells found in the CNS, which mediate immune response by acting as macrophages, removing the cell debris and dead neurons through phagocytosis process (Lawson et al., 1992). In addition to phagocytosis, microglia also activate the proin- flammatory cytokines such as IL-1α, IL-1β and TNF-α in the CNS, which paly a major role in neurodegenaration (Mrak and Griffin, 2005). Microglia intensively interact with macroglial cells such as Müller cells in the damaged retina (Fischer et al., 2014); however, this thesis will focus on the macroglial cells Müller cells.

Müller cells Müller cells are a type of glial cells found in the retina, which represent a minority of the total cells in the CNS. It is estimated that there are 200 bil- lions cells in the human CNS, of which only 8-10 millions of cells are Mül- ler cells. Thus, Müller cells constitute not more than 0.005% of the total cells in the CNS (Bringmann et al., 2006). However, in recent years Müller cells have become a more popular research subject. As a result of these studies, Müller cells were found to be a very peculiar and multipotent glial cell-type in the retina. In most vertebrates, Müller cells are the principal glial cells and the predominant supporting cells in the retina. In the retina, Müller cells span the entire thickness and envelop all types of neurons. They play important roles in retinal homeostasis and also maintain ion balance, neurotransmitter levels and nutritive support to retinal neurons (Bringmann et al., 2006). Mül- ler cells are living optical fibers involved in light guidance in the vertebrate retina (Franze et al., 2007). The somata of Müller cells lie within the inner nuclear layer and form a distinct sub-layer in the retina. The size and shape of the Müller cells varies among different species but most features are fairly similar (figure 2). In most vertebrate retina, Müller cells are connected to neighboring Müller cells and photoreceptor cells through tight junctions to

21 form the outer limiting membrane (OLM). The side branches of Müller cells form sheaths around the processes and synapses of neurons in two plexiform layers (IPL and OPL). In the nuclear layers, the lamellar processes of Müller cells form structures that wrap the cell bodies of neuronal cells (Reichenbach et al., 1989).

Figure 2: Müller cells in different species. (Left) Müller cells in different species, sketched from Golgi-stained preparations (Cajal 1892). (Right) Müller cells in chicken retina stained with Müller cell marker, 2M6 (white). Scale bar 20 µm.

Müller cells express a wide range of voltage-dependent ion channels, ligand receptors, trans-membrane transporters, and different types of neurotransmitter receptors such as gamma-aminobutyric acid (GABA) receptors and glutamate receptors, and also release different signaling molecules that interact with different neurons (Bringmann et al., 2006). Thus, one may consider that neurons in the CNS and glial cells share the same progenitors. Studies in drosophila have identified a gene called ‘glial cells missing’ (gcm), which acts as a binary genetic ‘switch’ for glia versus neurons. When this gcm gene is expressed, the presumptive neurons becomes glia and, in its absence, presumptive glia becomes neurons (Jones et al., 1995). In vertebrates, a similar type of ‘switch’ that influences protein expression profiles of glial and neuronal cells, but it is unknown whether such a mechanism can also influence final cell type in presumptive cells (Bringmann et al., 2006; Kriegstein and Alvarez-Buylla, 2009; Miller and Gauthier, 2007). Müller cells are born during the late stage of retinal neurogenesis when most of the retinal cell types are already in the process of generation. Thus, the retinal progenitors decide during their late histogenesis phase either to differentiate into neurons or glia (Boije et al., 2010; Rapaport et al., 2004).

22 Müller cells in neuroprotection Gliotic responses seem to be similar in the brain and the neural retina. After any pathological alteration of the retina such as photic damage, retinal trauma, ischemia, detachment, glaucoma, diabetic retinopathy or age related macular degeneration, Müller cells become activated (Bringmann et al., 2006). Reactive gliosis involves morphological, biochemical and physiological alterations of Müller cells. Müller cells protect the retinal neurons after retinal damage through the release of neurotrophic factors, free radical scav- engers, glutamate uptake and facilitation of neovascularization (Bringmann et al., 2009b; Bringmann et al., 2006). Müller cells also protect the retina against excitotoxic damage. Glutamate is an excitatory neurotransmitter, which can cause acute neurotoxicity if the extracellular concentration increases in the CNS (Choi, 1987; Frandsen and Schousboe, 1993; Heidinger et al., 1999). It is thought that excess amount of glutamate is primarily responsible for different types of injuries such as anoxia, ischemia, hypoglycemia, trauma and chronic neurodegenerative diseases in the CNS and retina (Choi, 1988; Meldrum and Garthwaite, 1990). Müller cells express the glutamate transporter, L-glutamate/L-aspartate transporter (GLAST) that is involved in clearing glutamate and protecting from death of RGCs (Heidinger et al., 1999; Kashiwagi et al., 2001; Otori et al., 1994). Muller cells clear glutamate by expressing glutamine synthetase (GS) that converts the potentially toxic glutamate to glutamine (Linser et al., 1984). In the retina, reactive oxygen species (ROS) are generated under various conditions such as ischemia and anoxia. Glutathione is one of the important substances that protects the retina against ROS. Müller cells express glutathione, thereby playing a critical role in regulating ROS in retina (Pow and Crook, 1995). In addition, Müller cells produce various types of neurotrophic factors such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), basic fibroblast growth factor (bFGF), Neurotrophins-3 and -4, and glial cell-derived neurotrophic factor (GDNF) under different in vivo and in vitro conditions (Garcia and Vecino, 2003; Oku et al., 2002; Seki et al., 2005; Taylor et al., 2003; Wilson et al., 2007). Neurotrophin receptors such as Trk tyrosine kinases (TrkA, B and C), and the p75 neurotrophin receptor (p75NTR) are also expressed in Müller cells (Oku et al., 2002). The release of the neurotrophic factor BDNF from Müller cells increases the survival of RGCs after optic nerve damage (Yan et al., 1999). Neuroprotective effects are observed when co-culturing of Müller cells with RGCs due to secretion of neurotrophic factors by Müller cells in the culture (Garcia et al., 2002). It has been suggested that BDNF released from Müller cells exerts a feed-forward loop that increases CNTF and bFGF production in Müller cells, subsequently increasing the survival of photoreceptors (Bringmann et al., 2009a).

23 Phagocytosis of cellular debris, exogenous particles and hemorrhagic products is an important mechanism in tissue repairing after injury (Garcia and Vecino, 2003). Several studies have shown that Müller cells involve in phagocytosis of erythrocytes debris and sub-retinal hemorrhage (Mano and Puro, 1990; Miller et al., 1986). Microglia and Müller cells are involved in phagocytosis of fragmenting DNA in developing retina. Müller cells have the ability to phagocytose dead cells in all layers of the retina, as microglia normally do not enter the outer nuclear layer of the retina and it is thought that Müller cells are responsible for the phagocytosis of dying photoreceptors in the retina (Egensperger et al., 1996).

Müller cells in regeneration Müller cells are a potential source of progenitor-like cells for retinal regeneration (Dyer and Cepko, 2000; Fischer and Reh, 2001). According to experiments on several different vertebrates including fish, birds, and mammals, Müller cells become activated and undergo dedifferentiation, proliferation and acquire a progenitor-like state after acute retinal injury (Bringmann et al., 2006; Dyer and Cepko, 2000; Fausett and Goldman, 2006; Fischer and Reh, 2001; Karl et al., 2008; Ooto et al., 2004; Stefansson et al., 1988) or in the presence of exogenous growth factors (Fischer et al., 2002). However, the regenerative capacity of Müller cells varies among different species and seems to be lower in warm-blooded animals. After acute retinal injury, Müller cells become gliotic (figure 3). The gliotic response of Müller cells includes dedifferentiation, proliferation, cell cycle re-entry, expression of various retinal progenitor cell markers like Pax6, Sox2, Chx10, Six3, Sox9 and Asc11a, and formation of retinal progenitor cells (Fischer and Omar, 2005; Fischer and Reh, 2001, 2003; Fischer et al., 2009a; Fischer et al., 2009b; Hayes et al., 2007; Roesch et al., 2008). The progenitor leads to the generation of new neurons in lower vertebrates like fish and amphibians (Fischer and Bongini, 2010; Wan et al., 2012). The gliotic response also sustains the functions of mature Müller cells that support and contribute homeostasis to retinal neurons. In mammals the capacity to form neurons (neurogenesis) has become limited and regeneration is much less or non-existent, however the mammalian Müller cells have been shown to become gliotic and dedifferentiate (Karl et al., 2008; Karl and Reh, 2012). Therefore, it has been hypothesized that a mammalian retina may be better- off if the Müller cells remain as a functional glia cell instead of becoming a retinal progenitor cell (Reichenbach and Bringmann, 2013). Müller cell proliferation is a fundamental step in becoming progenitor-like and dedifferenti- ated cells after injury. Most of the proliferating Müller cells remain undifferentiated progenitor-like cells whereas some differentiated into new Müller cells and new neurons (Fischer, 2005; Fischer and Reh, 2001, 2003). The remaining undifferentiated progenitor-like cells can be stimulated to differ-

24 entiate and regenerate retinal neurons (Fischer et al., 2009a; Fischer et al., 2009b). Understanding the neurogenic potential of Müller cells is key to identifying the modulatory factors, signaling pathways, and transcription factors that regulate and ultimately enable dedifferentiation, proliferation, and neurogenesis also in the human retina (Reichenbach and Bringmann, 2013).

Figure 3: Schematic representation of injury-induced gliotic response of Müller cell. Injury may lead to dedifferentiation and formation of retinal progenitors, and generation of new neurons in lower vertebrates (right blue arrow). The possibility to modulate the response (red arrow) may attenuate the dedifferentiation and retain the support by mature Müller cells (left blue arrow).

Factors that stimulate Müller cells in the retina Few factors have currently been shown to stimulate the Müller cells. Treat- ing uninjured retina with exogenous growth factors, including FGFs, epidermal growth factors (EGFs) or Wnts triggers dedifferentiation and proliferation of Müller cells (Ooto et al., 2004; Osakada et al., 2007; Wan et al., 2012). The process is dependent on the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2). The ERK1/2 are classical mitogen- activated protein kinases (MAPKs), which are widely expressed intracellular signaling molecules involved in many cellular programs, such as growth, development and proliferation. The ERK1/2 are catalytically inactive in their base form. To become active, they require phosphorylation of their activation loop, which contains a TxY (threonine-x-tyrosine) motif. Both the threonine and the tyrosine residues need to be phosphorylated to become phos-

25 phorylated-ERK1/2 (P-ERK1/2) (Boulton and Cobb, 1991; Pearson et al., 2001). Studies of species (e.g. zebrafish) with a retina that has regeneration potential have identified the epidermal growth factor receptor (EGFR) as a key regulator in Müller cell dedifferentiation and retina regeneration (figure 4) (Wan et al., 2012). Retinal injury triggers the expression of endogenous EGFR ligand heparin binding-EGF (HB-EGF) in Müller cells (Todd et al., 2015; Wan et al., 2012). HB-EGF mediates its effects through EGFR/MAPK signaling pathway, which up-regulates the expression of genes associated with retinal regeneration such as ascl1a and Pax6. These regeneration- associated genes activate the Wnt/β-catenin signaling pathway, which leads to progenitor proliferation (Wan et al., 2012). In excitotoxic chicken retina, MAPK signaling also stimulates Müller cells to proliferate and to re-enter the cell cycle and become progenitor-like cells (Fischer et al., 2009a; Fischer et al., 2009b). The active MAPK pathway also regulates both Notch and Wnt signaling in the Müller cell (Ghai et al., 2010). This signal makes the cell less prone to become a retinal progenitor and thus stay in its glial state. The glial fate is promoted by Notch-Delta signalling (Ghai et al., 2010). Sonic Hedgehog (SHH) signaling can stimulate proliferation of Müller cell through its receptors and target genes. SHH-treated Müller cells undergo dedifferentiation and express progenitor cell markers and regenerate to rod photoreceptor cells (Wan et al., 2007).

Figure 4: Injury-induced HB-EGF triggers Müller cell dedifferentiation and retina regeneration. Retinal injury induces the expression of HB-EGF in Müller cells. HB-EGF activates EGFR/MAPK signal transduction pathway, which up- regulates the regeneration associated genes: Ascl1a and Pax6, and consequently stimulates Wnt/β-catenin pathway that leads to progenitor proliferation. HB- EGF/EGFR/MAPK/Ascl1a-signaling cascade regulates injury-dependent stimulation of Notch signaling components: delta, Notch, Her4, 6, 9. Notch signaling feedback loop inhibits hb-egf and ascl1a gene expression and defines the zone of progenitor proliferation. This figure has been adapted from (Wan et al., 2012) and is used with permission from Elsevier.

26 Brimonidine and its effects in the retina Brimonidine (BMD) is a selective alpha2-adrenergic receptor (α2-ADR) agonist that was first discovered in 1996 (Angelov et al., 1996). BMD has been used clinically for its role in reducing intraocular pressure in the treatment of ocular hypertension and glaucoma (Cantor, 2000; Saylor et al., 2009). In addition to controlling intraocular pressure, BMD has also been reported to have neuroprotective activity in a variety of retinal injury models including experimental glaucoma, retinal ischemia and light damage (Lafuente et al., 2001; Vidal-Sanz et al., 2001; Wen et al., 1996; WoldeMussie et al., 2001; Yoles et al., 1999). These investigations indicate that BMD might have therapeutic effects if used clinically to treat optic neu- ropathies in humans. The neuroprotective effect of BMD treatment is dose dependent and α2-ADR specific (Lafuente et al., 2002). In ischemic injury, induced by ligation of ophthalmic artery, systemic administration of BMD rescued up to 33% of RGCs, while topical administration rescued up to 55% of RGCs (Lafuente et al., 2002). Long-term neuroprotective effects have been shown in photocoagulation-induced optic nerve ischemia in rats, where pre-treatment topically with BMD 7 days before the insult can prevent up to 5 months neuronal and axonal loss (Danylkova et al., 2007). The underlying mechanisms for these effects have been suggested to include attenuation of excitotoxicity by modulation of NMDA receptor signaling in RGCs or promotion of the trophic factor responses that contribute to increased neuronal survival or up-regulation of intrinsic cell survival signaling pathways and anti-apoptotic genes such as Bcl-2 and BCL-XL (Dong et al., 2008; Gao et al., 2002; Lonngren et al., 2006; Wheeler et al., 2003). However, the exact molecular mechanism behind the neuroprotection remains elusive. One of the immediate responses to α2-ADR-stimulation is a robust ERK1/2 activation in Müller cells (Peng et al., 1998) and this prompted us to further study the α2-ADR system in Müller cells. The effects of BMD on Müller cell activation in uninjured or excitotoxically-injured chicken retina are presented in this thesis in paper I, III and IV. Furthermore, in paper II, we studied α2-ADR activation by BMD in a human model system. We obtained human Müller cell line, MIO-M1 and studied the effects of BMD on this cell.

Alpha2-adrenergic receptors Alpha2-adrenergic receptors are G-protein coupled receptors (GPCRs), which mediate the effects of their ligands by modulating the activities of adenylyl cyclase, phospholipase C (PLC), phosphatidylinositide 3-kinase (PI3K) as well as by the MAPK pathway. They are present in virtually every internal organ of the mammalian body, and mediate a wide variety of physiological functions. The α2-ADRs modulate sympathetic transmission both

27 in the brain and in the spinal cord, with effects on hypotension, bradycardia, sedation, sleep and analgesia (Aantaa et al., 1995). The α2-ADRs mediate both contraction and relaxation of vascular smooth muscle and increase growth hormone release from the pituitary gland (Aantaa et al., 1995; Ruf- folo and Hieble, 1994). They also modulate the release of key hormones, such as insulin and adrenalin (Moura et al., 2006; Peterhoff et al., 2003), and neurotransmitters, such as serotonin and glutamate (Pan et al., 2002; Scheib- ner et al., 2001). There are three subtypes of α2-ADRs, namely α2A-ADR, α2B-ADR and α2C-ADR, which vary in their tissue distribution, pharmacological and signaling properties (Daunt et al., 1997; Hawes et al., 1995). The α2A-ADR is the predominant subtype in the CNS and BMD has the highest affinity for this receptor subtype (MacDonald et al., 1997). All subtypes of α2-ADRs are expressed in retina (Woldemussie et al., 2007). All α2-ADRs subtypes are negatively coupled to adenylate cyclase via Gi α subunit of GPCR. When Gi is activated by a α2-ADR, the level of cAMP decreases and protein kinase A is deactivated, which leads to inhibition of smooth muscle contraction (Aantaa et al., 1995). However, many physiological effects of α2-ADRs in different tissues cannot be attributed to a decrease in cAMP. Events like inhibition of insulin release, inhibition of neurotransmitter release, platelet aggregation and mitogenic signaling through MAPKs all ap- pear independent of the cAMP pathway. Cellular activation of PLC, Phos- pholipase A2 (PLA2) and Phospholipase D (PLD) by α2-ADRs is even more distant to a cAMP decrease (Nasman et al., 2001). Stimulation of α2-ADRs has been shown to transactivate EGFRs in different cellular systems (Daub et al., 1996; Taylor et al., 2003). Receptor transactivation refers to the ability of one receptor to activate another receptor via signaling cascades. There are various other growth factor receptors e.g. platelet derived growth factor receptor (PDGFR) and insulin growth factor -1 receptor (IGF-1R) that are trans-activated by GPCRs (Daub et al., 1996; Heeneman et al., 2000; Zahradka et al., 2004). Transactivation of these receptors can potentially occur through several different mechanisms. One of the mechanisms occurs via activation of intracellular protein tyrosine kinases, such as Src and PKC, which can phosphorylate the tyrosine residues of these receptors, thereby promoting their activation (Amos et al., 2005; Roelle et al., 2003). Once these receptors are activated, several cellular signaling proteins are phosphorylated and form receptor complexes composed of SHC, GRB2 and SOS, which in turn trigger the activation of downstream MAPK cascade (Pawson and Scott, 1997; Yang et al., 2005). The α2-ADR- induced transactivation of EGFR involves two-steps. In the first step, the activation of the intracellular protein tyrosine kinase Src that causes a release of the membrane bound EGFR ligand, heparin binding-epidermal growth factor (HB-EGF) by activating matrix metalloproteinase (MMPs) in an autocrine mode of action. In the second step, the free HB-EGF binds with EGFR

28 in a conventional manner; tyrosine kinase residues of the EGFR are phosphorylated and contribute directly to Ras-Raf-dependent ERK1/2 activation. Müller cells express EGFR (Roque et al., 1992), HB-EGF (Wan et al., 2012) and α2-ADRs (paper I and II) but it is not known if EGFR are en- gaged in the ERK/MAPK response after stimulation of α2-ADR on Müller cells. We studied the transactivation of EGFRs by α2-ADR stimulation in chicken and human Müller cells and the results are presented in this thesis in papers I and II.

Endothelins and their effects in the retina Endothelins (EDNs) are potent vasoconstrictor peptides and one of the growth-promoting factors that are primarily produced by vascular endothelial cells and, in some extent, by epithelial cells (Ling et al., 2013). In addition to vasoconstriction activity, EDNs have direct effects on both neurons and glial cells in the developing and adult nervous system (Baynash et al., 1994; Lahav et al., 1996; Rattner and Nathans, 2005). Three endothelin subtypes were identified, which are encoded by three genes: EDN1, EDN2 and EDN3. EDNs are expressed in the retina and their expression is induced by different retinal injuries (Rattner and Nathans, 2005; Rattner et al., 2013). EDNs are pleiotropic and have different and sometime opposite effects in various cell- types among different species. A number of studies suggest that EDN1 is involved in retinal pathogenesis such as glaucoma and diabetes retinopathy. It was found that some glaucoma patients had increased levels of EDN1 in aqueous humor (Chen et al., 2013; Choritz et al., 2012; Tezel et al., 1997) and EDN1 has been shown to induce RGC death in experimental glaucoma model (Mi et al., 2012; Oku et al., 2008). Apart from the adverse effects of EDN1 in some injury models, EDN2 has been shown to have neuroprotective properties for photoreceptors. Over-expression of EDN2 in mouse model of photoreceptor degeneration was found to prevent photoreceptor death (Bramall et al., 2013). Recently it has been shown that over-expression of Norrin in the retinal pigment epithelium leads to up-regulation of END2 expression in the retina and consequently protects the photoreceptors from light damage (Braunger et al., 2013). Furthermore, treatment with EDN2 has been found to induce gliotic gene expression in Müller cells, which are associated with intrinsic protective mechanism in the retina (Sarthy et al., 2015).

Endothelin receptors Endothelin receptors (EDNRs) are seven transmembrane GPCRs, which activate different signaling pathways in different cell-types. There are three sub-types of endothelin receptors: endothelin receptor A (EDNRA), endo-

29 thelin receptor B (EDNRB) and endothelin receptor B2 (EDNRB2) (Inoue et al., 1989). Different EDNs have different binding affinities to two main receptors; EDNRA and EDNRB (Inoue et al., 1989; Ling et al., 2013) The EDNRB2 has been found in non-mammalian vertebrates but it is less well characterized than EDNRA and EDNRB (figure 5) (Lecoin et al., 1998).

Figure 5: Endothelins and their receptors. (A) Interaction between endothelins (EDNs) and EDNRs, and their binding affinities. (B) Schematic tree illustrating orthologs and paralogs of endothelin receptors (EDNR) in Aves and Mammalia. The tree is obtained from Ensembl genome browser, Ensembl Gene tree ID: ENSGT00760000119177.

EDNRs couple to the different members of G-protein families such as Gi, Gq, Gs, and Gα12/13 (Grantcharova et al., 2006) and their activation modulates several effectors including adenyl cyclase, PLC, cyclooxygenases, nitric oxide synthase, PI3K and in some cells they also trigger ERK/MAPK signaling (Hawes et al., 1995; Sakurai et al., 1990; Takigawa et al., 1995). Both EDNRA and EDNRB receptors are expressed in normal retina, whereas EDNRB is expressed in Müller cells (Rattner and Nathans, 2005; Stitt et al., 1996; Torbidoni et al., 2006). Retinal phototoxicity up-regulates EDN2 in photoreceptors and EDNRB in Müller cells, and it is suggested that EDN2 mediates injury signaling between degenerating photoreceptors and Müller cells (Rattner and Nathans, 2005). However, the details of the cellular mechanisms by which Müller cells respond to injury-induced EDN2 remains elusive. Studies have shown that EDNRB transactivates EGFRs in vascular smooth muscle cells (Grantcharova et al., 2006; Li et al., 2010), but it is still unknown if stimulation of EDNRB transactivates the EGFR signaling in Müller cells. We studied the EDNRB-induced transactivation of EGFRs on both chicken and human Müller cells, and the results are presented in this thesis in paper V.

Epidermal growth factor receptors The EGFR is a transmembrane glycoprotein receptor, one of the four members of the ErbB family receptors. EGFR is also known as HER-1 (Human

30 epidermal receptor-1) or ErbB-1. Other members of this receptor family are HER-2 (ErbB-2), HER-3 (ErbB-3), and HER-4 (ErbB-4) (Yarden and Sli- wkowski, 2001). EGFR is a receptor tyrosine kinase (RTK) that possesses an extracellular domain containing a ligand-binding site, a transmembrane domain and an intracellular domain with a protein tyrosine kinase activity. The extracellular domain consists of 622 amino acids and is highly glycosylated. For ligand binding, the extracellular domain also contains two cysteine rich regions. The transmembrane domain is a 23 amino acids long α-helical peptide. The intracellular domain of EGFR is 542 amino acids long, which consists of a conserved protein tyrosine kinase domain and a C-terminal domain comprising the regulatory tyrosine residues (Carpenter, 1987). There are different types of ligands that bind to and activate EGFR, which belong to the EGF family of peptide growth factors. The EGF family growth factors have an EGF-like domain comprising of three disulfide-bonded intramolecu- lar groups, which confers ErbB binding specificity and have additional structural motifs such as heparin-binding sites, immunoglobulin-like domains and glycosylation sites. Depending on their affinity for different ErbBs, they are generally categorized into three groups. The first group consists of EGF, amphiregulin, and transforming growth factor alpha (TGF-α) that specifically bind to EGFR (ErbB-1). The second group consists of HB-EGF, epiregu- lin, and betacellulin, which have dual specificity for both ErbB-1 and ErbB-4 (Yarden, 2001). The third group comprises neuregulins (NRG1, NRG2; NRG3 and NRG4), whereas both NRG1 and NRG2 bind specifically on both ErbB-3 and ErbB-4, and NGR3 and NGR4 bind specifically on ErbB-4 (Ha- rari et al., 1999; Zhang et al., 1997). No direct ligand has been yet identified for ErbB-2 but a number of evidences suggested that ErbB-2 mainly acts as a coreceptor for other ErbB receptors (Graus-Porta et al., 1997; Tzahar et al., 1996). EGF and TGF-α are thought to be the most important type of ligands for EGFR.

Epidermal growth factor receptor signaling Binding of ligand with EGFR results in receptor homo- or heterodimerization at the cell surface, subsequently auto- or trans-phosphorylation of key tyrosine residues in the kinase domain. The phosphorylated tyrosine residues provide docking sites for proteins, which contain phosphotyrosine binding or Src homology 2 (SH2) domains (Shoelson, 1997). Generally these proteins include different types of adapter proteins such as growth factor receptor-bound protein 2 (Grb2), Src homology domain-containing adaptor protein C (Shc), and phospholipase C γ (PLCγ); various kinases such as Src, PI3K and Chk; and protein tyrosine phosphatases such as SHP1, PTP1B and SHP2 (Olayioye et al., 2000; Sebastian et al., 2006). The recruitment of these proteins by the activated EGFR activates different downstream signaling pathways, which regulate multiple biological processes such as cell growth,

31 development, proliferation, differentiation, survival, angiogenesis, adhesion, and migration (Franklin et al., 2002; Herbst, 2004; Morandell et al., 2008). For most of the ErbB family receptors, the Ras-Raf mediated MAPK/ERK pathway and PI3K mediated AKT pathway are two major signaling pathways (Hirsch et al., 2003; Singh and Harris, 2005). The other signaling pathways include c-Jun N-terminal kinase (JNK) and p38 MAPKs (Johnson et al., 2005), signal transducer and activator of transcription (STAT) (Andl et al., 2004; Kloth et al., 2002), Ca2+-calmodulin-dependent protein kinase (CaMK) (Sengupta et al., 2009) and PLCγ/ protein kinase C (PKC) (Santi- skulvong and Rozengurt, 2007). In this PhD project, I focused on MAPK-ERK and PI3K-AKT signaling pathways (paper I, II, III and V).

Mitogen activated protein kinase signaling The transduction of extracellular signals to their intracellular targets is mediated by a number of interacting molecules that regulate many cellular processes. MAPK signaling cascades play substantial roles in transducing extracellular signals to cellular responses (figure 6). There are four members of the MAPK family that have been identified, namely ERK1/2, p38, JNK, and ERK5 (Nishimoto and Nishida, 2006). The ERK1/2 signaling pathway is mainly responsive to growth factor stimulation, whereas JNK and p38 are known as stress-activated MAPKs (SAPKs) because of their stimulation by physiological, chemical, and physical stressors (Kyriakis and Avruch, 2012). The ERK5 signaling pathway is associated with both stress and growth factor signaling (Hayashi and Lee, 2004). MAPK signaling is also stimulated by activated GPCR. Activated GPCR stimulates cytosolic Src kinase, which in turn activates RTK and consequently activates MAPK singaling (Du et al., 2009). ERK1 and 2 are two isoforms of the classical MAPK and are the best characterized MAPKs. Following stimulation of RTKs by a variety of mitogens such as growth factors (e.g., EGF, FGF and NGF), a protein complex is formed, where the cytosolic adapter protein Grb2 binds to the specific phosphotyrosine residue of the activated receptor and to the cytosolic SOS protein. SOS then binds to its plasma membrane bound substrate Ras-GDP (inactive form). SOS functions as a guanine nucleotide exchange factor (GEF) for Ras and promotes dissociation of GDP from Ras. GTP binds to empty Ras molecule and becomes active Ras-GTP. The active Ras-GTP then release from SOS and binds to the N-terminal domain of Raf, a serine/threonine kinase. Cytosolic Raf is phosphorylated and bound in an inactive form to 14-3-3, a phosphoserine-binding protein. The interaction of Raf with Ras-GTP results in dephosphorylating of one of the serine residues, which binds to 14-3-3, subsequently phosphorylates other Raf residues and leads to activation of Raf. The hydrolysis of Ras-GTP to Ras-GDP releases active Raf from Ras and also from its complex with 14-3-3. The active Raf

32 kinase then activates MAP/ERK kinase (MEK) by phosphorylation of its kinase residues (Schlessinger, 2000; Simon, 2000). The active MEK then phosphorylates and activates another MAP kinase called serine/threonine kinase, which is also known as ERK. MEK1 and 2 are responsible for activation of ERK1 and 2 respectively. Activated ERK1/2 stimulates multiple sub- strates, such as transcription factors e.g., Elk1 and c-Myc; immediate early response transcription factors e.g., c-Fos and c-Jun; and protein kinase e.g., ribosomal S6 kinase (RSK) (Nishimoto and Nishida, 2006). p38 MAPKs are activated by a number of cellular stresses such as heat shock, UV irradiation, high osmotic shock, inflammatory cytokines (e.g. TNF-α, FASL, Interleukin-1), lipopolysaccharide and growth factors. There are four p38 isoforms have been identified: p38 α, p38 β, p38 γ and p38, δ. These isoforms are phosphorylated by different MAPK kinases (MKKs). MKK6 can phosphorylate all of these isoforms, where MKK3 can phosphorylate p38 α, p38 γ and p38 δ, and MKK4 can phosphorylate p38 α (Ichijo, 1999). Activated p38 MAPK phosphorylates and activates transcription factors such as ATF-2, Sap-1a and c-Myc, and can also activates non- transcription factor targets such as MAPK-activated protein kinases (MAP- KAPKs) (Wang and Ron, 1996). JNK MAPKs are multifunctional and are involved in numerous physiological processes. JNKs have a major role in apoptosis in various cell death programme. There are three types of JNK, which are encoded by three genes: Jnk1, Jnk2, and Jnk3. JNK1 and JNK2 are generally expressed in all tissues but the expression of JNK3 is mostly limited to CNS and cardiac muscle tissues (Yang et al., 1997). Like p38 MAPKs, JNKs are also responsive to stress stimuli such as UV irradiation, heat shock, inflammatory cytokines and osmotic shock. JNKs are phosphorylated and activated by two MAP kinases, MKK4 and MKK7 (Ip and Davis, 1998). Activated JNKs modulates the activity of various proteins that reside in the nucleus or in the mitochondria. JNKs activates numerous downstream molecules such as c- Jun, ATF-2, Sp-1a, Elk-1 and p53 (Vlahopoulos and Zoumpourlis, 2004). ERK5 MAPK is activated by stress stimuli such as oxidative stress and osmotic shock but it can also be activated by growth factor stimulation. ERK5 is phosphorylated and activated by MAPK kinase MKK5 and exerts its kinase activity on other protein kinases and transcription factors. The c- terminal domain of ERK5 contains a unique sequence that allows ERK5 to function directly as a transcriptional activator (Akaike et al., 2004). ERK5 activates a number of transcription factors such as myocyte enhancer factor- 2 (MEF2), c-fos, Fra1, and peroxisome proliferator activated receptor γ1 (PPARγ1) (Kasler et al., 2000).

Figure 6: MAPK and AKT signaling cascades. Growth factors stimulate RTK that leads to activate Ras-Raf mediated ERK1/2 MAPKs. Activated ERK1/2 stimulates transcription factors STAT, c-Jun, ELK and cFos. Stressors and proinflamatory cytokines stimulate other MAPKs ERK-5, JNK 1/2/3 and p38 α/β/γ/δ, which lead to stimulate different transcription factors Sp-1, c-Myc, ATF2 and c-Jun. Activated GPCR stimulates RTK via cytosolic Src kinase. Survival factors also activate RTK that leads to stimulate PI3K. Activated PI3K actives AKT. Activated AKT inacti- vates pro-apoptotic BAD, FOXO, and caspase 9 and consequently inhibits apoptosis. Activated AKT stimulates transcription factor CREB and also stimulates mTORC1 that leads to protein synthesis.

Phosphatidylinositol-3-kinase-AKT signaling The PI3K-AKT signaling cascade is highly conserved and is stimulated by activated growth factor receptors (RTKs), GPCR, B and T cell receptors, in- tegrins, and cytokine receptors (Hers et al., 2011) (figure 6). These activated

34 receptors directly stimulate PI3Ks, which are bound to these receptors via their regulatory subunit or adapter molecules. The activated PI3K triggers the con- version of plasma membrane bound phosphatidylinositol (3,4)-bis-phosphate (PIP2) lipids to phosphatidylinositol (3,4,5)-tris-phosphate (PIP3). These lipid molecules serve as plasma membrane docking site for proteins that have pleckstrin-homology (PH) domains such as AKT and its upstream activators, phosphoinositide-dependent kinase 1 (PDK1) and PDK2 (Hemmings and Restuccia, 2015; Partovian and Simons, 2004). AKT is a serine/threonine kinase, also known as protein kinase Balpha (PKB) that has three closely related isoforms AKT1, AKT2 and AKT3 (Hers et al., 2011). The AKT is normally inactive and unphosphorylated and its full activation requires two PIP3- dependent phosphorylation events (Partovian and Simons, 2004). AKT binds to PIP3 at the plasma membrane, which allows PDK1 binding and phosphorylates Thr-308 residue in the activation loop leading to partial AKT activation. This partial activation is sufficient to stimulate mechanistic target of rapamy- cin complex 1 (mTORC1) by direct phosphorylation and inactivation of proline-rich AKT substrate of 40 kDa (PRAS40) and tuberous sclerosis protein 1 (TSC1), TSC2. mTORC1 acts on eukaryotic translation initiation factor 4E binding protein 1 (4EBP1), and ribosomal protein S6 kinase 70 kDa, polypeptide 1 (p70S6K) and consequently phosphorylates the ribosomal protein S6 (RPS6) and leads to protein synthesis and cellular proliferation (Hemmings and Restuccia, 2015). Phosphorylation of AKT at Ser-473 in the C-terminal domain by PDK2 stimulates its full kinase activity (Hemmings and Restuccia, 2015; Partovian and Simons, 2004). Full kinase activity leads to additional substrate-specific phosphorylation events such as inhibitory phosphorylation of pro-apoptotic proteins like Bad or inhibition of pro-apoptotic signals induced by forkhead box O (FOXO) proteins or direct inhibition of caspase-9 activation (Guertin et al., 2006). There are various cellular functions are mediated by fully activated AKT including growth, proliferation, survival, metabolism, angiogenesis, protein synthesis, apoptosis, transcription, migration and invasion (Hemmings and Restuccia, 2015). There are negative regulators that can antagonize AKT singaling including phosphatase and tensin homolog (PTEN), which converts PIP3 to PIP2, protein phosphatase 2 (PP2A) and PH-domain leucine-rich- repeat-containing protein phosphatases (PHLPP1/2), which dephosphorylate the Thr-308 and Ser-473 residues of AKT respectively (Hemmings and Restuccia, 2015).

Regulation of epidermal growth factor receptor signaling EGFR signaling is essential for normal physiology and in many developmental processes. Therefore, the activity of EGFRs is tightly regulated at different molecular levels to ensure appropriate cellular responses. There are different mechanisms that control the EGFR signaling, including receptor endocytosis/degradation, dephosphorylation by protein tyrosine phosphatas-

35 es (PTPs), and negative feedback regulation. In this PhD project, I focused on dual specificity phosphatases (DUSPs) and negative feedback regulation of EGFR signaling.

Dual specificity phosphatases DUSPs are one of the groups of PTPs that can dephosphorylate both phosphotyrosine and phosphoserine/phosphothreonine residues within the similar substrate (Alonso et al., 2004; Pearson et al., 2001). Mitogen-activated protein kinase phosphatases (MKPs) are a sub-group of DUSPs that can dephosphorylate MAPKs. Different MKP type of typical DUSPs and their substrate specificity are listed in the table 1 (Patterson et al., 2009). Each MKP has substrate-specific preference for one or more of the MAPKs such as ERK, JNK or p38 that are phosphorylated in response to extracellular stimuli (Chang and Karin, 2001; Johnson and Lapadat, 2002; Pearson et al., 2001). MKPs are also regulated at different levels to fine-tune the major signaling pathways. Most of the MKPs are inducible or immediate early- response genes and their expression are rapidly increased upon appropriate stimulation such as stimulation by peptide growth factors, cytokines, or serum. Nevertheless, the kinetics and magnitude of the induction of each MKP is cell type- and context-specific. The induction is usually dependent on MAPK activation and is involved in down-regulation of mitogenic signaling (Brondello et al., 1997; Ekerot et al., 2008).

Table 1: List of typical chicken DUSPs and their substrate specificity. Adapted from (Patterson et al., 2009). Name Alternative name Substrate specificity DUSP1 MKP-1 p38 = JNK > ERK DUSP2 PAC-1 ERK = P38 > JNK DUSP4 MKP-2 ERK = JNK > p38 DUSP5 hVHR3 ERK DUSP6 MKP-3 ERK > JNK = p38 DUSP7 MKP-X, Pyst2 ERK > JNK = p38 DUSP8 hVH5, M3/6 JNK = p38 > ERK DUSP9 MKP-4 ERK > P38 > JNK DUSP10 MKP-5 p38 = JNK > ERK DUSP16 MKP-7 JNK = P38 > ERK

Negative feedback regulation Negative feedback regulation plays a pivotal role in controlling the activity of the EGFR, and ensures to generate a stable and reproducible signaling response. The most common negative feedback regulators that act on the ERK and RTK signaling pathways including mitogen inducing gene-6 (MIG-6),

36 sprouty homolog (Drosophila) (SPRY), and sprouty-related protein with an EVH domain (SPRED). The negative feedback regulation induced by these proteins is a delayed event because it requires de novo syntheses of mRNA and protein (Kim and Bar-Sagi, 2004; Zhang and Vande Woude, 2007). MIG-6 is also known as ERRF1, GENE-33 or RALT. It functions as a scaffold adaptor protein, which has several functional domains that are crucial for interaction with signaling molecules (Makkinje et al., 2000; Zhang and Vande Woude, 2007). Growth factors including EGF, fibroblast growth factor (FGF) and hepatocyte growth factor (HGF) can induce MIG-6 expression. The attenuation of EGFR signaling by MIG-6 occurs at two molecular levels: one on the receptor level and another on the downstream signaling molecules of the receptor (Zhang et al., 2007). Via EBR domain, MIG-6 directly binds to EGFR receptor and blocks the dimer interface thereby preventing EGFR activation. The second level of regulation involves binding of MIG-6 via proline- rich domain to the SH3 domain-containing protein Grb2, the key molecule in linking activated RTK to the intracellular signaling cascade. SPRY protein contains conserved functional cysteine-rich domain (SPR domain) in its C-terminal and an SH2 binding domain in its N-terminal. These conserved domains are essential for SPRY mediated regulation of RTK signaling (Christofori, 2003; Kim and Bar-Sagi, 2004). There are four SPRY family members (SPRY1-4) have been identified and their expression is induced by activated RTKs such as EGFR, FGFR, hepatocyte growth factor receptor (HGFR) and vascular endothelial growth factor receptor (VEGFR) (Kim and Bar-Sagi, 2004). SPRY2 is highly expressed in brain, lung and heart tissues (Tefft et al., 1999). In EGFR mediated RTK signaling SPRY2 exerts its inhibitory activity through interaction with downstream signaling molecules. The binding of SPRY2 via SH2 binding domain to Grb2 prevents the recruitment of Grb2-SOS, thereby inhibiting the downstream signaling molecules (Walsh and Lazzara, 2013). SPRY2 also has been shown to interact with Raf kinase via SPR domain and suppresses its activation (McClatchey and Cichowski, 2012). SPRED protein contains conserved C-terminal SPR domain like other sprouty proteins but does not contain the conserved tyrosine residue-binding domain and thus, does not allow for binding with Grb2. In addition to the SPR domain, SPRED protein also contains EVH1 domain in its N-terminal and a central c-Kit-binding domain (KBD) (Bundschu et al., 2007). Three SPRED proteins have been identified: SPRED1, 2 and 3, where SPRED3 does not contain KBD (Wakioka et al., 2001). Like other sprouty proteins, the expression of SPRED proteins is induced in response to a number of peptide growth factors, chemokines and cytokines. SPRED proteins have been shown to asso- ciate with Ras but do not inhibit Ras activation or membrane translocation of Raf. Instead SPRED proteins inhibit the MAPK signlaing by suppressing phosphorylation and activation of Raf kinase (Wakioka et al., 2001).

37 Aims of the thesis

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. The focus has been on the α2-adrenergic and endothelin receptor-induced modulation of Müller cell-properties after injury. Based on this knowledge, we hope that the results will 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.

The specific aims were:

Paper I: To study the expression of α2-ADRs in chicken Müller cells and to test the hypothesis that EGFRs on chicken Müller cells are transactivated by α2-ADRs.

Paper II: To test the hypothesis that α2-ADR agonist brimonidine stimulates ERK1/2 and AKT signaling via transactivation of EGFRs in MIO-M1 human Müller cells.

Paper III: To study the modulatory factors and signaling pathways that regulates the dedifferentiation of Müller cells. More specifically, (1) to study the effect of α2-ADR signaling on injury-induced ERK and Müller cell dedifferentiation and (2) to test the hypothesis that α2-ADR-stimulation triggers negative feedback regulation of the injury-induced ERK pathway that attenuates Müller cell dedifferentiation.

Paper IV: To study the RGC population in normal chicken retina and excitotoxically injured chicken retina after pretreatment with the α2-ADR agonist brimonidine. More specifically, to study the effects of α2-ADR signaling on injured chicken RGCs.

Paper V: To study whether endothelin receptor signaling can stimulate EGFR and ERK signaling in Müller cells.

38 Results and discussion

Paper I The α2-ADR agonist BMD has been used clinically in glaucoma and ocular hypertension treatments (Arthur and Cantor, 2011). In addition, BMD has been shown to have some neuroprotective effects on different types of experimental injuries (Lafuente et al., 2001; Wen et al., 1996; WoldeMussie et al., 2001; Yoles et al., 1999). There are different mechanisms that have been suggested for these neuroprotective effects such as inhibition of excitotoxicity by modulation of NMDA receptor signaling in RGCs or promotion of neurotrophic factors that can contribute to neuronal survival (Dong et al., 2008; Lonngren et al., 2006). However, the molecular mechanisms of neuroprotection are not still fully resolved. One of the instant responses to α2- ADR stimulation is a robust activation of ERK1/2 in Müller cells (Peng et al., 1998), which prompted us to further study the α2-ADR system in Müller cells. In this study we first characterized the expression of α2-ADRs in chicken retina and in cultured primary Müller cells, and then studied the transactivation of EGFRs on chicken Müller cells by α2-ADR stimulation. Our results show that α2A-ADRs are localized in the cell somata and on the processes of the chicken Müller cells. In addition, α2A-ADRs+ cells were also found in the GCL of the chicken retina. The pattern of α2A-ADRs in the chicken retina was similar to published data in the rat, monkey, and human retina (Kalapesi et al., 2005; Woldemussie et al., 2007). We also found α2A-ADR staining in the primary Müller cell cultures that supports our findings from chicken retina. We did not find α2B-ADRs either in chicken Müller cells or in the primary Müller cell cultures. We saw distinct α2B-ADR immunoreactivity in photoreceptor outer segments. In rodents, α2B-ADRs were present in all layers of the retina (Woldemussie et al., 2007). The pattern of α2B- ADR expression across species seems to be less well conserved than that of α2A-ADRs. The qRT-PCR analysis of primary Müller cells confirmed that α2A-ADR mRNA was highly expressed and the expression of α2B- and α2C-ADR mRNAs were low. We used the α2-ADR agonist BMD, to stimulate Müller cells. We analyzed the phosphorylation of ERK1/2 as an assay of Müller cell activation. BMD stimulation resulted in a robust activation of ERK/MAPK in Müller cells that are consistent with earlier studies showing ERK1/2 activation in rat

39 Müller cells after systemic administration of the α2-ADR agonist xylazine (Peng et al., 1998). The specificity of the ERKs activation by BMD was shown by the ability of the α2-ADR-antagonist yohimbine (Verwaerde et al., 1997), to completely block the transient increase in P-ERK. Studies in different cell types have shown that activation of ERK1/2 by GPCR involves transactivation of the EGFR (Daub et al., 1996). BMD- stimulation of primary Müller cells resulted in phosphorylation of tyrosine residues 1068 and 1173 of the EGFR that allows binding of adaptor proteins Grb2 and SHC with the receptor and mediates Ras-Raf activated ERK1/2 MAPK signaling. These results are consistent with dexmedetomidine- induced transactivation of EGFR in intact brain tissues and in astrocytes (Du et al., 2009; Li et al., 2008). Cytosolic Src-kinase has been shown to involve in α2-ADR-induced EGFR transactivation in different cell-types (Hanke et al., 1996). Based on the results from the Src-inhibition studies, our data show that BMD-induced transactivation of EGFR in Müller cells is strictly dependent on Src-kinase activity. Primarily, it was thought that α2-ADR- induced transactivation of EGFR mediated by a direct effect by Src-kinase. Nevertheless, matrix metalloproteinases (MMPs) have been shown to serve key roles in transactivation of EGFR signaling via the release of membrane bound EGFR ligand, HB-EGF (Daub et al., 1996; Prenzel et al., 1999) which activates EGFR in an auto- or paracrine mode of action (Karkoulias et al., 2006). Our primary Müller cell cultures express HB-EGF and we have seen that BMD-induced ERK1/2 activation in cultured primary Müller cells was significantly abrogated by MMP-inhibitor treatment, indicating a ligand- dependent mechanism for transactivation of EGFR in Müller cells. However, MMP-inhibitor unable to completely abolished the BMD-induced ERK1/2 activation, suggesting a ligand-independent activation of ERK1/2 in Müller cells via Src-kinase. Thus, our results demonstrate that chicken Müller cells express α2A-ADR and that stimulation by BMD triggers both Src-kinase and MMP mediated autocrine ligand-dependent transactivation of the EGFR in Müller cells.

Paper II In this study we used a human Müller cell-line MIO-M1, to replicate our results on chicken Müller cells (Paper I). In addition, we studied whether PI3K-AKT signaling is activated via transactivation of EGFR in MIO-M1 human Müller cells. We first characterized α2-ADRs in MIO-M1 human Müller cell culture and found that α2A-ADRs are localized in the cell body of MIO-M1 cells. Moreover, the pattern of α2A-ADRs staining was consistent with chicken primary Müller cells (Paper I). We found very low α2B-ADRs in the MIO- M1 cells, whereas Müller cell in the chicken retina or primary Müller cell

40 culture do not have any α2B-ADRs. Thus, this indicates that α2B-ADRs among the species seem to be less conserved than α2A-ADRs. A qRT-PCR analysis of all three α2-ADR subtypes confirmed the high mRNA levels of α2A-ADR, very low levels of α2B-ADR and moderate levels of α2C-ADR. Similar to chicken Müller cells (Paper I) we studied if stimulation of α2- ADR by BMD could activate the ERK1/2 signaling in MIO-M1 human Mül- ler cells. We also studied the activation of AKT signaling by analysis of AKT (Thr-308) phosphorylation in this system. Our results showed a robust ERK1/2 and AKT activation in MIO-M1 human cells after BMD treatment. These results are consistent with previous studies showing α2-ADR-induced ERK1/2 and AKT activation in pheochromocytoma (PC) 12 and epithelial cells (Buffin-Meyer et al., 2007; Karkoulias et al., 2006). We observed a delayed and longer MAPK response in MIO-M1 human Müller cells compared to primary chicken Müller cells. This could be explained by intrinsic cellular properties that may vary among different species. We studied whether BMD-induced ERK1/2 and AKT activation in MIO-M1 cells is associated with transactivation of EGFR. Our results show that BMD stimulation in MIO-M1 cells led to transactivation of EGFR by phosphorylation of its tyrosine residue 1173. Similarly to our study on chicken Müller cells (Pa- per I), we also analyzed whether cytosolic Src-kinase and MMPs are involved in transactivation of EGFR in MIO-M1 cells. Our results show that blocking of Src-kinase completely abrogated the BMD-induced ERK1/2 and AKT activation, and blocking of MMPs significantly abolished the ERK1/2 and AKT activation, indicating a ligand-dependent EGFR transactivation. Partial blockage of ERK1/2 and AKT activation by an MMP inhibitor also indicates the ligand-independent mechanism for EGFR transactivation in MIO-M1 human Müller cells. Thus, our results indicate that MIO-M1 human Müller cells express α2A- ADRs and stimulation of that receptor by BMD activates Src-kinase mediated both ligand-dependent and ligand-independent EGFR transactivation. In addition, we found that transactivation leads to activation of ERK1/2 and AKT signaling in MIO-M1 human Müller cells.

Paper III A role for EGFR as a regulator of Müller cells during retinal injury or disease is well established (Wan et al., 2012). In papers I and II, we identified a mechanism that modulates the mode of action of EGFR on Müller cells by α2-ADR stimulation. The results from papers I and II open up the possibility to modulate Müller cell responses after injury. The outcome of any injury or disease to the retina is dependent on how Müller cells respond. We investigated how the property of Müller cells can be regulated to either remain as a glia cell with homeostatic functions or to become a retinal progenitor. The

41 studies of retinal responses either to injury or to α2-ADR stimulation reveal a potential paradox. On one hand, retinal injury-induced ERK/MAPK signaling drives a gliotic response and neuronal loss (Fischer et al., 2009a; Fischer et al., 2009b). On the other hand, stimulation of α2-ADRs prevents neuronal loss (Lafuente et al., 2001; Wen et al., 1996) but also activates the ERK/MAPK signaling pathway (papers I and II). In this study, we addressed this paradox by studying the early ERK/MAPK response after an excitotoxic injury with or without α2-ADR stimulation. Our results show that BMD pretreatment in injured retina reduced phosphorylation of ERK in Müller cells and also reduced the increased expression of transitin, and retinal progenitor cell genes Pax6 and Sox2. These results indicated that BMD attenuates not only injury-induced ERK response but also the dedifferentiation of Müller cells in injured retina. Based on these results, we hypothesized that stimulation of α2-ADR triggers a negative feedback regulation of the ERK/MAPK pathway in Müller cells. Our results showed that the attenuation was concomitant with a synergistic up-regulation of several negative ERK-signal feedback regulators including ERK- phosphatases DUSP1 and 5, and MAPK interacting proteins MIG6 and SPRY2. Similar results were also seen in cultures of primary Müller cells. Several preclinical studies suggested that BMD might protect RGCs inde- pendently of its effects on lowering IOP. Therefore, the neuroprotective effect is an added benefit of its use in glaucoma and our results suggest an additional and prospective mechanism for how these effects can be utilized. Our data support the notion that Müller cell function or malfunction is a factor that may affect progression of glaucoma. Our results are consistent with studies showing that the progression of retinal injuries is ERK- dependent (Fischer et al., 2009a) and that increased expression of phosphatase negatively regulates the ERK/MAPK pathway (Jeffrey et al., 2007; Ostman and Bohmer, 2001). The extent and duration of intracellular ERK/MAPK signaling can regulate the differentiation of cell. For example in PC12 cells, transient versus sustained ERK-signaling directly regulates the fate of proliferation or differentiation after growth factor stimulation (Marshall, 1995). In this study, the attenuation of injury–increased number of Pax6+ and Sox2+ cells is an indication of altered differentiation. Pax6 expression is induced in Müller cell after injury (Fischer and Omar, 2005) and sustained Sox2 expression is required to maintain the progenitor state of Müller cells in mouse retina (Bhatia et al., 2011; Surzenko et al., 2013). Therefore, BMD-induced attenuation of these progenitor cell genes supports the hypothesis that α2-ADRs inhibit Müller cell dedifferentiation in injured retina. Modulation of adrenergic receptor activity affects progenitor cells in the adult hippocampus, and it has been shown that selective stimulation or inhibition of α2-ADR inhibits (Yanpallewar et al., 2010) or activates (Jhaveri et al., 2014) progenitor cells. It has also been shown that acute stimulation of

42 α2-ADR inhibits hematopoietic stem cells via the ERK signaling pathway (Schraml et al., 2009). Although the expression of negative regulators is transient with a duration of 4 to 6 hours, the attenuation of P-ERK levels remained to 48 hours or more. One interpretation could be that in order to achieve effective long-term protection by BMD, the levels of negative feedback regulators need to be elevated in Müller cells at the time of injury. Adrenergic stimulation during injury creates a fight or flight response that tells the physiological cellular system to rapidly respond to injury for survival. The biological rationale behind the empirical results showing that stimulation of α2-ADR during injury give neuroprotective effects is based on the hypothesis of an assumed cellular stress-response leading to increased damage control, which is mimicked by BMD stimulation.

Paper IV In this work we studied short- versus long-term EGFR-induced changes in Müller cells after injury. It is hypothesized that by altering the immediate response by Müller cells at the time of injury, intermediate and long lasting effects are achieved. The “trajectory” of the cells is altered, resulting in a major difference in their end-point. In paper III, we have seen that α2-ADR stimulation modulates Müller cell response to injury by attenuating their dedifferentiation properties. In this work, we studied the effects of this attenuation of Müller cell dedifferentiation on injured retina from the perspective of neuroprotection. We analyzed RGC survival after α2-ADR stimulation on NMDA-induced excitotoxic chicken retina. By using flat-mount whole retina immunohistochemistry technique we first analyzed the total population of RGC in embryonic (E) and post-natal (P) chicken retinas and then studied the effect of BMD on RGC survival in post-natal chicken retinas at 7 and 14 days post lesion. We used the RGC-specific transcription factor Brn3a marker to label flat-mounted retinas that were analyzed using automated cell counting. This method allowed us to study the complete Brn3a+ RGC population and generate RGC isodensity maps that showed the regional (e.g. ventral, dorsal, nasal and temporal) distribution of RGC in the retina. Our results show that Brn3a+ RGCs in E8 to E10 retinas are approximately 1.5x106, in E12 to E20 are approximately 1.8x106 and in P4 to P11 are approximately 1.9x106 cells per retina. Previously, in total 2.8x106 RGCs in the P2 chicken retina were estimated, based on the number of optic nerve fibers (Rager and Rager, 1978). We found consistently a total of 1.7 - 2x106 Brn3a+ cells in the E12 to P11 retina. A previous RGC estimation study reports higher RGC numbers in the embryonic retina than after hatching (Chen and Naito, 1999). A constant number of RCG from E12 to P11 is reliable with the idea that neurogenesis and naturally occurring death of RGCs occurs before E12 (Mayordomo et al., 2003). However, not all RGCs

43 express Brn3a. In rat retina, Brn3a+ labeling was found in 92% of all retrograde tracing-identified RGCs (Nadal-Nicolas et al., 2009). As a result, as- suming a similar efficacy in chicken, our method may underestimate the number of RGC. We calculated overall that 72% of all cells are Brn3a+ in the GCL, indicating that 28% are Brn3a negative RGC or displaced amacrine cells in the GCL. This fraction is consistent with previous data on displaced amacrine cells that accounted for 20 to 30% of the cells in the GCL (Layer and Vollmer, 1982). Isodensity mapping exposed the density and spatial distribution of RGCs in the retina at developmental ages from E8 to P11. We did not find any region with increased RGC density, and RGCs were distributed homogeneously across the retina at all ages with the excep- tion in the peripheral zone and the region of pectin oculli. RGC density decreases and the total number of RGC increases as the retina aged. We determined the NMDA dose for RGC lesions prior to study the effect of BMD on RGC survival. The NMDA doses that we determined produced a robust injury but not more severe than could be discerned from the effects of BMD. Our results showed that RGC loss was higher in the dorsal retina than in the ventral one, indicating that dorsal RGCs are more sensitive to NMDA. The effect of BMD was therefore more evident in the dorsal retina than in the ventral. The regional difference of RGC death by NMDA is consistent with previous studies that NMDA has less effect in the temporal aspect of the retina and eye (Fischer et al., 1998; Zeevalk et al., 1989). Our results show that BMD pretreatment significantly reduced the RGC death both at 7 and 14 days post lesion and support the notion that α2-ADR agonists have neuroprotective effects on RGCs in response to various retinal injuries including optic nerve crush, excitotoxicity, phototoxicity, ischemia, optic nerve transection and IOP injury (Lafuente et al., 2002; Ortin-Martinez et al., 2014; Wen et al., 1996; WoldeMussie et al., 2001; Yoles et al., 1999) and that these neuroprotective effects is not limited to the mammalian class of retina. The amount of BMD and the experimental condition were similar to our previous experiments, where BMD triggers an intracellular attenuation of the injury-response that involves negative ERK-signaling feedback leading to attenuated Müller cell dedifferentiation and gliosis (paper III). Our present results showed that α2-ADR agonist BMD protected RGCs from excitotoxic injury. Therefore, we assume that BMD-induced protection of RGCs in injured retina is due to enhancing the attenuation of the glial injury response and to sustaining mature glial functions.

Figure 7: Flow chart illustrating effects of α2-ADR stimulation in exicitotoxic retina. NMDA-induced excitotoxic injury up-regulates P-ERK1/2 and negative feedback regulators DUSP1, 5, MIG6 and SPRY2. Injury also triggers cell death. Up-regulated P-ERK1/2 leads to up-regulation of transitin and progenitor cell genes, Pax6 and Sox2, which in turn increase dedifferentiation of Müller cells. BMD pretreatment attenuates the injury-induced up-regulation of P-ERK1/2 and consequently attenuates transitin, Pax6 and Sox2 expression and attenuates Müller cell dedifferentiation. BMD pretreatment synergistically up-regulates the negative feedback regulators and also reduces cell death.

Paper V Glia cells have a pivotal role in maintaining homeostasis and supporting neuronal survival after injury but in some systems they also serve as progenitor cells contributing to neuronal regeneration (Wan et al., 2012). Regu- lation of the glia cell response immediately after injury is therefore crucial for the ultimate outcome after injury. In this work, we studied endothelins (EDNs)-induced intracellular signal transduction response in Müller cells with a focus on ERK/MAPK signaling. EDNs are well known for their potent vasoactive property but they have direct effects on both neurons and glia cells in the nervous system. Cells in the retina express EDNs and endothelin receptors (EDNRs) (Rattner and Nathans, 2005). Phototoxic injury induces EDN2 expression in photoreceptors and EDNRB in the Müller cells and it has been suggested that EDN2 mediates signaling between degenerating photoreceptors and Müller cells (Rattner and Nathans, 2005). Injury-induced Müller cell dedifferentiation is dependent on the activation of ERK/MAPK signaling downstream of EGFRs (Wan et al., 2012). We studied if the stimu-

45 lation of EDNR signaling can activate EGFR and ERK/MAPK signaling in Müller cells. We first analyzed the expression of the EDNs and EDNRs in chicken retina after excitotoxic injury. We found a robust increase of EDN1, EDN2 and the EDNRB mRNA expression in the excitotoxic chicken retina, giving support to the idea that up-regulation of EDNs and EDNRB is a common phe- nomenon after injury. We then confirmed previous findings of EDNRB expression in Müller cells by analyzing cultures of chicken Müller cells or the human MIO-M1 Müller cell-line. Stimulation of EDNRB with the agonist IRL1620 activated the MAPK signaling by increasing P-ERK levels in Mül- ler cells in the chicken retina in vivo as well as in both chicken- and human- cultured Müller cells. Our results showed that P-ERK-signaling is dependent on the Src-kinase that engages both ligand-dependent and ligand- independent transactivation of EGFRs. These results are consistent with previous studies showing EDN-induced EGFR transactivation in rat fibro- blasts cells and in vascular smooth muscle cells (Daub et al., 1996; Grant- charova et al., 2006). We found that in chicken Müller cells, EGFR transactivation occurs during two different phases, whereas in human Müller cells transactivation occurs simultaneously. The reason for this heterogeneity may be a species difference, reflecting differences in intrinsic cellular properties. It is well known that the regenerative capacity of mammalian Müller cells is reduced or non-existing in mammals compared to in birds. Chicken Müller cells can dedifferentiate and proliferate, which are the initial steps in regeneration, while the human Müller cells do not have that capacity. Therefore, this difference may be associated with the temporal differences of ERK signaling. EDNs are pleiotropic and produce several different and sometimes opposing effects in a variety of cells and among different species, which can be explained by the broad range of signal transduction effectors that are activated by the endothelin receptors (EDNRs). Our results are consistent with data showing that EDN2 is a signaling molecule between photoreceptors and Müller cells and that EDN2 induces genes associated with EGFR signaling and reactive gliosis in Müller cells (Bramall et al., 2013; Rattner et al., 2013; Sarthy et al., 2015). The neuroprotective effects of EDN2 and EDNRB that have been seen during photoreceptor degenerations (Bramall et al., 2013; Joly et al., 2008) may relate to the capacity of EDNRs to modulate the injury-response by Müller cells in a similar way as has been seen by α2-ADR agonist (paper III).

Figure 8: Schematic diagram illustrating summary of the results obtained in this thesis. BMD binds and stimulates α2-ADR. Stimulation of that receptor activates cytosolic Src kinase. Activated Src-kinase either stimulates the catalytic activity of MMPs in extracellular membrane, where MMPs cleavage membrane-bound pro HB-EGF ligand or directly interacts with tyrosine domain of EGFR and stimulates EGFR by phosphorylation of its tyrosine residues. Cleaved HB-EGF binds and stimulates EGFR in a conventional mode of action. Activated tyrosine kinase of EGFR allows binding of adapter proteins and consequently activates Ras-Raf mediated ERK1/2 MAPK signaling. Active MAPK signaling leads to differentiation, proliferation, growth and development. MAPK signaling also transcriptionally induces the expression of negative feedback regulators, DUSP1 and 5, MIG6 and SPRY2 and that downregulate the MAPK signaling. Activated tyrosine kinase of EGFR also stimulates PI3K mediated AKT phosphorylation and then phosphorylated AKT inhibits cellular apoptosis. EDNRB agonist IRL1620 stimulates EDNRB and stimulation of that receptor also activates cytosolic Src-kinase dependent MMP- mediated ligand-dependent or ligand-independent transactivation of EGFR. BQ-788, EDNRB blocker; Yohimbine, α2-ADR blocker; PP1/PP2, Src blockers; GM6001, MMP blocker; AG1478, EGFR kinase blocker.

47 Conclusions and perspectives

This thesis has focused on how the progenitor cell and homeostatic properties of Müller cells is regulated after injury. Knowledge of how these properties are regulated is pivotal for developing therapeutic strategies in different retinal degenerative diseases. To understand the regulation of these key properties, we investigated the intra- and extracellular signlaing of Müller cells and studied alpha2–adrenergic and endothelin receptor-induced modulation of Müller cell response to injury and the effects of this modulation from the perspective of neuroprotection. One of the goals of this PhD project was to reveal the mechanisms of how the α2-ADR agonist BMD exerts its neuroprotective effects in the retina. Our results from papers I and II, demonstrate that both chicken and human Müller cells express α2A-ADRs. Stimulation of those receptors by BMD triggers Src-kinase-dependent and both MMP-mediated ligand-dependent and ligand-independent transactivation of EGFRs. Transactivation leads to activation of ERK and AKT signaling in Müller cells. These data are important for understanding the effects of BMD, which is used in glaucoma treatment and the potential effects of EGFR transactivation in the context of neuroprotective and regenerative functions by Müller cells in chickens and, potentially in humans. In paper III, our objective was to study the effects of EGFR transactivation by α2-ADR stimulation in injured retina by investigating the dedifferentiation property of Müller cells. Our results demonstrated that stimulation of α2-ADR by BMD attenuates injury-induced activation and dedifferentiation of Müller cell by attenuating injury-induced ERK signalling. The adrenergic stress-signal triggers synergistic up-regulation of negative ERK- and RTK- feedback regulators during injury. The implications of this study are that adrenergic stress signals modulate glial responses during retinal injury and that α2-ADR pharmacology may be used to modulate responses to glial injury. The results lead to a better understanding of how the α2-ADR agonist BMD induces neuroprotection. In paper IV, our main goal was to study the effect of α2-ADR signaling on excitotoxic retina by analyzing RGC survival in post-natal chicken retina. In addition, we characterized the total RGC populations in chicken retina. Our results demonstrate that pretreatment with the α2-ADR agonist BMD protects RGCs against excitotoxic injury in the chicken retina in a fashion that is similar to neuroprotection in mammals. The total numbers of Brn3a+

48 RGCs in E8 to E10 retinas are approximately 1.5x106; in E12 to E20 are approximately 1.8x106 and in P4 to P11 are approximately 1.9x106 cells per retina. No high-density areas were found in the retina, in contrast to previous data. However, a dorsal region of the retina showed increased susceptibility to the excitotoxic injury. The regional difference was also seen in the effect of BMD. In paper V, we studied another molecule EDN, which is best known as vasoconstrictor but has direct effects on both neurons and glia cells. Our goal in this study was to investigate EDN-induced intracellular signal transduction response in both chicken and human Müller cells. Our results demonstrate that stimulation of the EDN receptor EDNRB by its agonist IRL1620 leads to Src-kinase mediated ERK1/2 activation that engages ligand- dependent and ligand-independent EGFR transactivation. Thus, our data outline a mechanism for how injury-induced EDNs may modulate Müller cell responses by transactivation of EGFRs. Our data support a view in which EDNs, among several other functions, serve as an injury signal that regulates the gliotic response of Müller cells. In conclusion, the results in this thesis provide support to the notion that Müller cell function or malfunction is an important factor that may affect the progression of any disease or injury in the retina. The knowledge that has been gained in this thesis is crucial for understanding how Müller cells can be used to develop neuroprotective treatments for retinal degenerative diseases or acute retinal injuries.

49 Future prospects

This thesis work holds greatly advances our knowledge of the functions of Müller cells in the retina. Nevertheless, this work suggests further exploration in some areas. One of the promising future prospects of this thesis work is cell transplantation therapy. Cell transplantation approaches can be used in retinal degenerative diseases including macular degeneration and glaucoma, where photoreceptor and retinal ganglion cells are progressively dead respectively. BMD-modulated Müller cells can be transplanted to reduce the progression of those diseases. However, prior to clinical study, more preclinical studies are required. For example, this cell transplantation approach could be studied in retinal degenerating animal models such as rds mouse, RCS rat and rat glaucoma models. Other signaling pathways may act as messengers of injury to Müller cells. Sonic hedgehog and Wnt signlaing have been shown to stimulate Müller cells by dedifferentiation and expression of retinal progenitor cell genes (Gallina et al., 2015; Todd and Fischer, 2015). To gain a further understanding of the mechanisms of BMD’s neuroprotective properties, we could study whether BMD regulates the sonic hedgehog and Wnt singling in similar way as we have seen in our system. Other MAPK signaling pathway such as the p38 MAPK pathway may also modulate the Müller cell response to injury. Previous studies have shown that p38 MAPK signaling acts upstream of leukemia inhibitory factor (LIF)- dependent neuroprotection during photoreceptor degeneration (Agca et al., 2013). LIF is an important factor for photoreceptor survival and is produced by Müller cells in response to photoreceptor injury. We can study whether BMD activates p38 MAPK signaling and stimulates its downstream effector LIF in Müller cells, thereby inducing its neuroprotective effects in the retina. In our study, we found expression of α2A-ADR in other cell types in the chicken retina rather than in Müller cells (paper I). For example, expression of α2A-ADR was found in the cells of ganglion cell layer. Therefore, it opens up the possibility that BMD may directly stimulate α2A-ADR in RGCs in order to exert its neuroprotective activity. To test this direct effect, we can perform an in vitro study by isolating RGCs from retina and can study BMD-induced intracellular signaling in RGCs. We can study the activation of neurotrophin receptor such as TrkB receptor in RGCs after α2- ADR stimulation by BMD treatment.

50 Our results from paper V show that injury-induced EDNs modulate Mül- ler cell response by transactivation of EGFRs. We can study the effect of this transactivation in the context of retinal damage or neuroprotection. Studies have shown that BMD induces neuroprotective effect in the EDN1-induced optic nerve ischemia model (Aktas et al., 2007). We can study whether BMD pretreatment modulates the injury-induced EDNs-mediated EGFR transactivation in Müller cells. Similarly, we can study the negative feedback regulation in this system.

51 Materials and methods

Animals All animals experiments in papers I, III, IV and V were performed in ac- cordance with the recommendations in the guide for the care and use of la- boratory animals of the Association for Research in Vision and Ophthalmol- ogy (ARVO) statement and were approved by the local committee on the Ethics of Animal Experiments by Uppsala djurförsökeriska nämnd. Ferti- lized White Leghorn chicken (Gallus gallus) eggs were obtained from OVA Produktion AB (Västerås, Sweden) and incubated at 38°C in a humidified egg-incubator (Grumbach, Germany). All hatched chickens (paper IV) were marked with a numbered plastic ring in the leg and moved to the animal facility at the National Veterinarian Institute (SVA) Uppsala, Sweden. Hatched chickens were fed ad libitum and kept under standard conditions according to the legislation of the Swedish Board of Agriculture.

Intraocular injection In papers I, III, IV and V, embryonic day (E) 18 embryos were injected in- traocularly in the dorsal quadrant of the eye using a Hamilton syringe (Bo- naduz, Switzerland) with 27-G needle supplied with a stopper. A hole was made in the eggshell, head pulled up with a bent glass rod and injections were made through the chorioallantoic membranes. BMD, NMDA and IRL1620 in the sterile saline solutions were injected in the experimental right eye and for control experiment saline solution (vehicle) was injected. All reagents used for intraocular injection were listed in the table 2. After injection, eggs were sealed and incubated for different periods of times as indicated in the figures in each paper.

Müller cell cultures In papers I, III and V, primary chicken Müller cell cultures were established from E14 chicken embryos. Retinas from E14 eyes were dissected, dissoci- ated and cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% new-born calf serum (NCS), 2 mM glutamine, 100 U/mL penicillin,

52 and 100 mg/mL streptomycin at 37°C in a humidified atmosphere of 5% CO2. Cells were grown for up to four weeks and cultures were ready to use when all neurons were gone and the cultures only contained Müller cells. In papers II and V, human Müller cell-line MIO M1 was obtained form E- LUCID, London, UK. MIO- M1 cell-line was cultured in the similar conditions as the primary chicken Müller cells. MIO-M1 cells were ready to use when reached to more that 90 % confluency. Prior to cell culture treatments, chicken primary Müller cells and human MIO-M1 cells were serum-starved for 5 and 16 h respectively. All reagents used for Müller cell culture treatments were listed in the table 2.

Table 2: List of reagents and their mode of action Reagent Target Source Action AG1478 EGFR-kinase 1276, Tocris Bioscience A highly potent EGFR- kinase inhibitor (Han et al., 1996). BMD α2-ADR 2466, Tocris Bioscience Selective α2-ADR agonist (Cambridge, 1981). BQ-788 EDNRB 1500, Tocris Bioscience Potent, selective EDNRB antagonist (Ishikawa et al., 1994). GM6001 MMPs BML-EI300-0001, A potent broad-spectrum ENZO Life Science MMP inhibitor (Santiskulvong and Rozengurt, 2003). IRL1620 EDNRB 1196, Tocris Highly selective Bioscience EDNRB agonist (Takai et al., 1992). NMDA NMDA recep- 0114, Tocris Bioscience NMDA receptor agonist. tor (Laube et al., 1997). PP1 Cytosolic Src P0040-5MG, Sigma A potent and highly selec- kinase Aldrich tive Src kinase inhibitor (Hanke et al., 1996). PP2 Cytosolic Src 1407, Tocris Bioscience A potent and highly selec- kinase tive Src kinase inhibitor (Hanke et al., 1996). EGF EGFR AF-100-15, Peprotech EGFR ligand. Stimulates EGFR signaling (Carpenter, 1987). FGF2 FGF receptor 100-18B, Peprotech FGFR ligand. Stimulates FGFR signaling (Hanke et al., 1996). Yohimbine α2-ADR 1127, Tocris Bioscience Selective α2-ADR antagonist (Goldberg and Robertson, 1983).

53 Immunohistochemistry and Cytochemistry For immunohistochemistry, eyes were dissected and fixed in 4% paraform- aldehyde (PFA) in phosphate-buffered saline (PBS) for 30 minutes at room temperature, incubated in 30% sucrose in PBS for 4 hours at 4°C, embedded in optimum cutting temperature (OCT) freezing medium (Tissue-TEK) or NEG 50 (Thermo Scientific) and sectioned (10 µm) in an orientation parallel to the center of the lens and through the optic nerve exit containing dorsal and ventral retina. For immunocytochemistry, the chicken primary Müller cells or human MIO-M1 cells on coverslips were fixed in 4% PFA in PBS for 15 minutes and then washed in PBS. Sectioned retina or flatten whole retina or fixed Müller cells were incubated with primary antibodies for over night at 4°C and secondary antibodies for 2 hours at room temperature. After that retinas or Müller cells were mounted with ProLong Gold antifade DAPI to visualize nuclei (Thermo Scientific). Antibodies used in this PhD studies were listed in the table 3.

Microscopy Microscopy was performed using Zeiss Axioplan2 microscope integrated with Axiovision software v4.8 (Jena, Germany) and LSM 510 confocal microscope (Jena, Germany). Photomicrographs were captured using same setting of exposure time for both the experimental and control groups. For flat-mounted retina, Zeiss AxioImager 2 (Jena, Germany) was used and photomicrographs were captured in tile acquisition mode with an overlap of 5% between frames.

54 Table 3: List of primary and secondary antibodies used in the studies Antibody Antigen Dilution and purpose Host Source Actin β isoform of 1:1000 for WB Rabbit 3850-100, BioVision actin Brn3a Brn3a 1:200 for IHC Mouse MAB1585, Chemicon CRALBP CRALBP 1:100 for ICC Mouse ab15051, Abcam EGFR EGFR 1:1000 for WB Rabbit sc-03, Santa Cruz GAPDH GAPDH 1:15000 for WB Mouse ab9482, Abcam GS GS 1:100 fro ICC Mouse MAB302, Chemicon Pax6 Pax6 transcrip- 1:200 for IHC Mouse PAX6, DSHB tion factor P-ERK Phospho- 1:200 for IHC/ICC, Rabbit 9101, Cell Signaling ERK1/2 MAPK 1:800 for WB P-EGFR(Y1068) Phospho-EGFR 1:200 for IHC/ICC Mouse 2236, Cell Signaling (Y1068) P-EGFR(Y1173) Phospho-EGFR 1:75 for IHC/ICC, Rabbit sc-12351-R, Santa (Y1173) 1:1000 for WB Cruz Sox2 Sox2 transcrip- 1:200 for IHC/ICC sc-17320, Santa Cruz tion factor Total ERK ERK1/2 MAPK 1:800 for WB Rabbit 4695, Cell Signaling Transitin Transitin inter- 1:50 for IHC Mouse EAP3-S, DSHB mediate filament Visinin Visinin 1:600 for IHC Mouse 7G4, DSHB α2A α2A receptor 1:100 for IHC/ICC Rabbit ab92560, Abcam α2B α2B receptor 1:100 for IHC/ICC Rabbit T3257, Epitomics α2C α2C receptor 1:100 for ICC Rabbit ab15051, Abcam 2M6 2M6 antigen 1:200 for IHC/ICC Mouse Paul Linser, University (TopAP), of Florida (Ochrietor et al., 2010) Goat 2nd anti- Goat IgG 1:1000 for IHC/ICC Donkey Alexa 568/647, Invi- body trogen Mouse 2nd anti- Mouse IgG 1:1000 for IHC/ICC Donkey Alexa 488/568/647, body Invitrogen Rabbit 2nd anti- Rabbit IgG 1:1000 for IHC/ICC Donkey Alexa 488/568, Invi- body trogen Rabbit 2nd anti- Rabbit IgG 1:25,000 for WB Donkey ab97064, HRP conju- body gated, Abcam Note: DSHB, Developmental studies hybridoma bank; HRP, Horseradish peroxidase; IHC, Immunohistochemistry; ICC, Immunocytochemistry; WB, Western blot.

Quantitative Reverse Transcription-PCR Total RNA was extracted with TRIzol (Invitrogen) and cDNA was synthe- sized from 1 µg DNase-treated RNA by using High Capacity RNA to cDNA synthesis kit (Applied Biosystems). The Quantitative Reverse Transcriptase- PCR analyses were performed using IQ SyBr Green Supermix and a C1000 Thermal Cycler (Bio-Rad). Primers were designed by using Primer Express V2.0 software with setting Tm 60°C, 50% G/C and product size minimum 100 base pairs. Primer efficiency, linearity and specificity were checked and

55 the expression levels were calculated from cycle threshold (Ct) and 2-ΔΔCt method (Livak and Schmittgen, 2001). The mRNA expression levels were normalized to housekeeping gene β-actin expression levels.

Western blot analysis Retinas were dissected or the treated cells were scraped off the dish and ho- mogenized in the lysis buffer containing Halt Protease and Phosphatase In- hibitor Cocktail (Thermo Scientific). Total protein concentration was meas- ured by using Dc Protein Assay kit (Bio-rad). Lysis buffer preparation and western blot analysis were performed according to the manufacturer’s in- struction (Bio-rad). Protein expression levels were normalized to β-actin or Glyceraldehyde 3-phosphate dehydrogenase expression levels. Protein densi- tometry was carried out using Image Lab v4.1 software (Bio-rad).

Statistical analysis GraphPad Prism 6 (GraphPad Software Inc.) software was used for statistical analysis. Differences among the multiple groups were tested by one-way analysis of variances (ANOVA) followed by Tukey’s post hoc test as indicated in the figure legends in each paper.

56 Acknowledgements

First of all, I would like to thank almighty Allah who has given me the strength and energy to complete my journey as a PhD student. This journey was full of struggle, ups and downs, some achievements and some failures. However, finally it is done. I would like to thank my supervisor Finn Hallböök for giving me the opportunity to be the PhD student in his group and his constant guidance and encouragements. I am grateful for all the efforts that he put for the accom- plishment of my PhD studies. I am also thankful for his help in developing my scientific writing skill. Thanks to all the past and present members of Retina group. Special thanks to Henrik Boije for introducing me in the retina lab and for all the scientific discussions. Thanks to Henrik Ring for helping me in the teaching and different issues in the lab. Thank you Shahrzad for nice talk and helping me with critical thinking for my project. Thanks to Maria for keeping the lab nice and orderly, and maintaining nice working environment. Minas, thanks for helping me in my project. Sonya, it was nice to have you as a colleague. I would like to convey my gratitude to Caridad for her help in my project especially for doing all the flat-mount immunohistochemistry. I can imagine how tricky it was to dissect out vitreous body from post-natal chicken retina! Thanks to all the students that I have supervised, especially, Marta and Dardan for contributing in my project. Thanks to Jonas and Alireza for helping me in the side projects. Thanks to Lotta for her encouragements and help in the embryo image teaching. My special thanks to David Wheatcroft for his tremendous efforts on proofreading my thesis. I am really grateful to him for his suggestions in thesis writing. Thanks to the Bangladeshi community in Uppsala and all of my friends for their encouragements and supports during my PhD studies. Thanks to my parents for their love and inspirations. Thanks to my young brothers, Rashid and Kowshik for giving more times to our parents in absence of me and I am really missing you guys. My special gratitude to Sultana for her love, continuous inspirations and supports and I am really grateful to have a wife like you. My daughter, Sneha you are my love and inspiration to see the next daylight and I promise to spend more time with you and listen to your stories.

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73 Acta Universitatis Upsaliensis Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1201 Editor: The Dean of the Faculty of Medicine

A doctoral dissertation from the Faculty of Medicine, Uppsala University, is usually a summary of a number of papers. A few copies of the complete dissertation are kept at major Swedish research libraries, while the summary alone is distributed internationally through the series Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine. (Prior to January, 2005, the series was published under the title “Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine”.)

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