Molecular analysis of the neuroprotective role of Norrin for photoreceptors in the mammalian retina DISSERTATION Zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) der Naturwissenschaftlichen Fakultät III Biologie und vorklinische Medizin der Universität Regensburg vorgelegt von Barbara Maria Braunger aus Biberach an der Riß Regensburg, 2013 Das Promotionsgesuch wurde eingereicht am: 8.10.2013 Die Arbeit wurde angeleitet von: Prof. E.R. Tamm Unterschrift: The human being, creature of eyes, needs the image. Leonardo da Vinci (1452 – 1519) Meinen Eltern Table of Contents 1 Introduction: .................................................................................................................................... 1 1.1 Retina....................................................................................................................................... 1 1.1.1 Retinal neurons and glia cells .......................................................................................... 1 1.1.1.1 Retinal ganglion cells ................................................................................................... 1 1.1.1.2 The inner nuclear layer (INL): ...................................................................................... 2 1.1.1.3 Neurons of the INL: amacrine cells, bipolar cells and horizontal cells ........................ 2 1.1.1.4 Retinal glia cells: astrocytes, Müller glia cells and microglia ....................................... 3 1.1.1.5 Cells of the outer nuclear layer (ONL): photoreceptors .............................................. 4 1.1.1.6 The retinal pigment epithelium ................................................................................... 6 1.1.2 Phototransduction and visual cycle ................................................................................. 7 1.1.2.1 Phototransduction ....................................................................................................... 7 1.1.2.2 Visual cycle .................................................................................................................. 8 1.1.3 Electrophysiology: Electroretinography (ERG) ................................................................ 9 1.1.4 Retinal vessels in development and adulthood ............................................................ 10 1.2 Norrin .................................................................................................................................... 12 1.2.1 Norrie disease: symptoms and genetics ........................................................................ 12 1.2.2 The protein Norrin ......................................................................................................... 12 1.2.3 The biological functions of Norrin ................................................................................. 13 1.2.3.1 Angiogenic properties of Norrin ................................................................................ 13 1.2.3.2 Neuroprotective properties of Norrin ....................................................................... 14 1.2.3.3 Norrin in reproduction and development ................................................................. 15 1.2.4 Norrin signaling and the Wnt/β-catenin pathway ........................................................ 16 1.3 Animal models to induce cell specific stress ......................................................................... 19 1.3.1 The retinopathia of prematurity (ROP) model .............................................................. 19 1.3.2 A damage model for photoreceptor degeneration: light damage ................................ 19 1.3.3 Neuroprotective signaling pathways in the context of photoreceptor degeneration .. 19 1.4 Aim of the current work ........................................................................................................ 22 2 Results ........................................................................................................................................... 25 2.1 Characterization of Rpe65-Norrin-2 mice ............................................................................. 25 2.1.1 Southern Blot: successful genomic insertion of the transgene construct .................... 25 2.1.2 Northern blot analysis: effective transcription of the construct ................................... 26 2.1.3 Western blot analysis: effective protein translation ..................................................... 27 2.1.4 Immunohistochemistry: retinal localization of the transgenic Norrin protein ............. 27 2.1.5 Rpe65-Norrin-2 mice and Wnt/β-catenin activation .................................................... 28 2.2 Phenotype analysis of Rpe65-Norrin-2 animals .................................................................... 31 2.2.1 Retinal morphology ....................................................................................................... 31 2.2.2 Ultrastructure of Rpe65-Norrin-2 animals .................................................................... 32 2.2.3 Vascular phenotype ....................................................................................................... 33 2.3 The retinopathia of prematurity (ROP) model ...................................................................... 35 2.4 Light damage experiments .................................................................................................... 40 2.4.1 Morphometric analysis .................................................................................................. 40 2.4.2 In vivo SLO and OCT imaging before and after light damage ........................................ 43 2.4.3 Functional analysis: electroretinography (ERG) analysis ............................................... 45 2.4.4 Analysis of the visual cycle ............................................................................................ 47 2.4.5 Light exposure and apoptosis ........................................................................................ 49 2.4.6 Wnt-/β-catenin signaling and Norrin mediated neuroprotection ................................ 51 2.5 Neuroprotective factors ........................................................................................................ 54 2.5.1 Transgenic Norrin overexpression and neuroprotective factors .................................. 54 2.5.2 Correlation between Norrin induced Wnt/β-catenin activation, EDN2 upregulation and light induced apoptosis .......................................................................................... 58 2.5.3 Brain-derived neurotrophic factor and the protective effects of Norrin ...................... 61 3 Discussion ...................................................................................................................................... 67 3.1 Norrin in the context of neuroprotection ............................................................................. 67 3.1.1 Characterization and morphology of Rpe65-Norrin-2 mice .......................................... 67 3.1.2 Norrin and light exposure .............................................................................................. 68 3.1.3 Norrin, Wnt/β-catenin and Edn2 signaling .................................................................... 69 3.1.4 Norrin neuroprotection and BDNF after light exposure ............................................... 70 3.2 Norrin in the context of angiogenesis ................................................................................... 73 3.2.1 Vascular phenotype of Rpe65-Norrin-2 animals ........................................................... 73 3.2.2 Norrin in the ROP model ............................................................................................... 74 3.3 Future directions ................................................................................................................... 75 4 Summary – Zusammenfassung ...................................................................................................... 77 4.1 Summary................................................................................................................................ 77 4.2 Zusammenfassung ................................................................................................................. 78 5 Material and methods ................................................................................................................... 79 5.1 Methods ................................................................................................................................ 79 5.1.1 Animal models. .............................................................................................................. 79 5.1.1.1 Rpe65-Norrin-2 mice ................................................................................................. 79 5.1.1.2 βB1-Crystallin Norrin mice ........................................................................................ 79 5.1.1.3 Wnt-Reportermice: TOPGAL mice ............................................................................. 79 5.1.2 DNA analysis .................................................................................................................. 80 5.1.2.1 DNA isolation ............................................................................................................. 80 5.1.2.2 Genotyping: Polymerase chain reaction (PCR) .........................................................