DISSERTATION S-NITROSYLATION MEDIATES SYNAPTIC PLASTICITY IN THE RETINA Submitted by Ryan E Tooker Department of Biomedical Sciences In partial fulfillment of the requirements For the Degree of Doctor of Philosophy Colorado State University Fort Collins, Colorado Summer 2015 Doctoral Committee: Advisor: Jozsef Vigh Michael Tamkun Shane Hentges Kim Hoke Copyright by Ryan Edward Tooker 2015 All Rights Reserved ABSTRACT S-NITROSYLATION MEDIATES SYNAPTIC PLASTICITY IN THE RETINA Over the course of an entire day, our visual system must accommodate intensities of light that can change by a factor of 1010. In order to do so, the retina adapts to large, daily changes in natural light intensity by shifting its dynamic range of coding. For example, as morning light intensity increases, the retina implements multiple strategies that result in decreases in overall sensitivity in order to avoid saturation. However, adaptation to bright environments poses the inherent risk of losing visual information carried by dim/weak signals in complex natural scenes. Here we studied whether the light-evoked increase in retinal nitric oxide (NO) production is followed by NO-mediated, direct post-translational modification of proteins called S- nitrosylation and if it contributes to the modulation of the dynamic range of vision. In the central nervous system, including the retina, S-nitrosylation has not been considered to be significant under physiological conditions, and instead, has been primarily associated with neurodegenerative diseases. In this study, we provide immunohistochemical and proteomic evidence for extensive S- nitrosylation that takes place in the goldfish and mouse retinas under physiologically relevant light intensities, in an intensity-dependent manner. Functionally, we report a novel form of activity-dependent synaptic plasticity via S-nitrosylation: a “weighted potentiation” that selectively increases the output of Mb-type bipolar cells in the goldfish retina in response to weak inputs but leaves the input-output ratio for strong stimuli unaffected. Importantly, the NO ii action resulted in a weighted potentiation of Mb output in response to small (≤-30 mV) depolarizations. Our data strongly suggest that in the retina, light-evoked NO production leads to extensive S-nitrosylation and that this process is a significant post-translational modification affecting a wide range of proteins under physiological conditions. S-nitrosylation may function to extend the dynamic range of vision by counteracting the decreases in retinal sensitivity during light adaptation ultimately preventing the loss of visual information carried by dim scotopic signals. Finally, our results may set the framework for exploring the role of S-nitrosylation in certain neurodegenerative retinal diseases that are associated with toxic levels of NO. iii ACKNOWLEDGEMENTS Over the course of my graduate career, I am fortunate and thankful to have been surrounded by a supportive and helpful environment, which played no small part in my ability to achieve success. I have learned an incredible amount from my advisor, Jozsef Vigh, who I appreciate for his commitement to integrity and humility. Mike Tamkun, Shane Hentges and Kim Hoke were an incredibly supportive advisory committee that challenged me and consistently offered valuabe and constructive input. I wish to thank all my co-workers in the Vigh lab, particularly Mikhail Lipin and Valerie Leuranguer for their patience and helpful nature over the years and Shannon Gallagher for his friendship and always being a reminder to value perspectives different than my own. I also wish to express appreciation to my family, whose love and encouragment were a constant reminder of the incredible support that was always behind me. Finally, I am forever grateful for my wife, Alex, who never batted an eye at the prospect of moving our life for me to pursue my dream. She has been my partner through all of this, sharing in the successes and failures, all the while reminding me that there is always something to celebrate. iv TABLE OF CONTENTS ABSTRACT .................................................................................................................................... ii ACKNOWLEDGEMENTS ........................................................................................................... iv LIST OF FIGURES ...................................................................................................................... vii Chapter 1: Introduction ....................................................................................................................1 1.1 The Retina ......................................................................................................................2 1.2 Mechanisms of Light- and Contrast Adaptation in the Vertebrate Retina .....................7 1.3 Nitric Oxide Signaling in the Vertebrate Retina ..........................................................10 1.4 Overall Hypothesis and Aims ......................................................................................16 Chapter 2: Light-Evoked S-nitrosylation in the Retina .................................................................17 2.1 Summary ......................................................................................................................17 2.2 Introduction ..................................................................................................................18 2.3 Materials and Methods .................................................................................................20 2.4 Results ..........................................................................................................................31 2.5 Discussion ....................................................................................................................51 Chapter 3: Nitric Oxide Mediates Activity-Dependent Plasticity of Retinal Bipolar Cell Output via S-nitrosylation ........................................................................................................58 3.1 Summary ......................................................................................................................58 3.2 Introduction ..................................................................................................................59 3.3 Materials and Methods .................................................................................................60 3.4 Results ..........................................................................................................................67 3.5 Discussion ....................................................................................................................99 v Chapter 4: Conclusion..................................................................................................................105 4.1 S-nitrosylation Occurs Under Physiological Conditions in the Retina ......................106 4.2 S-nitrosylation Mediates Weighted Potentiation of Mb Output in the Goldfish Retina .........................................................................................................................109 4.3 S-nitrosylation in Neurodegenerative Diseases .........................................................112 4.4 Final Remarks ............................................................................................................115 References ....................................................................................................................................116 Appendix I ...................................................................................................................................140 Appendix II ..................................................................................................................................148 Appendix III .................................................................................................................................162 Appendix IV.................................................................................................................................163 vi LIST OF FIGURES 1.1 Schematic of retinal anatomy .....................................................................................................3 1.2 Inherent challenges of a visual scene .........................................................................................8 1.3 Schematic of nitric oxide synthesis in neurons ........................................................................12 1.4 NO signaling pathways ............................................................................................................13 2.1 S-nitrosocysteine immunofluorescence in light- and dark-adapted goldfish retinas ...............32 2.2 S-nitrosocysteine immunofluorescence in light- and dark-adapted WT mouse retinas ..........33 2.3 S-nitrosocysteine immunofluorescence labeling pattern in the goldfish retina is shaped by light intensity ...........................................................................................................................35 2.4 Light intensity determines the S-nitrosocysteine immunofluorescence labeling pattern in the wild-type mouse retina .............................................................................................................38 2.5 Light-evoked S-nitrosylation in the goldfish and wild-type mouse retina is prevented by pre- incubation with N-Ethylmaleimide (NEM) .............................................................................42