Photochemical restoration of visual responses in blind mice by Aleksandra Polosukhina A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Vision Science in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Richard Kramer, Chair Professor Marla Feller Professor Diana Bautista Spring 2013 Photochemical restoration of visual responses in blind mice Copyright 2013 by Aleksandra Polosukhina Abstract Photochemical restoration of visual responses in blind mice by Aleksandra Polosukhina Doctor of Philosophy in Vision Science University of California, Berkeley Professor Richard Kramer, Chair This dissertation addresses two non-genetic therapeutic approaches for treating inher- ited retinal diseases such as retinitis pigmentosa and age related retinal degeneration. The firstapproachutilizesalightsensitive moleculeAzobenzene-acrylomide-quaternary amonium (AAQ) to restore light sensitivity to retinal degenerated (rd1) mice. AAQ molecules per- meate through the cell membrane and photosensitize voltage-gated K+ channels in response to different wavelengths of light. Consequently, we achieved robust photosensitization of most retinal cells ex-vivo upon application of AAQ on rd1 mouse retinal explants. We then characterized the effect of AAQ on mouse behavior and restored a pupillary light reflex and light elicited behavior in these blind rd1 mice. The third chapter addresses improvements to the AAQ therapeutic approach. We utilize Di-ethyl-amine-azobenzene quaternary ammonium (DeNAQ) that also confers light sensi- tivity on RGCs by photosensitizing voltage-gated channels in response to 480 nm light and silencing the channels in the dark (trans form). The property of DeNAQ being sensitive in the visible range is one of the major advantages of this therapeutic. The quick relaxation of DeNAQ in the dark is also an advantage, since only one wavelength is required for photosen- sitization. In addition to the red-shifted properties we found that DeNAQ can confer light sensitivity to retinal cells for several weeks and restores light elicited behavioral responses. The fourth chapter addresses the question of whether the physiological and anatomical changes that accompany retinal degeneration lead to functional changes in the retinal gan- glion cells (RGCs). We used a membrane impermeable Quarternary-azobenzene-quaternary molecule (QAQ) molecule that can only enter the cells and render them light sensitive through large pore openings, those seen during pore dilation of P2X and TRP channels. We characterized the path of entry of QAQ using pharmacology and found that photosensiti- zation of RGCs could be reduced by adding P2XR antagonists. In agreement with other studies suggesting that pore dilation has been associated with neurodegenerative processes, we show that QAQ can only confer light sensitivity to RGCs from rd1 and not from wild- type (WT) mice. We also demonstrate that QAQ does not photosensitize RGCs in glaucoma 1 mouse models, suggesting that this physiological change in permeability is specific to retinal degenerating disease. The therapeutic approaches addressed in this thesis apply to a variety of retinal disease genotypes. The high promiscuity of AAQ delivery allows us to optically stimulate any cell type in the retina. Additionally, the red-shifted molecule DeNAQ, combined with its ability to target only diseased tissue, offers a promising clinical alternative for vision restoration. Finally, using another photosensitive molecule, QAQ, we study the physiological impact of retinal degeneration on pore-dilating ion channel activity. Understanding the sources of physiological changes could help prevent further damage caused by degenerative retinal diseases. 2 Contents List of Figures ii 1 Introduction 1 1.1 Retina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.2 Inherited Retinal Degenerating Diseases . . . . . . . . . . . . . . . . . . . . . . . 2 1.3 Glaucoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1.4 Treatments for Retinal Degeneration . . . . . . . . . . . . . . . . . . . . . . . . . 6 2 Photochemical restoration of visual responses in blind mice 9 2.1 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 2.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 2.3 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 2.4 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 2.5 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 3 Restoring visual function to blind mice with red-shifted chemical pho- toswitches 35 3.1 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 3.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 3.3 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 3.4 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 3.5 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 4 Physiological Changes of Retinal Ganglion Cells during Retinal Degen- eration 53 4.1 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 4.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 4.3 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 4.4 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 4.5 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 4.6 Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 i Bibliography 71 ii List of Figures 2.1 AAQ imparts light sensitivity onto blind retinas from rd1 mice. . . . . . . . . . . . 16 2.2 Concentration-dependence of AAQ photosensitization of rd1 retinas. . . . . . . . 17 2.3 AAQ photosensitization abates slowly after treatment. . . . . . . . . . . . . . . . . 18 2.4 Multiple types of retinal neurons contribute to the AAQ-mediated light response of RGCs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 2.5 Blocking retinal synapses greatly reduces AAQ photosensitization. . . . . . . . . 21 2.6 The AAQ treated retina generates spatially precise light responses. . . . . . . . . . 22 2.7 Spectral and illuminance sensitivity of AAQ-mediated photocontrol of RGC firing. 24 2.8 AAQ restores the pupillary light response in mice lacking all retinal photoreceptors. 26 2.9 AAQ restores active light avoidance behavior in mice lacking all retinal photore- ceptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 2.10 AAQ restores light-modulated locomotor behavior in an open-field test. . . . . . . 29 3.1 Solubility of the photoswitch compounds in PBS. . . . . . . . . . . . . . . . . . . . 41 3.2 DENAQ imparts light sensitivity onto blind retinas from rd1 mice. . . . . . . . . . 42 3.3 BENAQ imparts light sensitivity onto blind retinas from rd1 mice. . . . . . . . . . 43 3.4 Intensity response and persistence durves of DENAQ and BENAQ. . . . . . . . . . 44 3.5 DENAQ has no effect on WT and TKO mouse retinas. . . . . . . . . . . . . . . . . 46 3.6 DENAQ is not toxic to WT retina after 1 week treatment in-vivo. . . . . . . . . . 47 3.7 BENAQ does not alter WT light responses. . . . . . . . . . . . . . . . . . . . . . . . 48 3.8 DENAQ restores active light avoidance behavior in mice lacking all retinal pho- toreceptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 3.9 DENAQ restores conditioned fear response. . . . . . . . . . . . . . . . . . . . . . . . 51 4.1 QAQ does not load into WT mouse RGCs. . . . . . . . . . . . . . . . . . . . . . . . 59 4.2 QAQ only photosensetizes rd1 RGCs. . . . . . . . . . . . . . . . . . . . . . . . . . . 60 4.3 bzATP does not enhance QAQ loading in WT RGCs. . . . . . . . . . . . . . . . . . 62 4.4 bzATP enhances QAQ loading in rd1 RGCs. . . . . . . . . . . . . . . . . . . . . . . 63 4.5 TNP-ATP decreases QAQ loading in rd1 RGCs. . . . . . . . . . . . . . . . . . . . 64 4.6 RR greatly reduces QAQ photosensitization. . . . . . . . . . . . . . . . . . . . . . . 66 4.7 One and 10 weeks of IOP elevation is not sufficient to induce QAQ loading in WT RGCs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 iii Acknowledgments First and foremost, I would like to thank my advisor Richard Kramer for providing me with scientific guidance, encouragement, and patiently providing advice and inspiration; I learned immeasurably from his creative way of pursuing questions. I would also like to thank Marla Feller, who introduced me to electrophysiology and provided priceless guidance throughout my graduate studies, Diana Bautista and John Flannery for additional guidance and support, I am grateful for Udi Isacoff’s invaluable encouragement and career advice, my collaborators Dirk Trauner and Russ Van Gelder. Special thanks to my undergraduate advisor Marcelo Camperi who introduced me to neuroscience. I would like to thank my collaborators over the years, Jeff Litt for scientific discussions, advice, encouragement, and for being a wonderful scientist and friend. Thank you to Alexis Fedorchak, Alex Mourot, Doris Fortin, Christian Herold, and the rest of the lab for their companionship, making my graduate experience fun and motivating, and for teaching me how to excel in volleyball. Semyon Dyatlov, Justin Elstrott, Anastasia Anishchenko for their guidance with analysis. Thank you to our lab manager Rachel Montpetit who has provided invaluable support, advice, and friendship. I want to thank the administrative staff in the Vision Science Graduate Group, Inez Bailey for her energy, help, and support over the years. I dedicate this dissertation to my family, friends, and Pierre, for always being there for me and for being incredibly wonderful human beings. iv Chapter 1 Introduction Visionisoneofthemostvital senses, allowing usnotonlytoeffortlessly navigatethroughour daily life, but to also appreciate the beauty of this world. This visual information consists of movement, contrast, brightness, and color, each of which is encoded in different neural circuits in the retina. Once extracted, the retinal ganglion cells send this information to higherorderprocessesofthebrain. However, manydiseasesoftheretinadisruptthisintricate circuitry rendering the person completely blind. It is crucial to enhance our understanding of these diseases in order to find cures and develop more precise tools that could restore visual sensitivity. 1.1 Retina The retina is the first image processing unit in our body that extracts information from the light reflecting off the objects. Vision begins when light is absorbed by the visual pigment molecules in rod and cone photoreceptors. The visual pigment is found primarily in the outer segments of photoreceptor cells and consists of an opsin protein that is covalently attached to 11-cis-retinal. In the dark, photoreceptors rest at -40 mV and maximally release glutamate. When a photon is absorbed, the 11-cis retinal is isomerized to all-trans and activates thevisual pigment. The visual pigment activates the Gprotein, where theGt alpha subunit in turn activates the phosphodiesterase (PDE) enzyme to break down cyclic GMP (cGMP). The decrease in cGMP leads to the closure of cGMP-gated cation channels causing hyperpolarization of the outer segment plasma membrane. Due to the hyperpolarization, the photoreceptors decrease the amount of neurotransmitter (glutamate) released. These changes in glutamate release drive information processing by bipolar cells, horizontal cells, and amacrine cells in the inner nuclear layer (INL). Finally, all the signals converge onto retinal ganglion cells (RGCs), which send information via action potentials to the visual cortex and the lateral geniculate nucleus. Photoreceptors synapse onto bipolar cells in the outer plexiform layer which then trans- mit the signals to retinal ganglion cells in the inner plexiform layer. Horizontal cells play 1 an important role inregulating the signals from photoreceptors to bipolar cells and in for- mation of bipolar cell receptive fields. Photoreceptors release glutamate in the dark thereby depolarizing the OFF bipolar cells, located in the OFF lamina and hyperpolarizing the ON bipolar cells. When photoreceptors are exposed to light, they hyperpolarize and cease re- leasing glutamate, thereby depolarizing the ON bipolar cells and hyperpolarizing the OFF bipolar cells (Nelson et al., 1978; Raviola and Dacheux, 1983). Signals from bipolar cells to retinal ganglion cells are modulated by amacrine cells. The rod bipolar cells connect to ON and OFF cone bipolar cells via the AII amacrine cell. The AII amacrine cell excites ON cone bipolar cells through gap junctions and inhibits the OFF cone bipolar cell through glycine release. Amacrine cells play a role in extracting contrast, color, movement, as well as brightness. Ganglion cells then process the signals in terms of action potentials and send them via the optic nerve to the higher order processes of the brain. However, diseases like retinitis pigmentosa could slowly degenerate the sensitive photoreceptor layer, rendering the rest of the circuitry mostly anatomically in tact but unable to respond to light. 1.2 Inherited Retinal Degenerating Diseases 1.2.1 Retinitis Pigmentosa Millions of people suffer from retinal degenerating diseases, including retinitis pigmentosa, age related macular degeneration, glaucoma, Lebers congenital amaurosis and many others. Retinitis pigmentosa is the most commonly inherited retinal degeneration worldwide, occur- ring in approximately 1 in 5000 individuals, affecting around 1.5 million people in the world (Shintani et al., 2009). RP can develop due to a mutation in up to as many as 100 different genes, 13% inphosphodiesterase-b. This disease can be inherited as an autosomal dominant (30-40% of cases), autosomal recessive (50-60%), x-linked trait (5-15%), but though there are genetic variations of this disease, they all share a common phenotype (Hartong et al., 2006). RP is clinically characterized by lack of night vision and often progresses over time to tunnel vision and then to complete blindness. These effects occur due to the progressive degeneration of rod and then cone photoreceptors, followed by degeneration of the retinal pigment epithelium layer. 1.2.2 Stages of Degeneration The rd1 mouse model has a mutation in phosphodiesterase-b, and photoreceptor degenera- tion begins around postnatal day (P)14 and by P17 only 2% of rod photoreceptors remain (Carter-Dawson et al., 1978). This model goes through three main stages of degeneration. The first phase of degeneration begins before eye opening, around P8. By P30-P40, only the light insensitive photoreceptor bodies remain connected to bipolar cells. The dendritic glutamate receptor expression of iGluRs and mGluRs in bipolar cells is down-regulated, but the expression of iGLuRs on bipolar cells is double than in WT mouse retinas (Marc et al., 2