Medicine, Faculty of
Medicine, Department of
DSpace
UBCV
Skitsko, Dominic
Master of Science - MSc
University of British Columbia
GNAT1 is the α-subunit of a heterotrimeric G-protein present in rod photoreceptors that plays a critical role in phototransduction. Photoactivated rhodopsin binds GNAT1, which stimulates phosphodiesterase to hydrolyze cGMP, closing ion channels and hyperpolarizing the cell. Mutations in human GNAT1 cause Congenital Stationary Night Blindness. Experiments in knock-out GNAT1-/- mice have observed an absent rod ERG with largely unaffected morphology (Calvert et al., 2000). We sought to generate GNAT1-KO Xenopus laevis, taking advantage of their genetic similarity to humans and large photoreceptor size to study anatomically normal but non-functional photoreceptors. We have previously generated F0 GNAT1-KO animals with CRISPR technology, but these animals are genetically chimeric. Therefore, we interbred these F0 animals to produce F1 animals of defined genotypes. Investigation of F1 animals via western blot, histology, electroretinography (ERG), and DNA sequencing has identified true and functional knockouts, a relationship between GNAT1 expression and phototransduction kinetics, and mutations that alter trafficking. X. laevis tadpoles with nonsense mutations in all four GNAT1 alleles presented absent scotopic ERGs. Histology and western blot confirmed the absence of GNAT1 protein, indicating full knockout. Reduction in the level of GNAT1 protein led to a suppression of the ERG, however the relationship was not as direct as expected. We also noted a delay in the timing of the A and B-waves. Greater reduction in GNAT1 protein was associated with greater suppression and delay of the ERG, indicating a second functional role of GNAT1 in addition to signal transduction. We identified two distinct mutations tied to unexpected phenotypes. GNAT1.L-Q184L conferred an ERG phenotype similar to full knockout animals but with minimally reduced GNAT1 protein. A three base pair deletion (T183,Q184→K183) in GNAT1.S caused abnormal rod morphology at the connecting cilium, diminished ERG response, altered return of GNAT1 to the outer segment following light exposure, and defective dark adaptation. The morphological and electrophysiological effects of the Δ3-GNAT1.S mutation were dominantly inherited and required a population of wildtype GNAT1 present to exert their maximum effect.
Characterization of Retinal Function and Morphology in GNAT1-Edited Xenopus laevis by Dominic Skitsko B.Sc., University of Alberta, 2020 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Experimental Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) May 2024 © Dominic Skitsko, 2024 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: Characterization of Retinal Function and Morphology in GNAT1-Edited Xenopus laevis submitted by Dominic Skitsko in partial fulfilment of the requirements for the degree of Master of Science in Experimental Medicine Examining Committee: Dr Orson Moritz, Professor, Department of Ophthalmology and Visual Sciences, UBC Supervisor Dr Robert Molday, Professor, Department of Biochemistry and Molecular Biology, UBC Supervisory Committee Member Dr Hakima Moukhles, Associate Professor, Department of Cellular and Physiological Sciences, UBC Additional Examiner Additional Supervisory Committee Members: Dr Myeong Jin Ju, Assistant Professor, Department of Ophthalmology and Visual Sciences and School of Biomedical Engineering, UBC Supervisory Committee Member iii Abstract GNAT1 is the a-subunit of a heterotrimeric G-protein present in rod photoreceptors that plays a critical role in phototransduction. Photoactivated rhodopsin binds GNAT1, which stimulates phosphodiesterase to hydrolyze cGMP, closing ion channels and hyperpolarizing the cell. Mutations in human GNAT1 cause Congenital Stationary Night Blindness. Experiments in knock-out GNAT1-/- mice have observed an absent rod ERG with largely unaffected morphology (Calvert et al., 2000). We sought to generate GNAT1-KO Xenopus laevis, taking advantage of their genetic similarity to humans and large photoreceptor size to study anatomically normal but non-functional photoreceptors. We have previously generated F0 GNAT1-KO animals with CRISPR technology, but these animals are genetically chimeric. Therefore, we interbred these F0 animals to produce F1 animals of defined genotypes. Investigation of F1 animals via western blot, histology, electroretinography (ERG), and DNA sequencing has identified true and functional knockouts, a relationship between GNAT1 expression and phototransduction kinetics, and mutations that alter trafficking. X. laevis tadpoles with nonsense mutations in all four GNAT1 alleles presented absent scotopic ERGs. Histology and western blot confirmed the absence of GNAT1 protein, indicating full knockout. Reduction in the level of GNAT1 protein led to a suppression of the ERG, however the relationship was not as direct as expected. We also noted a delay in the timing of the A and B-waves. Greater reduction in GNAT1 protein was associated with greater suppression and delay of the ERG, indicating a second functional role of GNAT1 in addition to signal transduction. iv We identified two distinct mutations tied to unexpected phenotypes. GNAT1.L-Q184L conferred an ERG phenotype similar to full knockout animals but with minimally reduced GNAT1 protein. A three base pair deletion (T183,Q184àK183) in GNAT1.S caused abnormal rod morphology at the connecting cilium, diminished ERG response, altered return of GNAT1 to the outer segment following light exposure, and defective dark adaptation. The morphological and electrophysiological effects of the D3-GNAT1.S mutation were dominantly inherited and required a population of wildtype GNAT1 present to exert their maximum effect. v Lay Summary The protein rod a-transducin, or GNAT1, is a key component of the pathway that converts light into signals interpretable by our brains. Mutations in the gene coding for this protein cause a variety of inherited blinding conditions in humans. Here, we have utilized Xenopus laevis, or the African Clawed Frog, as a model to investigate the effect of several mutations in GNAT1 on the structure and function of the retina, with a focus on the rod photoreceptors. We generated animals with no functional GNAT1 protein and investigated the effect of this absence. In the process, we made note of several interesting mutations that arose. These results deepen our understanding of the fundamental processes underlying our vision, and how changes in these processes may be involved in a variety of diseases. vi Preface All data collection and analysis presented within were conducted by myself. Super resolution confocal microscopy displayed in Figure 3.5 was obtained with the assistance of Beatrice Tam. Figures 1.9, 1.10, 1.11, and 1.12 are used with permission from the copyright holder, Dr. Orson Moritz. Research goals were designed in collaboration with my supervisor, Dr. Orson Moritz. At the time of submission, no publications have arisen from the work presented in this dissertation. All procedures involving animals were approved by the UBC animal care committee under certificates A22-0145 Xenopus Protocol – Experimental, and A22-0146 Xenopus Protocol – Breeding issued to Orson L. Moritz. vii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ........................................................................................................................ vii List of Tables ..................................................................................................................................x List of Figures ............................................................................................................................... xi List of Abbreviations ................................................................................................................. xiv Acknowledgements ................................................................................................................... xvii Chapter 1: Introduction ................................................................................................................1 1.1 The Eye ........................................................................................................................... 1 1.1.1 The Retina ............................................................................................................... 1 1.1.2 Rod and Cone Photoreceptors ................................................................................. 3 1.1.3 Compartmentalization of Photoreceptors ............................................................... 6 1.1.4 Mechanisms of Phototransduction in Rods ............................................................. 8 1.1.5 Kinetics of Phototransduction ............................................................................... 11 1.1.6 a-transducin (GNAT1) ......................................................................................... 12 1.1.7 Disorders of Phototransduction and a-transducin Mutants .................................. 21 1.2 Xenopus laevis as a Model for Retinal Development and Disease ............................... 26 1.3 CRISPR/Cas9 Gene Editing ......................................................................................... 27 1.4 Electroretinography ....................................................................................................... 30 1.5 Rationale and Objectives .............................................................................................. 34 viii Chapter 2: Generation of GNAT1-Knockout Xenopus laevis ..................................................41 2.1 Introduction ................................................................................................................... 41 2.2 Methods......................................................................................................................... 42 2.2.1 Animal Rearing ..................................................................................................... 42 2.2.2 DNA Processing and Analysis .............................................................................. 42 2.2.3 Electroretinogram Screening of F1 Tadpoles ....................................................... 43 2.2.4 Western Blotting and Analysis ............................................................................. 44 2.2.5 Confocal Microscopy ............................................................................................ 46 2.3 Results ........................................................................................................................... 47 2.3.1 Identification of Promising F0 CRISPR-Edited X.laevis ...................................... 47 2.3.2 Identification of F1 GNAT1-KO X. laevis ............................................................ 49 2.3.3 Relative Expression of GNAT1.S and GNAT1.L ................................................... 55 Chapter 3: Investigation of GNAT1-Edited X. laevis .................................................................56 3.1 Introduction ................................................................................................................... 56 3.2 Methods......................................................................................................................... 57 3.2.1 Characterization of Mutants .................................................................................. 57 3.2.2 Development of Primers for PCR Genotyping ..................................................... 57 3.2.3 Light Induction of GNAT1 Translocation ............................................................ 58 3.2.4 GNAT1 Localization Measurements .................................................................... 59 3.2.5 Dark Adaptation Electroretinography ................................................................... 59 3.2.6 Super Resolution Microscopy ............................................................................... 60 3.3 Results ........................................................................................................................... 62 3.3.1 Reduction in GNAT1 Expression Produces Delays in the ERG Response ........... 62 ix 3.3.2 The One Base Pair Substitution Causing GNAT1.L-Q184L Produces Functional GNAT1-Knockouts ............................................................................................................... 66 3.3.3 A Three Base Pair Deletion Produces a Dominant Morphological and Electrophysiological Phenotype ............................................................................................ 69 3.3.3.1 Initial Identification of D3-GNAT1.S Phenotype .............................................. 69 3.3.3.2 The Effect of the D3-GNAT1.S Mutation is Dominant ..................................... 71 3.3.3.3 The D3-GNAT1.S Phenotype is Only Produced by the ERG Light Protocol .... 74 3.3.3.4 The Photopic Flicker is Critical in the Production of the D3-GNAT1.S Phenotype. ......................................................................................................................... 77 3.3.3.5 Light Intensity Alone Does Not Explain the Difference in Phenotype Generation Between Lighting Protocols ........................................................................... 77 3.3.3.6 GNAT1 Concentrates Alongside a-tubulin Within the Inner Segment in GNAT1.S-D3 Mutant Animals .......................................................................................... 79 3.3.3.7 A Minimum Level of Normally Functioning GNAT1 is Required to Produce the D3-GNAT1-S-Associated Morphological Phenotype .................................................. 81 3.3.3.8 Dark Adaptation is Altered in D3-GNAT1.S Animals ...................................... 88 Chapter 4: Conclusions ...............................................................................................................94 References ...................................................................................................................................105 x List of Tables Table 3.1 The Functional Knockout Phenotype Associated with Q184L-GNAT1.L is Recessive in Inheritance ..................................................................................................................................... 68 Table 3.2 Light Intensity Measurements Across Different Treatments Used .............................. 78 Table 3.3 Characterization of Effect of Mutations Present in D3-GNAT1.S F1 x F0 Mating ...... 87 xi List of Figures Figure 1.1 Overview of Retinal Anatomy ....................................................................................... 2 Figure 1.2 Comparison of Rod and Cone Structure ........................................................................ 5 Figure 1.3 Activation of the Phototransduction Cascade .............................................................. 10 Figure 1.4 Alpha-Transducin Protein Structure ............................................................................ 15 Figure 1.5 Alpha-Transducin Localization and Transport Under Differing Lighting Conditions 20 Figure 1.6 Overview of CRISPR-Cas9 DNA Editing .................................................................. 29 Figure 1.7 Averaged Wildtype Scotopic ERG Trace .................................................................... 32 Figure 1.8 Representative Wildtype ERG Trace Demonstrating Oscillatory Potentials .............. 33 Figure 1.9 P23H-Rhodopsin Increases Light-Induced Autophagy in X. laevis ............................ 37 Figure 1.10 Knockout of GNAT1 Reduces Light-Induced Autophagy in X. laevis ...................... 38 Figure 1.11 Bleaching of T4K and T17M Rhodopsin Produces Abnormal Persistent Photoproducts ............................................................................................................................... 39 Figure 1.12 Knockout of GNAT1 Exacerbates T4K-RHO-Induced Retinal Degeneration .......... 40 Figure 2.1: Standard Curve for Quantification of GNAT1 Expression from Western Blots ........ 45 Figure 2.2 Representative Examples of Sequence Traces Used in Characterization ................... 48 Figure 2.3 Characterization of GNAT1-Knock Out X. laevis ....................................................... 50 Figure 2.4 ERG Magnitude is Highly Suppressed in GNAT1-KO X. laevis ................................. 51 Figure 2.5 GNAT1 Expression in X. laevis Identified as Knock Outs is Significantly Reduced Compared to Wild Type ................................................................................................................ 52 Figure 2.6 Representative Example of GNAT1.L and GNAT1.S Sequences of Knock Out Animals....................................................................................................................................................... 53 xii Figure 2.7 Alternate GNAT1-KO ERG Phenotypes ..................................................................... 54 Figure 3.1 Characterization of X. laevis with Reduced Expression of GNAT1 ............................ 63 Figure 3.2 ERG Wave Magnitude Does Not Correlate Strongly with GNAT1 Expression ......... 64 Figure 3.3 Implicit Time of the ERG Response Strongly Correlates to GNAT1 Expression ....... 65 Figure 3.4 A One Base-Pair Substitution Producing the Mutation Q184L in GNAT1.L Results in a Functional Knock Out of GNAT1 .............................................................................................. 67 Figure 3.5 A Three Base-Pair Deletion in GNAT1.S Producing the Mutation T183-Q184 à K183 Is Linked to the Development of Abnormal Morphological Structures with Minimal Alteration of the ERG Response ........................................................................................................................ 70 Figure 3.6 Mating of an F1 Animal to Wild Type Reveals a Dominant Morphological and Electrophysiological Phenotype Associated with D3-GNAT1.S .................................................. 72 Figure 3.7 GNAT1 Localization is Significantly Altered in D3-GNAT1.S Animals .................... 73 Figure 3.8 Use of the ERG Lighting Protocol is Required to Generate Structural Abnormalities in D3-GNAT1.S Animals ................................................................................................................... 75 Figure 3.9 The Extent of GNAT1 Mislocalization in D3-GNAT1.S Animals is Dependent on Lighting Conditions ...................................................................................................................... 76 Figure 3.10 GNAT1 Co-Localizes with a-Tubulin at the Base of the Connecting Cilia in D3-GNAT1.S Animals. ........................................................................................................................ 80 Figure 3.11 In D3-GNAT1.S Animals with no Wild Type Alleles GNAT1 Localizes to the Corners of the Rod Inner Segment, Increasing in Prominence with Age ..................................... 82 Figure 3.12 In D3-GNAT1.S Animals with no Wild Type Alleles GNAT1 is Mislocalized to the Rod Inner Segment Under all Lighting Conditions ...................................................................... 84 xiii Figure 3.13 Sibling Animals Expressing Mutations other than D3-GNAT1.S Have More Inner Segment GNAT1 Labeling than Wild Type ................................................................................. 85 Figure 3.14 GNAT1 Return to the Outer Segment is Significantly Altered in D3 x WT-GNAT1.S Animals ......................................................................................................................................... 90 Figure 3.15 Measurement of the Mean Signal Intensity of Rod Inner and Outer Segments Shows that Changes in the OS:IS GNAT1 Ratio are due to Light-Induced Movement Between Compartments ............................................................................................................................... 91 Figure 3.16 Electroretinography Shows Dark Adaptation is Disrupted in D3 x WT-GNAT1.S Animals ......................................................................................................................................... 93 Figure 4.1 Proposed Pathway for the Interaction Between GNAT1 and Rhodopsin Photointermediates ........................................................................................................................ 97 xiv List of Abbreviations bp: Base pair Cas9: CRISPR associated 9 CC: Connecting cilium cGMP: Guanosine 3’,5’-cyclic monophosphate CRISPR: Clustered regularly interspaced short palindromic repeats CSNB: Congenital stationary night blindness DNA: Deoxyribonucleic acid dpf: Days post fertilization ELM: External limiting membrane ER: Endoplasmic reticulum ERG: Electroretinogram/Electroretinography ERP: Early receptor potential GAP: GTPase-activating protein GCAP1: Guanylyl cyclase activating protein 1 GCL: Ganglion Cell Layer GDP: Guanosine 3’-diphosphate GEF: Guanine nucleotide exchange factors Glu: Glutamate GNAT1: Rod a-transducin GPCR: G protein coupled receptor GTP: Guanosine 3’-triphosphate xv HCG: Human chorionic gonadotropin HDR: Homology directed repair ILM: Internal limiting membrane Indel: Insertion or deletion INL: Inner nuclear layer IPL: Inner plexiform layer ipRGC: Intrinsically photosensitive retinal ganglion cell IRD: Inherited retinal dystrophy IS: Inner segment KO: Knock-out Meta I/II/III: Metarhodopsin I/II/III NFL: Nerve fibre layer NHEJ: Non-homologous end joining ONL: Outer nuclear layer OPL: Outer plexiform layer OS: Outer segment PAM: Promoter adjacent motif PCR: Polymerase chain reaction PDE: Phosphodiesterase RD: Retinal degeneration RNA: Ribonucleic Acid RP: Retinitis Pigmentosa sgRNA: single-guide RNA xvi WGA: Wheat germ agglutinin WT: Wildtype X. laevis: Xenopus laevis xvii Acknowledgements I would like to express my sincere gratitude to my supervisor, Dr. Orson Moritz, for providing the opportunity to conduct research in his lab, and his support and guidance throughout the process. His wealth of knowledge coupled with endless curiosity, optimism, and a genuine passion for learning and research has inspired me throughout my time in the lab and will continue to do so. I would also like to thank the members of my supervisory committee, Dr. Robert Molday and Dr Myeong Jin Ju, for their thoughtful discussion, extremely helpful feedback, and encouragement throughout the process of my MSc. My thanks to all the current and former members of the Moritz Lab who I had the pleasure to share time with: Beatrice Tam, Colette Chiu, Aaron Loewen, James Hui, Dr. Brittany Carr, Farhad Ghasmeminejad, Ross Scharbach, Cristina Novakovic, and Eric Reich. From taking time out of pandemic-tightened schedules to train me when I arrived, to endless helpful discussions both formal and thrown out across lab benches, none of this work would have been possible without their help and I will be forever grateful. Finally, a special thanks to all my family and friends for their support and encouragement. This thesis would double in length if I tried to list every way they have helped me, but know that it means the world to me. To my parents, who have done more for me than I could ever repay. For supporting me no matter where life takes me, thank you. 1 Chapter 1: Introduction 1.1 The Eye 1.1.1 The Retina The retina is a highly organized central nervous tissue lining the back wall of the globe of the eye. It is composed of five major cell types which integrate to detect light, produce an electrical impulse, and pass this signal along to the brain where it can be interpreted (Sung & Chuang, 2010; Fig. 1.1). Photoreceptors detect incoming light and produce a stimulus in response. Horizontal and amacrine cells are interneurons that refine this signal, integrating information from multiple photoreceptors and laterally inhibiting to improve contrast. Bipolar cells transmit information to ganglion cells, which pass neural impulses to the primary visual cortex of the brain via the optic nerve, a bundle of ganglion cell axons exiting the eye (Gregg et al., 2023). The retina itself is segmented into a series of clearly divided layers starting from the photoreceptors and moving inwards through the external limiting membrane (ELM), outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), ganglion cell layer (GCL), nerve fiber layer (NFL), and internal limiting membrane (ILM) (Gregg et al., 2023). Underlying the photoreceptors is the melanated retinal pigment epithelium (RPE), a layer providing a range of supportive functions to the neural retina including metabolism, light absorption, and containing visual cycle components (Gregg et al., 2023). 2 Figure 1.1 Overview of Retinal Anatomy A graphical overview of the layered architecture of the retina. Incoming light enters through the pupil, traveling through the nervous layers of the inner retina before reaching the photoreceptor layer where it is detected by the rods and cones. Signals are passed from these cells to the brain via the bipolar and ganglion cells, with amacrine and horizontal cells supporting and enhancing this process. Retinal pigment epithelium (RPE) cells play a major supportive role in the function and health of the photoreceptors. Adapted from “Structure of the Retina”, by BioRender.com (2024). Retrieved from https://app.biorender.com/biorender-templates 3 1.1.2 Rod and Cone Photoreceptors Vertebrate photoreceptors can be divided into two classes based on function and, to a varying degree in different organisms, morphology (Fain et al., 2010; Fig. 1.2). Rods are light-sensitive to the single photon level with a peak absorption of approximately 500 nm, expressing one visual pigment, and have a low capacity for adaptation (Athanasiou et al., 2018). Cones are less sensitive by ~100-fold but are highly adaptable, allowing them to detect brighter stimuli continuously. Cones express one of a range of visual pigments, allowing the total population to respond to light between ~350-560 nm (Athanasiou et al., 2018). Broadly, rods are responsible for dim light and peripheral vision, while cones enable bright light color vision (Hoon et al., 2014). Structurally, the photoreceptors share common elements. The cells are highly polarized and can be divided into an outer segment (OS) and inner segment (IS) joined by a connecting cilium (CC), a nucleus and a synaptic terminal (Perkins & Fadool, 2010). The rod OS is an elongated modified cilium packed with enclosed membranous discs containing the components of phototransduction, including the light-sensitive G protein coupled receptor (GPCR) rhodopsin and its heterotrimeric G protein partner transducin (Arikawa et al., 1992). Proteins are trafficked to the OS via the connecting cilium from the IS, which contains the other organelles of the cell (Mustafi et al., 2009). While the morphological distinctions between the cell types vary by species, a major difference is that the discs of rods are closed off to the surrounding cytoplasm, while many cone discs remain open (Arikawa et al., 1992). 4 As the site of phototransduction, outer segment discs accumulate damage due to reactive byproducts (Young & Bok, 1969); Young, 1976). As a result, photoreceptors replace up to 10% of their discs daily, shedding old discs for recycling by the supporting retinal pigment epithelium cells and generating new discs at the base of the outer segment (Besharse et al., 1977); Young, 1967; Young & Bok, 1969; Young & Droz, 1968). 5 Figure 1.2 Comparison of Rod and Cone Structure Rods and cones share a similar overall structure. Both are structurally and functionally divided into an outer segment (OS), inner segment (IS), and synaptic terminal. The OS is a modified primary cilium containing stacks of discs, which in rods are closed off and discontinuous from the cytoplasm while in cones they remain open on one end. The IS contains the biosynthetic machinery of the cell and is continuous with the synaptic terminal, the region interacting with bipolar cells. Outer and inner segments are joined by a connecting cilium, facilitating protein transport between the two. The extent to which the morphology of the cell varies across species. In the case of X. laevis, rods are significantly larger with columnar outer segments, while cones are more conical in shape. Adapted from “Morphology of Photoreceptors”, by BioRender.com (2024). Retrieved from https://app.biorender.com/biorender-templates 6 1.1.3 Compartmentalization of Photoreceptors As mentioned above photoreceptors are divided into distinct compartments, each serving their own function (Fig. 1.2). The membranous discs contained within the outer segment are the site of phototransduction, the inner segment is packed with mitochondria in a region termed the ellipsoid zone and contains the biosynthetic machinery of the cell, while the synaptic terminal hosts proteins involved in interaction with the neurons of the inner retina (Malhotra et al., 2021). This structural and functional compartmentalization requires a high degree of control over the localization of proteins, maintaining the health and proper function of the cell by condition-dependent movement of proteins. Phototransduction proteins are manufactured by the organelles within the inner segment and must be trafficked to the site of the phototransduction cascade in the outer segment discs. The constant replacement of old discs means these proteins are produced and transported in massive quantities, especially rhodopsin at around 300 million molecules per day per X. laevis rod (Malhotra et al., 2021). An intrinsic membrane protein, the seven trans-membrane domain GPCR rhodopsin is transported from the ER to the Golgi where it is packaged into vesicles (Malhotra et al., 2021). The small GTPase Arf4 binds the rhodopsin C-terminal VxPx OS localization signal (Wang et al., 2017). This cues the recruitment of Rabin8 and formation of a Rab11-Rabin8-Rab8 module through several intermediaries (Wang & Deretic, 2014), which recruits the VAMP7 SNARE protein to allow membrane fusion and release of the vesicular contents at the base of the connecting cilium (Kandachar et al., 2018). 7 From here, rhodopsin and other vesicular contents are transported to the OS discs through mechanisms which remain unclear, with both active transport and passive diffusion being proposed (Malhotra et al., 2021). Peripheral membrane proteins, including the subunits of the heterotrimeric G protein transducin, reversibly interact with membranes depending on a variety of factors. Additionally, several of these proteins move between compartments in a light-dependent manner. These facts mean that proper localization of these proteins requires mechanisms on top of the standard secretory pathway, with lipid modification to control membrane affinity being critical (Maza et al., 2019). The single lipid anchor of a-transducin is insufficient to induce strong membrane association and as such, its membrane affinity depends on its association with other proteins. In its heterotrimeric form, the anchors of bg-transducin anchor the G protein to the membrane where it can interact with rhodopsin (Kosloff et al., 2008; Rosenzweig et al., 2007). This increase in membrane affinity when associated with bg-transducin means that transport of a-transducin to the OS from the IS requires dissociation. This transport is facilitated by chaperones which bind and hide the membrane anchor of a-transducin, solubilizing the protein for transport to the connecting cilia (Frederick et al., 2020). Many peripheral outer segment proteins are similarly dependent on the activity of small chaperones within the inner segment. For example, the protein RD3 serves to inactivate and traffic GCAP1 (Guanylyl cyclase activating protein 1) from the ER to the connecting cilia (Molday et al., 2014). GCAP1 serves to increase cGMP concentration and is key in the deactivation and recovery from phototransduction within the outer segment (Molday et al., 2014). 8 Deactivation of the protein within the inner segment helps maintain proper functional compartmentalization, preventing GCAP1 from operating within the inner segment and decreasing local GTP concentration (Molday et al., 2014). Specifics of the chaperone-based trafficking of a-transducin will be discussed below. 1.1.4 Mechanisms of Phototransduction in Rods Phototransduction is the highly sensitive signaling cascade in the rod OS producing electrical signals from a light stimulus. Incoming photons stimulate the 7-transmembrane G-protein-coupled receptor (GPCR) rhodopsin, composed of rod opsin and a bound 11-cis retinal chromophore (Park, 2014), isomerizing the 11-cis into all-trans retinal. This proceeds through several spectroscopically distinct intermediates, passing through the inactive metarhodopsin I into the active metarhodopsin II, with a portion of the Meta I population undergoing a reversible decay into metarhodopsin III (Zimmermann et al., 2004; Vogel et al., 2004). Structurally, Meta I is largely identical to dark state rhodopsin (Rothschild et al., 1987). The critical change in conversion to Meta II that accompanies the spectral shift is the deprotonation of the retinal Schiff base and protonation of its counterion Glu 113, disrupting a salt bridge and allowing conformational changes (Bartl & Vogel, 2007). The transition to Meta III involves an isomerization of this Schiff base from all-trans 15-anti to all-trans 15-syn (Bartl et al., 2001; Vogel et al., 2003; Vogel et al., 2004), with the chromophore remaining in the binding pocket (Heck et al., 2003). Meta I and Meta II rhodopsin exist in a temperature and pH-dependent equilibrium after activation, with this being shifted in favor of Meta II by the presence of a-transducin (Emeis et al., 1982). 9 Meta III is extremely persistent in vitro but much shorter lived in vivo, being converted to Meta II by direct interaction with the a subunit of transducin (Zimmermann et al., 2004). It is theorized to function as a form of retinal storage, regulating the amount of retinal entering the visual cycle for regeneration (Zimmermann et al., 2004). The rhodopsin conformation change to Meta II enables interaction with the heterotrimeric G-protein transducin, promoting a GDP à GTP exchange and dissociation of the a subunit (GNAT1) from the bg complex. Activated rhodopsin (R*) goes on to activate additional transducin proteins while GNAT1-GTP binds cGMP-phosphodiesterase (PDE), displacing the inhibitory g-subunit and freeing the protein to convert cGMP to 5’-GMP. This decrease in the cGMP concentration of the cell leads to the closing of cGMP-gated cation channels and hyperpolarization of the cell, closing voltage-gated calcium channels (Yue et al., 2019). As a result, the photoreceptor ceases its steady release of glutamate (Glu) from the synapse, releasing inhibition of second order bipolar cells and beginning the passage of the visual signal to the brain (Leskov et al., 2000; Fig. 1.3). 10 Figure 1.3 Activation of the Phototransduction Cascade Light stimulation of rhodopsin induces interaction with the G protein transducin. The alpha subunit exchanges GDP for GTP and separates from the bg complex, allowing it to bind and activate cGMP phosphodiesterase (PDE). The resulting decrease in intracellular [cGMP] closes cGMP gated cation channels, hyperpolarizing the cell. At the level of the synaptic terminal this change in cellular polarization stops the photoreceptor from releasing glutamate, removing the tonic inhibition of second order neurons that occurs in the dark, allowing signaling to the brain. Reproduced from “Phototransduction in Photoreceptors”, by BioRender.com (2024). Retrieved from https://app.biorender.com/biorender-templates 11 Following the transmission of a visual impulse, the phototransduction cascade must be deactivated rapidly to allow for responses to subsequent stimuli. This requires shut down of active signaling components and subsequent recovery of intracellular cGMP concentration, opening cation channels, and re-starting the dark current. R* undergoes multiple phosphorylations, inducing binding of visual arrestin which completes the quenching (Ohguro et al., 1993; Xu et al., 1997). PDE deactivation occurs when the GAP (GTPase activating protein) RGS-9 increases the rate of GNAT1-GTP hydrolysis, allowing its replacement by the inhibitory PDE-g subunit (Arshavsky & Wensel, 2013; Tsang et al., 1998). With the removal of this stimulus, the protein GCAPs (guanyl cyclase activating protein), whose activity is stimulated by decreased calcium levels caused by channel closure, can activate retinal guanlylyl cyclase to restore normal levels of cGMP and bring about recovery of the dark current (Palczewski et al., 2004). 1.1.5 Kinetics of Phototransduction As the initiation point for vision, aspects of phototransduction are tightly regulated, including the rate at which it proceeds. This is reflected by the consistency of the single photon response, the highly reproducible signal detected upon minimal stimulation of an individual rod. While most signals generated from single molecule stimulation fluctuate greatly due to variance in the activity of the molecule, the rod single photon response occurs with ~20% of the trial-by-trial variance expected for first-order processes (Baylor et al., 1979; Rieke & Baylor, 1998;Whitlock & Lamb, 1999), indicating mechanisms exist to control this randomness. This reduction is due to the multiple steps involved in rhodopsin shut off. 12 Variance increases drastically with each phosphorylation site removed from rhodopsin (Doan et al., 2006). The multiple steps involved in the shutdown of rhodopsin decrease the overall variance of the process by compensating for any differences in the timing of individual steps across different trials. Additional contributions to the decreased variance of phototransduction may come from saturation of the transducin portion of the cascade. Overexpression of R9AP, the anchor protein for RGS9, results in significantly accelerated inactivation of the GNAT1-PDE complex along with increased speed of single photon responses and faster recovery following stimulation (Sundermeier et al., 2014). Knockout of the protein had the opposite effect on phototransduction, presenting with normal response amplitude but a 10-fold increase in recovery time (Keresztes et al., 2004). Additionally, human mutations to GNB5 present with normal appearance on OCT but significantly reduced ERG amplitude, with the specific signaling defects resembling bradyopsia (Shao et al., 2020), or delayed visual response. 1.1.6 a-transducin (GNAT1) While Rhodopsin is the initiator of the phototransduction cascade, the G protein transducin is the first amplifier, with each R* activating twelve to fourteen transducin proteins (Yue et al., 2019). The heterotrimeric protein is composed of an a, b, and g subunits, with bg functioning as a complex (Lambright et al., 1996; Fig. 1.4). a-transducin, the active signaling component as discussed above, is encoded by the gene GNAT1 in rods and GNAT2 in cones (G. Lin et al., 2013). GNAT1, along with the other transducin subunits are produced within the inner segment. 13 Post-translational modification of these proteins is essential for membrane association and OS targeting, allowing for transport of the protein to the outer segment discs to play its essential role in phototransduction. Rod g-transducin is farnesylated at the C-terminus (Kassai et al., 2005). Typical G protein a-subunits are myristrolated (C14) at the far N-terminus and/or palmitolated at an N-terminal cysteine residue (Kerov et al., 2007). Rod g-transducin is farnesylated at the C-terminus, while rod GNAT1 is unique in its heterogeneous N-terminal acetylation with either lauroyl (C12:0), myristoyl (C14:0), (cis-delta5)-tetradecaenoyl (C14:1) or (cis,cis-delta5,delta8)-tetradecadienoyl (C14:2) found attached (Neubert & Hurley, 1998). These modifications are essential for proper localization of GNAT1 and play a key role in its ability to translocate between compartments when required (Kerov et al., 2007; Lobanova et al., 2007). The overall structure of a-transducin can be divided into GTPase, a-helical, and N-terminal helix domains (Lambright et al., 1996; Fig 1.4). The ras-like GTPase domain is made up of a b sheet surrounded by six helices and contains the three switch regions of the protein which change conformation upon GDP à GTP exchange (Lambright et al., 1996). The switch I (residues 172-183) and II (227-238) regions, as well as the N-terminal helix of a-transducin are critical in interaction with the bg subunits, as well as other effectors (Lambright et al., 1996) As with other phototransduction proteins, a-transducin is synthesized within the inner segment before transport to the outer segment, as described above. Within the inner segment it interacts with several proteins aside from the other transducin subunits, most of which are involved with ensuring proper trafficking. 14 An additional interactor with unclear purpose is Ric8A, a GEF known to interact with GDP-bound a-subunits that associates with rod a-transducin in vitro (Srivastava et al., 2020). Despite a proposed role as a chaperone, rod-specific knockout of Ric8A causes only a slight reduction in a-transducin expression and no electrophysiological deficits (Srivastava et al., 2020) indicating its GEF activity may be more important, with its precise role unclear. 15 Figure 1.4 Alpha-Transducin Protein Structure Heterotrimeric transducin interacts with activated rhodopsin stimulating guanine nucleotide exchange and release of the a-subunit. On the structure of active GTP-bound bovine a-transducin several important residues are marked to identify regions critical in protein interactions, including with the bg complex and effectors of a-transducin. (PDB 6OYA (Gao et al., 2019) / 1TND (Noel et al., 1993)). These residues are conserved in X. laevis GNAT1 (Hellsten et al., 2010). Created with BioRender.com 16 Under dark adapted conditions the full heterotrimeric G-protein is bound to GDP and associated with the rod OS membranes through the combined strength of the three subunits’ lipid anchors (Kosloff et al., 2008). Upon rhodopsin activation of transducin, the a subunit dissociates from bg to activate PDE as described above. Following signaling, the a-transducin-PDE complex must be deactivated. The intrinsic GTPase activity of a-transducin is enhanced by RGS-9, its membrane anchor R9AP, and its partner Gb5. The hydrolysis of GTP allows for displacement of a-transducin and re-association of the inhibitory g-PDE subunit (Arshavsky & Wensel, 2013). The majority of GNAT1 within a rod is activated only when a minimum light threshold is met (Sokolov et al., 2002), with this threshold being linked to the ability of the cell to activate the GNAT1-GTPase complex (Kerov et al., 2005). When this is surpassed and GNAT1 is produced in excess of the rod’s ability to inactivate it, the insufficient membrane affinity of free GNAT1 results in mass translocation of up to 80% of all molecules to the inner segment with a t1/2 of 3-5 minutes (Calvert et al., 2006; Artemyev, 2008; Fig. 1.5). The effect of phosducin KO supports this. The protein serves to sequester bg transducin in the outer segment, increasing the lifetime of active GNAT1. Knockout of phosducin decreases movement of GNAT1 to the inner segment by allowing bg to interact with a, decreasing the amount of free GNAT1 available to move to the IS (Sokolov et al., 2004). This process serves several functions. Mass GNAT1 translocations prevents overstimulation of the photoreceptor under periods of intense stimulation, avoiding related toxicity, and allows rods to avoid saturation by reducing their gain, recovering a portion of their dark current and avoiding long-lasting channel closure (Frederiksen et al., 2021). 17 GNAT1 presence in the inner segment appears to enhance bipolar cell transmission (Majumder et al., 2013) as does the presence of its trafficking partner Unc119 (Fehlhaber et al., 2023), although these two phenomena may be linked. Additionally, GNAT1 movement to the inner segment may serve to regulate the retinal stores of the cell by controlling the clearance rate of Meta III rhodopsin (Zimmermann et al., 2004). In periods of prolonged darkness, the translocated GNAT gradually returns to the rod OS via a combination of facilitated diffusion and active transport (Frederick et al., 2020; Fig. 1.5). As described above, the compartmentalization of GNAT1 is partially dependent on its membrane affinity, altered through association with other proteins (Kosloff et al., 2008). Unc119 is a small protein first identified in C. elegans and is highly expressed in the inner segment and synaptic terminal of photoreceptors (Swanson et al., 1998). Knockout of Unc119 dramatically slows transport of GNAT1 to the outer segment (Zhang et al., 2011), while overexpression also results in mislocalization of GNAT1 (Chaya et al., 2019). Within the rod inner segment Unc119 binds myristrolated cargo, notably a-transducin, with high affinity. Binding of Unc119 occludes bg-transducin forcing the subunits to dissociate and hides the N-terminal lipid anchor of a-transducin, solubilizing it for transport (Cheguru et al., 2015). Release of a-transducin from Unc119 is triggered by the microtubule-associated small GTPase Arl3 (Grayson et al., 2002), present in the photoreceptor cell body and connecting cilia but absent from the OS (Zhou et al., 2006; Lokaj et al., 2015; Grayson et al., 2002; Hanke-Gogokhia et al., 2016; Wright et al., 2016). 18 Arl3 activity is controlled by its GEF Arl13B who’s expression is restricted to within the connecting cilia, restricting Arl3 activity to this location (Gotthardt et al., 2015). Association with Arl13B and guanine nucleotide exchange allows Arl3-GTP to induce release of a-transducin from Unc119 within the connecting cilia, where it is free to diffuse to the outer segment (Jaiswal et al., 2016). Once there, a-transducin is free to bind its bg subunits, allowing the heterotrimer to associate with the membrane where it will interact with rhodopsin upon photoactivation. Following its action on Unc119, Arl3-GTP is deactivated by the highly specific GAP RP2. Highly enriched at the transition zone but absent within the connecting cilium itself, this also serves to restrict the location of activity of Arl3 on Unc119 and its cargo (Jaiswal et al., 2016) Evidence also exists for active transport of a-transducin to the outer segment in addition to the Unc119-facilitated diffusion. Specific inhibition of cytoskeletal components which form the scaffolding for vesicular transport slows the return of GNAT1 to the OS during dark adaptation (Peterson et al., 2005; Reidel et al., 2008). Recent studies suggest this transport pathway may be related to interactions between GNAT1 and the proteins Gpsm2 (G-protein signaling modulator 2, LGN) and Frmpd1 (FERM and PDZ domain containing 1, two proteins known to interact with cytoskeletal components and expressed within the rod inner segment (Campla et al., 2019; Campla et al., 2022). Frmpd1 and Gpsm2 are known to associate in vivo (Pan et al., 2013). Gpsm2 is a GDI (guanosine nucleotide dissociation inhibitor) best known for its role in cell division (Zheng et al., 2013; Pirovano et al., 2019) and expressed in rod inner segments where it stabilizes the inactive GDP-bound form of a-transducin in vitro (Kerov et al., 2005; McCudden 19 et al., 2005; Nair et al., 2005). Members of the Frmpd family have been linked to a role in coordinating anchoring of cytoskeletal and signaling complexes with membranes (Moleirinho et al., 2013; Harris & Lim, 2001), but investigation into Frmpd1 itself has been limited. In the retina, Frmpd1 is transcribed from an alternate promotor, where it is expressed specifically in rods but not cones (Campla et al., 2019). Interestingly, in cones GNAT2 does not undergo light-induced translocation (Chen et al., 2007). Knockout of Frmpd1 delays the return of a-transducin to the outer segment following bleaching (Campla et al., 2022). Knockout of Frmpd1 and Gpsm2 do not significantly alter phototransduction from wildtype under complete dark or light adaptation, with bipolar cell sensitivity decreased under bright light conditions. These animals do however show significant delays in their dark adaptation, with ERG A wave amplitudes being significantly smaller in both groups compared to wildtype at ~30 minutes, and smaller in only the Frmpd1 KO at 1 hour (Campla et al., 2022). This is consistent with histological examination of GNAT1 localization in these rods (Campla et al., 2022). These results implicate Frmpd1 and Gpsm2 in the return of GNAT1 to the outer segment following translocation. Gpsm2 is proposed to sequester GNAT1-GDP within the inner segment before interacting with Frmpd1 at the plasma membrane. Alongside the information that the involved proteins interact with cytoskeletal elements, this implicates Frmpd1 and Gpsm2 in the active transport of a-transducin to the rod outer segment. 20 Figure 1.5 Alpha-Transducin Localization and Transport Under Differing Lighting Conditions When light-induced activation of rhodopsin generates enough transducin to saturate the available PDE, the excess free a-transducin diffuses to the inner segment due to its decreased membrane affinity when not bound to other proteins. Return to the outer segment in the dark occurs through a combination of facilitated diffusion and active transport. The carrier protein Unc119 dissociates a-transducin from bg and solubilizes it, carrying it to the connecting cilium where Arl3 induces release and allows a-transducin to diffuse to the outer segment. Additionally, GPSM2 binds free GDP-bound a-transducin and in combination with FRMPD1 brings a-transducin into association with the cytoskeleton at the connecting cilium for active transport to the outer segment. Created with https://biorender.com/ 21 1.1.7 Disorders of Phototransduction and a-transducin Mutants Dysfunction in phototransduction cascade components contribute to many of the 317 retinal disease-causing genes and loci (RetNet, 2024). Often mutations in these proteins, notably rhodopsin, result in progressive degeneration of the retina, especially photoreceptor cells, termed inherited retinal dystrophies (IRD). A majority have their effects isolated to the retina while a less prevalent subset, syndromic IRDs, manifest with extra-ocular phenotypes (Diakatou et al., 2019). Non-syndromic IRDs are typically classified based on the cells affected. Macular dystrophies specifically target their namesake region (Berger et al., 2010), while rod-cone and cone-rod dystrophies are named for the order of cell type affected. The most common rod-cone dystrophy, and inherited retinal dystrophy in general, is Retinitis Pigmentosa (RP). A classification with more than 80 identified disease-causing genes, rod loss with subsequent cone loss in RP manifests as a progressive loss of low-light vision with narrowing of the visual field (Diakatou et al., 2019). RP cases may affect the entire retina or, in cases termed sector RP, be restricted to specific quadrants (Cideciyan et al., 1998). It is suggested that these differences in severity across the retina are due to differences in light exposure (Tam & Moritz, 2007; 2009). Light-induced retinal degeneration is a common method for inducing retinal damage in animal models (Remé et al., 1996). Animal models expressing P23H Rhodopsin, an RP-causing mutation which misfolds and causes endoplasmic reticulum (ER) stress, have their retinal degeneration phenotype rescued by dark rearing (Tam & Moritz, 2007). Retinal degeneration induced by T4K and T17M rhodopsins is aggravated by light exposure, likely through the production of deleterious byproducts after photoactivation 22 (Tam et al., 2024). Research into the effect of lighting conditions on different models of retinal degeneration provides valuable insight into the mechanism of degeneration, as well as information on possible strategies to mitigate disease progression. Of the transducin subunits, a-transducin has by far the most disease-causing mutations identified, with one recent case of rod cone dystrophy associated with b-transducin (Yang et al., 2023). The condition most associated with GNAT1 mutations is congenital stationary night blindness (CSNB), a non-progressive lack of vision in low light conditions due to loss of rod function. Inheritance may be autosomal dominant or recessive depending on the specific mutation (Kubota et al., 2019). CSNB is identified by the absence of the scotopic electroretinogram (ERG), a measure of the electrical impulse created by the retina in response to light. Specifically, GNAT1-linked CSNB cases are associated with Riggs-type ERGs identified via attenuation of the A-wave, indicating photoreceptor dysfunction at the root of the condition rather than communication with bipolar cells (Marmor & Zeitz, 2018). The Gnomad database lists 9 pathogenic or likely pathogenic mutations in GNAT1 present in their data from human populations, with an additional 125 variants of unknown concern (Karczewski et al., 2020). Many dominant CSNB cases occur through dominantly inherited constitutively active GNAT1 mutants like p.Gln200Glu, which decreases transducin’s inherent GTPase rate to the point that PDE becomes constitutively active as a result, leaving the cell unable to signal (Szabo et al., 2007). Another mechanism is revealed by the Gly38Asp mutation underlying autosomal dominant Nougaret CSNB, named for the proband Jean Nougaret, which is unable to bind and activate gPDE (Dryja et al., 1996; Muradov & Artemyev, 2000). 23 Other notable mutations associated with human CSNB cases are the recessive D129G mutation observed clinically by Asif Naeem et al., although the underlying mechanism remains unclear (Naeem et al., 2012), and a dominant case associated with I52N which affects a nuclear localization signal, inducing CSNB through uncertain mechanisms (Zeitz et al., 2018). Recently, rare GNAT1 mutations have been linked to rod-cone dystrophies and autosomal recessive RP in clinical studies. Kubota et al. described an in-frame deletion DK273 causing a dominant CSNB phenotype with fundus and ERG resembling Oguchi disease that they suggest may in reality be early rod cone dystrophy (Kubota et al., 2019). Carrigan et al. observed an 80-year-old patient homozygous for Q302* who presented with an RP-like phenotype resembling the late slow retinal degeneration seen in GNAT1-KO mice (Carrigan et al., 2016). This mutation produces a premature stop codon which eliminates a set of highly conserved C terminal residues. Méjécase et al. describe a 32-year-old patient with a similar C321* mutation who presented with severe rod cone dystrophy, which pedigree showed to be recessively inherited. The suggested mechanism underlying this mutation is decreased guanine nucleotide affinity, leaving the protein in an unstable intermediary conformation (Méjécase et al., 2016). Mutations in trafficking partners of a-transducin have also been implicated in human disease. Mice transgenically expressing a dominant K57* mutant producing truncated Unc119 identified in a patient with late rod-cone dystrophy possess a similar phenotype to the patient, who presented with poor night vision after 40 years old, macular atrophy, and abnormal ERG (Kobayashi et al., 2000). Zenteno et al. identified a 55-year-old presenting with bilateral macular drusen-like deposits diagnosed with rod-cone dystrophy heterozygous for a E201* mutation in 24 Unc119 (Zenteno et al., 2023). Huang et al. report a child identified with central retinal atrophy and pigmentary deposits heterozygous for a D87N missense mutation in Unc119 (HUANG et al., 2013), and de Castro-Mirro describe a family with dominant RP linked to a V3* mutation (de Castro-Miró et al., 2016). In addition to Unc119 disease-associated mutations, the associated Arl3 protein has been linked to cases of autosomal dominant retinal degeneration (Travis & Pearring, 2023). Notable examples include the description of a Y90C mutation in Arl3 producing dominant RP in human patients by Strom et al. and Holtan et al., predicted to act through destabilization of interactions with RP2 and Unc119 (Strom et al., 2016; Holtan et al., 2019), and a missense mutation causing D67V causing a non-syndromic retinal degeneration via disruption of effector interactions in a dominant-negative manner (Ratnapriya et al., 2021). Mutations in RP2, the GAP of Arl3, are strongly associated with X-Linked Retinitis Pigmentosa (Lyraki et al., 2016). Responsible for 7-18% of cases, the second most following RPGR (Sahel, 2015), human mutations in RP2 cause a severe atypical RP phenotype with pronounced macular involvement (Dandekar et al., 2004; Jayasundera et al., 2010). The site of interaction with Arl3 is a particular hotspot for disease-causing mutations (Veltel et al., 2008). Unlike other animal models with mutations in phototransduction cascade components, GNAT1-KO mice display normal retinal structure with no degeneration until late time points (Calvert et al., 2000). However, the intact rods in GNAT1-KO mice lack an ERG response (Calvert et al., 2000). 25 The absence of morphological changes suggests no structural role for the protein despite its abundance. In the GNAT1-KO mouse model, heterozygous animals show reduced expression of GNAT1 and rod ERGs of decreased magnitude with otherwise normal appearance (Calvert et al., 2000). Interestingly despite the lack of response from photoreceptors, responses to light stimuli have been observed in the visual cortex of GNAT1/GNAT2 KO mice, likely due to the presence of intrinsically photosensitive retinal ganglion cells (Flood et al., 2022). The non-progressive disruption of low-light vision, mirroring the typical human phenotype, makes GNAT1-knockout animal models promising for the study of anatomically normal but non-functional photoreceptors. This allows the investigation of effects of phototransduction and its byproducts under normal and abnormal conditions. In addition to the GNAT1-KO mouse, a murine model of GNAT1-G38D-induced congenital stationary night blindness has been developed (Moussaif et al., 2006). Homozygous expressing mice did produce light responses but with dramatically reduced sensitivity, requiring a 40-fold brighter light for stimulation. Heterozygotes required 40% brighter light. Response recovery time was dramatically increased. No constitutive activity nor a dominant negative effect on WT GNAT1 were apparent. Sensitivity changes were described as likely due to an impairment in activation of PDE, ascribed to the fact that the mutant GNAT is so deficient in its activating ability that a large stimulus was required to achieve any activation, upon which the delayed response time became apparent (Moussaif et al., 2006). 26 1.2 Xenopus laevis as a Model for Retinal Development and Disease Xenopus laevis (X. laevis), or the African Clawed Frog, has been a common model organism in developmental biology due to its large embryo size allowing easy observation, rapid growth, and large number of offspring with each mating (Horb et al., 2019). These large embryos also facilitate manipulation, allowing for straightforward genetic editing (Wheeler & Brändli, 2009). When paired with the animal’s fully sequenced genome containing at least 1700 orthologs for human disease (Hellsten et al., 2010), this means human inherited diseases can be modeled in X. laevis. The retina of X. laevis is especially well suited to study. It contains the same overall cell types with the same architecture as the human retina. While the X. laevis retina lacks a macula, it does contain highly morphologically distinct rods and cones at an approximately 1:1 ratio in contrast to the rod-dominant mouse retina. These photoreceptors, especially rods, are dramatically larger than other commonly used models, at 45 µm long and 7 µm wide (Röhlich & Szél, 2000) compared to the 24 µm x 1.4 µm observed in mice (Fu & Yau, 2007). This large size greatly simplifies a number of techniques, including microscopy and electrophysiology, and allows for the study of subcellular operations including the trafficking and localization of proteins. When working with X. laevis a challenge is posed by the pseudo-tetraploid nature of their genetics. Due to a genome duplication event, the 2n = 36 X. laevis possess two sub genomes, termed S (short chromosomes) and L (long chromosomes) (Elurbe et al., 2017). The a-transducin gene in X. laevis has two copies, GNAT1.S and GNAT1.L (Session et al., 2016). 27 1.3 CRISPR/Cas9 Gene Editing CRISPR/Cas9 is a gene editing system allowing for induction of desired edits into the genome of models, including X. laevis (Hsu et al., 2014). The ability to knock-in (add a new gene) or knock-out (prevent the expression of a gene) allows for deeper in-vivo study of mechanisms and functions and facilitates the creation of highly specific disease models for study. CRISPR (Clustered Regularly Interspaced Palindromic Repeats) was identified as an RNA-based source of adaptive immunity in prokaryotes, protecting against invading nucleic acids by inducing double stranded DNA breaks (Rath et al., 2015). CRISPR and the accompanying DNA cleaving enzyme cas9 (CRISPR Associated Protein 9) have been optimized for experimental use, applying the ability to induce targeted breaks in double stranded DNA to edit the genome (Zaboikin et al., 2017). A guide RNA (sgRNA) designed to specifically recognize a region of interest directs cas9, which induces double stranded breaks 3bp upstream of a PAM site (Promotor Adjacent Motif). Once DNA is broken innate repair mechanisms, typically non-homologous end joining (NHEJ) or homology-directed repair (HDR), can be taken advantage of to insert DNA of interest or prevent the expression of a gene (Zaboikin et al., 2017; Fig. 1.6). These processes, NHEJ especially, are error- prone and often result in insertions and deletions (indels) of the DNA. If these induced mutations result in a shift of the reading frame, the gene may be knocked out, with no functional protein produced as nonsense mediated decay (NMD) systems prevent transcription of these altered genes (Baker & Parker, 2004). 28 Alternatively, in frame mutations may result in transcribed proteins with altered structure or function. Among these, genes are referred to as functionally knocked out when the protein is produced but is entirely non-functional (Bialk et al., 2018). As the specific indel produced is essentially random, different end mutations will result in each cell exposed. If using a multicellular model, like X. laevis, the final F0 animal will be genetically chimeric. These F0 animals can be studied but are often characterized and mated to produce genetically distinct F1 offspring for more predictable results. 29 Figure 1.6 Overview of CRISPR-Cas9 DNA Editing Designed guide RNA sequences lead cas9 to designated sites just upstream of a PAM sequence within the DNA being edited where it induces double stranded breaks. If cells are exposed to a desired foreign dsDNA sequence, homology-directed repair will join the ends of the DNA of interest to the edges of the double stranded break, inserting a new region of DNA to be expressed. The alternative DNA repair method of non-homologous end joining (NHEJ) is more error prone and results in insertions or deletions (indels) of the original sequence. Depending on whether these indels alter the reading frame, the cells may have the gene knocked out or otherwise altered in its final structure or ability to function. Reproduced from “CRISPR/Cas9 Gene Editing”, by BioRender.com (2024). Retrieved from https://app.biorender.com/biorender-templates 30 1.4 Electroretinography Electroretinography (ERG) is a non-invasive technique used to measure retina function both clinically and in research. The layered structure of the retina means that electrical impulses created by its component cells are aligned, and as such can be detected from the cornea with an electrode and sufficient amplification (Perlman, 1995). A typical ERG response involves two principal components. Hyperpolarization of photoreceptors in response to light causes an initial negative deflection called the A wave, followed by a larger positive response arising from depolarization of inner retina neurons termed the B wave (Brandli & Stone, 2015); Fig. 1.7). Changes to the magnitude and timing of the components of the ERG wave reflect the health and function of the retina. Additional information can be derived from less frequently analyzed aspects of the ERG response. Notable among these additional responses, the C wave is a slow positive deflection arising from the retinal pigment epithelium in response to decreases in extracellular potassium concentration caused by photoreceptor activity (Oakley & Green, 1976; (Green & Oakley, 1978). Oscillatory potential refers to low amplitude oscillations within the rising B wave on especially bright stimulation (Fig. 1.8). They are thought to arise from negative feedback loops between inner retina neurons (Heynen et al., 1985). Early receptor potential (ERP) occurs immediately following stimulus onset, preceding the A wave, and likely relates to initial photoactivation of rhodopsin (Murakami & Pak, 1970). 31 Depending on the lighting regimen used, rod or cone function can be investigated more specifically. Scotopic ERGs are a measure of rod function and are conducted on dark-adapted subjects using dim flashes of blue or white light (Benchorin et al., 2017). Conversely, photopic ERGs investigate cone function and utilize bright flashes of white light, occasionally superimposed over a background light, and a light-adapted subject (Li et al., 2020). Using these setups as a base, protocols can be developed to investigate specific aspects of retinal function. For example, dark adaptation can be investigated through repeated exposure to low intensity stimuli following a bleaching stimulus, looking for changes in the response over time as the rods recover. Use of designed lighting protocols and analysis of the components of the ERG wave can help identify the root cause of specific cases of vision loss. CSNB is typically identified by the absence of a scotopic ERG. Cases can be further divided based on the nature of the A and B waves. A decreased A wave with an intact B wave indicates photoreceptor dysfunction and is termed Riggs Type CSNB. An intact A wave with attenuated B wave, called Schubert-Bornstein CSNB, indicates bipolar cell dysfunction (Marmor & Zeitz, 2018). While ERG allows non-intrusive investigation into the site of retinal dysfunction, the technique does have limits. ERG lacks sensitivity and resolution when compared to single cell electrophysiological measurements like patch clamps recordings but can be conducted in vivo unlike these measurements. As the detected signal is averaged across the retina, specific techniques including multi-focal ERG must be used in conditions like sector RP where only isolated regions of the retina are affected (Giambene et al., 2020). 32 Figure 1.7 Averaged Wildtype Scotopic ERG Trace Averaged response of n=5 wildtype X. laevis tadpoles to a flash of 2500 cd•s/m2. Each animal had five trials repeated and averaged. Highlighted are critical aspects of the ERG response referred to throughout this text. The small negative A wave is the portion of the response attributable to photoreceptors, while the larger positive B wave represents the following inner retina reaction. Their respective amplitudes and implicit times are indicative of specific aspects of the retinas response to light. 33 Figure 1.8 Representative Wildtype ERG Trace Demonstrating Oscillatory Potentials Representative trace selected from a wildtype X. laevis tadpole. Five flashes of 2500 cd•s/m2 were repeated and averaged. The oscillatory potentials on the rising portion of the B wave are highlighted. 34 1.5 Rationale and Objectives Previous work in our lab developed and optimized a CRISPR-Cas9 system for use in genetically modifying Xenopus laevis, producing several genetically chimeric F0 gene knock out animals for use in research. Among these, the F0 GNAT1-KO animals were used to investigate the mechanisms underlying two distinct models of retinitis pigmentosa linked to rhodopsin mutations, P23H and T4K. The P23H rhodopsin mutation produces misfolded rhodopsin which is retained in the ER, an effect mitigated by chromophore binding which acts as a chaperone during protein folding (Noorwez et al., 2003; Athanasiou et al., 2018). P23H-RHO-linked retinal degeneration is exacerbated by light exposure (Tam & Moritz, 2007) and deprivation of vitamin A, upon which production of chromophore depends (Tam et al., 2010). Results from our lab show that light exposure induces an increase in autophagy in WT-RHO animals and massive degeneration in P23H-RHO, with autophagic structures enriched with P23H-RHO labeling present in apoptotic cells (Wen et al., 2019; Fig 1.9). P23H-RHO produces misfolded and mislocalized rhodopsin. When paired with the increase in demand for autophagic clearance of WT-RHO upon light exposure, the presence of the misfolded variant overwhelms the rods capacity to clear the protein, causing cell death (Lobanova et al., 2013; Wen et al., 2019). These results are supported by 2022 work from Intartaglia et al. in mouse models of P23H-RHO degeneration (Intartaglia et al., 2022). In F0-GNAT1-KO animals light-induced autophagy levels were significantly reduced, with two-way ANOVA confirming a specific relationship between GNAT1-KO and autophagy 35 level (Wen et al., 2019; Fig 1.10). These results suggest this autophagic pathway is dependent on the activity of the phototransduction pathway and that knockout of GNAT1 in P23H-RHO animals may significantly reduce degeneration. Retinal degeneration linked to T4K-RHO is mitigated by dark rearing as in P23H-RHO (Tam & Moritz, 2009; Tam et al., 2014) but occurs through a different mechanism. T4K-RHO is not retained in the ER like P23H-RHO and prevention of chromophore binding prevents degeneration while it exacerbates it in P23H-RHO animals (Tam & Moritz, 2007; Tam et al., 2010; Tam et al., 2014). Recent preprinted work from our lab shows that degeneration in these animals only occurs under cyclic lighting conditions. Constant dark or constant light prevents degeneration and increased cycling time up to 20 minutes light/60 minutes dark exacerbates cell death (Tam et al., 2024). CRISPR-based knockout of GNAT1-KO in X. laevis expressing T4K-RHO significantly increases the level of degeneration (Fig. 1.12; Tam et al., 2024). This suggests that T4K-based retinal degeneration does not occur through persistent phototransduction activation. Alternatively, degeneration may be induced by production of abnormal toxic photointermediates of T4K-RHO (Fig. 1.11; Tam et al., 2014). As noted above in Section 1.1.6, GNAT1 has established interactions with rhodopsin photointermediates under normal conditions (Zimmermann et al., 2004) and as such may convert this hypothesized T4K product to a less toxic conformation or otherwise prevent accumulation of this product to toxic levels. 36 Further investigation of interactions between GNAT1 specifically and activation of the phototransduction cascade more generally would be aided by the ability to express rhodopsin mutants over a genetically distinct GNAT1 knockout background. As such, we sought to generate and characterize these knockout animals using the F0-CRISPR modified X. laevis produced in the experiments described above. As these F0 animals are genetically chimeric, mutations that produce knockout animals are accompanied by a host of in-frame edits. Analysis of the effects of these mutations on the GNAT1 protein can provide valuable information regarding human disorders linked to GNAT1-mutations as well as the trafficking and function of the protein. 37 Figure 1.9 P23H-Rhodopsin Increases Light-Induced Autophagy in X. laevis Quantification of autophagic puncta seen in the confocal micrograms of rod inner segments displayed below the chart shows an increase in light-induced autophagy in P23H-RHO animals compared to WT-RHO controls. The color of the puncta changes from green to yellow to red as autophagy proceeds and the GFP component of the marker is degraded, leaving only RFP. Reproduced with permission from Wen et al., 2019 (Wen et al., 2019). 38 Figure 1.10 Knockout of GNAT1 Reduces Light-Induced Autophagy in X. laevis In CRISPR-edited F0 GNAT1-KO X. laevis, formation of autophagic structures is highly reduced upon light exposure compared to control animals. Modified with permission from Wen et al., 2019 (Wen et al., 2019). 39 Figure 1.11 Bleaching of T4K and T17M Rhodopsin Produces Abnormal Persistent Photoproducts After expression in cell culture and bleaching with >495 nm light in the presence of excess 11-cis-retinal, WT-RHO has its absorbance at 500 nm drop to 0 before recovering to 80% over the course of an hour. Neither T4K nor T17M-RHO has its absorbance decline to 0, rather forming persistent products with altered absorbance. No recovery is seen from either of these rhodopsin mutants, suggesting light exposure causes destabilization of the protein which subsequently cannot return to the native conformation under these conditions. Modified with permission from Tam et al., 2014 (Tam et al., 2014). 40 Figure 1.12 Knockout of GNAT1 Exacerbates T4K-RHO-Induced Retinal Degeneration GNAT1 is successfully knocked out via exposure to Cas9 and the sg2 guide RNA. sgN serves as an inactive control (A/B), with exposure to sg2 resulting in sequence changes (A) and significant reduction in GNAT1/Rod Opsin via western blot (A/B). Knockout of GNAT1 was confirmed via electroretinography, displaying significant reduction of the ERG response compared to wildtype (C, represented as dose-response curve in D). Knockout of GNAT1 as confirmed in panels A-D potentiates T4K-RHO-induced retinal degeneration (E, representative histology in F). Reproduced with permission from Tam et al., 2024 (Tam et al., 2024). 41 Chapter 2: Generation of GNAT1-Knockout Xenopus laevis 2.1 Introduction As it is a point of amplification for the phototransduction cascade, GNAT1 makes for a promising target for genetic editing to produce animals with non-functional rods, especially given the fact that animal models show minimal retinal degeneration (Calvert et al., 2000). This in combination with the large size of X. laevis rod photoreceptors makes a putative GNAT1-KO X. laevis attractive for study of the relationship between phototransduction and the health of retinal cells. Human mutations in GNAT1 typically result in congenital stationary night blindness (CSNB), as well as rare cases of rod-cone dystrophy. We have previously observed that GNAT1-KO suppresses light-induced autophagy in P23H Rhodopsin mutants (Wen et al., 2019) and exacerbates T4K-rhodopsin-induced retinal degeneration (Tam et al., 2024), although these observations were made on genetically chimeric F0 CRISPR-edited animals. As such, we sought to breed these animals to generate F1 animals of defined genotype for future study of the role of the phototransduction cascade in different models of retinitis pigmentosa. 42 2.2 Methods 2.2.1 Animal Rearing Selected GNAT1-edited F0 frogs were selected and primed for mating with subcutaneous injections of human chorionic gonadotropin (HCG). Following mating, healthy, fertilized embryos were selected and transferred to transparent 4L bins kept in an 18 degree C incubator and exposed to 12h on 12h off cyclic light of 1700 lux. Once tadpoles began to swim independently, they were fed ground frog brittle (Nasco). Animals were raised to a predesignated time point, typically 14 days or 6 weeks post fertilization depending on the nature of the experiment, before use. Following experimental procedures, tadpoles were sacrificed by pithing. Eyes were dissected with the right being fixed in 4% paraformaldehyde with 0.1M sodium phosphate buffer pH 7.4. The left was solubilized in 100 µL of 1:1 PBS and SDS-PAGE loading buffer with 1mM EDTA and 100µg/mL PMSF. Tail clippings were collected for DNA analysis. 2.2.2 DNA Processing and Analysis Tail clippings obtained as described above were prepared as in Wen et al 2019 and used as template DNA for PCR reactions. Amplified DNA was Sanger sequenced (Genewiz, Azenta Life Sciences) and analyzed for the presence of mutations using Snapgene (www.snapgene.com). 43 2.2.3 Electroretinogram Screening of F1 Tadpoles Once F1 offspring were generated from the selected pair of CRISPR-modified F0 animals, these tadpoles were raised up to Nieuwkoop and Faber stage 58 (Nieuwkoop & Faber, 1994; approximately 6 weeks of age) before screening by scotopic Ganzfeld electroretinography (ERG) for evaluation of rod function. Tadpoles were dark adapted overnight and fed the night prior to recording. Animals were anesthetized in 0.1% tricaine in 0.1X MMR until unresponsive. At this point, animals were placed on a stage mounted to a base electrode. A micromanipulator was then used to position an electrode, a silver wire immersed in 1X MMR contained by a glass microcapillary, in contact with the cornea. A lubricating drop (Genteal) was placed on the eye and the stage was placed under a Ganzfeld dome. Electrodes were connected to a Model 1800 AC amplifier (AM systems), the output of which connected to an Espion ERG unit (Diagnosys). This system was used to record the electrical impulse at the cornea following flashes of 2.5, 25. 250, 750, 1250, and 2500 cd•s/m2, each of which was repeated 5 times and averaged. The protocol finished with a photopic flicker, pulsing a 2.5 cd•s/m2 with a frequency of 10 ms and a period of 4Hz over a 6500K white background. Following ERG recording, animals were sacrificed as described above. A slightly modified lighting protocol, using 3 averaged trials at 250, 750, 1250 cd•s/m2 was developed for use in selecting potential knockout animals to raise to adulthood. The shorter length meant that tadpoles could be returned to ringer after screening and selected based on ERG results. Selection of animals was validated through Sanger sequencing of blood samples drawn from adult frogs or tail clippings of tadpoles, depending on the age of the animal sequenced. 44 2.2.4 Western Blotting and Analysis Western blots were carried out on solubilized eyes, generated as described previously. 12µL of sample was loaded per well, electrophoresed through a 10% SDS polyacrylamide gel, and transferred to a PVDF membrane overnight. Membranes were blocked with 1% skim milk powder in PBS for 30 minutes before washing with PBS and labeling with anti-GNAT1 (1:1000, rabbit polyclonal) and anti-acetylated a-tubulin (1:3000), incubating for 4 hours at room temperature. After another wash, membranes were incubated for 2 hours at room temperature with IR800 goat anti-rabbit and IR700 goat anti-mouse at 1:10,000 of 1 mg/mL solution (Rockland). Blots were then imaged using the Li-Cor Odyssey imaging system and the resulting images were used to quantify protein expression levels. Use of western blots for quantification was validated by running and imaging a serially diluted wildtype sample to obtain a standard curve, showing the intensity was linear until ~6.5% where a sudden drop occurred (Fig. 2.1). Image files were analyzed using ImageJ to measure mean intensity values within defined areas. Background values were subtracted from our protein bands and GNAT1 sample values were normalized based on a-tubulin measurements. Expression level was quantified by comparing normalized GNAT1 intensity measurements to the value for the average of n=5 WT. Measured values were only directly compared between samples from the same blot. 45 Figure 2.1: Standard Curve for Quantification of GNAT1 Expression from Western Blots Serial dilution of a solubilized wildtype eye labeled for a-transducin shows that western blot intensity is linear for expression values above ~6.25%, below which the measured intensity on the blot dramatically decreases. 46 2.2.5 Confocal Microscopy After being placed in fixative for 4 hours as described above, the right eyes of the sacrificed animals were sucrose infiltrated in a 20% sucrose solution. After infiltration eyes were embedded in OCT (Sakura Finetek), frozen, and stored at -20 before sectioning. 12µm sections were labeled with rabbit anti-GNAT1 (1:1000) overnight. Secondary labeling was performed with 1:750 Cy3-conjugated anti-rabbit (Jackson Immunoresearch), 1:100 AlexaFluor 488-conjugated wheat germ agglutinin (WGA; Life Technologies), and 1:1000 Hoechst-32342 (Sigma Aldrich), incubating for four hours. Coverslips were mounted with MOWIOL 4-88 (EMD Milipore). Labeled sections were imaged with a Zeiss 800 laser scanning confocal microscope and Zen software, using a 10x air and 40x water immersion NA 1.2 objective lenses. Using the 40x objective, 3D Z-stack images were collected by assembling optical sections with a consistent spacing of 0.3 microns per optical section. Maximum intensity projections were generated from these Z stacks using the Zen software (Zeiss) for analysis. Within each experiment, all images were acquired with the same gain and contrast settings. For experiments involving quantification of confocal micrographs, comparisons were only made between images acquired with the same settings. Images were post-processed and analyzed in Adobe Photoshop. Hoechst and WGA channels were adjusted non-linearly to improve image clarity, while GNAT1 labeling was adjusted linearly when necessary to improve signal quality. 47 2.3 Results 2.3.1 Identification of Promising F0 CRISPR-Edited X.laevis Generation of knockouts began with the identification of suitable breeding pairs from the genetically chimeric CRISPR-modified F0 animals previously generated by Wen et al. (Wen et al., 2019). Male F0 animals were characterized first due to the lack of required rest time between matings. These animals were crossed with wildtype females and the genomic DNA from the fertilized embryos underwent PCR amplification and Sanger sequencing. Superimposed sequence traces were compared to a wildtype example (Fig. 2.2A/B) to identify the mutations present, and by comparing differences in the end point of the different sequences based on the large terminal ‘A’ peak (Fig. 2.2 C). Animals were selected as good candidates for knockout generation based on the percentage of offspring produced with frame-shift mutations. Once a promising male candidate was identified, this animal was crossed with the F0 GNAT1-edited females. These animals were characterized as described below and the sequencing process was repeated, albeit with two edited sequences superimposed (Fig. 2.2D). As the edits contributed by the male parent were established prior, the contributions of the female could be deciphered and the combination of animals producing F1 offspring with a frameshift mutation in each of the four GNAT1 alleles could be identified. 48 . Figure 2.2 Representative Examples of Sequence Traces Used in Characterization DNA from a wildtype animal was used as a base to determine the edits present (A). Male F0 animals were mated with wild type females allowing differentiation between their edited DNA and the wildtype background based on mismatches between nucleotides (B) and differences in the length of the sequence (C). Once promising males were identified these were mated with uncharacterized F0 females and the process was repeated, using the mutations identified in the males to determine those present in the females (D). 49 2.3.2 Identification of F1 GNAT1-KO X. laevis F1 tadpoles generated by mating of our selected candidate F0 animals were raised to 6 weeks of age before undergoing electroretinography. This process identified a subset of tadpoles with significantly reduced rod ERGs. Among this group some animals had essentially flat responses at all stimulus intensities (Fig. 2.3A), while others showed a low amplitude highly delayed response at high flash intensities (Fig. 2.6). No electronegativity, meaning a larger A wave than B wave, was observed. This was as expected based on our interference with a phototransduction protein rather than a neural signaling factor and previous data from GNAT1-KO models and human mutations (Calvert et al., 2000). Following electroretinography, these animals were sacrificed with one eye fixed for histology and the other solubilized for protein content analysis. Immunohistochemical analysis confirmed the absence of GNAT1 labeling (Fig. 2.3B) in either inner or outer segments of the cells. This absence was confirmed through SDS-PAGE electrophoresis and western blot of the solubilized eyes (Fig. 2.3C). Based on the results of our serial dilution of a wildtype sample, we treated bands presenting with less than 6% of wildtype after quantification as possibly equivalent to 0% expression. The normalized intensity of these bands was highly significantly different from wildtype (Fig 2.4; p<0.0001 according to student’s t-test, n=16 KO and n=6 WT). Sanger sequencing of these animals identified frameshift mutations in all four GNAT1 alleles (Fig. 2.5), confirming our successful generation of GNAT1-knockout X. laevis. 50 Figure 2.3 Characterization of GNAT1-Knock Out X. laevis Potential GNAT1-knockout animals were first identified through electroretinography (A) where they presented with absent scotopic ERGs (average of n=6 WT, average of n=8 KO in red). Results were verified immunohistochemically (B), with GNAT1-labeling (green) absent in knockouts. Red represents WGA while blue is Hoechst. This absence of GNAT1was further confirmed through western blotting of solubilized eyes (C), confirming the lack of GNAT1 protein in these animals. Acetylated a-tubulin was used as a loading control and for normalization. 51 Figure 2.4 ERG Magnitude is Highly Suppressed in GNAT1-KO X. laevis A dose response curve generated for the magnitude of the B wave compared to the log stimulus intensity (n=6 WT, n=8 KO), showing a significant reduction in B wave response amplitude (two-way ANOVA, p<0.0001 for all factors). 52 Figure 2.5 GNAT1 Expression in X. laevis Identified as Knock Outs is Significantly Reduced Compared to Wild Type Following gel electrophoresis and western blot of solubilized eyes, the intensity of the a-transducin and anti-tubulin bands were measured using ImageJ and compared between knockout (n=16) and wildtype (n=6) animals, revealing highly significant differences (student’s t test, p<0.0001) and further demonstrating the absence of GNAT1 expression in these eyes. 53 Figure 2.6 Representative Example of GNAT1.L and GNAT1.S Sequences of Knock Out Animals Example DNA sequence of a knockout animal with frameshift mutations (deleted nucleotides marked in red) in all four GNAT1 alleles. 54 Figure 2.7 Alternate GNAT1-KO ERG Phenotypes Of animals confirmed to have no expression of GNAT1 by immunohistochemistry, sequencing, and western blot (Fig. 2.3), some had completely suppressed scotopic ERG (A), while others showed an extremely reduced and delayed residual response (B). Black traces represent the average of n=6 WT X. laevis tadpoles, while red traces are individual representative GNAT1-KO X. laevis. 55 2.3.3 Relative Expression of GNAT1.S and GNAT1.L Alongside the full KO animals, X. laevis with frame shift mutations in only the GNAT1.S or GNAT1.L paralogue were produced. This allowed us to compare measured expression values between these animals and wildtype, determining GNAT1.L is responsible for approximately 60% of total produced GNAT1 protein in X. laevis (not shown). 56 Chapter 3: Investigation of GNAT1-Edited X. laevis 3.1 Introduction Inherent in the process of genetic engineering with CRISPR/Cas9 is the production of genetically heterogeneous F0 animals. As the specific indel resulting from DNA cleavage is random, dependent on DNA repair mechanisms, a range of unique mutations occur. While some of these may be frame-shift mutations preventing protein production and generating gene knockouts as described above, in-frame mutations abound and produce unique phenotypes via dominant gain of function mechanisms. Additionally, the pseudo-tetraploid nature of X. laevis provides more opportunities for combinations of mutations and a broader range of possible expression values when mated to generate F1 animals for analysis. In the process of generating GNAT1-KO X. laevis, we observed and characterized several interesting phenotypes among the animals produced with edits in GNAT1 that did not result in a complete knockout. The animals generated may reveal an additional functional role for GNAT1, prove useful for the study of the trafficking of a-transducin to the outer segment and its role in dark adaptation, and potentially provide new insight into the underlying mechanisms of both dominant and recessively inherited disease-causing mutations in GNAT1. 57 3.2 Methods 3.2.1 Characterization of Mutants Initial identification and characterization of GNAT1-edited animals proceeded using the methods described above: DNA sequencing, western blots, immunofluorescence microscopy, and electroretinography. For histology show in in Figure 3.9, slides were labeled with rabbit polyclonal anti-GNAT1 (gift of W.D Smith, 1:1000) and mouse anti-acetylated a-tubulin (1:200) overnight. Secondary labeling was performed with Cy3-conjugated anti-rabbit (1:750), AlexaFluor 488-conjugated a-mouse (Jackson Immunoresearch), AlexaFluor 647-conjugated wheat germ agglutinin (WGA; Life Technologies) and Hoechst-32342 (Sigma Aldrich), incubating for four hours. 3.2.2 Development of Primers for PCR Genotyping Two sets of additional PCR primers were designed to be specific for two S gene mutations of interest that we encountered, a D3 and a D12. These were used to facilitate PCR genotyping of offspring during experiments to be later validated by Sanger sequencing. A gradient PCR was preformed to confirm ideal conditions of 96°C for 1 minute followed by 30 cycles of 94°C-10s, 50°C-15s, 72°C-20s, and a final 72°C for 2 minutes for the D3 oligomers. 58 These results were confirmed using the D12-specific oligomers and a similar protocol, adjusting the annealing temperature to 53°C. Genotyping PCRs were tested on previously sequenced and analyzed genomic DNA samples to confirm their specificity. Results were confirmed through Sanger sequencing (Genewiz, Azenta Life Sciences). 3.2.3 Light Induction of GNAT1 Translocation Based on previous work investigating light-induced GNAT1 trafficking in X. laevis photoreceptors, which exposed animals to ~800 lux for 45 minutes (Peterson et al., 2005), we believed that exposure to ~1700 lux for 10 minutes within our animal incubators would induce a noticeable movement of GNAT1 from OS to IS. This time was also chosen to resemble the time course of our standard ERG protocol. We tested this on a set of age-matched wild type animals and were able to induce significant translocation of GNAT1 to the inner segment. Light intensity was measured with an ILT700 light meter (International Light) using an SED033 detector, 28925R objective, and ZCIE scotopic filter. Measurements were integrated over the course of their respective experiment times. Light exposure within the incubator was measured with the detector facing up, down, left, right, forwards, and backwards, and averaged across these 6 measurements. 59 3.2.4 GNAT1 Localization Measurements Localization of GNAT1 was quantified using ImageJ. Maximum intensity projections were generated from stacks collected with the Zeiss 800 laser scanning confocal microscope and Zen software as described in Section 2.2.5. Outlines of outer and inner segments were drawn, and measurements were collected moving left to right across the image until a pre-determined number of cells were analyzed. Data was then transferred to Graphpad PRISM for plotting and statistical analysis. 3.2.5 Dark Adaptation Electroretinography As previously, animals were raised to Nieuwkoop and Faber stage 58 (approximately 6 weeks of age) before screening by scotopic Ganzfeld electroretinography (ERG) for evaluation of rod function. Tadpoles were dark adapted overnight and fed the night prior to recording. Animals were anesthetized in 0.1% tricaine in 0.1X MMR until unresponsive. At this point, animals were placed on a stage mounted to a base electrode. A micromanipulator was then used to position an electrode, silver wire immersed in 1X MMR contained by a glass microcapillary, in contact with the cornea. A lubricating drop (Genteal) was placed on the eye and the stage was placed under a Ganzfeld dome. Electrodes were connected to a Model 1800 AC amplifier (AM systems), the output of which connected to an Espion ERG unit (Diagnosys). This system was used to record the electrical impulse at the cornea. 60 To measure the time course of dark adaptation, animals were exposed to 6 flashes of 250 cd•s/m2 blue light with 20 s between each flash to verify connection of the electrode and cornea, 5 flashes of 75 cd•s/m2 6500K white light over a 6500K white background of 250 cd/m2 with 6 s between each flash to interrupt dark adaptation, followed by 8 rounds of 4 flashes of 250 cd•s/m2 blue light, with 20 seconds between each round and 20 seconds between each trial. Following ERG recording, animals were sacrificed as described above. 3.2.6 Super Resolution Microscopy Slides were prepared as described in section 2.2.5 before imaging with a Zeiss 880 LSM Airyscan microscope and Zen software with a 40x water immersion NA 1.2 objective lens. In traditional confocal microscopy the pinhole is used to reject out of focus light, increasing resolution as the pinhole shrinks but decreasing the total signal as less light reaches the detector (Huff, 2015). Airyscan technology uses a point detector array with 32 elements combining to form a hexagonal detector with the central detector aligned with the optical path of the system and peripheral elements displaced. Each element is small enough to act as its own pinhole, rejecting out of focus light at its location. Deconvolution of these signals provides spatial information about the signal from each element. Information from the entire array is then weighed and combined to form a final image. The use of this detector allows for samples to be imaged with a wider pinhole due to the element’s rejection of out of focus light at different spatial locations, dramatically increasing the signal to noise ratio while also increasing resolution by integrating spatial information gathered by the detector elements (Huff, 2015). 61 Z-stacks were acquired with a thickness of 0.3 microns per optical section. Maximum intensity projections were generated from these stacks using the Zen software (Zeiss) before export and analysis. 62 3.3 Results 3.3.1 Reduction in GNAT1 Expression Produces Delays in the ERG Response The knowledge that the mix of mutations passed on by the F0 parents led to a wide range of expression values led us to investigate the relationship between protein expression and aspects of our electrophysiological data. While we expected the most obvious relationship to be between the magnitude of the waves of the ERG and the level of GNAT1 expression, this was quite variable until reaching very low expression levels (Fig. 3.2), with an R2 of 0.02 and 0.11 for linear regressions of the A and B wave respectively. While conducting ERGs we observed a great degree of variability in the implicit time (time to peak) of both the A and B waves at a variety of magnitudes, with many responses being highly delayed at all flash intensities (Fig. 3.1A). These changes were most apparent at 2500 Cd/m2. 69±9.1 ms (n=10, ± S.D.) while partial GNAT1-KO implicit times ranged from 62 ms to 196 ms. The average wildtype B wave implicit time was 178±13.4 ms (n=10) while partial knockouts ranged from 176 ms to 386 ms. When the implicit time of the ERG was quantified and plotted against the GNAT1 expression values relative to wildtype derived from the western blots, a clear relationship emerged (Fig. 3.3). The A wave implicit time fit a first order exponential decay model with an R2 of 0.82 and a GNAT1 expression producing half-maximal effect of 6.45%, while the B wave fit a linear relationship with an R2 of 0.57. 63 Figure 3.1 Characterization of X. laevis with Reduced Expression of GNAT1 In the process of identifying the knockout animals described above, animals with varying reduction in ERG magnitude but extremely delayed responses were identified (A; n=6 WT, representative delayed trace in red). Immunohistochemistry showed reduction of GNAT1 labeling intensity compared to WT (B), a result confirmed by western blotting (C; a representative range of expression levels identified in screening). These western blots were used to obtain the expression levels graphed in Figures 3.2 and 3.3. 64 Figure 3.2 ERG Wave Magnitude Does Not Correlate Strongly with GNAT1 Expression While knockout and extremely low expression of GNAT1 did result in dramatic reduction of the scotopic ERG, across the n=24 samples examined we did not identify a strong correlation between expression levels derived from our western blot and normalized to a tubulin and the magnitude of the ERG response. Values plotted here were obtained from the same animals displayed in Figure 3.1. Among extremely low expressing animals (<5%), those without discernable A and B waves were excluded. 65 Figure 3.3 Implicit Time of the ERG Response Strongly Correlates to GNAT1 Expression When the implicit time, or time to reach the maximum value, of the ERG waves were plotted against the normalized expression levels of GNAT1 a relationship emerges. This is especially prominent in the A wave implicit time, the measure of the photoreceptor response, which fits a sigmoidal relationship with an R2 of 0.821 and a half maximal effect of 6.45%. The B wave response is linear with an R2 of 0.5685. Values plotted here were obtained from the same animals displayed in Figure 3.1. Among extremely low expressing animals (<5%), those without discernable A and B waves were excluded from this analysis. 66 3.3.2 The One Base Pair Substitution Causing GNAT1.L-Q184L Produces Functional GNAT1-Knockouts During further characterization of animals selected based on their significantly reduced ERGs (Fig. 3.4A), immunohistochemistry revealed a subset of animals with near wildtype levels of GNAT1, albeit mislocalized to the IS under dark conditions (Fig. 3.4B). Western blots confirmed that the level of GNAT1 in these animals was only slightly reduced from WT (Fig. 4C), despite their significantly reduced ERG responses, marking these animals as functional GNAT1-KO’s. Sanger sequencing identified a one base pair substitution causing the point mutation Q184L in GNAT1.L in X. laevis with the functional KO phenotype. This was accompanied by a 5 base pair deletion in the other GNAT1.L in these animals. D5, along with the GNAT1.S mutations in these animals, were present in other animals that lacked this phenotype while Q184L only appeared in animals with the functional knockout phenotype (Table 3.1; n=3 FKO out of n=9 edited animals in the initial mating, p=0.000911) by Chi test). The functional knockout phenotype only occurred in animals with edits in all GNAT1 alleles, including a frame shift edit in the other GNAT1.L allele. When observed in animals with wildtype alleles or mutations established to produce functional GNAT1 protein, the ERG phenotype is not present (Table 3.1), marking the Q184L-associated functional knock out as recessive. 67 Figure 3.4 A One Base-Pair Substitution Producing the Mutation Q184L in GNAT1.L Results in a Functional Knock Out of GNAT1 Among animals selected as possible GNAT1 knockouts due to their absent ERG (A; average of n=6 WT, average n=3 FKO in red), immunohistochemistry identified a small subset with abundant GNAT1 labeling, albeit confined to the rod inner segment (B). Western blotting confirmed the presence of GNAT1 within these samples (C). 68 GNAT1.L 1 GNAT1.L 2 GNAT1.S 1 GNAT1.S 2 ERG? Localization D5 Sub1 (FKO) D12 D12 No Inner Segment D5 Sub1 (FKO) i4D1 i4D1 No Inner Segment D5 i1D12 D2 D7sub1 No KO D5 D5 D12 D4 Yes Outer Segment D12 i27D7 D12 D12 No Inner Segment D12 D12 D3 D12 Yes Inner Segment D12 Sub1 (FKO) D3 D12 Yes OS with IS retention Table 3.1 The Functional Knockout Phenotype Associated with Q184L-GNAT1.L is Recessive in Inheritance Initial identification of the functional KO phenotype occurred in animals with frame-shift mutations in the other three GNAT1 alleles (first two rows). Sub1 is the 1 base pair substitution causing Q184L identified as causing the functional knockout phenotype. These mutations were observed in animals without the functional KO phenotype while Q184L was not (third and fourth rows). A future mating revealed that when accompanied by mutations established to produce an ERG successfully, Q184L does not manifest the full functional KO phenotype (bottom four rows). D represents a deletion, i an insertion, and sub a substitution of nucleotides. 69 3.3.3 A Three Base Pair Deletion Produces a Dominant Morphological and Electrophysiological Phenotype 3.3.3.1 Initial Identification of D3-GNAT1.S Phenotype During screening of the F1 animals produced by mating F0 CRISPR-edited animals, a group of tadpoles were identified based on their abnormal histological appearance (Fig. 3.5A). These animals presented with large GNAT1-positive structures at the base of the rod outer segment which did not stain with the post-Golgi membrane label wheat germ agglutinin (WGA). GNAT1-labeling in these animals was restricted to the inner segment and these structures, unlike wildtype animals where it properly localized to the outer segment. Genotyping linked this phenotype with a three base pair deletion in the GNAT1-S gene causing the mutation T183-Q184 à K183. The morphological phenotype was apparent in 5 animals expressing this three-base pair deletion, and all other mutations present in these animals appeared in the 6 other animals lacking this distinct phenotype (n=5 with phenotype out of n=11 total, p=0.000911 by Chi test). All animals with the observed phenotype also had at least one wildtype allele. These animals presented with minimal alteration to the ERG (Fig. 3.5B). 70 Figure 3.5 A Three Base-Pair Deletion in GNAT1.S Producing the Mutation T183-Q184 à K183 Is Linked to the Development of Abnormal Morphological Structures with Minimal Alteration of the ERG Response Examination of samples generated from the mating of F0 animals via airyscan super resolution microscopy (Zeiss 880) revealed a group of X. laevis tadpoles with GNAT1 confined to the rod inner segment and abnormal morphological structures adjacent to the outer segment which did not stain with WGA (A). These animals possessed a mild ERG phenotype (B; average of n=6 WT in black, n=6 edited animals in red). 71 3.3.3.2 The Effect of the D3-GNAT1.S Mutation is Dominant Following the identification of the D3-GNAT1.S phenotype, mature F1 animals had blood drawn for DNA analysis, seeking the presence of the D3 mutation. The identified F1 animal expressing D3-GNAT1.S was mated with a wildtype animal. The F2 offspring were analyzed, sacrificing animals following electroretinography measurements and processing the tadpoles as described above. F2 offspring positive for the D3-GNAT1.S mutation developed the morphological phenotype observed in F1 animals following exposure to the ERG protocol, with GNAT1 labeling strongly confined to the rod inner segment and the abnormal morphological structures present (n= 5; Fig. 3.6C). Differences in the localization of GNAT between wildtype animals and D3-GNAT1.S were statistically significant (Fig. 3.7; p<0.0001, student t-test) and these animals also manifested a significantly reduced ERG compared to WT (Fig. 3.6D), suggesting dominant inheritance of the D3-GNAT1.S phenotype. The siblings of these animals expressing the other GNAT1.S mutation carried by the edited parent, a 12-base pair deletion (D12-GNAT1.S), presented with minimal ERG alteration and GNAT present in the outer segment, albeit with more GNAT1 labeling in the inner segment than typical in wildtype animals (n=6; Fig. 3.6 A/B). This could indicate a degree of misfolding and resulting mislocalization caused by the D12 mutant. 72 Figure 3.6 Mating of an F1 Animal to Wild Type Reveals a Dominant Morphological and Electrophysiological Phenotype Associated with D3-GNAT1.S When a D3-GNAT1.S-carrying F1 animal was crossed with wildtype offspring carrying the alternative D12-GNAT1.S mutation presented with abnormal GNAT1 labeling within the IS, although a majority was properly localized in the OS (A). ERGs of these animals were essentially WT (B; average of n=5 WT, n=5 D12). D3-GNAT1.S offspring manifested the same morphological phenotype as the F1 animals generated by cross of F0-edited animals (A) and presented with significantly reduced ERG amplitude (D; average of n=5 WT, n=5 D3). 73 Figure 3.7 GNAT1 Localization is Significantly Altered in D3-GNAT1.S Animals Localization of GNAT1 was determined using ImageJ, outlining the outer and inner segments of rods, and measuring the mean intensity of the GNAT1 channel of 5 cells from two animals for each group (n=10 rods total per group). In WT animals the OS signal intensity exceeded that of the IS as expected, while D3-GNAT1.S animals had this pattern reversed to a statistically significant extent (student’s T-test, p<0.0001), showing the extent of mislocalization of GNAT1 in these animals. 74 3.3.3.3 The D3-GNAT1.S Phenotype is Only Produced by the ERG Light Protocol Using siblings of the F2 animals described above, we attempted to determine whether the morphological phenotype occurring in D3-GNAT1.S animals was produced by any change in lighting following dark adaptation. We exposed animals to 10 minutes of constant 1700 lux light before returning them to the dark, sacrificing dark adapted animals, animals at t0 following light adaptation, and a series of increasing times upon returning to the dark. We observed GNAT1 labeling in the OS of all animals (n=15), those expressing D3-GNAT1.S and the alternate D12-GNAT1.S mutation, under dark conditions and through the process of dark adaptation (Fig. 3.8, Fig. 3.9). The location of the structures formed under ERG lighting conditions adjacent to the connecting cilia of the cells, as well as the observation of GNAT labeling in the outer segment of D3-GNAT1.S animals not exposed to the ERG protocol, suggests that these structures and the mislocalization of GNAT1 to the inner segment are caused by a defect in the trafficking of GNAT1, rather than misfolding of the protein. 75 Figure 3.8 Use of the ERG Lighting Protocol is Required to Generate Structural Abnormalities in D3-GNAT1.S Animals Exposure to a steady light source induces translocation of wildtype GNAT1 to the inner segment, while under ERG conditions the majority of GNAT1 remains in the outer segment. With D3-GNAT1.S animals, exposure to the ERG protocol, specifically the photopic flicker portion at a minimum, induces mass translocation to the inner segment and formation of abnormal GNAT1-positive structures (n=15). This effect is not seen in these animals when exposed to a steady light source for a time equivalent to the ERG protocol (n=15). 76 Figure 3.9 The Extent of GNAT1 Mislocalization in D3-GNAT1.S Animals is Dependent on Lighting Conditions Quantification of histology in Figure 3.8 shows D3-GNAT1.S presence within the outer segment relative to the inner segment is significantly reduced upon exposure to steady light, but not to the extent of mislocalization occurring following exposure to the ERG lighting protocol (n=10 rods per group; p<0.0001 for all differences by one-way ANOVA). 77 3.3.3.4 The Photopic Flicker is Critical in the Production of the D3-GNAT1.S Phenotype. After determining that steady light was not sufficient to produce the D3-GNAT1.S phenotype, we returned to the ERG protocol. We repeated the full protocol and successfully reproduced the phenotype in our mutant animals (not shown). Following this confirmation, we investigated whether specific aspects of the protocol were critical by exposing sets of animals to specific portions of the whole lighting regimen. Our phenotype was produced by all combinations of steps containing the photopic flicker phase (n=15) including the flicker alone (n=5). We determined the photopic flicker phase was the minimum requirement to induce the D3-GNAT1.S phenotype (Fig. 3.8). 3.3.3.5 Light Intensity Alone Does Not Explain the Difference in Phenotype Generation Between Lighting Protocols The above result led us to perform light intensity measurements summarized in Table 3.2, seeking to determine whether light intensity was the key difference in the production of the structures or if another factor was key. The full ERG protocol was measured at 3495 cd/m2 (5.825 cd/s/m2), and the photopic flicker at 1525 cd/m2 (69.318 cd/s/m2). The average measurement from all positions of the detector within the incubator was 169 942 cd/m2 (283 cd/s/m2), indicating differences in light intensity alone did not explain the production of our morphological phenotype only under ERG lighting conditions, and that the flickering quality of light was essential. 78 Incubator Average Full Protocol Photopic Flicker Integrated Intensity (cd/m2) 169 942 cd/m2 3495 cd/m2 1525 cd/m2 Intensity (cd/s/m2) 283 cd/s/m2 5.825 cd/s/m2 69.318 cd/s/m2 Table 3.2 Light Intensity Measurements Across Different Treatments Used Light intensity measurements of different courses of exposure used were obtained using the ILT700 as described above. The photopic flicker involves exposure to 2.5 cd•s/m2 flashes of 6500K white light with a frequency of 10 Hz over a 25 cd/m2 6500K white background. 79 3.3.3.6 GNAT1 Concentrates Alongside a-tubulin Within the Inner Segment in GNAT1.S-D3 Mutant Animals We proceeded to characterize offspring generated by mating the D3-positive F1 male back with its genetically chimeric F0 female parent. Examination of D3-positive offspring exposed to the ERG lighting protocol revealed GNAT1 labeling to be highly concentrated in the corners of the inner segment adjacent to the outer segment where it co-localized with acetylated a-tubulin in the region of the connecting cilium (Fig. 3.10). This localization, along with the fact that trafficking partners of GNAT1 are microtubule-associated (Harris & Lim, 2001; Grayson et al., 2002; Moleirinho et al., 2013), lends support to the idea that these structural abnormalities are generated due to a trafficking defect. 80 Figure 3.10 GNAT1 Co-Localizes with a-Tubulin at the Base of the Connecting Cilia in D3-GNAT1.S Animals In 18 day old animals generated by crossing our D3-GNAT1.S-positive F1 male back to its parent F0 female, GNAT1 labeling localized strongly to the boundary between inner and outer segments (B). Here it colocalized with acetylated a-tubulin (A,C), suggesting association with the connecting cilium and supporting a role for trafficking in the development of our observed phenotype. Arrows mark an example of a connecting cilium labeled in both channels. GNAT1 signal intensity was linearly adjusted for clarity. 81 3.3.3.7 A Minimum Level of Normally Functioning GNAT1 is Required to Produce the D3-GNAT1-S-Associated Morphological Phenotype We exposed animals generated by mating the D3-positive F1 male back with its genetically chimeric F0 female parent to the ERG lighting routine without recording to expedite sample generation, before sacrificing and processing as above. Histological examination found that GNAT1 labeling was concentrated in the corners of the IS adjacent to the OS (Fig. 3.11), the site of formation of the abnormal structures previously observed. We repeated this examination with increasingly older tadpoles to investigate whether the phenotype could be age dependent. Regardless of the age of animals examined, abnormalities on the same scale as previously observed were never seen in offspring produced by this mating (n=24 tadpoles across 4 time points; Fig 3.11). At late time points the concentration of GNAT1 described above resembled smaller version of the structures observed in the initial identification and the wild-type cross but were markedly less prominent (Fig. 3.11). 82 Figure 3.11 In D3-GNAT1.S Animals with no Wild Type Alleles GNAT1 Localizes to the Corners of the Rod Inner Segment, Increasing in Prominence with Age As animals age the concentration of GNAT1 to the corners of the IS adjacent to the OS increases, developing into what appear to be a less prominent form of our previously observed structures. These animals express no wildtype GNAT1. GNAT1 signal intensity was linearly adjusted for clarity. 83 Based on the apparent involvement of GNAT1 translocation in the generation of our phenotype, we attempted to investigate whether the D3-GNAT1.S mutation affected the rate of return of GNAT1 to the outer segment, exposing dark adapted animals to our translocation lighting protocol before returning to the dark and collecting samples at increasing time points. Surprisingly, immunohistochemistry showed GNAT1 to be localized to the IS in all D3-positive animals analyzed, including those sacrificed before light exposure (n= 12; Fig. 3.12). Analysis of siblings without the D3-GNAT1.S mutation exposed to the same conditions revealed a greater degree of IS GNAT1 labeling than expected when compared to wildtype, as previously observed in analysis of D12-GNAT1.S animals produced in our wildtype cross (Fig. 3.13). This suggests that other mutations present in animals from the mating between our D3-GNAT1.S F1 animal and the F0 parent produce GNAT1 with partially altered localization and/or function. 84 Figure 3.12 In D3-GNAT1.S Animals with no Wild Type Alleles GNAT1 is Mislocalized to the Rod Inner Segment Under all Lighting Conditions An attempt to measure differences in the rate of return of mutant and wildtype GNAT1 to the outer segment failed, as in animals from this mating the protein is strongly confined to the rod inner segment under all lighting conditions. GNAT1 signal intensity was linearly adjusted for clarity. 85 Figure 3.13 Sibling Animals Expressing Mutations other than D3-GNAT1.S Have More Inner Segment GNAT1 Labeling than Wild Type Sibling animals from this mating, also with four mutant GNAT1 alleles but expressing D12-GNAT1.S from the F1 parent rather than D3-GNAT1.S show a degree of inner segment labeling greater than expected based on wildtype results without developing the complete confinement seen in D3-GNAT1.S animals following the ERG protocol, suggesting other mutations present in these animals are partly defective in their localization. GNAT1 signal intensity was linearly adjusted for clarity. 86 The observation of mislocalized GNAT1 under all lighting conditions alongside differences in the development of the D3-GNAT1.S phenotype when compared to tadpoles produced by crossing with a WT parent led us to analyze the animals generated by this mating in greater depth, recording ERG traces before sacrificing, immunohistochemically labeling the eyes for fluorescence confocal microscopy, and completing full Sanger sequencing and analysis of all alleles. Results from this analysis are summarized in Table 3.3. DNA sequencing revealed a set of mutations, GNAT1.S-D12, GNAT1.L-D12, and GNAT1.L-D27/i7, which were not capable of generating ERG traces on their own and produced GNAT1 mislocalized to the IS (n=3). Two animals expressing the combinations of the above mutations in three alleles alongside D3-GNAT1.S in the fourth allele did generate ERGs and had GNAT1 localized to the IS, as observed previously in animals expressing D3-GNAT1.S. The remaining animals analyzed (n=3) expressed combinations of the mutations described above alongside others that did not appear to significantly alter GNAT1 localization or function. These animals generated ERGs and histological examination showed GNAT1 in the OS with varying degrees of IS retention. These results lead us to conclude that manifestation of the full D3-GNAT1.S phenotype is dependent on the presence of some level of normally functioning GNAT1, consistent with dominant-negative inheritance of the D3-GNAT1.S mutation. 87 GNAT1.L 1 GNAT1.L 2 GNAT1.S 1 GNAT1.S 2 ERG? Localization i27D7 D12 D12 D12 No IS i27D7 D12 D12 D12 No IS D12 D12 D12 D12 No IS D12 D12 D3 D12 Yes IS D12 sub1 D3 D12 Yes IS Table 3.3 Characterization of Effect of Mutations Present in D3-GNAT1.S F1 x F0 Mating Analysis of the mutations present in the offspring of this mating and their effect on the ERG reveal a set of mutations that produce non-functional GNAT1 that localizes to the inner segment. It is likely that the trafficking defects in these mutations prevent the full development of the D3-GNAT1.S phenotype, as this is dependent on the effect of D3-GNAT1.S GNAT1 on the remainder of the GNAT1 population. 88 3.3.3.8 Dark Adaptation is Altered in D3-GNAT1.S Animals After obtaining surprising results in our attempts to investigate D3-GNAT1.S trafficking in animals generated by crossing an F1 animal back to its CRISPR-modified F0 parent, we decided to repeat the experiment on animals generated by crossing our D3-GNAT1.S-F1 with wildtype, as D3-GNAT1.S has previously been observed in the outer segment of these animals under dark conditions (Fig. 3.8). We dark adapted tadpoles overnight, exposed them to our translocation lighting regimen, then returned them to the dark. We sacrificed and collected eyes from overnight dark adapted, light adapted, and 4/32/60/120-minute dark adapted animals (n=4 per time point), labeling them as described above and quantifying the labeling using ImageJ. Comparing changes in the ratio of the mean GNAT1 signal intensity in the outer and inner segments for wildtype and D3-GNAT1.S animals revealed that the rate of return and endpoint were dramatically reduced in D3-GNAT1.S retinas (Fig. 3.14), indicative of obstructed trafficking of GNAT1 to the outer segment. These differences were highly significant by two-way ANOVA (n=10, 5 rods from two animals each per treatment; P interaction = 1.03•10-11, P time = 3.06•10-11, P genotype = 9.19•10-14). To confirm that our observed changes in OS/IS ratio were due to protein movement and not (for example) consistently high IS expression of GNAT1 in the D3-GNAT1.S group, we compared the mean signal intensity for each compartment under fully dark and light adapted conditions. 89 Light adapted animals presented with increased GNAT1 within the inner segment and decreased GNAT1 signal in the outer segment for both groups (Fig. 3.15), and all light-induced changes within genotypes were significant by two-way ANOVA (WT x WT OS p=0.0002, IS p<0.0001; D3 x WT OS p=0.0071, IS p=0.0006). This result confirmed that the cause of the altered GNAT1 signal ratios in Fig. 3.15 was light-induced translocation of GNAT1 between the outer and inner segments, with the decreased rate of recovery and endpoint in D3-GNAT1.S animals reflective of impaired translocation of GNAT1 to the outer segment. 90 Figure 3.14 GNAT1 Return to the Outer Segment is Significantly Altered in D3 x WT-GNAT1.S Animals Plotting the ratio of the mean signal intensity for the outer and inner segments of rods at increasing time points following light exposure (n=5 rods from two animals for each time point) shows that the return of GNAT1 to the outer segment during dark adaptation is significantly reduced in D3 x WT-GNAT1.S animals by two-way ANOVA (p<0.0001 for genotype, time, and the interaction between the two). 91 Figure 3.15 Measurement of the Mean Signal Intensity of Rod Inner and Outer Segments Shows that Changes in the OS:IS GNAT1 Ratio are due to Light-Induced Movement Between Compartments Comparison of the mean signal intensity of the rod segments shows that changes in the GNAT1 signal ratio described in Figure 3.14 are due to significant light-induced changes in mean GNAT1 intensity within both segments (WT x WT OS p=0.0002, IS p<0.0001; D3 x WT OS p=0.0071, IS p=0.0006). This confirms GNAT1 translocation still occurs in our D3 x WT-GNAT1.S mutants, albeit in an altered manner compared to WT. Mean IS intensity is elevated vs WT in both light and dark-adapted conditions (dark p<0.0001, light p=0.0004) while no significant difference in the OS was observed (dark p=0.3432, light p=0.7853). *= p<0.05, **= p<0.01, ***= p<0.001, ****= p<0.0001. 92 After confirming that our D3-GNAT1.S mutation alters trafficking of GNAT1 during dark adaptation, we decided to investigate whether this change manifested electrophysiologically through a specialized ERG protocol. Described in detail in section 3.2.5, dark-adapted animals were exposed to a bleaching stimulus followed by regularly spaced low intensity flashes. As expected, wildtype responses increased in magnitude progressively as the rods dark adapted (Fig. 3.16A). While light adapted D3-GNAT1.S (n=10) animals displayed a similar response magnitude to WT controls (n=5; Fig. 3.16A/B/C), their B wave magnitude did not significantly increase over the course of dark adaptation. Comparing wildtype, D3-GNAT1.S, and D12-GNAT1.S (n=4) animals revealed a highly statistically significant interaction between genotype and time via two-way ANOVA (P interaction = 4.54•10-5, P time = 3.17•10-6, P genotype = 5.34•10-12; Fig. 3.16). Tukey post-hoc comparison revealed no significant differences at t=0 or t=100. D12-GNAT1.S was not significantly different from wildtype until t=400 (p=1.52•10-2), while D3-GNAT1.S significantly differed from both groups at t=200 (P wildtype = 4.66•10-5, P D12-GNAT1.S = 7.31•10-3), t=300 (P wildtype = 5.00•10-9, P D12-GNAT1.S = 1.72•10-2), and t=400 (P wildtype = 4.66•10-5, P D12-GNAT1.S = 7.31•10-3). These results confirm that D3-GNAT1.S exhibits a significant effect on dark adaptation distinct from deficits caused by other GNAT1 mutations. 93 Figure 3.16 Electroretinography Shows Dark Adaptation is Disrupted in D3 x WT-GNAT1.S Animals ERG recordings of repeated low-intensity light stimulation following a bleaching stimulus show that dark adaptation of D3 x WT-GNAT1.S animals (n=10) is significantly reduced when compared to wildtype (n=5; A/B/C) and D12 x WT-GNAT1.S siblings (n=5; A/B/D). 94 Chapter 4: Conclusions We successfully generated, identified, and characterized both GNAT1-knock-out and otherwise edited Xenopus laevis. As discussed in Chapter 2 above, GNAT1-KO Xenopus laevis, behaved as expected based on previous reports from other models (Calvert et al., 2000), presenting with an absent scotopic ERG, no GNAT1 presence by western blot, no labeling of GNAT1 via immunofluorescence microscopy, and frame-shift mutations in all GNAT1 alleles according to Sanger sequencing. That some knockout animals presented with subtle ERG responses at high intensities is not completely unexpected, as previous work in our lab has observed similar responses from RPE65-KO and F0 GNAT1-KO xenopus which should also lack scotopic responses (Tam et al., 2024). This response could be due to some activation of cones in these animals or the presence of intrinsically photosensitive retinal ganglion cells (ipRGC) or another photosensitive cell type. Our GNAT1 knock-out animals will provide a useful background for future investigation of the role of phototransduction in retinal degeneration by expressing disease-causing transgenes on the GNAT1-KO background. In the process of generating these knock-out animals we obtained several unexpected results providing insight into the function of GNAT1. There is precedence for both altered GNAT1 lifetime and low GNAT1 expression altering the kinetics of phototransduction. As described in section 1.1.5 Kinetics of Phototransduction, altering the expression of proteins controlling shutdown of GNAT1 alter the rate of the response. In their 2019 paper Yue et al. transgenically expressed wildtype a-transducin at ~6% of normal levels on a GNAT1-KO background (Yue et al., 2019), a value remarkably close to our observed concentration inducing half maximal effect 95 of 6.45%. These mice presented with a ~1.5x increase in the integration time, time to peak, and recovery time constant; 587, 391, and 231 ms respectively for wildtype and 880, 557, 426 ms for GNAT1 transgenic animals (Yue et al., 2019). This range agrees with our observed ~1.75x increase in A wave implicit time at ~6% GNAT1 expression. The presence of this delay when WT transducin is expressed at low levels suggests that the effect is directly related to decreased GNAT1 expression rather than the presence of a mutant protein with altered function. The fact that reduction in GNAT1 levels alters the timing of the ERG response as well as its magnitude suggests a second functional role for GNAT1 in addition to the amplification of the phototransduction cascade. 1982 work from Emies et al. shows that in isolated discs, the presence of GNAT1 results in the production of excess active Meta II rhodopsin compared to the intermediate Meta I form (Emeis et al., 1982). Additionally, a 2004 paper from Zimmerman et al. demonstrates that that in vitro, the rate of conversion of the retinal storage photointermediate Meta III Rhodopsin into the active signaling Meta II Rhodopsin form is dependent on the concentration of GNAT1 present, with evidence for a direct interaction between GNAT1 and Meta III (Zimmermann et al., 2004). It is possible that the delayed ERG response we observe is a manifestation of these effects in vivo (Fig. 4.1). With decreased expression of GNAT1, initial photoactivation of Rhodopsin produces less Meta II Rho, favouring Meta I, delaying the photo-response. Additionally, the Meta III rhodopsin generated is not efficiently converted to Meta II in the absence of GNAT1, amplifying this effect. As these persistent intermediates gradually convert into Meta II, at a delayed rate the photoresponse is prolonged, resulting in an overall delayed ERG time course. 96 This effect becomes prominent at extremely low expression levels, as seen in our observed concentration inducing half-maximal effect of 6.45% and the transgenic mouse of Yue et al. expressing at ~6% (Yue et al., 2019), explaining why it was not observed in heterozygous GNAT1-KO mice in which an approximately 25% reduction in expression level was observed (Calvert et al., 2000). The existence of a direct relationship between GNAT1 and intermediates of rhodopsin raises interesting questions as to a role for GNAT1 in light-induced retinal degeneration. Notably previous work from our lab implicates toxic photointermediates in T4K-rhodopsin induced retinal degeneration, with knockout of GNAT1 dramatically exacerbating this effect (Tam et al., 2024). As discussed above, GNAT1 shifts the ratio of Meta I to Meta II produced upon light stimulation (Emeis et al., 1982) and directly interacts with Meta III rhodopsin, converting it to the Meta II form from which it can degrade to opsin and retinal (Zimmermann et al., 2004). GNAT1 may serve a similar function with regards to toxic intermediates produced by T4K rhodopsin upon activation, facilitating their conversion into the stable Meta II conformation for clearance and preventing accumulation of these toxic intermediates within the outer segment. The models generated by the work within this thesis, including the GNAT1-KO and X. laevis with reduced expression, will be applied to the study of how GNAT1 interacts with these photointermediates and its role in the pathways involved in inherited retinal degenerations. 97 Figure 4.1 Proposed Pathway for the Interaction Between GNAT1 and Rhodopsin Photointermediates Following light activation of rhodopsin, the Meta I and Meta II isoforms exist in an equilibrium partially dependent on the presence of GNAT1. Meta II, the active signaling form, interacts with transducin in the phototransduction pathway before decaying to opsin and all-trans retinal. A portion of the Meta I population converts into the persistent retinal storage form Meta III, the lifetime of which is dependent on GNAT1 concentration which interacts directly and enables conversion into Meta II, re-entering the phototransduction pathway. We propose that our observed delayed phototransduction in animals with reduced GNAT1 expression relates to the role of GNAT1 in maintaining proper levels of these rhodopsin photointermediates, with reduction in GNAT1 leading to reduced and delayed production of active Meta II rhodopsin. Created with BioRender.com 98 Alternatively, the ERG delay could be related to the packing structure of rhodopsin and transducin within the outer segments. Electron microscope analysis of rod discs reveals rhodopsin to be organized into tracks of dimers (Gunkel et al., 2015). Among other possible reasons for this structure a role in the maintenance of phototransduction kinetics has been proposed, with consistency being upheld by the preassembly of consistent numbers of a-transducin in complex with each rhodopsin track. In this scenario, decreases in a-transducin expression would result in delays of phototransduction with activated rhodopsin needing to recruit a-transducin from neighbouring tracks, introducing escalating delays in the pathway (Gunkel et al., 2015). According to 2019 work by Yue et al., twelve to fourteen GNAT1 are activated per active rhodopsin (Yue et al., 2019). With an expression of 6.45% for the relationship between GNAT1 expression and A wave timing according to our results in Chapter 3, this means that the greatest impact on phototransduction in our model is seen when on average under one GNAT1 is activated per rhodopsin, agreeing with predictions made by Gunkel et al. (Gunkel et al., 2015). The relationship between decreased a-transducin expression and the implicit time of the ERG response could be further validated through patch clamp recordings of individual rods, avoiding the averaging of the entire retina that occurs when measuring the electroretinogram from the cornea. Additionally, our two proposed mechanisms could be differentiated through microspectrophotometry. As the Metarhodopsin photointermediates are all spectroscopically distinct, differences in the amount and rate of production and clearance of each could be measured in animals expressing varying levels of GNAT1 and compared to wildtype values. 99 It is likely our observation of this relationship was aided by our use of X. laevis, specifically CRISPR-edited animals, as a model. Their pseudotetraploid nature, while complicating the generation of specific mutant models, increases the possible range of expression values for a protein. This is especially prominent when investigating the F1 offspring of genetically heterogeneous CRISPR animals with their diverse array of mutations expressed in combination. By a remarkable coincidence, a mutation equivalent to the GNAT1.L-Q184L was associated with our functional-KO phenotype described in Chapter 3 has been linked to a silencing phenotype in the yeast G protein a-subunit analogue GPA1 (Clark et al., 1993; Whiteway et al., 1994). They describe this mutation suppressing the effects of a constitutively active bg mutant by altering interactions between the a and bg subunits to increase affinity. Q184L is located between the switch I and II regions of the protein which change conformation in response to GTP binding and dissociation from bg-transducin (Sondek et al., 1994). Therefore, it is likely that this mutation produces a conformational change that specifically alters binding of a-transducin to bg, preventing transport to the rod OS without causing other biosynthetic defects. This proposed mechanism also aligns with the recessive nature of the phenotype in our model; the Q184L mutation does not affect the entire transducin population of the cell and as such its mislocalization is compensated for by the expression of other GNAT1 alleles. Additionally, by searching the GNOMAD database we found that an identical GNAT1 mutation is present in the human population (Karczewski et al., 2020), where it is not associated with any known disease cases. However, based on our results we predict this is likely a disease-causing variant that would be associated with autosomal recessive CSNB. 100 Several results indicate that our D3-GNAT1.S phenotype is related to deficits in the trafficking of GNAT1 to the outer segment rather than a misfolding-induced permanent mislocalization. D3-GNAT1.S mutant displays its morphological phenotype under specific lighting conditions, with GNAT-1 observed in the outer segment of animals when dark adapted (Fig. 3.8/3.9). Within the inner segment, GNAT1 co-localizes with a-tubulin near the connecting cilium in mutant animals (Fig. 3.10). Following light adaptation, GNAT1 returns to the OS in both groups, but to a much lesser extent in D3-GNAT1.S X. laevis than in WT (Fig 3.14/3.15). We also observed deficits in dark adaptation in these animals electrophysiologically (Fig. 3.16). That obstructions in GNAT1 return to the OS are accompanied by impaired dark adaptation is supported by 2022 results by Campla et al. (Campla et al., 2022). T183-Q184 à K183, the three base pair deletion producing structural and electrophysiological abnormalities, occurs in a region of GNAT1 pulled down in association with UNC119 (Zhang et al., 2011) adjacent to the switch II region. This site is known to stabilize interactions between the N-terminal myristylation site with UNC119 (Cheguru et al., 2015). As UNC119 is essential for trafficking of GNAT1 to the outer segment, and the region of our mutation of interest is known to be involved with this interaction, issues with association between GNAT1 and UNC119 may be the cause of the structural abnormalities observed. Expression of a dominant constitutively active Arl3 mutant which induces premature release of cargo results in mislocalization of transducin subunits, among other proteins, and formation of deposits within the inner segment (Wright et al., 2016). We suspect that the D3-GNAT1.S protein interacts abnormally with Unc119, preventing this GNAT1 from effectively diffusing to the OS. 101 We propose that by occupying the available Unc119, D3-GNAT1.S blocks this transport pathway along the connecting cilium for the remainder of the GNAT1 population of the cell. When light exposure induces movement of GNAT1 to the IS it attempts to return to the OS, pushing against the D3-GNAT1.S blockade and deforming the IS or connecting cilium membrane, generating the abnormal morphological structures seen in our animals. The repeated flashes of the photopic flicker may induce multiple small waves of GNAT1 translocation, which return quickly to the outer segment under normal conditions but are blocked by the D3-GNAT1.S protein, accumulating to form the structures observed and explaining the absence of GNAT1 labeling within the OS. Our observed morphological abnormalities could also be caused by interference with the Gpsm2/Frmpd1 active transport pathway. Knockout of Frmpd1 results in decreased return of GNAT1 to the OS and deficient dark adaptation measured via ERG (Campla et al., 2022), as is observed in our mutants. Under normal conditions, trace amounts of GNAT1 translocated to the inner segment may be rapidly moved back to the outer segment to minimize desensitization. We hypothesize that in our mutants this pathway becomes saturated rapidly under photopic flicker lighting, forming the observed structures in a similar fashion as described above for interactions with Unc119. Gpsm2 is present at a ~1:15 concentration compared to GNAT1 (Nair et al., 2005), so saturation could occur with only one mutant allele. Unfortunately, the specific region of the GNAT1 protein interacting with Gpsm2 and FRMPD1 remains unclear. 102 Further work, including pull-down assays investigating proteins associated with wildtype and mutant GNAT1 under specific conditions, requires identification or generation of antibodies that successfully bind these proteins in X. laevis. Selective poisoning of cytoskeletal elements in mutant animals could also elucidate which trafficking mechanism is being interfered with in the D3-GNAT1.S animals. As this mutation site overlaps with that edited in our functional knockout it is also possible that this mutation alters interactions with the GNATb-g subunits, although the explanation for the new morphological phenotype is unclear in this case. While it is possible that other mutations located in GNAT1.L and GNAT1.S respectively may be responsible, this seems unlikely as all other mutations present were also observed in animals without the morphological phenotype and the phenotype can be generated in offspring produced by an animal carrying the D3-GNAT1.S mutation. The two mutations differ in their specific gene of origin, GNAT1.S for D3 and GNAT1.L for Q184L, and in their inheritance, with D3-GNAT1.S being dominant and Q184L-GNAT1.L appearing recessive. These differences suggest the mechanism of action of the two likely differ. Regardless, the D3-GNAT1.S mutation provides an interesting model for study of the role of light-induced movement of GNAT1 and its return to the outer segment with regards to its role in cellular health and dark adaptation. Our observed phenotype may also represent a new dominant-negative mechanism for dominantly inherited GNAT1-related disorders, including congenital stationary night blindness and rod-cone dystrophy. 103 The results described within this thesis provide a backbone for future study into the fundamental processes underlying vision, the behavior of the proteins required for these processes, and how misfunction in the most basic operations within photoreceptors can contribute to human disease. The generation of a genetically distinct line of GNAT1-KO X. laevis will form a useful background for study of the role of the phototransduction cascade in different retinal degenerations, notably expanding upon previous experiments with transgenic P23H-RHO and T4K-RHO X. laevis from our lab (Wen et al., 2019; Tam et al., 2024). Knockout X. laevis, alongside edited animals with variable expression, can be studied to answer questions about a direct role for GNAT1 in preventing retinal degeneration in T4K-RHO-like models of RP through the buildup of toxic photointermediates. Further investigation into the mechanisms of the relationship between GNAT1 expression and ERG response timing will help expand our knowledge of the fundamental processes underlying our vision. Work on these animals may answer questions regarding the physiological relevance of biochemical studies into the effect of GNAT1 on the production of metarhodopsin isoforms (Emeis et al., 1982; Zimmermann et al., 2004), and could provide support for theories related to the packing structure of rhodopsin (Gunkel et al., 2015). 104 Results obtained from our functional knockout animals suggest that the Q184L-GNAT1.L mutation, which has been observed in human populations (Karczewski et al., 2020), may be a disease-causing variant that would likely by associated with autosomal recessive congenital stationary night blindness. Our D3-GNAT1.S animals provide an opportunity to investigate the trafficking of GNAT1 to the outer segment and its physiological importance. Clarification of whether interaction with the Unc119 or Frmpd1/Gpsm2 pathway is altered by this mutation and poisoning of cytoskeletal components, as in precious work investigating GNAT1 trafficking (Calvert et al., 2006, could provide evidence for active or passive transport as the primary contributor to our phenotype, and by extension trafficking of GNAT1 to the outer segment. The fact that rapid flashing, as in the photopic flicker, appears to be required to induce the phenotype raises interesting questions as to the timeline of GNAT1 return to the outer segment following stimulation that could be investigated in these animals. Additionally, that dark adaptation is dramatically reduced in these animals suggests an important role for GNAT1 trafficking between compartments in the initial stages of dark adaptation. 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Thesis/Dissertation
eng
Experimental Medicine
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Moritz, Orson L.
Characterization of retinal function and morphology in GNAT1-edited Xenopus laevis
Text