ABSTRACT RATE-LIMITING STEP OF CONE PHOTOTRANSDUCTION RECOVERY AND OGUCHI DISEASE MECHANISMS By Frank Sungping Chen Advisor: Ching-Kang Jason Chen, Ph.D. Retinal photoreceptors provide the first gateway in which light information from the environment is transformed into neuronal signals. The cone and rod photoreceptors are responsible for day and night vision, respectively. Understanding rod and cone phototransduction is to figure out how these cells differ in their temporal and spatial sensitivities to allow perception of a broad dynamic range of stimuli. Phototransduction is mediated through a Gprotein signaling cascade. Light absorption by visual pigment triggers the isomerization of 11- cis-retinal covalently attached to these pigments, which are heptahelical transmembrane Gprotein- coupled receptors. Isomerization of 11-cis-retinal to all-trans-retinal activates the receptor, which catalyzes the exchange of GDP for GTP on the α subunit of heterotrimeric Gprotein called transducin. Activated transducin relieves inhibitory constraint on cGMP-PDE, leading to rapid hydrolysis of cGMP, closure of cGMP gated cation channels, and membrane hyperpolarization. In order for photoreceptor to be responsive to light again, this robust phototransduction pathway must be deactivated in a timely fashion and this involves several reactions simultaneously. First, the activated opsin must be phosphorylated by G-protein-coupled receptor kinases (GRKs) and capped by arrestin binding. Second, activated transducin must hydrolyze bound GTP through intrinsic GTPase activity, which is accelerated by a GTPase accelerating protein (GAP) complex comprised of RGS9-1/Gβ5-L/R9AP. Mutations in human genes involved in these reactions cause various visual defects. Cone, by and large, uses the same set of genes for pigment and transducin deactivations but it has lower sensitivity and faster kinetics than rod and is responsible for high visual acuity. During phototransduction recovery in which multiple reactions take place, the slowest reaction will determine the overall rate of recovery. In rod, this so-called, rate-limiting step has been determined to be transducin deactivation. It is unknown whether cone transducin deactivation also controls the timing of conerecovery, although we and others have shown that cone possesses a higher level of GAP concentration. In this thesis, the rate-limiting step in cone phototransduction recovery has been unequivocally determined by overexpressing RGS9-1 by 2.7 fold in mouse cones, which results in accelerated cone recovery. Complementarily, we find that ectopically expressing a human cone opsin kinase GRK7 in mouse cones does not affect cone recovery. These results altogether demonstrate that the rate-limiting step of cone recovery is the GTP-hydrolysis of cone transducin, not the opsin phosphorylation by GRKs. By elucidating the rate-limiting step of photoreceptor recovery, we have revealed the importance of G-protein cycling in timing of both rod and cone photoreceptors. This may further be generalized to other physiological processes controlled by heterotrimeric G-proteins. The proper shutoff of phototransduction is essential for normal vision as recovery defects lead to visual impairment. Even though the reaction catalyzed by GRK1 is not rate-limiting, mutations of this important gene render rhodopsin phosphorylation and deactivation the slowest step in rod recovery and create a pathological condition. GRK1 mutations have been found in Oguchi disease patients, who suffer from congenital stationary night blindness. One of the mutations, V380D, is investigated in detail in this study. Transgenic expression of GRK1 V380D mutant in rods reveals a kinase with reduced expression and catalytic activity. While V380D GRK1 is found capable of inactivating rhodopsin, the reduction in kinase activity leads to a delayed dark adaptation, and is congruent with the night blindness phenotype observed in Oguchi disease patients. Finally, we have also investigated the role of post-translational isoprenylation on GRK1 function. We found that isoprenylation is required for GRK1 membrane association and outer segment targeting. Altogether our data add significantly to understanding the structure and function of GRK1, which is one of the least understood molecules involved in vertebrate phototransduction.

Cichlid visual system is highly adaptive to the environment. Fish visual abilities are deter- mined by composition of opsin-based photosensitive pigments located in photoreceptor cells (rods and cones) as well as their distribution. In this thesis, four species of Lake Barombi Mbo cichlids were targeted: Stomatepia mariae, Konia eisentrauti, Konia dikume and Myaka myaka. Shallow-water cichlids K. eisentrauti and S. mariae express these types of cone opsin genes: LWS, RH2A (RH2Aβ more than RH2Aα), SWS2A and SWS2B. Contra- rily, both seasonally deep-water M. myaka and deep-water K. dikume lack expression of SWS2B and LWS in their retinae, but they express SWS2A and RH2Aα more than RH2Aβ, which corresponds to modified light conditions in deep water - a dimmer habitat lacking marginal parts of the spectrum (i.e., ultraviolet and red wavelengths). The photoreceptor distribution of selected species was investigated by means of fluorescent in situ hybridi- zation (FISH) in order to understand the performance and evolution of their visual per- ception with emphasis on the effect of depth. Distinctively, cichlid cones are arranged in a retinal mosaic consisting of short-wavelength sensitive single cones, each surrounded by four long-wavelength sensitive double cones. In this thesis, the same arrangement is...

Planar cell polarity (PCP) is the organization of cellular characteristics within the plane of a tissue. PCP manifests both structurally, as in the directionality of insect bristles or mammalian skin hair, or dynamically, as in vertebrate neurulation, gastrulation, and oriented cell division in the kidney. Two well-conserved pathways are known to regulate PCP in invertebrates and in vertebrates: the Frizzled/PCP pathway and the Fat-Dachsous (Ft-Ds) pathway. The latter consists of the cadherins Ft and Ds, along with the Golgi kinase Four-jointed (Fj) and the transcriptional co-repressor Atrophin (Atro). Ft and Ds can bind each other, suggesting a mechanism for signal transduction. Fj phosphorylates Ft and Ds, modulating their binding affinities for each other. Atro is proposed to link Ft-Ds signaling with downstream events in the nucleus during eye development. The details of Ft-Ds binding, and the consequences of their interactions with other members of the pathway are poorly understood. In this work, I quantitatively analyzed Ft-Ds pathway mutant clones for their effects on ommatidial polarity in the Drosophila eye. My findings suggest that the Ft-Ds pathway regulates PCP independently of asymmetric cellular accumulation of Ft or Ds. I found that Atro has a position-specific role in regulating polarity in the eye, that Fj dampens clonal polarity signals, and that asymmetric accumulation of the atypical myosin Dachs is not essential for production and propagation of a long-range PCP signal. My observations suggest that Ft and Ds interact to modulate a secondary signal that regulates long-range polarity, that signaling by the Ds intracellular domain is dependent on Ft, and that ommatidial fate specification is genetically separable from long-range signaling.

Planar cell polarity (PCP) is the organization of cellular characteristics within the plane of a tissue. PCP manifests both structurally, as in the directionality of insect bristles or mammalian skin hair, or dynamically, as in vertebrate neurulation, gastrulation, and oriented cell division in the kidney. Two well-conserved pathways are known to regulate PCP in invertebrates and in vertebrates: the Frizzled/PCP pathway and the Fat-Dachsous (Ft-Ds) pathway. The latter consists of the cadherins Ft and Ds, along with the Golgi kinase Four-jointed (Fj) and the transcriptional co-repressor Atrophin (Atro). Ft and Ds can bind each other, suggesting a mechanism for signal transduction. Fj phosphorylates Ft and Ds, modulating their binding affinities for each other. Atro is proposed to link Ft-Ds signaling with downstream events in the nucleus during eye development. The details of Ft-Ds binding, and the consequences of their interactions with other members of the pathway are poorly understood. In this work, I quantitatively analyzed Ft-Ds pathway mutant clones for their effects on ommatidial polarity in the Drosophila eye. My findings suggest that the Ft-Ds pathway regulates PCP independently of asymmetric cellular accumulation of Ft or Ds. I found that Atro has a position-specific role in regulating polarity in the eye, that Fj dampens clonal polarity signals, and that asymmetric accumulation of the atypical myosin Dachs is not essential for production and propagation of a long-range PCP signal. My observations suggest that Ft and Ds interact to modulate a secondary signal that regulates long-range polarity, that signaling by the Ds intracellular domain is dependent on Ft, and that ommatidial fate specification is genetically separable from long-range signaling.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 95-101).

Title from first page of PDF document. Includes bibliographical references (p. 26-29).

L’ataxie spinocérébelleuse de type 7 (SCA7) est une maladie neurodégénérative à transmission autosomale dominante, causée par une expansion toxique de polyglutamine (polyQ) dans la protéine Ataxine-7. Elle se caractérise par une dégénérescence des photorécepteurs en cônes et en bâtonnets, ainsi que des cellules cérébelleuses de Purkinje et granuleuses. La nature sélective de cette dégénérescence reste peu claire, l’expression d’Ataxine-7 étant ubiquitaire. Dans ce contexte, nous avons exploré la fonction de l’orthologue d’Ataxine-7 chez le poisson-zèbre au cours du développement de l’œil. L’inactivation d’atxn7 chez le poisson-zèbre – par des approches utilisant des oligonucléotides anti-sens ou par CRISPR/Cas9 – résulte principalement en un colobome, malformation structurelle de l’œil causée par un défaut de fermeture de la fissure choroïde. Les morphants atxn7 présentent une altération du motif proximo-distal de la vésicule optique causée par une élévation de la signalisation Hedgehog (Hh). Une étude minutieuse des photorécepteurs révèle un défaut de la morphogénèse des segments externes. La sensibilité de l’œil aux variations de fonction d’atxn7 pourrait expliquer la phyiopathologie SCA7. Notre étude suggère également qu’une perte de fonction d’atxn7 contribuerait au développement du colobome chez l’Homme. / Spinocerebellar ataxia type 7 (SCA7) is an autosomal-dominant neurodegenerative disorder caused by a toxic polyglutamine (polyQ) expansion in Ataxin-7 which leads to degeneration of cone and rod photoreceptors. The selective nature of degeneration remains unclear since Ataxin-7 is ubiquitously expressed. Here, we have explored the function of the Ataxin-7 ortholog in zebrafish during eye development. Inactivation of atxn7 in zebrafish primarily resulted in a coloboma defect, a structural malformation of the eye caused by failure of the choroid fissure to close. atxn7 morphants displayed altered proximo-distal patterning of the optic vesicle, caused by elevated Hedgehog (Hh) signaling. Careful examination of the photoreceptors reveals a defect in the morphogenesis of the outer segments. The eye sensitivity to variations in atxn7 function could account for SCA7 physiopathology. Our study also suggests that atxn7 loss of function may contribute to the development of human coloboma.

Age-related macular degeneration (AMD) is the leading cause for visual impairment in the elderly. The etiology of AMD remains unclear. Clinical and histopathological studies suggest that photoreceptors play a role in disease progression. Here, we found that photoreceptors of AMD patients show adaptive changes in gene expression, suggestive of a nutrient shortage. To study the effect of these changes, we mimicked the metabolic alteration in mouse photoreceptors, by disruption of the Tuberous Sclerosis Complex (TSC). This led to AMD hallmarks, including the advanced stages of geographic atrophy (GA) and choroidal neovascularization (CNV). Furthermore, we found that disease onset requires the activity of the mammalian target of rapamycin complex 1 (mTORC1). To study the contribution of photoreceptors to disease, we profiled retinal phospholipids as photoreceptors are rich in phospholipids. We found a reduction in two docosahexaenoic acid (DHA)-containing phospholipids. Feeding DHA to mutant mice, alleviated most AMD-associated hallmarks. To study the inflammatory complications seen with current anti-vascular endothelial growth factor (VEGF) treatments for CNV we used rAAV-mediated gene transfer to overexpress an anti-VEGF protein. We found that inhibition of VEGF can promote retinal inflammation. The data suggests that targeting photoreceptor metabolism may provide novel therapies to treat AMD.

The signal transduction pathway of halophilic archaea remains a fascinating example of adaptation to extreme environments. Despite similarities with bacterial taxis systems, its structural and dynamics patterns during signal relay remain debatable. The currently investigated SRII/HtrII phototaxis system of Natronomonas pharaonis shows remarkable similarities with chemoreceptors in its membrane and HAMP domains functioning design. By combining site-directed spin labeling (SDSL) with electron paramagnetic resonance (EPR) spectroscopy we investigate the kinase control domain (i.e. signaling domain) of NpSRII/HtrII both in terms of dynamic and structural properties. Our data, as provided by continuous wave and pulse (DEER) EPR techniques, builds on current dynamics based signaling models for HAMP domains (such as the “frozen–dynamic” or two-state equilibrium models). We present an expanded mechanism for signal propagation throughout the signaling domain, where salt and temperature variations trigger subtle shifts in dynamics. Extreme dynamics motional ranges (compact or highly-dynamic) associate with a specific flagellar signaling state, here the kinase-off response, where a more moderate dynamics motion (dynamic) associates with the kinase-on response. Structurally, we reference our data on PML and ND reconstituted NpSRII/HtrII to the EcTsr crystal structure and the NpHtrII homology model. We show that, despite a difference in packing, NpHtrII oligomerizes in a similar manner as EcTsr, even in the absence of stabilizing structures such as the CheA/CheW baseplate. The presence of trimers-of-dimers but also dimers-of-dimers in membrane sheet samples exposes the high affinity with which NpHtrII signaling domains interact. We hope our structural and dynamics details will push further not just drug design but also environmental preservation efforts where taxis systems drive colonization and virulence of pathogens in plants, animals and humans alike.

Die Hauptursache für die Erblindung von Erwachsenen in Industrieländern ist die fortschreitende Degeneration von Photorezeptoren (PR). Die PR-Degeneration tritt in Krankheiten, wie Retinitis Pigmentosa (RP) und Altersabhängiger Makuladegeneration (AMD), auf. Photobiomodulation (PBM) ist ein vielversprechender Ansatz, um die Degeneration der PR zu verzögern. Bestrahlung mit Licht, im roten bis nahinfraroten Bereich (600-1000 nm) und geringen Intensitäten (1-500 mW), induzierte antiinflammatorische, antiapoptotische Effekte und verbesserte Mitochondrienfunktion in retinalen Schädigungsmodellen. Klinische Studien belegten übereinstimmend ein erhöhtes Sehvermögen von Patienten mit AMD, RP oder diabetischer Retinopathie durch PBM-Therapie. Die basierenden zellulären Auswirkungen, die die Degeneration der PR verzögern, sind nur unzureichend beschrieben. Retinale Studien konzentrieren sich nahezu ausschließlich auf die Effekte von 670-nm-Rotlicht. Die aktuelle Leithypothese fokussiert sich auf den mitochondrialen Atmungskettenkomplex IV, die Cytochrom-c-Oxidase (CCO). CCO wirkt als primärer Photoakzeptor und Effektor von PBM, dessen Aktivierung zu erhöhter ATP-Synthese, d.h. einem erhöhten mitochondrialen Energiemetabolismus führt. Aktuelle Erkenntnisse lassen zusätzliche Photoakzeptoren vermuten. Allerdings wurde die direkte Wirkung auf andere Atmungskettenkomplexe bislang kaum untersucht. Eine Genregulation und molekularbiologische Mechanismen, die die zellulären Veränderungen erklären, sind weitgehend unbekannt. In dieser Arbeit wurde die neuroprotektive Wirkungsweise von PBM auf geschädigte PR untersucht, die dessen Degeneration verzögert. Es erfolgte eine Charakterisierung der zellulären sekundären Effekte und regulatorischer Mechanismen von RL (670 nm) und NIRL (810 nm). Die Analysen wurden in einem organotypischen ex-vivo-PR-Schädigungsmodell durchgeführt. Mithilfe von Blaulicht (BL) -Bestrahlung (405 nm) über 9 h wurde oxidativer Stress in PR induziert, wodurch diese degenerieren und teilweise absterben. Im Anschluss der Schädigung wurde RL oder NIRL für 10 min appliziert. Nach dieser RL/NIRL-Therapie konnte mittels TUNEL-Assay eine 40-50 %ige Reduktion der Apoptoserate der PR gegenüber BL-geschädigten PR nachgewiesen werden. Dies bestätigt, in Übereinstimmung mit bisherigen PR-spezifischen PBM-Studien, eine PBM-induzierte Neuroprotektion der PR. Zusätzliche positive zelluläre Auswirkungen durch PBM zeigten sich anhand von geringerem oxidativen Zellstress und verbesserter Mitochondrienfunktion. Der oxidative Stress wurde folgendermaßen detektiert: Reaktive Sauerstoffspezies (ROS) in den Innen- (IS) und Außensegmenten (OS) der PR wurden mittels Vitalfärbung nachgewiesen. PBM-Behandlung reduzierte die durch BL-Exposition massiv erhöhte ROS-Bildung. Die relativen ROS-Level sanken von 2,7 (BL) auf 1,2 (RL/NIRL) im OS und von 1,9 (BL) auf 1,3 (RL/NIRL) im IS und näherten sich der normalisierten Kontrolle ungeschädigter Zellen an. PBM inhibierte die Bildung der Lipidperoxidationsprodukte 4-HNE und HEL in den IS und OS. Weiterhin reduzierte sich durch PBM die mtDNA-Schädigung und PBM initiierte mtDNA-Reparaturmechanismen in den IS. Neben dem bereits beschriebenen CCO als Photoakzeptor von PBM, stehen weitere Atmungskettenkomplexe als primäre Photoakzeptoren zur Diskussion. In in-situ-Aktivitätsassays konnten die Atmungskettenkomplexe I und II als direkte Photoakzeptoren von RL und NIRL identifiziert werden. Die, durch BL-Bestrahlung bedingte, 40-50%ige Inhibierungen ihrer Aktivitäten wurde durch RL- bzw. NIRL-Behandlung nahezu komplett wiederhergestellt. Zudem wurde erstmalig belegt, dass PBM, neben den Atmungskettenkomplexen der Mitochondrien in den IS, auch die Aktivität der funktionellen ektopischen Atmungskettenkomplexe in den OS gleichermaßen stimuliert. Als Konsequenz konnte, in Übereinstimmung mit früheren Studien, ein gesteigerter retinaler ATP-Gehalt nachgewiesen werden. Vermutlich sind die Mitochondrien mit ihren Atmungskettenkomplexen die Hauptakzeptoren der PBM. Der gesteigerte Energiemetabolismus bewirkt im Allgemeinen zelluläre Verbesserungen. Wahrscheinlich wird durch PBM der Kreislauf zwischen ROS-Bildung und Schädigung der Atmungskettenkomplexe unterbrochen. Die funktionelle Verbesserung der ektopischen Komplexe kann ebenso zu dem verringerten oxidativem Stress der PR beitragen. Neben der verbesserten Mitochondrienfunktion durch PBM, belegten die Bax-, Bcl-2- und Caspase-9-Proteinlevel eine reduzierte mitochondrieninduzierte Apoptose. Um molekularbiologisch regulatorische Vorgänge aufzuklären, die den veränderten zellulären Phänotyp bewirken, erfolgte eine umfassende Genexpressionsanalyse an isolierten PR, sowie Validierung ausgewählter verändert exprimierter Gene mittels in-situ-Hybridisierung und immunohistochemischer Analyse der kodierten Proteine. Die Ergebnisse der Genregulation durch PBM identifizierten besonders die Gene cryaa und cryab, aus der Klasse der α-Crystalline, die nach RL- und NIRL-Behandlung überexprimiert vorlagen. Als Negativregulatoren der mitochondrieninduzierten Apoptose und der ROS-Produktion können cryaa und cryab eine Reduktion des oxidativen Zellstresses in PR initiieren. Insgesamt demonstriert die vorliegende Arbeit die neuroprotektive Wirkungsweise von PBM auf BL-geschädigte PR. Sie zeigt einen PBM-Wirkmechanismus, der auf Atmungskettenkomplexe und die Genregulation von cryaa und cryab abzielt. Dies führt sekundär zu zellulären Effekten mit reduziertem oxidativen Zellstress. Diese grundlegenden Ergebnisse können helfen, PBM umfassend in klinische Anwendungen von Patienten mit PR-Schädigung zu transferieren.