Russell Van Gelder, M.D., Ph.D - US grants

The candidate's long-term aims is to understand non-visual photoreception in the eye, and its relationship to modulation of circadian rhythmicity and ocular immune function. Retinal degenerate mice, although blind, retain functional ocular photoreception for two phenomena: the resetting of the behavioral circadian clock, and the modulation of ocular inflammation (ACAID). This finding suggests the existence of ocular photoreceptors outside rods and cones. The identity of the photoreceptor(s) underlying these phenomena are unknown, but as both phenomena share a common spectral sensitivity, a single photoreceptor molecule is implicated. A new family of flavin-based blue-light photoreceptors - the cryptochromes - have been described, and are expressed in the eye. These molecules have been implicated in the light-resetting of the circadian clock in plants, and are well conserved in mammals. Their role in the modulation of immune function or circadian rhythmicity in mammals is unknown. Identification of this phototransduction pathway has broad import in understanding both the ocular contributions to circadian rhythms and their disease states, such as insomnia and seasonal depression, and in understanding the mechanisms of pathogenesis of ocular inflammation. The candidate proposes to study the molecular biology and genetics of this protein family in the sponsor's laboratory. He will (1) determine the temporal and spatial expression of the cryptochromes in the murine eye and suprachiasmatic nucleus of the brain, (2) determine the gene structure of members of the cryptochrome family, and (3) generate loss- and gain-of-function mutations in the members of this family and study their effects on circadian rhythm entrainment and modulation of intraocular immune response.

DESCRIPTION (provided by applicant): Recent experiments have demonstrated that mice with complete outer retinal degeneration still retain the ability to synchronize their circadian rhythms to exogenous light-dark cycles, constrict their pupils in response to light, and suppress important hormonal signals (such as melatonin) with light. A subset of retinal ganglion cells have recently been shown to be directly photoresponsive. The photopigment(s) underlying these responses are presently unknown. The cryptochromes are a family of flavin-based proteins related to photolyase that are potential photopigments in the inner retina. Retinal degenerate mice lacking cryptochrome function show markedly decreased sensitivity to light for behavioral rhythmicity and pupillary responsiveness. Using a combination of genetic and physiologic approaches, the investigators propose testing the hypothesis that cryptochromes function as photopigments in the inner retina of retinal degenerate mice. Four specific aims are proposed: 1.) Determine the action spectrum, kinetics, and bleaching properties of the photopigment(s) for pupillary responsive-ness in retinal degenerate (rd/rd) mice with and without cryptochrome function; 2.) Compare inner retinal physiology and direct ganglion cell photoresponsive-ness between mice with and without cryptochrome function; 3.) Establish genetic rescue paradigms for the eye-specific expression of mammalian cryptochromes in genetically null backgrounds and perform systematic mutagenesis to delineate essential domains of cryptochrome function in the mouse eye; and 4.) Utilize the yeast two-hybrid system to characterize the light-dependent interaction of mammalian cryptochrome with potential downstream signaling molecules. The long-term objective of this work is to understand the mechanisms of non-visual ocular phototransduction, from photopigment to neural signal transduction. The full range of physiology subserved by this irradiance detection pathway is unknown but likely includes synchronization of the master circadian pacemaker to the external light-dark cycle, seasonal hormone fluctuations, and light-modulation of the sleep-wakefulness cycle. Subsets of patients with ophthalmologic disease are known to be at high risk for sleep disorders arising from circadian desynchronization; understanding the precise mechanisms by which the eye communicates with the subcortical brain centers responsible for these behaviors will greatly enhance understanding of the pathophysiology of these disorders.

[unreadable] DESCRIPTION (provided by applicant): Inflammatory eye disease is a major cause of decreased vision and blindness worldwide. Relatively few animal models of uveitis exist, which has limited understanding of disease pathogenesis. No biologically relevant system for screening of drugs active against uveitis is available. The few existing mouse models of uveitis do not take advantage of the burgeoning set of resources of modem mouse genetics. We propose creating a new model for uveitic disease by generating sets of transgenic mice engineered to express specific immunogenic proteins (derived from ocular pathogens) in specific subsets of ocular tissue. We predict that such transgenic mice will develop ocular inflammatory disease when challenged with those pathogens; that is, a synthetic uveitis model can be generated by molecular mimicry. This research will optimize the yeast Gal4-UAS transactivating system for use in mammalian cells and in transgenic mice, and add rheostat function with the mammalian-adapted lac-repressor system; create transgenic mice expressing the Gal4 transcriptional activator under the control of ocular tissue-specific promoters, and transgenic mice expressing specific proteins under the control of the UAS upstream recognition sequence; and analyze offspring of these mouse strains for spontaneous inflammation as well as inflammation following immunization with pathogen and pathogen protein. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Cryptochromes are a family of flavoproteins that function as blue light photoreceptors in plants and animals. Drosophila Cryptochrome functions as a deep-brain, non-ocular photoreceptor for photic entrainment of circadian rhythms. In vivo, Cryptochrome mediates light-dependent degradation of the circadian clock gene product Timeless;by a separate mechanism, it also triggers its own degradation following light exposure. We have established an in vitro assay for studying light-dependent Cryptochrome degradation in cell culture, by fusing the bioluminescence enzyme luciferase to full-length Cryptochrome. Greater than 80% of luciferase activity is lost within one hour of light exposure. The conferral of light-triggered degradation to a functional protein fused to Cryptochrome is a potentially very powerful way to study the function of specific proteins in cells. We will determine the minimal fragment of Cryptochrome necessary to mediate light-dependent degradation, and determine the range of proteins which can be made light-labile by fusion with Cryptochrome. We will apply this method to the study of proteins in whole flies. We will identify proteins required for light- mediated protein degradation, and attempt to use these components to port this system to mammalian cell culture. Results from these studies will enhance our understanding of the mechanisms of light-mediated protein degradation, and will additionally provide a valuable new method for studying specific proteins'function in vitro and in vivo. Project Narrative: Cryptochromes are blue light photoreceptors found in plants and animals. Drosophila Cryptochrome undergoes light-dependent degradation in vivo and in vitro. Fusion proteins containing Cryptochrome also undergo light-dependent degradation. We propose experiments to employ this phenomenon as a general tool for studying protein function.

DESCRIPTION (provided by applicant): Ocular infectious diseases, including microbial keratitis, conjunctivitis, and endophthalmitis, remain a significant cause of potentially blinding disease. Traditional microbial culture methods are unable to identify causative organisms in many cases; yields for cultures of corneal ulcers, for example, are around 55%. Most infectious organisms causing ocular disease originate in the ocular surface. However, the constituents of this surface have been incompletely characterized to date. Modern molecular biologic methods including recently available ?deep sequencing? methods allow unprecedented analysis of the ocular surface microbiome. Recent pilot studies from both principal investigator?s laboratories utilizing 16S ribosomal sequencing have demonstrated that 1.) many culture-negative corneal ulcers are associated with novel or unusual organisms, and 2.) the diversity of the ?normal? ocular surface biome is far greater than has been appreciated by traditional, culture-based methods. Taken together, these findings provide an impetus for performing a definitive characterization of the ocular surface (OS) microbiome. Our overarching hypothesis is that, similar to other normally-colonized sites on the body, the OS microbiome is a homeostatically controlled environment which is normally protective of deleterious infection; and that perturbations in this microbiome will predispose to pathologic conditions. In the first Aim of thes studies, two state-of-the-art techniques will be employed in parallel to study the OS microbiome. The first, deep 16S/5.8S sequencing using 454-based pyrosequencing will be used to generate a catalog of bacterial and fungal genera found in the conjunctiva of 100 healthy subjects, diversified for geographic location (from Miami to Seattle), ethnicity, and gender. We will additionally look at the stability of the microbiome across time in a subset of these subjects. The second technique, Biome Representational in Silico Karyotyping, is a novel method for discovery of previously uncharacterized species, utilizing Illumina-based deep sequencing of a defined genomic representation of a metagenomic sample. Using this technique, novel bacteria, viruses, fungi, phage, or parasites can be discovered and molecular diagnostic tests for these organisms can be devised. In Aims 2 and 3, the effect of common clinical scenarios that may disrupt the normal OS microbiome (specifically, treatment with topical antibiotics, topical corticosteroids, and use of soft contact lenses) will be tested using these same techniques. At the conclusions of these studies we anticipate we will have derived a definitive description of the ocular surface microbiome, and determined its variability amongst individuals, its stability within an individual between eyes and over time, and understood the response of this biome to treatment with medications and soft contact lens wear. This information will be essential to subsequent studies aimed at understanding the influence of the microbiome on ocular health. The principal investigators of this study have many years experience in molecular diagnostics. By collaboration, and with inclusion of a bioinformatics expert, expertise in the two complementary approaches to biome characterization can be brought to bear on this problem. Additionally, the geographic separation of the two groups provides excellent opportunity for understanding the effect of locale on the OS biome.

DESCRIPTION (provided by applicant): Photoreceptor cell death from age-related or hereditary retinal degeneration remains the leading cause of blindness in the developed world. The inner retina is largely spared in these diseases. Photoswitch chemicals are specific pharmacologic agents whose activity can be modulated using visible light. We propose utilizing this class of compounds as an approach to reverse blindness from outer retinal degeneration. Preliminary work from our laboratories has demonstrated that the photoswitchable voltage gated potassium channel antagonist AAQ is able to restore electrophysiological retinal responses to several mouse models of outer retinal blindness, and is able to restore pupillary light responses and behavioral responses to light in blind mice. However, AAQ has limitations in terms of its wavelength sensitivity, kinetics, and potential toxicity. Two 'second generation' photoswitch compounds, DENAQ and PhENAQ, show improved spectral response, kinetics, and tolerance in the eye. We propose rigorously testing these two compounds against each other in vitro and in in vivo in mouse, rat, dog, and primate models to determine which to take forward into clinical development; propose an in depth analysis of their effects on the primate retina in vitro, in order to understand their potential for rescue of human blindness; and propose performing required preclinical toxicology and efficacy tests with the goal of applying for a new drug application to allow human clinical studies at the conclusion of this grant.

PROJECT SUMMARY/ABSTRACT Circadian rhythms are the near-24-hour rhythms of physiology ubiquitous to almost all eukaryotic life. Dysfunction of circadian rhythms underlies a variety of common sleep disorders and is thought to contribute to other conditions ranging from psychiatric disease to cancer. The mammalian retina serves a critical function in synchronizing the master circadian pacemaker (the suprachiasmatic nucleus) to the daily light-dark cycle. Work over many years has also demonstrated that the retina itself is a strong circadian oscillator. Indeed, many critical retinal functions, including visual sensitivity, the pupillary light reflex, the electroretinogram, and the expression of hundreds of retinal genes, are under strong circadian control; and that loss of retinal circadian rhythms results in impaired retinal function. Our preliminary data have demonstrated that: 1) the retinal circadian clock can be entrained to light- dark cycles in culture ex vivo, 2) this entrainment is not dependent on the classical rods and cones or the melanopsin-expressing, intrinsically-photosensitive retinal ganglion cells, 3) the orphan opsin neuropsin (OPN5) is necessary for this photoentrainment, and the orphan opsin encephalopsin (OPN3) affects this process, 4) the retina utilizes a light-dependent, diffusible substance to synchronize its rhythms, and 5) the cornea also contains a circadian clock which, remarkably, can be entrained to light- dark cycles as well via an OPN5-dependent mechanism. We propose experiments to elucidate the signaling mechanisms of OPN5 and OPN3; experiments to characterize the diffusible signal(s) emanating from the retina, and experiments to elucidate the mechanism by which non-retinal tissues in the eye maintain circadian rhythmicity and entrain to light-dark cycles. These data will provide a critical basis for understanding how the circadian clock modulates retinal function as well as mechanistic insights into two novel ocular photoreceptors.

Adenoviral conjunctivitis is one of the most common conditions in all of medicine. Despite being a common cause of morbidity world-wide, there are no known host or pathogen factors that predict clinical outcomes in adenoviral keratoconjunctivitis (AdV KC). A recent, large, international clinical study of adenovirus-related KC revealed that approximately 10% of patients suffer from sustained visual loss. Preliminary deep DNA sequencing of AdV has revealed an unexpected sequence diversity in the viral genome, with ~8% sequence divergence even amongst samples with the same hexon-defined molecular type. Remarkably, 20% of the patients in this study lacked any polymerase chain reaction (PCR) or deep DNA sequencing evidence for adenovirus. Given the unexpected molecular variation of adenovirus and a large spectrum of clinical outcome in KC, we propose to analyze host and viral factors contributing to poor outcome (Aim 1). Using next generation DNA sequencing, we propose to catalog the molecular variants of adenovirus and correlate these with clinical outcomes as well (Aim 2). The results of these studies will expand our understanding of the molecular pathogenesis of viral conjunctivitis, and may provide biomarkers for predicting outcomes from this condition. These advances will facilitate future efforts toward developing therapies for this common condition.

ABSTRACT Adenoviral keratoconjunctivitis is one of the most common conditions in all of medicine. Despite being a common cause of morbidity world-wide, there are no known host or pathogen factors that predict clinical outcomes in this condition. A recent, large, international clinical study of adenovirus-related conjunctivitis revealed that approximately 10% of patients suffer from long term visual loss. A limited deep DNA sequencing study of AdV D8 clinical samples conducted under our previous R21 (1R21EY027453) revealed unexpected sequence diversity in the viral genome, with approximately 600 sequence variants among 87 samples within the same hexon-defined molecular type. These variants assorted into three subtypes with different propensity to poor outcome. Remarkably, using machine learning approaches, we found we were able to predict one critical outcome ? the development of subepithelial infiltrates ? from knowledge of the viral sequence alone. Our previous study sequenced samples from the placebo arm of the NVC-422 clinical trial. We have an additional 157 AdV D8 samples that have not been sequenced. In Aim 1 we propose sequencing the adenovirus of these samples in order to a.) further characterize the sequence diversity of AdV D8 and b.) validate our machine learning method for predicting development of subeptithelial infiltrates While AdV D8 was the most prevalent cause of AKC in our study worldwide, unexpectedly we found in the United States that AdV E4 was the most prevalent type. In Aim 2, we propose fully sequencing all 36 samples in the study from type E4, as well as 23 samples from type B3, 9 samples from D19, and a total of 35 samples distributed between type D53, D56, and D64 to determine their molecular diversity, and to apply the same machine learning methods to this set of samples to determine if outcomes can be predicted from viral sequence variants for these types as for AdVD8. The results of these studies will expand our understanding of the molecular pathogenesis of viral conjunctivitis, and will provide biomarkers for predicting outcomes from this condition. These advances will facilitate future efforts toward developing therapies for this common condition.