This laboratory is appropriately titled Translational Research, as we use inherited retinal degenerations identified in the clinic as both a source of clues about retinal function and dysfunction and a target for research in therapeutic intervention. The broad direction for our laboratory involves the biology of photoreceptor rescue and repair and opportunities to initiate human clinical rescue trials for RP and allied diseases based on animal studies. We have studied a number of mouse and rat models of human retinal degeneration diseases to elucidate the mechanisms of retinal neural signaling deficiencies and degeneration leading to blindness. We use normal rodents and rodents that are genetically altered to mimic human retinal disease to study the characteristics (phenotype), molecular genetics, physiological mechanisms and possible treatments of these inherited retinal degenerations. Our laboratory applies the techniques of light and electron microscopy, immunohistochemistry, biochemistry, and molecular biology to human and animal retinal tissue, as well as the electroretinogram (ERG), ocular coherence tomography (OCT) and behavioral measurements in living animals to access retinal structure and function in ways similar to those used to evaluate human vision in the clinic. These studies address human conditions of retinal and macular degenerations and age-related macular degeneration. Mechanisms of Retinal Degeneration: A critical facet of retinal neurodegenerative disease involves the structural changes, particularly to the photoreceptor outer segments (OS), that precede photoreceptor death, causing loss of vision. As photoreceptor cells undergo primary degeneration through progressive outer segment (OS) shortening in many of these conditions, a critical question is whether the outer segment may exhibit sufficient structural plasticity to support elongation of OS that have been shortened by disease states and whether this would promote survival of the photoreceptor cell. The goal of the work is to investigate the molecules that are important in the regulation of OS length under light stress and genetic degenerative conditions. We are focusing on neurotrophic factors, such as CNTF, and on small molecules that regulate cytoskeletal growth, including Rac1. Role of Rac1 in retinal plasticity, development and oxidative damage: This year we continue a molecular approach to studying retinal disease mechanisms by investigating the role Rac1 in photoreceptor plasticity and homeostasis and cell death by oxidative stress in normal and diseased retinas using Rac1 transgenic and conditional knockout mice. Rac1 is a protein that can function as an intracellular molecular switch, which is activated by various types of membrane receptors and produce a variety of downstream biological effects in many different cell types. We use a method call conditional gene targeting to modify the gene for Rac1 to learn about its role in photoreceptors. By this method only the gene in these cells is altered, leaving the Rac1 gene in other cell types unaffected. One of the photoreceptor specific functions of Rac1 in invertebrate photoreceptors is to regulate photoreceptor morphogenesis, and in particular the photoreceptive membrane analogous to outer segments in mammals. This was discovered using conditional gene targeting to produce depletion of Rac1 or constitutive activation of Rac1 in photoreceptors. We showed that conditional knockdown of Rac1 in mouse photoreceptors protected them from cell death resulting from overexposure to light, which indicates Rac1 is involved in one form of oxidative damage in photoreceptors. This may be useful in understanding the mechanisms of some types of inherited or environmental retinal degenerations and in designing treatments. To further explore the role of Rac1 in mammalian photoreceptors, we used conditional gene targeting to make a mouse which expresses a constitutively active form of Rac1 in rod photoreceptors. This transgenic Rac1 was constructed so that its expression in photoreceptors coincided with the major outer segment protein rhodopsin, which begins about postnatal day 4. This allowed us to test its effect on postnatal development. Three lines of mice expressing different levels of this transgenic Rac1 are being studied. Constitutively active RAC1 had a profound negative effect on mouse rod cell viability and development. Rod photoreceptors in the CA RAC1 retina exhibited a defect in polarity and migration. Constitutively active RAC1 disrupted rod morphogenesis and gave a phenotype resembling that found in the Crumbs mutant. The outer segment portion of the displaced cells was either absent or severely shortened. Our study implicates an important role for RAC1 in the process of initial polarization and morphogenesis during rod photoreceptor development. We are exploring this further with CA RAC1 at different ages. In addition, we are investigating the biochemical pathways leading to RAC1 induced degeneration of photoreceptor cells and the neuroprotective effect of blocking those pathways. Retinoschisnin Function in Photoreceptors: Mutations in the gene for retinoschisin protein (RS1) found on the X chromosome cause X-linked retinoschisis (XLRS). XLRS is an inherited retinal disease and is a leading cause of juvenile macular degeneration in human males. The RS1 is found primarily on the outer membrane of photoreceptor inner segments. However, the role of RS1 in photoreceptor function is not known. We showed that young mice lacking retinoschisin have a specific defect in how their photoreceptors respond to light. While their electrical response to a light flash measured with the ERG is normal, the process of light activated protein translocation in photoreceptors (the movement of proteins from one compartment of the cell to another) in response to continuous illumination is ten times less sensitive in these mice at a young age than in litter mates who have the RS1. When the mice are a few weeks older, however, the light sensitivity of translocation is near normal. Furthermore, during this period, the photoreceptor outer segments in the mice lacking RS1 grow from much shorter than normal to near normal. This suggests that the photoreceptors in these mice have a delay in their maturation. Our published report describes how these changes may be related to changes in transcription factors which determine the level of the proteins involved in photoreceptor transduction during maturation. In addition, we are finding out that RS1 may play an important role in the localization of proteins at the synaptic connection between photoreceptors and the next neuron in the chain of neurons passing visual information on to the brain. Dysfunction at this connection would help explain some of the vision loss and abnormal electrophysiological response in XLRS patients. Treating the Rs1-KO mouse model of XLRS with a vector delivering the missing gene partial restores the synaptic proteins to their normal location.

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7
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2015
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Deafness & Other Communication Disorders
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Marangoni, Dario; Yong, Zeng; Kjellström, Sten et al. (2017) Rearing Light Intensity Affects Inner Retinal Pathology in a Mouse Model of X-Linked Retinoschisis but Does Not Alter Gene Therapy Outcome. Invest Ophthalmol Vis Sci 58:1656-1664
Zeng, Yong; Petralia, Ronald S; Vijayasarathy, Camasamudram et al. (2016) Retinal Structure and Gene Therapy Outcome in Retinoschisin-Deficient Mice Assessed by Spectral-Domain Optical Coherence Tomography. Invest Ophthalmol Vis Sci 57:OCT277-87
Song, Hongman; Vijayasarathy, Camasamudram; Zeng, Yong et al. (2016) NADPH Oxidase Contributes to Photoreceptor Degeneration in Constitutively Active RAC1 Mice. Invest Ophthalmol Vis Sci 57:2864-75
Cukras, Catherine; Flamendorf, Jason; Wong, Wai T et al. (2016) LONGITUDINAL STRUCTURAL CHANGES IN LATE-ONSET RETINAL DEGENERATION. Retina 36:2348-2356
Bush, Ronald A; Zeng, Yong; Colosi, Peter et al. (2016) Preclinical Dose-Escalation Study of Intravitreal AAV-RS1 Gene Therapy in a Mouse Model of X-linked Retinoschisis: Dose-Dependent Expression and Improved Retinal Structure and Function. Hum Gene Ther 27:376-89
Bush, Ronald A; Wei, Lisa L; Sieving, Paul A (2015) Convergence of Human Genetics and Animal Studies: Gene Therapy for X-Linked Retinoschisis. Cold Spring Harb Perspect Med 5:
Ou, Jingxing; Vijayasarathy, Camasamudram; Ziccardi, Lucia et al. (2015) Synaptic pathology and therapeutic repair in adult retinoschisis mouse by AAV-RS1 transfer. J Clin Invest 125:2891-903
Veleri, Shobi; Lazar, Csilla H; Chang, Bo et al. (2015) Biology and therapy of inherited retinal degenerative disease: insights from mouse models. Dis Model Mech 8:109-29
Song, Hongman; Bush, Ronald A; Vijayasarathy, Camasamudram et al. (2014) Transgenic expression of constitutively active RAC1 disrupts mouse rod morphogenesis. Invest Ophthalmol Vis Sci 55:2659-68
Ziccardi, Lucia; Vijayasarathy, Camasamudram; Bush, Ronald A et al. (2014) Photoreceptor pathology in the X-linked retinoschisis (XLRS) mouse results in delayed rod maturation and impaired light driven transducin translocation. Adv Exp Med Biol 801:559-66

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