We study inherited retinal degenerations in animal models of human diseases to develop therapeutic interventions. Our laboratory studies the biology of photoreceptor rescue and repair which are relevant for Retinitis Pigmentos (RP) and allied diseases based on animal studies. We have studied the disease models to elucidate the mechanisms of retinal neural signaling deficiencies and degeneration leading to blindness. We use genetically altered rodents to mimic human retinal disease, and we study the characteristics, molecular genetics, physiology and possible treatments of these inherited retinal degenerations. We apply techniques of light and electron microscopy, immunohistochemistry, protein and lipid biochemistry, RNAseq, 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: Retinal degenerative disease involves structural changes of photoreceptor outer segments (OS), that precede photoreceptor death, causing loss of vision. Photoreceptor cells undergo primary degeneration through progressive outer segment (OS) shortening in many of these conditions. Hence a critical question is whether the outer segment may exhibit enough structural plasticity to support elongation of OS that have been shortened by disease states, and whether this can promote survival of the photoreceptor cells. Our work investigates molecules that are important in the regulating of OS length under light stress and genetic degenerative conditions. We focus on neurotrophic factors, such as CNTF, and on small molecules that regulate cytoskeletal growth, including molecules such as Rac1. Dominant-active Rac1 is reported to rescue photoreceptor structure in Drosophila rhodopsin (Rho)-null mutants. From this, we hypothesized that constitutively active (CA) Rac1 might restore rod outer segment (ROS) formation in Rhodopsin knock-out (Rho-KO) photoreceptors in mice. We developed a mouse model using AAV8-pOpsin-CA Rac1introduced into Rho KO mice during later retinal development (> P4). Electron microscopy (EM) shows small and thin rudimentary OS membranes formed in Rho KO rods at P21. In contrast, we observed a few of enlarged ROS membrane sacs emerging from ciliary plasma membranes in EM images of Tg CA Rac1-Rho KO rods. This suggests that constitutively active Rac1 promotes outgrowth of rudimentary OS disc membranes and plasma membrane in Rho KO rods, but is insufficient to develop normal OS structures in Rho KO rods. Internal limiting membrane (ILM) in wild type (WT) mouse eye impedes AAV vector penetration: Studies in the Rs1-knockout mouse model provided proof of principle that an RS1 adeno-associated virus vector can enter the retina after intravitreal delivery, lead to closure of the schisis cavity, and restore the retinal architecture as well as give functional improvement to synaptic visual signaling. Expression was not achieved in wild-type retina after intravitreal injection, thus implicating XLRS disease pathology changes the ILM barrier. Since gene delivery into the vitreous cavity would be preferable for many ocular indications, is less invasive than subretinal delivery, and would reach more of the retina, we developed a method of applying a small and safe electric current across the intact eye in vivo for a brief period following intravitreal vector administration. This significantly improved AAV-mediated transduction of retinal cells in wild type mice eye following intravitreal delivery, with gene expression in retinal pigment epithelium and photoreceptor cells. The low-level current had no adverse effects on retinal structure and function. This method should be generally applicable for other AAV serotypes and may have broad application in both basic research and clinical studies. Retinoschisin Structure and Function: 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 plasma membrane of photoreceptor inner segments, where the energy and protein producing machinery of the cell reside. However, the role of RS1 in photoreceptor function is not known. We showed that young mice lacking retinoschisin have delayed maturation of outer segments. This also may be related to changes in transcription factors, which determine the level of proteins involved in photoreceptor transduction. Another indicator that RS1 is important in photoreceptor function are the observed changes in mitochondrial structure in Rs1-knockout mice and changes found in OCT reflective bands after gene delivery into Rs1-KO mice. Thus, OCT may be a useful marker for tracking effectiveness of treatment in the clinic. RS1 protein is also present in the synaptic and inner nuclear layers of the retina where it plays a role in maintaining structure of the tissue and proper cell to cell signaling. We found that lack of RS1 affects the localization of proteins at the synaptic connection between photoreceptors and the next neuron in the chain 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 partially restores the synaptic proteins to their normal location. To better understand the mechanism by which RS1 restores synaptic protein localization, we have initiated studies using high pressure freezing and cryo-electron microscopy of vitreous sections from Rs1-KO mice that have received intraocular gene delivery of the RS1 transgene. These studies are intended to provide high resolution 3-dimensional images of RS1:RS1 assembly and interactions. More recently using RNASeq strategies, RNA profiles were compared between wild-type and Rs1-KO mouse eyes. There are several interesting differences that we are exploring further. And lastly, a Rs1-KO rat is being generated to determine if the features observed in the Rs1-KO mouse also will be present in other species.
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