Lecture XV

Recognize the role of the RPE in the Visual Cycle. Remember that the light transduction stars with photoabsorption by rhodopsin. The process of light absorption involves the stereochemical change of 11cis-retinal into all-trans-retinal (because it requires a LOT of energy to change the double bond). Alltrans-retinal is metabolized into all-trans-retinol and transported to the retinal pigment epithelium. In the retinal pigment epithelium, retinol is reisomerized to 11-cis-retinal and then redelivered to the photoreceptors. Here is a more detailed reaction progress (from slides): The visual cycle is a pathway of enzyme reactions responsible for the recycling of retinoids that are used during light detection in the photoreceptor cells. The activation of the photoreceptor pigment rhodopsin by light occurs through the isomerisation of the bound chromophore, 11-cis-retinal, to alltrans-retinal. While the phototransduction cascade continues, the all-trans-retinal is released from rhodopsin, reacts with the membrane lipid phosphatidyl-ethanolamine (PE) and is transported to the cytoplasm by ABCA4. After modification to all-trans-retinol by retinol dehydrogenase (RDH), it is transported to the RPE and trans-isomerised to 11-cisretinal through the actions of LRAT, RPE65 and 11-cis-RDH. After transport back to the photoreceptor cell, 11-cisretinal binds rhodopsin, rendering it sensitive to light. Retinoidbinding proteins IRBP, CRBP and CRALBP are involved in the transport of the hydrophobic retinoids in an aqueous environment. Interruption of the visual cycle by mutations in the RPE65 protein results in a lack of 11-cis-retinal to bind to Rhodopsin. In the absence of active visual pigment, vision is impaired. This has led towards efforts in nutritional strategies towards the limiting the symptoms of retinitis pigmentosa. Remember that retinitis pigmentosa encompasses several inherited diseases with the major symptoms are progressive retinal degeneration and night blindness in young adults. Visual examination may reveal bone spicules that appear on the fundus. Nutritionally, it involves vitamin A and docosahexaenoic acid (DHA). Why does this work? Retinal is just a form of vitamin A (along with retinol and retinoic acid depending on the redox state at the terminal C15). The precursor for this is -carotene from ainly vegables. Understand in general terms the principles of gene therapy for retinal disease and the factors that make the gene therapy for the eye a reasonable objective. The substantive candidacy of gene therapy for retinal disease is mainly held in three main points: 1. It is an enclosed space for gene delivery. 2. Blood does not immediately disperse deposited meterials. 3. Retinal cells have little or no turnover. The strategy of the treatment involves addressing certain mutations. Some mutations can involve the loss of certain proteins necessary for the eye to function. One form of RP-like retinal disease is caused by a mutation that results in loss of RPE65. The following displays the considerable potential targets for gene therapy: Mutations Target proteins Consequence Affecting renewal and - Rhodopsin

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shedding of the rod outer segment Affecting the visual transduction cascade Affecting retinal (vitamin A) metabolism

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Peripherin cGMP phosphodiesterase cGMP-gated cation channel (CNCG) Cellular retinaldehyde binding protein (CRalBP) RPE 65

There are several potential targets for gene replacement. Target Affected Proteins Defects in Visual Cycle - Lecithin retinol acetyltransferase RPE65 - ABC transporter A4 (ABCA4) - Retinal dehydrogenase 12 (RDH12) Phototransduction - Rod cGMP phosphodiesterase (PDE6B) Protein defects Cilium Protein Defects - Myosin VIIA (MYO7A) - RPGRIP - Retinitis pigmentosa GTPase (RPGR) Structural Protein - Peripherin (RDS/PRPH2) Defects Phagocytosis Defects - MERTK So far a new research trial will involve a receptor protein called MERTK that is expressed in the retinal pigment epithelium. Humans with the loss of MERTK function have a defect in phagocytosis. AS a result of this defect, debris accumujlates between the photoreceptors and retinal pigment epithelium, resulting in death of photoreceptors and loss of vision. The therapy will introduce a MERTK gene to the RPE.

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