ABSTRACT During the previous grant period we showed that 11-cis retinal, the light-detecting chromophore of the visual pigment, is the primary source of the toxic deposits that accumulate in the Retinal Pigment Epithelium (RPE) as lipofuscin in the human retina. We also showed that central RPE, the area underlying the macula, contains low levels of these toxic deposits. Defects of retinoid processing have long been linked to diseases of the retina, and the pivotal role played by 11-cis retinal as a mediator of acute and long-term damage suggests specific points in the processing pathway as potential therapeutic targets. In the previous funding period we focused exclusively on rods, which comprise more than 90% of the photoreceptor cells of the human retina. Here, we shift our attention to cone photoreceptors, which are the ones supporting vision for most of our daily activities, and are concentrated in the macula, the part of the retina responsible for high acuity vision. Both the 11-cis and all-trans isomers of retinal are highly reactive aldehydes and photosensitizers, and their reactions with photoreceptor components give rise to the toxic deposits that accumulate in the RPE in the form of lipofuscin. In contrast to rods, which use 11-cis retinal, cones use 11-cis retinol as the source for their visual pigment chromophore. 11-Cis retinol is oxidized to 11-cis retinal within the cell and is far less toxic than either 11-cis or all-trans retinal. We will determine and compare the damage mediated by all-trans retinal, 11- cis retinal and 11-cis retinol in single living cone photoreceptors isolated from monkey and human donor eyes. We will use fluorescence imaging of single photoreceptors to measure oxidative damage and the formation of lipofuscin fluorophore precursors. The aims of the research are: Specific Aim #1: Determine the damage-causing potential of all-trans retinal in cone outer segments. Specific Aim #2: Determine the damage-causing potential of 11-cis retinal in cone outer segments. Specific Aim #3: Determine the damage-causing potential of 11-cis retinol in cone outer segments. Results from these studies will provide new insights into the basic pathogenic mechanisms operating in the macula and underlying vision loss in diseases like Age-related Macular Degeneration (AMD). They will investigate the specialized mechanisms employed by cone photoreceptors to protect the macula from retinaldehyde toxicity. They will allow the evaluation of the potential toxicity of therapies for visual pigment chromophore deficiencies that depend on boosting the chromophore supply; they will also provide a measure for the expected effectiveness of the opposite type of therapies, which aim to limit lipofuscin formation by slowing down the generation of 11-cis retinal.