The retinal pigment epithelium (RPE) is involved in many processes necessary to maintain photoreceptor function and health. The RPE is also involved in the regulation of the photoreceptor outer segment turnover (disk shedding and phagocytosis) a process that has been shown to be under circadian control. We have recently developed an RPE-choroid preparation in which we can monitor, in real-time, the circadian clock using the PER2::LUC knock-in mouse, a transgenic mouse model where the PER2 oscillation is faithfully reported via a firefly luciferase. This model is unique in that it reflects both transcription and post-translational events, providing a powerful tool to investigate circadian clock function in a specific tissue and/or cell. Using this new preparation we have demonstrated that the mouse RPE contains a circadian clock that is entrained by the neuromodulator dopamine (DA). In the present proposal (Specific Aim 1) we will test the hypothesis that DA, via D2-like receptors located in the RPE, entrains the circadian clock in the RPE, thus synchronizing the daily burst in phagocytosis of rod outer segment disks. Then we will identify the molecular mechanisms by which DA synchronizes the circadian clock in the RPE.
In Specific Aim 2 we will test the prediction that removal of D2R signaling will affect the daily rhythm of phagocytosis thus leading to lipofuscin accumulation and reduced photoreceptor viability during aging. Finally, in Specific Aim 3 we will define the roles of RPE and inner retinal clocks in the regulation of the daily rhythm in RPE phagocytic activity. To reach this goal we will disrupt circadian clocks selectively in RPE and neural retina and assess the consequences on the daily rhythm of phagocytosis. The experiments described in this research proposal will determine the role that DA and its associated receptors play in the regulation of the circadian rhythms in the RPE and the role of retinal/RPE circadian clocks in the regulation of the daily rhythm of RPE phagocytic activity.
Photoreceptor outer segment renewal is vital to the health and survival of these primary sensory neurons. This process has been shown to be under circadian control, but the mechanisms controlling this circadian rhythm are poorly understood. The experiments proposed in our application will elucidate how the circadian clock and Dopamine regulate this important process.
