Recent experiments have demonstrated that mice with complete outer retinal degeneration still retain the ability to synchronize their circadian rhythms to exogenous light-dark cycles, constrict their pupils in response to light, and suppress important hormonal signals (such as melatonin) with light. A subset of retinal ganglion cells have recently been shown to be directly photoresponsive. The photopigment(s) underlying these responses are presently unknown. The cryptochromes are a family of flavin-based proteins related to photolyase that are potential photopigments in the inner retina. Retinal degenerate mice lacking cryptochrome function show markedly decreased sensitivity to light for behavioral rhythmicity and pupillary responsiveness. Using a combination of genetic and physiologic approaches, the investigators propose testing the hypothesis that cryptochromes function as photopigments in the inner retina of retinal degenerate mice.
Four specific aims are proposed: 1.) Determine the action spectrum, kinetics, and bleaching properties of the photopigment(s) for pupillary responsive-ness in retinal degenerate (rd/rd) mice with and without cryptochrome function; 2.) Compare inner retinal physiology and direct ganglion cell photoresponsive-ness between mice with and without cryptochrome function; 3.) Establish genetic rescue paradigms for the eye-specific expression of mammalian cryptochromes in genetically null backgrounds and perform systematic mutagenesis to delineate essential domains of cryptochrome function in the mouse eye; and 4.) Utilize the yeast two-hybrid system to characterize the light-dependent interaction of mammalian cryptochrome with potential downstream signaling molecules. The long-term objective of this work is to understand the mechanisms of non-visual ocular phototransduction, from photopigment to neural signal transduction. The full range of physiology subserved by this irradiance detection pathway is unknown but likely includes synchronization of the master circadian pacemaker to the external light-dark cycle, seasonal hormone fluctuations, and light-modulation of the sleep-wakefulness cycle. Subsets of patients with ophthalmologic disease are known to be at high risk for sleep disorders arising from circadian desynchronization; understanding the precise mechanisms by which the eye communicates with the subcortical brain centers responsible for these behaviors will greatly enhance understanding of the pathophysiology of these disorders.
