Cryptochrome function in non-visual photoreception

描述(申请人提供)：最近的实验表明，患有完全性视网膜外部变性的小鼠仍然保持着将它们的昼夜节律与外源性明暗周期同步的能力，对光的反应收缩它们的瞳孔，并用光抑制重要的激素信号(如褪黑素)。视网膜神经节细胞的一个子集最近被证明是直接光响应的。这些反应背后的感光色素(S)目前尚不清楚。隐色素是一类基于黄素的蛋白质，与光解酶有关，是视网膜内部潜在的光色素。缺乏隐色素功能的视网膜退化小鼠表现出对光的行为节律性和瞳孔反应性的敏感度显著降低。 通过结合遗传学和生理学方法，研究人员提出了一种假设，即隐色素在视网膜退化小鼠的视网膜内部起着光色素的作用。提出了四个具体目标：1)目的：1.测定具有和不具有隐色素功能的视网膜变性(RD/RD)小鼠的光致色素(S)对瞳孔反应的作用光谱、动力学和漂白特性。比较具有和不具有隐色素功能的小鼠视网膜内部生理和直接神经节细胞的光响应性；建立遗传救援范例 在基因缺失的背景下，哺乳动物隐花色素的眼睛特异性表达，并进行系统的突变，以描绘小鼠眼睛中隐花色素功能的基本结构域；利用酵母双杂交系统来表征哺乳动物隐花色素与潜在的下游信号分子的光依赖相互作用。这项工作的长期目标是了解非视觉眼部光传导的机制，从光色素到神经信号转导。这种辐照度检测途径所支持的全部生理过程尚不清楚，但可能包括主昼夜节律起搏器与外部明暗周期的同步、季节性激素波动以及睡眠-觉醒周期的光调制。众所周知，眼科疾病患者的亚群是由昼夜节律去同步化引起的睡眠障碍的高危人群；了解眼睛与负责这些行为的皮质下大脑中心沟通的准确机制将极大地提高对这些疾病的病理生理学的理解。

DESCRIPTION (provided by applicant): 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.