Molecular characterization of mouse melanopsin and second messenger pathway.

描述(申请人提供)：哺乳动物生理和行为的许多方面表现出每天24小时的节律。这些每日的振荡是昼夜节律，由被称为视交叉上核(SCN)的大脑结构控制。哺乳动物的生物钟随着环境光的到来而不断地被重置。时钟的光携带需要来自视网膜的输入，视网膜通过一小部分视网膜神经节细胞(RGC)的轴突投射与SCN通信。令人惊讶的是，视杆和视锥感受器并不是必需的；相反，投射到SCN的视网膜节细胞似乎作为自主的昼夜节光感受器发挥作用，因为它们表现出独立于视杆和视锥驱动的突触输入的光反应。这些投射SCN的视网膜节细胞也表达黑素，这是一种新的脊椎动物视蛋白，在这些细胞中启动光反应是必要的。在所有已知的脊椎动物视蛋白中，黑素蛋白是独一无二的；它与无脊椎动物横纹体感光细胞视蛋白的序列相似性高于其他脊椎动物视蛋白。目前对黑素的生化性质的了解还很简单，它在体内的第二信使系统还没有最终建立和广泛的表征。这项研究的总体目标是表征黑素的光谱和生化特性，并确定介导SCN投射的视网膜节细胞内在光反应的生化途径。我们假设黑素形成一种光色素，它具有与横纹体光感受器相关的视觉色素相似的生化特征，并激活基于GQ的信号通路。此外，我们假设黑素经历了光依赖的修饰，这有助于黑素依赖的信号级联的光适应。为了验证这些假设，我们提出了一种结合生物化学、电生理学、分子遗传学和行为学的跨学科方法。这一方法旨在确定黑素的光化学，阐明由黑素激活的第二信使途径的性质，并确定是否存在光依赖的黑素翻译后修饰。公共卫生相关性：哺乳动物生理和行为的许多方面表现出每天24小时的节律。这些每日的振荡是昼夜节律，由被称为视交叉上核的大脑结构控制。哺乳动物的生物钟随着环境光的到来而不断地被重置。时钟的光携带需要来自视网膜的输入，视网膜通过一小部分视网膜神经节细胞的轴突投射与视交叉上核通信。令人惊讶的是，经典的光感受器、视杆和视锥并不是必需的；相反，视交叉上投射核的视网膜神经节细胞起着自主光感受器的作用。这些神经节细胞还表达一种新的脊椎动物视觉色素，称为黑色素，它是在这些细胞中启动光反应所必需的。这项拟议的研究项目旨在对黑色素进行表征。了解黑素激活的信号级联反应对于感觉生物学和昼夜节律领域具有重要的意义。此外，昼夜节律紊乱通常伴随神经退行性疾病、睡眠障碍和失明，更常见的是跨子午线旅行和轮班工作的结果。在未来，对基于黑素的信号级联的阐明将使我们能够开发成功的药物治疗这些疾病。

DESCRIPTION (provided by applicant): Many aspects of mammalian physiology and behavior exhibit a daily 24 hour rhythm. These daily oscillations are circadian rhythms and are controlled by a brain structure known as the suprachiasmatic nucleus (SCN). The mammalian circadian clock is constantly being reset by the onset of environmental light. Light entrainment of the clock requires input from the retina, which communicates with the SCN via the axonal projections of a small subset of retinal ganglion cells (RGCs). Surprisingly, rod and cone photoreceptors are not required; instead, RGCs that project to the SCN appear to function as autonomous circadian photoreceptors as they exhibit light responses independent of rod- and cone-driven synaptic input. These SCN-projecting RGCs also express melanopsin, a novel vertebrate opsin, which is necessary for initiating the light response in these cells. Among all known vertebrate opsins, melanopsin is unique; it shows greater sequence similarity to invertebrate rhabdomeric photoreceptor opsins than to other vertebrate opsins. The current understanding of melanopsin's biochemical properties is rudimentary and its in vivo second messenger system has yet to be conclusively established and extensively characterized. The overall goal of the proposed research is to characterize the spectral and biochemical properties of melanopsin and to determine the biochemical pathway that mediates the intrinsic light responses of SCN-projecting RGCs. We hypothesize that melanopsin forms a photopigment that has biochemical characteristics similar to visual pigments associated with rhabdomeric photoreceptors and activates a Gq-based signaling pathway. Furthermore, we hypothesize that melanopsin undergoes light-dependent modifications that contribute to light adaptation of the melanopsin-dependent signaling cascade. To test these hypotheses, we propose an interdisciplinary approach combining biochemistry, electrophysiology, molecular genetics and behavioral studies. This approach is designed to determine melanopsin's photochemistry, to elucidate the nature of the second messenger pathway activated by melanopsin, and to determine if there are light-dependent post-translational modifications of melanopsin. PUBLIC HEALTH RELEVANCE: Many aspects of mammalian physiology and behavior exhibit a daily 24 hour rhythm. These daily oscillations are circadian rhythms and are controlled by a brain structure known as the suprachiasmatic nucleus. The mammalian circadian clock is constantly being reset by the onset of environmental light. Light entrainment of the clock requires input from the retina, which communicates with the suprachiasmatic nucleus via the axonal projections of a small subset of retinal ganglion cells. Surprisingly, classical photoreceptors, rods and cones are not necessary; instead, suprachiasmatic nucleus - projecting retinal ganglion cells function as autonomous photoreceptors. These ganglion cells also express a novel vertebrate visual pigment, known as melanopsin, which is necessary for initiating the light response in these cells. The proposed research project aims to characterize melanopsin. Understanding the signaling cascade activated by melanopsin is of great interest and significance for the field of sensory biology and circadian rhythms. In addition, disorders of the circadian system often accompany neurodegenerative diseases, sleep disorders, and blindness, and are more commonly experienced as a result of transmeridian travel and shift work. In the future, elucidation of the melanopsin-based signaling cascade should allow us to develop successful pharmacological treatments for these disorders.