Molecular characterization of mouse melanopsin and second messenger pathway.

描述（由申请人提供）：哺乳动物生理和行为的许多方面都表现出每天24小时的节律。这些每日的振荡是昼夜节律，由一种被称为视交叉上核（SCN）的大脑结构控制。哺乳动物的生物钟不断被环境光线重置。生物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（RGCs）的轴突投射与SCN通信。令人惊讶的是，杆状和锥状光感受器并不是必需的；相反，投射到SCN的RGCs似乎作为自主的昼夜节律光感受器发挥作用，因为它们表现出独立于杆状和锥状驱动的突触输入的光反应。这些scn投射的RGCs也表达黑视素，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，黑视蛋白是独一无二的；它与无脊椎动物横纹肌光感受器视蛋白的序列相似性大于与其他脊椎动物视蛋白的序列相似性。目前对黑视素的生化特性的了解是初步的，其体内第二信使系统尚未最终建立和广泛表征。本研究的总体目标是表征黑视素的光谱和生化特性，并确定介导scn投射RGCs内在光响应的生化途径。我们假设黑视素形成一种光色素，具有与横纹肌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，我们假设黑视素经历光依赖性修饰，有助于黑视素依赖性信号级联的光适应。为了验证这些假设，我们提出了一种跨学科的方法，结合生物化学，电生理学，分子遗传学和行为研究。该方法旨在确定黑视素的光化学，阐明黑视素激活的第二信使途径的性质，并确定黑视素是否存在光依赖性翻译后修饰。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，由一种被称为视交叉上核的大脑结构控制。哺乳动物的生物钟不断被环境光线重置。生物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交流。令人惊讶的是，经典的光感受器，视杆细胞和视锥细胞并不是必需的；相反，视交叉上核投射视网膜神经节细胞作为自主光感受器。这些神经节细胞也表达一种新的脊椎动物视觉色素，称为黑视素，这是在这些细胞中启动光反应所必需的。拟议的研究项目旨在表征黑视素。了解由黑视素激活的信号级联在感觉生物学和昼夜节律领域具有重要意义。此外，昼夜节律系统的紊乱通常伴随着神经退行性疾病、睡眠障碍和失明，并且更常见的是由于穿越子午线的旅行和轮班工作。在未来，对基于黑视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.