Molecular Characterization of Mouse Melanopsin, Circadian Photopigment

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。有趣的是，这些光敏视网膜神经节细胞的光诱发去极化的作用谱是由最大吸收波长为484nm的光色素描述的。黑视素是一种新的视蛋白样蛋白，在一些投射到SCN的视网膜神经节细胞中表达，基于几条线索的证据似乎是难以捉摸的昼夜节律光色素。黑视素在光敏的RGCs中表达，在小鼠中，黑视素基因的破坏会消除scn投射的RGCs的内在光反应，并损害昼夜节律。然而，这些优雅的实验并没有解决黑视素是直接负责产生光反应的光色素，还是仅仅是在单独的光色素上再生发色团所需的异构酶的问题。罗宾逊实验室的数据首次证明了黑视素确实能形成一种功能性的光色素。这导致了一种假设，即黑视素在哺乳动物RGCs中具有独特的作用，参与昼夜节律光干扰，光激活的黑视素触发了g蛋白级联反应，这是这些含黑视素的RGCs产生的光反应的基础。基于其与无脊椎视蛋白的同源性，预测黑视蛋白激活了一个基于gq的信号通路。这项拨款建议使用分子和生化方法进一步表征黑视素。黑视素是一种新发现的与昼夜光干扰有关的哺乳动物视网膜视觉色素。它似乎具有许多与横纹肌无脊椎动物眼睛中所具有的视觉色素相似的特性。这使得这种色素在哺乳动物的视觉色素中独一无二，值得研究。本基金提出的方法是新颖的。罗宾逊实验室是唯一一个明确采用分子和生化方法研究这种视觉色素的实验室，并准备在未来两年内在理解这种视觉色素方面取得重大进展。更广泛的影响：这项拨款的私人顾问敏锐地意识到国家科学基金会在21世纪对美国科学劳动力中代表性不足群体的教育和发展的承诺。私家侦探目前是合作私家侦探。UMBC作为一个致力于将女性和少数民族纳入科学领域的机构。P.I.已经建立了一个包容性的实验室环境，让代表性不足的学生，无论是研究生还是本科生，都有一个积极的研究经历。本基金中描述的部分研究将由一名非裔美国男性进行。在可能的情况下，这些本科生将包括有才华的少数民族和女性本科生，这些本科生来自全国认可的迈耶霍夫学者计划。调查团还将继续通过公开演讲和访问当地高中等方式，向公众宣传研究成果。

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. Interestingly, the action spectrum of the light-evoked depolarization of these photosensitive retinal ganglion cells is described by a photopigment with a wavelength of maximum absorbance of 484 nm.Melanopsin, a novel opsin-like protein expressed in some of the retinal ganglion cells that project to the SCN, appears to be the elusive circadian photopigment based on several lines of evidence. Melanopsin is expressed in the light-sensitive RGCs and disruption of the melanopsin gene in mice abolishes the intrinsic light response of the SCN-projecting RGCs and impairs circadian entrainment. However, these elegant experiments do not address the question of whether melanopsin is a photopigment directly responsible for generating the light response, or simply an isomerase required for chromophore regeneration on a separate photopigment. Data from the Robinson laboratory was the first to demonstrate that melanopsin does indeed form a functional photopigment. This led to the hypothesis that melanopsin has a unique role in mammalian RGCs involved in circadian photoentrainment and the light-activated melanopsin triggers a G-protein cascade that underlies the photic response generated by these melanopsin-containing RGCs. Based on its homology with invertebrate opsins, the prediction is that melanopsin activates a Gq-based signaling pathway. This grant proposes to use molecular and biochemical approaches to further characterize melanopsin.The proposed research is significant because melanopsin is a novel and newly discovered mammalian retinal visual pigment involved in circadian photoentrainment. It appears to have many properties similar to visual pigments that have been characterized in rhabdomeric invertebrate eyes. This makes this pigment unique among mammalian visual pigments and fascinating to study. The approaches proposed in this grant are novel. The Robinson lab is the only laboratory that has taken a decidedly molecular and biochemical approach to study this visual pigment, and is poised to make significant progress on understanding this visual pigment within the next two years. Broader Impacts: The P.I of this grant is acutely aware of NSF's commitment to education and the development of underrepresented groups in the United States' Scientific workforce in the 21 st century. The P.I is currently a co-P.I. on a NSF institutional ADVANCE award to UMBC which as an institution is committed to the inclusion of both women and minorities in science. The P.I. has established an inclusive laboratory environment where underrepresented students, both graduate and undergraduate, have a positive research experience. Part of research described in this grant will be conducted by an African-American male. When possible, these undergraduates will include talented minority and women undergraduates identified form the nationally recognized Meyerhoff Scholars Program. The P.I. will also continue the practice of disseminating research results to the public through public lectures and visits to local high schools.