Photic and Circadian Regulation of Retinal Melatonin

描述(申请人提供)：哺乳动物的视网膜包含生物钟，通过驱动基因表达、光感受器外节段膜周转和视觉敏感性的昼夜节律来调节视网膜功能的多个方面。先前的工作表明，褪黑素通过g蛋白偶联的褪黑素受体(MTRs)在调节多种视网膜昼夜节律中发挥关键作用，褪黑素参与了衰老过程中视网膜细胞活性的调节。在本应用中，我们将通过去除两种不同类型的g蛋白偶联褪黑素受体(MTR)的褪黑素熟练小鼠(C3H-f/)来进一步研究MTR去除对视网膜的影响。我们实验室开发的这些新的动物模型为研究褪黑激素和MTR在视网膜中的作用提供了一个独特而强大的工具。我们的初步数据表明，褪黑激素在某些视网膜细胞中的作用可能是通过褪黑素受体1和2异构体(MTRh)的形成而介导的。本申请包括三个具体目标。在特定的目标1中，我们将确定在视网膜细胞中褪黑素受体1和褪黑素受体2是否形成异构体。在特定的目标2中，我们将研究褪黑素及其相关受体在褪黑素熟练小鼠和褪黑素受体敲除小鼠椎间盘脱落的日常节律调节中的作用。最后，在特定的目标3中，我们将确定外源性褪黑素是否通过激活MTRs来增加视网膜对光诱导损伤的敏感性，并将剖析褪黑素在光感受器和RPE细胞中激活的信号通路。在我们的研究中，我们将使用一系列新的和技术先进的技术，如实时定量Q-RT-PCR、生物发光共振能量转移(BRET)、激光捕获解剖显微镜和微阵列分析。

DESCRIPTION (provided by applicant): The mammalian retina contains circadian clocks that regulate multiple aspects of retinal function by driving circadian rhythms of gene expression, photoreceptor outer segment membrane turnover, and visual sensitivity. Previous work has shown that melatonin acting via g protein-coupled melatonin receptors (MTRs) plays a key role in the regulation of a wide variety of retinal circadian rhythms and melatonin is involved in the modulation of retinal cell viability during aging. In the present application we will further investigate the effect of MTR removal on the retina by using melatonin proficient mice (C3H-f+/+) mouse in which the two different types of g- protein coupled melatonin receptors (MTRs) have been removed. These new animal models developed in our laboratory provide a unique and powerful tool with which to investigate the role of melatonin and MTRs in the retina. Our preliminary data suggest that melatonin action within the some retinal cells is likely to be mediated via the formation of melatonin receptor 1 and 2 heteromers (MTRh). The present application comprises three specific aims. In specific aim 1, we will determine whether melatonin receptor type 1 and melatonin receptor type 2 form heteromers in retinal cells. In Specific aim 2, we will investigate the role of melatonin and its associated receptors in the regulation of the daily rhythms in disc shedding in melatonin proficient mice and melatonin receptor knock-out mice. Finally, in specific aim 3 we will determine whether administration of exogenous melatonin increase the sensitivity of the retina to light-induced damage via activation of MTRs and we will dissect the signaling pathways activate by melatonin in the photoreceptor and RPE cells. In our research, we will use a wide array of new and technologically advanced techniques, such as quantitative real time Q-RT-PCR, Bioluminescence Resonance Energy Transfert (BRET), laser capture dissecting microscopy, and microarray analysis.