Circadian Photoentrainment in Mammals

描述(申请人提供)：黑素表达固有的光敏视网膜神经节细胞(mRGCs或ipRGCs)是使动物的非成像行为和生理适应环境光条件所必需的。黑素感光色素使用一种与经典视杆/视锥视蛋白不同的信号机制。人们对黑素激活后的分子事件知之甚少。具体地说，调节功能光色素脱敏、内化、降解或再生的分子和机制尚不清楚。受体功能的这些步骤决定了阈值敏感性、适应性和时间整合参数。我们已经确定黑素的C末端细胞质区域在多个位置被磷酸化，并且是其与arrestin功能相互作用和随后的脱敏所必需的。本申请中提出的实验将评估黑素磷酸化和arrestin相互作用在黑素介导的体内光反应中的相关性。在培养的细胞中，我们将测试光激活的黑素是否经历内吞和降解，并评估黑素磷酸化和arrestin相互作用在这一过程中的作用。随后，我们将制定策略，特异性地干扰成年小鼠mRGC中的基因表达。这种方法将有助于评估正常非成像光反应所需的黑素蛋白和黑素蛋白表达神经节细胞的最低阈值。使用这种ipRGC特异的基因表达策略，我们将通过改变激酶活性或表达具有突变的磷酸化位点的黑素来干扰黑素的磷酸化。接下来，我们将评估缺乏个别β-抑制素的小鼠的黑素功能。黑素蛋白磷酸化、激酶活性或arrestin表达受扰的小鼠将接受各种测试，以评估光依赖性黑素降解、ipRGCs的反应动力学和对光的行为适应。这些实验的结果将阐明黑素蛋白的活性依赖的磷酸化在行为和生理对光的正常适应中的作用。 与公共健康相关：视网膜内部的黑素表达细胞直接感知光线，并帮助有机体适应周围光线的行为和生理。本申请中提出的实验将帮助我们了解光激活黑素后的分子事件如何影响其整体信号特性，并调节对光的行为适应。

DESCRIPTION (provided by applicant): The melanopsin expressing intrinsically photosensitive retinal ganglion cells (mRGCs or ipRGCs) are necessary for adapting non-image forming behavior and physiology of the animal to the ambient light conditions. Melanopsin photopigment uses a signaling mechanism that is distinct from that of the classical rod/cone opsins. Very little is known about the molecular events following melanopsin activation. Specifically, molecules and mechanisms mediating photopigment desensitization, internalization, degradation or regeneration of functional photopigment are unknown. These steps in receptor function determine the threshold sensitivity, adaptation and temporal integration parameters. We have determined the C-terminus cytoplasmic region of melanopsin is phosphorylated at multiple sites and is required for its functional interaction with arrestin and subsequent desensitization. Experiments proposed in this application will evaluate the relevance of melanopsin phosphorylation and arrestin interaction in melanopsin mediated photoresponses in vivo. In cultured cells we will test whether light activated melanopsin undergoes endocytosis and degradation and assess the role of melanopsin phosphorylation, and arrestin interaction in this process. Subsequently, we will develop strategies to specifically perturb gene expression in the mRGCs of adult mice. This approach will help evaluate the minimum threshold of melanopsin protein and melanopsin expressing ganglion cells required for normal non-image forming photoresponses. Using this ipRGC specific gene expression strategy we will perturb melanopsin phosphorylation by altering kinase activity or by expressing melanopsin with mutated phosphorylation sites. Next, we will assess melanopsin function in mice lacking individual beta-arrestins. Mice with perturbations in melanopsin phosphorylation, kinase activity or arrestin expression will be subject to various tests to evaluate light dependent melanopsin degradation, response kinetics of ipRGCs, and behavioral adaptation to light. Results from these experiments will illustrate the role of activity dependent phosphorylation of melanopsin in normal adaptation of behavior and physiology to light. PUBLIC HEALTH RELEVANCE: The melanopsin expressing cells of the inner retina directly sense light and help the organism adapt its behavior and physiology to the ambient light. Experiments proposed in this application will help us understand how molecular events after light activation of melanopsin affects its overall signal properties and modulate behavioral adaptation to light.