Circadian Photoentrainment in Mammals

描述（由申请人提供）：表达内在光敏视网膜神经节细胞（mRGCs或ipRGCs）的黑视素是使动物的非图像形成行为和生理适应环境光条件所必需的。黑视素光色素使用的信号机制与经典的杆状/锥状视蛋白不同。人们对黑视素激活后的分子事件知之甚少。具体来说，介导光色素脱敏、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。我们已经确定黑视素的c端细胞质区域在多个位点磷酸化，并且是其与抑制蛋白和随后的脱敏功能相互作用所必需的。本应用中提出的实验将评估黑视素磷酸化和抑制蛋白相互作用在黑视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视素是否经历内吞和降解，并评估黑视素磷酸化的作用，以及在这一过程中的抑制素相互作用。随后，我们将开发策略来特异性地干扰成年小鼠mRGCs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视素蛋白和表达黑视素的神经节细胞的最低阈值。使用这种ipRGC特异性基因表达策略，我们将通过改变激酶活性或通过磷酸化位点突变表达黑视素来扰乱黑视素磷酸化。接下来，我们将评估缺乏单个β -抑制素的小鼠的黑视素功能。黑视素磷酸化、激酶活性或抑制蛋白表达受到干扰的小鼠将接受各种测试，以评估光依赖性黑视素降解、iprgc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视素活性依赖性磷酸化在正常行为和生理对光的适应中的作用。

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.