Circadian Photoentrainment in Mammals

描述（由申请人提供）：表达黑视素的内在光敏视网膜神经节细胞（mRGC或ipRGC）对于使动物的非成像行为和生理适应环境光条件是必需的。黑视蛋白色素使用不同于经典视杆/视锥视蛋白的信号传导机制。关于黑视素激活后的分子事件知之甚少。具体地，介导功能性色素的色素脱敏、内化、降解或再生的分子和机制是未知的。受体功能中的这些步骤决定阈值灵敏度、适应性和时间整合参数。我们已经确定黑视素的C-末端胞质区域在多个位点磷酸化，并且是其与抑制蛋白的功能性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视蛋白磷酸化和抑制蛋白相互作用在体内黑视蛋白介导的光响应中的相关性。在培养的细胞中，我们将测试光激活的黑视蛋白是否经历内吞作用和降解，并评估黑视蛋白磷酸化和抑制蛋白相互作用在此过程中的作用。随后，我们将开发策略，专门干扰成年小鼠mRGCs的基因表达。这种方法将有助于评估正常非图像形成光响应所需的黑视素蛋白和表达黑视素的神经节细胞的最小阈值。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表达具有突变的磷酸化位点的黑视蛋白来干扰黑视蛋白磷酸化。接下来，我们将评估黑视素在缺乏单个β-抑制蛋白的小鼠中的功能。具有黑视蛋白磷酸化、激酶活性或抑制蛋白表达扰动的小鼠将经受各种测试以评估光依赖性黑视蛋白降解、ipRGC的响应动力学和对光的行为适应。这些实验的结果将说明黑视蛋白的活性依赖性磷酸化在行为和生理对光的正常适应中的作用。

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.