beta-arrestins在自感光神经节细胞光信号转导中的作用和机制研究

Perception of light is important not only for image-forming, but also many biological processes including photoentrainment of circadian rhythm, sleep regulation, cognition and mood. These functions are thus termed “non-image forming vision”, and they are largely mediated by a group of retinal ganglion cells, the intrinsically photosensitive retinal ganglion cells (ipRGCs). Phototransduction of ipRGCs is distinct from that of the traditional photoreceptor rods and cones, using G-protein coupled receptor and PLC pathways to activate TRPC6/7 and generate positive photocurrent. Their photo response also exhibit a slower onset and a slower decay compared to rods and cones. Such characteristics bestow ipRGCs the unique ability to integrate light over a prolonged period of time, which is of great physiological relevance considering many functions ipRGC carry out. We are hence interested in the details involved in the transduction and dynamics of ipRGC photo-responses, in particular, roles of beta-arrestins in this process. In preliminary attempt we observed that ipRGCs lacking Arrb1/2 (from conditional knock-out mice) showed normal response to single photo stimulation, but required significantly longer dark adaption before they could respond again, indicating deficits in recovery from prior response. We therefore propose to investigate in further detail the cellular mechanisms involved in the beta-arrestin associated recovery. We plan to use electrophysiology approaches aided by various pharmacological tools, in combination with molecular and behavioral examinations, to reveal the site of action of beta-arrestin in the photo-transduction/recovery of ipRGCs, and the molecular basis for their interactions. We expect to broaden our knowledge of the cellular processes underlying light-detection by ipRGC and provide insights for a better understanding of the physiological signals coded by light via ipRGCs.

对光的感知除形成图像外，也影响睡眠节律和认知等多种生理过程，这些非成像视觉功能是由自感光视网膜神经节细胞（ipRGC）所介导的。ipRGC被光激活后，经GPCR-PLCb4通路产生光电流。其光响应具特殊的动力学特征，与ipRGC所介导的生理功能密切相关。我们预测在7TMR信号转导中极为重要的beta-arrestins可能参与ipRGC的光感受动力学，并繁育了在ipRGC内条件性敲除Arrb1/2的小鼠。双敲小鼠ipRGC在给予重复刺激时恢复响应所需的暗适应时间大幅度延长，表明Arrbs对ipRGC响应持续性光输入有重要意义，但作用路径和靶点有待阐明。本申请将使用电生理、药理学、和分子及行为水平的验证相结合的研究手段，解析Arrbs在ipRGC光信号转导中的作用位点和分子机制。实验结果将帮助我们理解ipRGC编码光信号的分子过程，并可能揭示beta-arrestins在信号转导中的新功能。

感光是生物体包括人类获得外界信息最重要的途径之一。对光的感知不仅仅形成视觉，也会影响很多不涉及视觉的生理功能，比如节律和睡眠，甚至高级认知行为。 这些非成像视觉功能被认为主要是由新近发现的一类自感光视网膜神经节细胞（intrinsically photosensitive Retinal Ganglion Cells, ipRGCs）所介导的。 IpRGCs对光的响应有别于经典的感光细胞视锥和视杆，时程较长且延迟，可持续响应连续的光信号。这种动力学特征对于ipRGCs进行光信号的整合以正确的行使功能至关重要，但是其背后的分子生物学机制仍不明确。本研究中，我们重点关注一类对G蛋白偶联受体的信号转导非常重要的脚手架蛋白——arrestins (捕获蛋白，Arrb)在ipRGCs光反应动力学中的角色和作用。通过构建Arrb1/2在ipRGCs中条件性敲除的转基因小鼠，使用单细胞光电生理记录的手段，我们证实了Arrb1/2的双缺失使得ipRGCs持续感光能力出现缺陷，无法在一次光响应后及时恢复而对重复光刺激产生连续响应。我们进一步证实，Arrb1/2缺失导致的ipRGC持续光反应缺陷可以通过重新表达Arrb1蛋白而修复，说明这种缺陷的确是由Arrb蛋白的敲除引起的。在分子机制方面，本研究发现Arrb1/2与视黑素melanopsin (ipRGC的感光蛋白)的互作很可能依赖于melanopsin碳末端的磷酸化位点。 Melanopsin碳末端磷酸化位点的缺失可以引起细胞水平上与Arrb1/2缺失相似的感光异常，也可以引起动物生理功能水平的缺陷。本研究的成果揭示了脚手架蛋白Arrb1/2在ipRGCs光信号转导过程中的具体作用和潜在分子机制,拓宽了我们对ipRGCs如何编码光信号以介导非成像视觉功能的理解。