The roles of Gbeta5 and R7 RGS protein in vision

描述(申请人提供)：由异三聚体G蛋白介导的信号转导在自然界中广泛存在，最好的代表是视网膜视杆中的光转导。然而，三聚体G蛋白如何在光感受器外削弱脊椎动物的视觉仍然知之甚少。这项应用将通过阐明第五GB亚单位(GB5)及其专有伙伴R7调节G蛋白信号(RGS6、RGS7、RGS9、RGS11)在小鼠视网膜中的作用来填补这一空白。这一建议是基于在GB5-/-小鼠的视觉系统中发现的缺陷，这些缺陷包括：1)罕见的自发视网膜波~2)不精细的视网膜生成投射~3)外丛状层(OPL)与未结合的去极化双极细胞(DBC)树突的异常带状突触~4)缺乏视网膜电图(ERG)b波~5)星爆无长突细胞(SAC)树突野减少，6)节律性振荡SAC膜电位，以及7)光传导恢复时间延长。我们证明，GB5只通过稳定视网膜中的R7RGS蛋白发挥作用，因此，在它缺失的情况下，GBO的信号状态被假设为病理上延长了导致这些视觉缺陷的原因。此外，我们还发现，正常DBC功能所需的GB5和R7RGS发生在视网膜发育过程中，并且在成年DBC的树突尖端，GB5的翻转可能非常缓慢。因此，解决上述缺陷的潜在机制(S)将促进对视网膜发育和功能的了解。我们建议使用分子生物学、生物化学、组织学、小鼠遗传学、ERG和单细胞电生理学的综合方法来实现以下目标。AIM-1将测试延长的GBO信号是否会延缓GB5-/-小鼠OPL三元带状突触的形成。AIM-2将定义视觉系统发育需要GB5和R7 RGS蛋白的关键时期。它还将测定DBC中OPL突触的稳定性和GB5、GBO、Gb13和R7RGS蛋白的周转率。AIM-3将研究哪些R7 RGS蛋白以及如何参与第二阶段的胆碱能视网膜波，延长的GBO激活是否是SAC树突域减少的基础，以及成年GB5-/-SAC的突触起源和振荡机制。通过完成这些目标，我们将知道在不同的视网膜神经元中，除了光转导之外，哪些三聚体G蛋白以及如何在视觉中使用G蛋白。这里将制作有用的小鼠线，并将其描述为便于将来研究视网膜中新获得的知识对中枢视觉系统和/或其他中枢神经系统回路的适用性。最后，了解正常的视觉机制对于有效预防、保护和恢复患有各种致残致盲疾病的患者的视力是必不可少的。 与公共健康相关：光感受器下游的视网膜神经元计算视觉信息并将其传递给大脑。许多遗传性人类致盲疾病不仅影响光感受器，还影响视网膜下游神经元。这项申请旨在推进关于异三聚体G蛋白在视网膜中的作用的知识，超越光感受器的光传导，并加强知识库，以提供新的治疗和治疗方式，以改善或保护人类视力。

DESCRIPTION (provided by applicant): Signal transduction mediated by heterotrimeric G-proteins occurs throughout nature and is best represented by phototransduction in retinal rods. However, how trimeric G-proteins subserve vertebrate vision outside photoreceptors is still poorly understood. This application will fill this gap by elucidating the roles of the fifth Gb subnit (Gb5) and its obligate partners R7 Regulators of G-protein Signaling (RGS6, RGS7, RGS9, RGS11) in mouse retina. The proposal is based on defects found in the visual system of Gb5-/-mice, which include: 1) infrequent spontaneous retinal waves~ 2) poorly refined retinogeniculate projections~ 3) abnormal ribbon synapses at outer plexiform layer (OPL) with unincorporated depolarizing bipolar cells (DBC) dendrites~ 4) lack of electroretinography (ERG) b-waves~ 5) reduction of starburst amacrine cell (SAC) dendritic field, 6) rhythmic oscillation SAC membrane potentials, and 7) prolonged phototransduction recovery. We demonstrate that Gb5 works exclusively through stabilizing R7 RGS proteins in retina and hence in its absence the signaling state of Gbo is hypothesized to be pathologically prolonged to cause these visual defects. Furthermore, we find that the requirement of Gb5 and R7 RGS for normal DBC function takes place during retinal development and that Gb5 may be very slowly turned over at dendritic tips of adult DBC. Solving the underlying mechanism(s) of the aforementioned defects will thus advance knowledge concerning the development and function of retina. We propose here to use an integrated approach combining molecular biology, biochemistry, histology, mouse genetics, ERG, and single cell electrophysiology to achieve the following goals. Aim-1 will test whether prolonged Gbo signaling retards formation of OPL triadic ribbon synapses in Gb5-/- mice. Aim-2 will define a critical period during which Gb5 and R7 RGS proteins are needed for visual system development. It will also measure the stability of OPL synapse and turnover rate of Gb5, Gbo, Gb13 and R7 RGS proteins in DBC. Aim- 3 will examine which and how R7 RGS proteins are involved in stage II cholinergic retinal waves, whether prolonged Gbo activation underlies the reduction of SAC dendritic fields, and the synaptic origin and oscillatory mechanism of adult Gb5-/- SAC. By completing these aims we shall know which and how trimeric G-proteins are used in vision beyond phototransduction in different retinal neurons. Useful mouse lines will be made here and characterized to facilitate future inquiry of the applicability of newly gained knowledge in the retina to central visual system and/or other CNS circuits. Finally, knowing the normal visual mechanism is indispensable for efficient prevention, preservation, and restoration of vision in patients suffering from various debilitating blinding diseases. PUBLIC HEALTH RELEVANCE: Retinal neurons downstream of photoreceptors compute and relay visual information to brain. Many hereditary human blinding diseases affect not only photoreceptors but also downstream retinal neurons. The application seeks to advance knowledge on the roles of heterotrimeric G-proteins in retina beyond phototransduction of photoreceptors and to strengthen the knowledge base to provide novel treatment and therapeutic modalities to improve or preserve human vision.