The roles of Gbeta5 and R7 RGS protein in vision

描述（由申请人提供）：由异源三聚体G蛋白介导的信号转导在整个自然界中发生，最好的代表是视网膜杆中的光转导。然而，三聚体G蛋白如何在光感受器之外辅助脊椎动物视觉仍然知之甚少。本申请将通过阐明小鼠视网膜中第五Gb亚基（Gb 5）及其专性伙伴R7 G蛋白信号转导调节因子（RGS 6、RGS 7、RGS 9、RGS 11）的作用来填补这一空白。该提案基于Gb 5-/-小鼠视觉系统中发现的缺陷，其中包括：1）自发性视网膜波不频繁~ 2）精细的视网膜小突投射~ 3）外丛状层（OPL）异常的带状突触与未并入的去极化双极细胞（DBC）树突~ 4）缺乏视网膜电图（ERG）b波~ 5）星状无长突细胞（SAC）减少树突状场，6）节律振荡SAC膜电位和7）延长的光转导恢复。我们证明，Gb 5的工作完全通过稳定视网膜中的R7 RGS蛋白，因此在其缺乏的情况下，Gbo的信号状态被假设为病理性延长，导致这些视觉缺陷。此外，我们发现，正常的DBC功能的需求发生在视网膜发育过程中的Gb 5和R7的RGS和Gb 5可能会非常缓慢地在成人DBC的树突状尖端翻转。解决上述缺陷的潜在机制将因此推进关于视网膜发育和功能的知识。我们建议在这里使用一个综合的方法结合分子生物学，生物化学，组织学，小鼠遗传学，视网膜电图，和单细胞电生理学，以实现以下目标。Aim-1将测试 延长的Gb 0信号传导延迟Gb 5-/-小鼠中OPL三联带状突触的形成。Aim-2将定义一个关键时期，在此期间，视觉系统发育需要Gb 5和R7 RGS蛋白。还将测量DBC中OPL突触的稳定性和Gb 5、Gbo、Gb 13和R7 RGS蛋白的周转率。Aim- 3将研究哪些R7 RGS蛋白以及R7 RGS蛋白如何参与II期胆碱能视网膜波，是否延长Gbo激活是SAC树突状场减少的基础，以及成年Gb 5-/- 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蛋白以及如何用于视觉。有用的小鼠线将在这里和特点，以促进未来的调查的适用性，新获得的知识，在视网膜中央视觉系统和/或其他中枢神经系统的电路。最后，了解正常的视觉机制是必不可少的有效预防，保护和恢复视力的患者患有各种衰弱性致盲疾病。 公共卫生相关性：视网膜神经元下游的光感受器计算和中继视觉信息到大脑。 许多遗传性人类致盲疾病不仅影响光感受器，而且影响下游视网膜神经元。该申请旨在推进关于异源三聚体G蛋白在视网膜中的作用的知识，超越光感受器的光转导，并加强知识基础，以提供新的治疗和治疗方式来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.