The roles of Gbeta5 and R7 RGS protein in vision

描述（由申请人提供）：异源三聚体g蛋白介导的信号转导在自然界中普遍存在，最好的代表是视网膜棒中的光转导。然而，三聚体g蛋白是如何在光感受器之外为脊椎动物的视觉提供服务的，人们仍然知之甚少。本应用将通过阐明第五个Gb亚基（Gb5）及其伴生伙伴R7 g蛋白信号调节因子（RGS6, RGS7, RGS9, RGS11）在小鼠视网膜中的作用来填补这一空白。该建议是基于Gb5-/-小鼠视觉系统中发现的缺陷，包括：1)自发性视网膜波不常见；2)视网膜原状突起不精细；3)外丛状层（OPL）带状突触异常，伴有未合并的去极化双极细胞（DBC）树突；4)视网膜电图（ERG） b波缺乏；5)星爆无突细胞（SAC）树突场减少，6)SAC膜电位节律振荡，7)光导恢复时间延长。我们证明Gb5仅通过稳定视网膜中的R7 RGS蛋白起作用，因此在缺乏Gb5的情况下，假设Gbo的信号状态在病理上延长，从而导致这些视觉缺陷。此外，我们发现Gb5和R7 RGS对正常DBC功能的需求发生在视网膜发育过程中，并且Gb5可能在成人DBC的树突尖端非常缓慢地翻转。解决上述缺陷的潜在机制将有助于进一步了解视网膜的发育和功能。在此，我们建议采用结合分子生物学、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将检测Gb5-/-小鼠中延长的Gbo信号是否会延缓OPL三联带突触的形成。Aim-2将确定视觉系统发育需要Gb5和R7 RGS蛋白的关键时期。同时检测OPL突触的稳定性和DBC中Gb5、Gbo、Gb13和R7 RGS蛋白的周转率。Aim- 3将研究R7 RGS蛋白参与II期胆碱能视网膜波的哪些和如何参与，延长的Gbo激活是否导致SAC树突场的减少，以及成人Gb5-/- SAC的突触起源和振荡机制。通过完成这些目标，我们将知道哪些三聚g蛋白以及如何在不同视网膜神经元的光导之外用于视觉。我们将在这里制作有用的小鼠细胞系，并对其进行特征描述，以促进未来对视网膜新获得的知识在中央视觉系统和/或其他中枢神经系统回路中的适用性的研究。最后，了解正常的视觉机制对于有效预防、保护和恢复各种致残性致盲疾病患者的视力是必不可少的。

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.