Mechanism of Cone Degeneration Resulting from CNG Channel Deficiency

描述（由申请人提供）：锥形环核苷酸门控（CNG）通道对中央视觉和色觉以及视觉灵敏度至关重要。两个通道亚基CNGA3和CNGB3的突变与色盲和进行性锥体营养不良有关。CNGA3和CNGB3缺乏的小鼠模型以及色盲或锥体营养不良患者的锥体退化。目前还没有治愈这些疾病的治疗方法，部分原因是缺乏对疾病病理生物学基础机制的理解。本研究的首要目标是了解由于CNG通道功能丧失而导致的锥体变性的机制。具体来说，我们将确定CNG通道缺乏模型中cGMP的积累是否会导致锥体变性。我们将使用CNGA3-/-/Nrl-/-和CNGB3-/-/Nrl-/-小鼠系进行拟议的研究。使用这些小鼠模型提供了一个独特的机会来研究只包含锥体的视网膜中的锥体变性。提出了三个具体目标。第一个具体目的是确定cGMP的积累是否导致CNGA3缺乏症的锥体变性。我们将测试鸟苷酸环化酶（GC）抑制剂在通道缺陷小鼠中减少锥体变性的能力。在一个验证实验中，我们还将通过利用RNAi和/或遗传方法（生成CNGA3-/-/Nrl-/-/retGC-1-/-）消除通道缺陷视锥细胞中retGC-1（视网膜guanlyate cyclase）的表达来验证这一假设。第二个具体目标是确定CNGA3缺乏时锥体是如何死亡的。如果cGMP是导致锥体细胞死亡的原因，我们将研究cGMP的积累如何导致锥体细胞死亡。我们将研究cGMP依赖性蛋白激酶（PKG）在锥体变性中的作用。我们将确定与Nrl-/-视网膜相比，CNGA3-/-/Nrl-/-视网膜中的PKG活性是否更高，并将测试使用PKG抑制剂/敲低PKG表达是否可以改善锥体变性。通过使用微阵列分析研究通道缺陷小鼠（与WT相比）基因表达的改变，也将研究锥体变性的其他机制。第三个具体目的是通过使用CNGB3-/-/Nrl-/-小鼠来确定CNGB3缺乏是否导致锥体中cGMP的积累。在完成拟议的研究后，我们将更好地了解由于CNG通道缺乏而导致的锥体退化的机制。提高退化锥体的寿命是成功治疗遗传性锥体疾病的必要条件。在这项研究中获得的知识对于预防或延缓这种退化的治疗策略的发展至关重要，无论是在致病基因是通道蛋白之一的情况下，还是在更一般的锥体营养不良病例中。

DESCRIPTION (provided by applicant): The cone cyclic nucleotide-gated (CNG) channel is essential for central and color vision and for visual acuity. Mutations in the two channel subunits, CNGA3 and CNGB3, are associated with achromatopsia and progressive cone dystrophy. Cones degenerate in mouse models of CNGA3 and CNGB3 deficiency and in patients with achromatopsia or cone dystrophy. There are currently no curative treatments for these diseases, in part due to a lack of understanding of the mechanisms underlying the disease pathobiology. The overarching objective of this study is to understand the mechanism(s) of cone degeneration resulting from loss of CNG channel function. Specifically, we will determine whether the accumulation of cGMP in models of CNG channel deficiency causes cone degeneration. We will perform the proposed studies using the CNGA3-/-/Nrl-/- and CNGB3-/-/Nrl-/- mouse lines. Using these mouse models provides a unique opportunity to study cone degeneration in a retina that contains only cones. Three specific aims are proposed. The first specific aim is to determine whether the accumulation of cGMP is responsible for cone degeneration in CNGA3 deficiency. We will test the ability of guanylate cyclase (GC) inhibitors to reduce cone degeneration in the channel-deficient mice. In a corroborating experiment, we will also test this hypothesis by abolishing expression of retGC-1 (retinal guanlyate cyclase) in channel deficient cones by utilizing RNAi and/or genetic methods (generation of CNGA3-/-/Nrl-/-/retGC-1-/-). The second specific aim is to determine how cones die in CNGA3 deficiency. If cGMP is responsible for cone cell death, we will examine how the accumulation of cGMP leads to cone cell death. We will investigate the role of cGMP- dependent protein kinase (PKG) in cone degeneration. We will determine whether PKG activity is higher in the CNGA3-/-/Nrl-/- retina compared to the Nrl-/- retina, and will test whether the use of PKG inhibitors/knockdown of PKG expression can ameliorate cone degeneration. Alternate mechanisms for cone degeneration will also be investigated by studying altered gene expression in channel deficient mice (compared to WT) by using microarray analysis. The third specific aim is to determine whether CNGB3 deficiency leads to an accumulation of cGMP in cones by using CNGB3-/-/Nrl-/- mice. Upon completion of the proposed study we will have a better understanding of the mechanism(s) underlying cone degeneration resulting from CNG channel deficiency. Improving the lifespan of degenerating cones is necessary for successful treatment of inherited cone diseases. The knowledge gained in this study is crucial for the development of therapeutic strategies to prevent or retard this degeneration, both in cases in which the causative gene is one of the channel proteins and in more general cases of cone dystrophy. PUBLIC HEALTH RELEVANCE: Congenital achromatopsia and progressive cone dystrophy are debilitating hereditary vision disorders for which there are currently no curative treatments. Mutations in genes encoding the cone cyclic nucleotide- gated (CNG) channel subunits account for 70% of all known cases of achromatopsia. The proposed study will probe the mechanisms underlying the cone degeneration that leads to loss of vision in CNG channel- associated achromatopsia. The results will provide significant insights into both the normal cell biology of cones and the pathobiology of cone dominant inherited disease. This knowledge is essential for developing therapeutic interventions to treat cone diseases and retard or rescue vision loss.