Mechanism of Cone Degeneration Resulting from CNG Channel Deficiency

描述（由申请人提供）：视锥细胞环核苷酸门控（CNG）通道对于中心视觉和色觉以及视力至关重要。两个通道亚基CNGA 3和CNGB 3的突变与色盲和进行性视锥细胞营养不良有关。在CNGA 3和CNGB 3缺陷的小鼠模型中以及在色盲或视锥细胞营养不良的患者中，视锥细胞退化。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机制的了解。本研究的主要目的是了解CNG通道功能丧失导致的视锥细胞变性的机制。具体而言，我们将确定CNG通道缺乏模型中cGMP的积累是否会导致视锥细胞变性。我们将使用CNGA 3-/-/Nrl-/-和CNGB 3-/-/Nrl-/-小鼠系进行拟定的研究。使用这些小鼠模型提供了一个独特的机会，研究在视网膜中，只包含锥细胞的视锥细胞变性。提出了三个具体目标。第一个具体目标是确定cGMP的积累是否是CNGA 3缺乏的视锥细胞变性的原因。我们将测试鸟苷酸环化酶（GC）抑制剂减少通道缺陷小鼠视锥细胞变性的能力。在确证实验中，我们还将通过利用RNAi和/或遗传方法（CNGA 3-/-/Nrl-/-/retGC-1-/-的产生）消除通道缺陷视锥细胞中retGC-1（视网膜鸟苷酸环化酶）的表达来测试该假设。第二个具体目标是确定CNGA 3缺乏时视锥细胞如何死亡。如果cGMP是视锥细胞死亡的原因，我们将研究cGMP的积累如何导致视锥细胞死亡。我们将研究cGMP依赖性蛋白激酶（PKG）在视锥细胞变性中的作用.我们将确定PKG活性在CNGA 3-/-/Nrl-/-视网膜中是否比在Nrl-/-视网膜中更高，并且将测试PKG抑制剂的使用/PKG表达的敲低是否可以改善视锥变性。还将通过使用微阵列分析研究通道缺陷小鼠（与WT相比）中改变的基因表达来研究视锥细胞变性的替代机制。第三个具体目的是通过使用CNGB 3-/-/Nrl-/-小鼠确定CNGB 3缺乏是否导致cGMP在视锥细胞中的积累。在完成拟议的研究后，我们将有一个更好的了解的机制（S）的锥体变性导致CNG通道不足。提高退化的视锥细胞的寿命对于成功治疗遗传性视锥细胞疾病是必要的。在这项研究中获得的知识是至关重要的治疗策略，以防止或延缓这种退化的发展，无论是在案件中的致病基因是通道蛋白之一，并在更一般的情况下锥营养不良。 公共卫生相关性：先天性色盲和进行性视锥细胞营养不良是使人衰弱的遗传性视力障碍，目前没有治愈性治疗。编码视锥环核苷酸门控（CNG）通道亚基的基因突变占所有已知色盲病例的70%。本研究将探讨CNG通道相关性色盲患者视锥细胞变性导致视力丧失的机制。这些结果将为视锥细胞的正常细胞生物学和视锥显性遗传疾病的病理生物学提供重要的见解。这些知识对于开发治疗性干预措施以治疗视锥细胞疾病和延缓或挽救视力丧失至关重要。

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.