The Role of Endoplasmic Reticulum Calcium Channels in Cone Degeneration Resulting from CNG Channel Deficiency

摘要 视锥细胞光感受器环核苷酸门控(CNG)通道是视锥细胞光信号转导所必需的。 编码锥体通道亚单位的CNGA3和CNGB3基因突变约占全部突变的80% 5例色觉减退，并伴有进行性视锥细胞营养不良。患者体内和体内的锥体 CNG通道缺陷的小鼠模型会随着时间的推移而退化。CNG通道缺陷(CCD)小鼠 模型，我们发现CNG通道缺陷导致内质网(ER)应激相关锥体 死亡。我们还观察到，CCD视网膜显示出内质网钙离子的活性和表达水平增加。 三磷酸肌醇受体(IP3R)和兰尼定受体(RyR)的释放通道及其治疗 使用IP3R和RyR抑制剂可减少内质网应激和视锥细胞死亡。这项研究的目的是了解 CNG通道缺陷时内质网钙通道相关锥体死亡的机制。我们将决定 功能性CNG通道的丧失是否导致内质网钙稳态受损/内质网钙耗竭， 蛋白质加工受损和内质网应激，以及抑制内质网钙通道是否会降低内质网 应激/锥体死亡。三个具体目标将解决我们的问题。目标1是确定内质网钙的作用 CCDER应激和锥体死亡中的通道。我们将评估IP3R和RyR抑制对ER的影响 压力和锥体死亡。条件基因敲除和腺相关病毒(AAV)介导的CRISPR/Cas9 基因组编辑方法将被用于耗尽内质网钙通道。目标2是确定ER的作用 错误抽奖中的钙离子通道。小鼠表现为视锥视盘错配和内质网 IP3R的伴侣和抑制剂促进视锥细胞的运输。我们将评估ER钙的作用 通道抑制/耗竭对视锥细胞视蛋白和其他视锥外段蛋白细胞定位的影响 Ccd小鼠。我们还将调查是否促进ER蛋白加工/ER相关蛋白 降解改善了锥体蛋白的运输。目的3是确定cGMP/PKG(依赖cGMP)如何 蛋白激酶)信号诱导内质网应激和锥体死亡，以及内质网钙通道是否是 重要的目标。CCD视网膜cGMP/PKG信号升高，cGMP/PKG信号抑制 减少内质网压力和锥体死亡。我们将研究cGMP/PKG信号对其表达和 内质网钙通道的活性。我们还将评估cGMP/PKG信号对其他未折叠基因的影响 蛋白质反应/内质网应激成分。在建议的研究完成后，我们会更了解 CNG通道缺陷的视锥细胞变性机制。具体地说，我们将知道ER钙是否 通道在内质网应激和锥体死亡中起作用。这一信息对内质网钙的发展至关重要 基于通道的光感受器保存治疗策略。

ABSTRACT The cone photoreceptor cyclic nucleotide-gated (CNG) channel is essential for cone phototransduction. Mutations in the CNGA3 and CNGB3 genes encoding the cone channel subunits account for about 80% of all cases of achromatopsia, and are associated with progressive cone dystrophies. Cones in patients and in mouse models of CNG channel deficiency degenerate over time. Using CNG channel-deficient (CCD) mouse models, we found that CNG channel deficiency leads to endoplasmic reticulum (ER) stress-associated cone death. We also observed that CCD retinas display increased activity and expression levels of the ER calcium- releasing channels inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR), and treatment with inhibitors of IP3R and RyR reduces ER stress and cone death. The objective of this study is to understand the mechanisms of ER calcium channel-associated cone death in CNG channel deficiency. We will determine whether the loss of functional CNG channels leads to impaired ER calcium homeostasis/ER calcium depletion, impaired protein processing, and ER stress, and whether suppressing ER calcium channels will reduce ER stress/cone death. Three specific aims will address our questions. Aim 1 is to determine the role of ER calcium channels in CCD ER stress and cone death. We will evaluate the effects of IP3R and RyR inhibition on ER stress and cone death. Conditional knockout and adeno-associated viral (AAV)-mediated CRISPR/Cas9 genome editing approaches will be used to deplete ER calcium channels. Aim 2 is to determine the role of ER calcium channels in CCD cone opsin mistrafficking. CCD mice display cone opsin mistrafficking, and ER chaperons and inhibitors for IP3R improve cone opsin trafficking. We will evaluate the effects of ER calcium channel inhibition/depletion on the cellular localization of cone opsin and other cone outer segment proteins in CCD mice. We will also investigate whether promoting ER protein processing/ER-associated protein degradation improves cone protein trafficking. Aim 3 is to determine how cGMP/PKG (cGMP-dependent protein kinase) signaling induces CCD ER stress and cone death, and whether ER calcium channels are the significant targets. CCD retinas show elevated cGMP/PKG signaling, and suppressing cGMP/PKG signaling reduces ER stress and cone death. We will examine the effects of cGMP/PKG signaling on the expression and activity of the ER calcium channels. We will also evaluate the effects of cGMP/PKG signaling on other unfolded protein response/ER stress components. Upon completion of the proposed study, we will better understand the mechanisms of cone degeneration in CNG channel deficiency. Specifically, we will know whether ER calcium channels play a role in ER stress and cone death. This information is vital for the development of ER calcium channel-based therapeutic strategies for photoreceptor preservation.