Splice Variant of Retinal G-Protein-Coupled Receptor Deletion-Mediated Dysregulation of Autophagy Increases the Susceptibility to Age-Related Macular Degeneration-Like Defects.

IF 2 4区 医学 Q2 OPHTHALMOLOGY
Ophthalmic Research Pub Date : 2024-01-01 Epub Date: 2024-10-15 DOI:10.1159/000541991
Yue Guo, Ningda Xu, Huichao Yan, Jiarui Li, Lvzhen Huang, Li Zhu, Wei Du, Zhiming Liu, Mingwei Zhao
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引用次数: 0

Abstract

Introduction: The splice variant of retinal G-protein-coupled receptor deletion (RGR-d) is a persistent component of drusen and may be involved in the pathogenesis of dry age-related macular degeneration (AMD). Increasing evidence has demonstrated the critical role of autophagy in AMD. In this study, we investigated whether RGR-d disrupts autophagy in early dry AMD in vivo and in vitro.

Methods: Fundus imaging and fluoroscopy were performed on RGR-d mice created by multiplex gene editing. The retina microstructure was evaluated by performing hematoxylin and eosin (H&E) staining as well as transmission electron microscopy (TEM). Retinal function was assessed by full-field electroretinography (ERG). After lentivirus transfection and stimulation, the permeability, phagocytosis, and tight junctions of ARPE-19 cells were evaluated. Western blotting of ATG5, Beclin-1, LC3II/I, and P62 was performed to detect the changes in autophagy pathways.

Results: Atrophy and patchy penetrating hyperfluorescent foci, consistent with early AMD-like defects, were observed in the fundus of 12-month-old RGR-d mice. H&E staining of retinal tissues indicated thinning of each layer of the retinal structure. H&E staining of retinal tissues indicated thinning of each layer of the retinal structure. TEM analysis showed some diffuse granular deposits. And the morphology of choroidal microvascular endothelial cells was degraded and distorted. The morphology of the photoreceptor outer segments showed structural damage, and Bruch's membrane was thickened. ERG indicated that the photoreceptor of RGR-d mice were dysfunctional. Changes in autophagy-related protein expression were observed in the retinal pigment epithelium and retinal neurepithelium, and autophagy regulation was decreased. Palmitic acid (PA) stimulation caused permeability, phagocytosis, and tight junction dysfunction in cells overexpressing RGR-d. Beclin-1 and LC3II/I expression levels were significantly decreased and that of P62 was elevated in RGR-d cells after PA stimulation.

Conclusion: RGR-d disrupts the autophagy pathway, causing the development of an early AMD-like pathophysiology.

视网膜 G 蛋白偶联受体缺失(RGR-d)的剪接变体介导的自噬失调增加了老年性黄斑变性样缺陷的易感性。
导言:视网膜 G 蛋白偶联受体缺失(RGR-d)的剪接变体是色素沉着的一种持久性成分,可能与干性老年性黄斑变性(AMD)的发病机制有关。越来越多的证据表明,自噬在黄斑变性中起着关键作用。在这项研究中,我们探讨了RGR-d是否会在体内和体外破坏早期干性AMD的自噬作用:方法:对通过多重基因编辑创建的 RGR-d 小鼠进行眼底成像和透视检查。通过苏木精和伊红(H&E)染色以及透射电子显微镜(TEM)评估视网膜的微观结构。视网膜功能通过全视野视网膜电图(ERG)进行评估。慢病毒转染和刺激后,评估了 ARPE-19 细胞的通透性、吞噬功能和紧密连接。对 ATG5、beclin-1、LC3II/I 和 P62 进行 Western 印迹,以检测自噬途径的变化:结果:在 12 个月大的 RGR-d 小鼠眼底观察到萎缩和斑点状穿透性高荧光病灶,这与早期 AMD 样缺陷一致。视网膜组织的 H&E 染色显示视网膜结构的每一层都变薄了。视网膜组织的 H&E 染色显示视网膜结构的每一层都变薄了。TEM 分析显示有一些弥漫性颗粒沉积。脉络膜微血管内皮细胞的形态发生退化和扭曲。感光体外节段的形态显示出结构性损伤,布鲁氏膜增厚。ERG显示,RGR-d小鼠的感光器功能障碍。在视网膜色素上皮细胞和视网膜神经上皮细胞中观察到自噬相关蛋白表达的变化,自噬调节能力下降。棕榈酸(PA)刺激会导致过表达 RGR-d 的细胞出现通透性、吞噬和紧密连接功能障碍。PA刺激后,RGR-d细胞中Beclin-1和LC3II/I表达水平显著下降,P62表达水平升高:结论:RGR-d破坏了自噬途径,导致了早期AMD样病理生理的发展。
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来源期刊
Ophthalmic Research
Ophthalmic Research 医学-眼科学
CiteScore
3.80
自引率
4.80%
发文量
75
审稿时长
6-12 weeks
期刊介绍: ''Ophthalmic Research'' features original papers and reviews reporting on translational and clinical studies. Authors from throughout the world cover research topics on every field in connection with physical, physiologic, pharmacological, biochemical and molecular biological aspects of ophthalmology. This journal also aims to provide a record of international clinical research for both researchers and clinicians in ophthalmology. Finally, the transfer of information from fundamental research to clinical research and clinical practice is particularly welcome.
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