Supplementary Material for: Splice variant of retinal G-protein-coupled receptor deletion (RGR-d)-mediated dysregulation of autophagy increases the susceptibility to age-related macular degeneration-like defects

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