Retbindin mediates light-damage in mouse retina while its absence leads to premature retinal aging.

Vision requires the transport and recycling of the pigment 11-cis retinaldehyde (retinal) between the retinal pigment epithelium (RPE) and photoreceptors. 11-cis retinal is also required for light-mediated photoreceptor death in dark-adapted mouse eye, probably through overstimulation of rod cells adapted for low light. Retbindin is a photoreceptor-specific protein, of unclear function, that is localized between the RPE and the tips of the photoreceptors. Unexpectedly, young Rtbdn-KO mice, with targeted deletion (KO) of retbindin, showed delayed regeneration of retinal function after bleaching and were strongly resistant to light-induced photoreceptor death. Furthermore, bio-layer interferometry binding studies showed recombinant retbindin had significant affinity for retinoids, most notably 11-cis retinal. This suggests that retbindin mediates light damage, probably through a role in transport of 11-cis retinal. In Rtbdn-KO mice, retinal development was normal, as were amplitudes of rod and cone electroretinograms (ERG) up to 4 months, although implicit times and c-waves were affected. However, with aging, both light- and dark-adapted ERG amplitudes declined significantly and photoreceptor outer segments became disordered, However, in contrast to other reports, there was little retinal degeneration or drop in flavin levels. The RPE developed vacuoles and lipid, protein and calcium deposits reminiscent of age-related macular degeneration. Other signs of premature aging included loss of OPN4+ retinal ganglion cells and activation of microglia. Thus, retbindin plays an unexpected role in the mammalian visual cycle, probably as an adaptation for vision in dim light. It mediates light damage in the dark-adapted eye, but also plays a role in light-adapted responses and in long term retinal homeostasis.

Amelotin is expressed in retinal pigment epithelium and localizes to hydroxyapatite deposits in dry age-related macular degeneration.

Deposition of hydroxyapatite (HAP) basal to the retinal pigment epithelium (RPE) is linked to the progression of age-related macular degeneration (AMD). Serum-deprivation of RPE cells in culture mimics some features of AMD. We now show that serum-deprivation also leads to the induction of amelotin (AMTN), a protein involved in hydroxyapatite mineralization in enamel. HAP is formed in our culture model and is blocked by siRNA inhibition of AMTN expression. In situ hybridization and immunofluorescence imaging of human eye tissue show that AMTN is expressed in RPE of donor eyes with geographic atrophy ("dry" AMD) in regions with soft drusen containing HAP spherules or nodules. AMTN is not found in hard drusen, normal RPE, or donor eyes diagnosed with wet AMD. These findings suggest that AMTN is involved in formation of HAP spherules or nodules in AMD, and as such provides a new therapeutic target for slowing disease progression.

Correlation of phenotype with genotype and protein structure in RYR1-related disorders.

Variants in the skeletal muscle ryanodine receptor 1 gene (RYR1) result in a spectrum of RYR1-related disorders. Presentation during infancy is typical and ranges from delayed motor milestones and proximal muscle weakness to severe respiratory impairment and ophthalmoplegia. We aimed to elucidate correlations between genotype, protein structure and clinical phenotype in this rare disease population. Genetic and clinical data from 47 affected individuals were analyzed and variants mapped to the cryo-EM RyR1 structure. Comparisons of clinical severity, motor and respiratory function and symptomatology were made according to the mode of inheritance and affected RyR1 structural domain(s). Overall, 49 RYR1 variants were identified in 47 cases (dominant/de novo, n = 35; recessive, n = 12). Three variants were previously unreported. In recessive cases, facial weakness, neonatal hypotonia, ophthalmoplegia/paresis, ptosis, and scapular winging were more frequently observed than in dominant/de novo cases (all, p < 0.05). Both dominant/de novo and recessive cases exhibited core myopathy histopathology. Clinically severe cases were typically recessive or had variants localized to the RyR1 cytosolic shell domain. Motor deficits were most apparent in the MFM-32 standing and transfers dimension, [median (IQR) 85.4 (18.8)% of maximum score] and recessive cases exhibited significantly greater overall motor function impairment compared to dominant/de novo cases [79.7 (18.8)% vs. 87.5 (17.7)% of maximum score, p = 0.03]. Variant mapping revealed patterns of clinical severity across RyR1 domains, including a structural plane of interest within the RyR1 cytosolic shell, in which 84% of variants affected the bridging solenoid. We have corroborated genotype-phenotype correlations and identified RyR1 regions that may be especially sensitive to structural modification.

Serum starvation of ARPE-19 changes the cellular distribution of cholesterol and Fibulin3 in patterns reminiscent of age-related macular degeneration.

Retinal pigment epithelium (RPE) has been implicated as key source of cholesterol-rich deposits at Bruch's membrane (BrM) and in drusen in aging human eye. We have shown that serum-deprivation of confluent RPE cells is associated with upregulation of cholesterol synthesis and accumulation of unesterified cholesterol (UC). Here we investigate the cellular processes involved in this response. We compared the distribution and localization of UC and esterified cholesterol (EC); the age-related macular degeneration (AMD) associated EFEMP1/Fibulin3 (Fib3); and levels of acyl-coenzyme A (CoA): cholesterol acyltransferases (ACAT) ACAT1, ACAT2 and Apolipoprotein B (ApoB) in ARPE-19 cells cultured in serum-supplemented and serum-free media. The results were compared with distributions of these lipids and proteins in human donor eyes with AMD. Serum deprivation of ARPE-19 was associated with increased formation of FM dye-positive membrane vesicles, many of which co-labeled for UC. Additionally, UC colocalized with Fib3 in distinct granules. By day 5, serum-deprived cells grown on transwells secreted Fib3 basally into the matrix. While mRNA and protein levels of ACTA1 were constant over several days of serum-deprivation, ACAT2 levels increased significantly after serum-deprivation, suggesting increased formation of EC. The lower levels of intracellular EC observed under serum-deprivation were associated with increased formation and secretion of ApoB. The responses to serum-deprivation in RPE-derived cells: accumulation and secretion of lipids, lipoproteins, and Fib3 are very similar to patterns seen in human donor eyes with AMD and suggest that this model mimics processes relevant to disease progression.

PKCζ-dependent upregulation of p27kip1 contributes to oxidative stress induced retinal pigment epithelial cell multinucleation.

Retinal pigment epithelial (RPE) cells increase in size and multinucleate during aging. We have shown using human and mouse cell lines that oxidised photoreceptor outer segments (oxPOS)-induced cytokinesis failure is related to RPE cell multinucleation, although the underlying mechanism remains unknown. This study investigated the role of the PKC pathway in oxPOS-induced RPE multinucleation using ARPE19 cells. oxPOS treatment promoted PKC activity and upregulated the mRNA expression of PKC α, δ, ζ, ι and µ. Inhibition of PKCα with GÓ§6976 resulted in a 33% reduction of multinucleate ARPE19 cells, whereas inhibition of PKCζ with GÓ§6983 led to a 50% reduction in multinucleate ARPE19 cells. Furthermore, oxPOS treatment induced a PKCζ-dependent upregulation of the Cdk inhibitor p27kip1, its inhibition using A2CE reduced oxPOS-induced ARPE19 multinucleation. Our results suggest that oxPOS-induced ARPE19 cytokinesis failure is, at least in part, due to the upregulation of p27kip1 through activating the PKC, particularly PKCζ pathway. Targeting the PKCζ-p27kip1 signalling axis may be a novel approach to restore RPE repair capacity during aging.

Zinc Protects Oxidative Stress-Induced RPE Death by Reducing Mitochondrial Damage and Preventing Lysosome Rupture.

Zinc deficiency is known to increase the risk of the development of age-related macular degeneration (AMD), although the underlying mechanism remains poorly defined. In this study, we investigated the effect of zinc on retinal pigment epithelium (RPE) survival and function under oxidative conditions. Zinc level was 5.4 µM in normal culture conditions (DMEM/F12 with 10% FCS) and 1.5 µM in serum-free medium (DMEM/F12). Under serum-free culture conditions, the treatment of RPE cells with oxidized photoreceptor outer segment (oxPOS) significantly increased intracellular ROS production, reduced ATP production, and promoted RPE death compared to oxPOS-treated RPE under normal culture condition. Serum deprivation also reduced RPE phagocytosis of oxPOS and exacerbated oxidative insult-induced cathepsin B release from lysosome, an indicator of lysosome rupture. The addition of zinc in the serum-free culture system dose dependently reduced ROS production, recovered ATP production, and reduced oxidative stress- (oxPOS- or 4-HNE) induced cell death. Zinc supplementation also reduced oxidative stress-mediated cathepsin B release in RPE cells. Our results suggest that zinc deficiency sensitizes RPE cells to oxidative damage, and zinc supplementation protects RPE cells from oxidative stress-induced death by improving mitochondrial function and preventing lysosome rupture.

Retinal pigment epithelial cell multinucleation in the aging eye - a mechanism to repair damage and maintain homoeostasis.

Retinal pigment epithelial (RPE) cells are central to retinal health and homoeostasis. Dysfunction or death of RPE cells underlies many age-related retinal degenerative disorders particularly age-related macular degeneration. During aging RPE cells decline in number, suggesting an age-dependent cell loss. RPE cells are considered to be postmitotic, and how they repair damage during aging remains poorly defined. We show that RPE cells increase in size and become multinucleate during aging in C57BL/6J mice. Multinucleation appeared not to be due to cell fusion, but to incomplete cell division, that is failure of cytokinesis. Interestingly, the phagocytic activity of multinucleate RPE cells was not different from that of mononuclear RPE cells. Furthermore, exposure of RPE cells in vitro to photoreceptor outer segment (POS), particularly oxidized POS, dose-dependently promoted multinucleation and suppressed cell proliferation. Both failure of cytokinesis and suppression of proliferation required contact with POS. Exposure to POS also induced reactive oxygen species and DNA oxidation in RPE cells. We propose that RPE cells have the potential to proliferate in vivo and to repair defects in the monolayer. We further propose that the conventionally accepted 'postmitotic' status of RPE cells is due to a modified form of contact inhibition mediated by POS and that RPE cells are released from this state when contact with POS is lost. This is seen in long-standing rhegmatogenous retinal detachment as overtly proliferating RPE cells (proliferative vitreoretinopathy) and more subtly as multinucleation during normal aging. Age-related oxidative stress may promote failure of cytokinesis and multinucleation in RPE cells.