bims-mideyd 2023-08-20 papers

Sci Rep. 2023 08 14. 13(1): 13239

Inhibition of CysLTR1 reduces the levels of aggregated proteins in retinal pigment epithelial cells.

Andreas Koller, Susanne Maria Brunner, Julia Preishuber-Pflügl, Daniela Mayr, Anja-Maria Ladek, Christian Runge, Herbert Anton Reitsamer, Andrea Trost.

The endosomal-lysosomal system (ELS), which carries out cellular processes such as cellular waste degradation via autophagy, is essential for cell homeostasis. ELS inefficiency leads to augmented levels of damaged organelles and intracellular deposits. Consequently, the modulation of autophagic flux has been recognized as target to remove damaging cell waste. Recently, we showed that cysteinyl leukotriene receptor 1 (CysLTR1) antagonist application increases the autophagic flux in the retinal pigment epithelial cell line ARPE-19. Consequently, we investigated the effect of CysLTR1 inhibition-driven autophagy induction on aggregated proteins in ARPE-19 cells using flow cytometry analysis. A subset of ARPE-19 cells expressed CysLTR1 on the surface (SE+); these cells showed increased levels of autophagosomes, late endosomes/lysosomes, aggregated proteins, and autophagy as well as decreased reactive oxygen species (ROS) formation. Furthermore, CysLTR1 inhibition for 24 h using the antagonist zafirlukast decreased the quantities of autophagosomes, late endosomes/lysosomes, aggregated proteins and ROS in CysLTR1 SE- and SE+ cells. We concluded that high levels of plasma membrane-localized CysLTR1 indicate an increased amount of aggregated protein, which raises the rate of autophagic flux. Furthermore, CysLTR1 antagonist application potentially mimics the physiological conditions observed in CysLTR1 SE+ cells and can be considered as strategy to dampen cellular aging.

DOI: https://doi.org/10.1038/s41598-023-40248-9

Curr Biol. 2023 Aug 10. pii: S0960-9822(23)00970-3. [Epub ahead of print]

Optineurin tunes outside-in signaling to regulate lysosome biogenesis and phagocytic clearance in the retina.

Li Xuan Tan, Colin J Germer, Thushara Thamban, Nilsa La Cunza, Aparna Lakkaraju.

Balancing the competing demands of phagolysosomal degradation and autophagy is a significant challenge for phagocytic tissues. Yet how this plasticity is accomplished in health and disease is poorly understood. In the retina, circadian phagocytosis and degradation of photoreceptor outer segments by the postmitotic retinal pigment epithelium (RPE) are essential for healthy vision. Disrupted autophagy due to mechanistic target of rapamycin (mTOR) overactivation in the RPE is associated with blinding macular degenerations; however, outer segment degradation is unaffected in these diseases, indicating that distinct mechanisms regulate these clearance mechanisms. Here, using advanced imaging and mouse models, we identify optineurin as a key regulator that tunes phagocytosis and lysosomal capacity to meet circadian demands and helps prioritize outer segment clearance by the RPE in macular degenerations. High-resolution live-cell imaging implicates optineurin in scissioning outer segment tips prior to engulfment, analogous to microglial trogocytosis of neuronal processes. Optineurin is essential for recruiting light chain 3 (LC3), which anchors outer segment phagosomes to microtubules and facilitates phagosome maturation and fusion with lysosomes. This dynamically activates transcription factor EB (TFEB) to induce lysosome biogenesis in an mTOR-independent, transient receptor potential-mucolipin 1 (TRPML1)-dependent manner. RNA-seq analyses show that expression of TFEB target genes temporally tracks with optineurin recruitment and that lysosomal and autophagy genes are controlled by distinct transcriptional programs in the RPE. The unconventional plasma membrane-to-nucleus signaling mediated by optineurin ensures outer segment degradation under conditions of impaired autophagy in macular degeneration models. Independent regulation of these critical clearance mechanisms would help safeguard the metabolic fitness of the RPE throughout the organismal lifespan.

Keywords: TFEB; TRPML1; lysosomes; macular degeneration; phagocytosis; photoreceptors; retinal pigment epithelium; trogocytosis

DOI: https://doi.org/10.1016/j.cub.2023.07.031

Mol Med Rep. 2023 Oct;pii: 185. [Epub ahead of print]28(4):

Updates on RPE cell damage in diabetic retinopathy (Review).

Min Li, Meimei Tian, Yuling Wang, Huijie Ma, Yaru Zhou, Xinli Jiang, Yan Liu.

Diabetic retinopathy (DR) is a microvascular complication of diabetes. The retinal pigment epithelium (RPE) forms the outer layer of the blood‑retinal barrier and serves a role in maintaining retinal function. RPE cell injury has been revealed in diabetic animal models, and high glucose (HG) levels may cause damage to RPE cells by increasing the levels of oxidative stress, promoting pro‑inflammatory gene expression, disrupting cell proliferation, inducing the endothelial‑mesenchymal transition, weakening tight conjunctions and elevating cell death mechanisms, such as apoptosis, ferroptosis and pyroptosis. Non‑coding RNAs including microRNAs, long non‑coding RNAs and circular RNAs participate in RPE cell damage caused by HG levels, which may provide targeted therapeutic strategies for the treatment of DR. Plant extracts such as citrusin and hesperidin, and a number of hypoglycemic drugs, such as sodium‑glucose co‑transporter 2 inhibitors, metformin and glucagon‑like peptide‑1 receptor agonists, exhibit potential RPE protective effects; however, the detailed mechanisms behind these effects remain to be fully elucidated. An in‑depth understanding of the contribution of the RPE to DR may provide novel perspectives and therapeutic targets for DR.

Keywords: cell injury; diabetic retinopathy; drugs; non‑coding RNAs; retinal pigment epithelium cell

DOI: https://doi.org/10.3892/mmr.2023.13072

Cell Rep. 2023 Aug 15. pii: S2211-1247(23)00993-2. [Epub ahead of print]42(8): 112982

Rapid RGR-dependent visual pigment recycling is mediated by the RPE and specialized Müller glia.

Aleksander Tworak, Alexander V Kolesnikov, John D Hong, Elliot H Choi, Jennings C Luu, Grazyna Palczewska, Zhiqian Dong, Dominik Lewandowski, Matthew J Brooks, Laura Campello, Anand Swaroop, Philip D Kiser, Vladimir J Kefalov, Krzysztof Palczewski.

In daylight, demand for visual chromophore (11-cis-retinal) exceeds supply by the classical visual cycle. This shortfall is compensated, in part, by the retinal G-protein-coupled receptor (RGR) photoisomerase, which is expressed in both the retinal pigment epithelium (RPE) and in Müller cells. The relative contributions of these two cellular pools of RGR to the maintenance of photoreceptor light responses are not known. Here, we use a cell-specific gene reactivation approach to elucidate the kinetics of RGR-mediated recovery of photoreceptor responses following light exposure. Electroretinographic measurements in mice with RGR expression limited to either cell type reveal that the RPE and a specialized subset of Müller glia contribute both to scotopic and photopic function. We demonstrate that 11-cis-retinal formed through photoisomerization is rapidly hydrolyzed, consistent with its role in a rapid visual pigment regeneration process. Our study shows that RGR provides a pan-retinal sink for all-trans-retinal released under sustained light conditions and supports rapid chromophore regeneration through the photic visual cycle.

Keywords: 11-cis-retinal; CP: Cell biology; CP: Neuroscience; Müller cells; chromophore; cone opsin; photic visual cycle; photoisomerization; retina; retinal pigmented epithelium; vision; visual cycle

DOI: https://doi.org/10.1016/j.celrep.2023.112982

bioRxiv. 2023 Aug 06. pii: 2023.08.04.552052. [Epub ahead of print]

Characterization and contribution of RPE senescence to Age-related macular degeneration in Tnfrsf10 knock out mice.

Iori Wada, Kenichiro Mori, Parameswaran G Sreekumar, Rui Ji, Christine Spee, Elise Hong, Keijiro Ishikawa, Koh-Hei Sonoda, Ram Kannan.

Background: Retinal pigment epithelial cells (RPE) play vital role in the pathogenesis of age-related macular degeneration (AMD). Our laboratory has shown that RPE cellular senescence contributed to the pathophysiology of experimental AMD, and SASP members are involved in this process. Recently, we presented confirmatory evidence to earlier GWAS studies that dysregulation of tumor necrosis factor receptor superfamily 10A (TNFRSF10A) dysregulation leads to AMD development and is linked to RPE dysfunction. This study aims to investigate the contribution of RPE senescence to AMD pathophysiology using TNFRSF10A silenced human RPE (hRPE) cells and Tnfrsf10 KO mice.
Methods: Sub-confluent primary hRPE cells and TNFRSF10A silenced hRPE were exposed to stress-induced premature senescence with H2O2 (500 μM, 48h), and senescence-associated markers (βgal, p16, and p21) were analyzed by RT-PCR and WB analysis. The effect of H2O2-induced senescence in non-silenced and silenced hRPE on OXPHOS and glycolysis was determined using Seahorse XF96 analyzer. Male C57BL/6J Tnfrsf10 KO ( Tnfrsf10 -/- ) mice were used to study the regulation of senescence by TNFRSF10A in vivo . Expression of p16 and p21 in control and KO mice of varying ages were determined by RT-PCR, WB, and immunostaining analysis.
Results: The senescence-associated p16 and p21 showed a significant ( p < 0.01) upregulation with H2O2 induction at the gene (1.8- and 3-fold) and protein (3.2- and 4-fold) levels in hRPE cells. The protein expression of p16 and p21 was further significantly increased by co-treatment with siRNA ( p < 0.05 vs. H2O2). Mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) (pmol/min/total DNA) increased with senescence induction by H2O2 for 48h in control RPE, and knockdown of TNFRSF10A caused a further increase in OCR and ECAR. In addition, co-treatment with PKC activator significantly improved all parameters. Similarly, in vivo studies showed upregulation of p16 and p21 by RT-PCR, WB, and immunostaining analysis in RPE/choroid of Tnfrsf10 KO mice. When subjected to examination across distinct age groups, namely young (1-3 months), middle (6-9 months), and old (12-15 months) mice, a discernible age-related elevation in the expression of p16 and p21 was observed.
Conclusions: Our findings suggest that TNRSF10A is a regulator of regulates in RPE senescence. Further work on elucidating pathways of senescence will facilitate the development of new therapeutic targets for AMD.

DOI: https://doi.org/10.1101/2023.08.04.552052

J Vis Exp. 2023 Jul 28.

LipidUNet-Machine Learning-Based Method of Characterization and Quantification of Lipid Deposits Using iPSC-Derived Retinal Pigment Epithelium.

Zander Esh, Sharanya Suresh, Davide Ortolan, Mitra Farnoodian, Devika Bose, Jiwon Ryu, Andrei Volkov, Kapil Bharti, Ruchi Sharma.

The retinal pigment epithelium (RPE) is a monolayer of hexagonal cells located at the back of the eye. It provides nourishment and support to photoreceptors and choroidal capillaries, performs phagocytosis of photoreceptor outer segments (POS), and secretes cytokines in a polarized manner for maintaining the homeostasis of the outer retina. Dysfunctional RPE, caused by mutations, aging, and environmental factors, results in the degeneration of other retinal layers and causes vision loss. A hallmark phenotypic feature of degenerating RPE is intra and sub-cellular lipid-rich deposits. These deposits are a common phenotype across different retinal degenerative diseases. To reproduce the lipid deposit phenotype of monogenic retinal degenerations in vitro, induced pluripotent stem cell-derived RPE (iRPE) was generated from patients' fibroblasts. Cell lines generated from patients with Stargardt and Late-onset retinal degeneration (L-ORD) disease were fed with POS for 7 days to replicate RPE physiological function, which caused POS phagocytosis-induced pathology in these diseases. To generate a model for age-related macular degeneration (AMD), a polygenic disease associated with alternate complement activation, iRPE was challenged with alternate complement anaphylatoxins. The intra and sub-cellular lipid deposits were characterized using Nile Red, boron-dipyrromethene (BODIPY), and apolipoprotein E (APOE). To quantify the density of lipid deposits, a machine learning-based software, LipidUNet, was developed. The software was trained on maximum-intensity projection images of iRPE on culture surfaces. In the future, it will be trained to analyze three-dimensional (3D) images and quantify the volume of lipid droplets. The LipidUNet software will be a valuable resource for discovering drugs that decrease lipid accumulation in disease models.

DOI: https://doi.org/10.3791/65503