bims-mideyd 2023-08-13 papers

bims-mideyd

Biomed News

on Mitochondrial dysfunction in eye diseases

Issue of 2023–08–13
seven papers selected by
Rajalekshmy “Raji” Shyam, Indiana University Bloomington

Comparative mechanistic study of RPE cell death induced by different oxidative stresses.
The Role of Mitophagy in Glaucomatous Neurodegeneration.
Neuroprotective effects of idebenone on hydrogen peroxide-induced oxidative damage in retinal ganglion cells-5.
Redox Regulation in Age-Related Cataracts: Roles for Glutathione, Vitamin C, and the NRF2 Signaling Pathway.
Purification of retinal ganglion cells using low-pressure flow cytometry.
Impaired Lysosome Reformation in Chloroquine-Treated Retinal Pigment Epithelial Cells.
Clearance phagocytosis by the retinal pigment epithelial during photoreceptor outer segment renewal: Molecular mechanisms and relation to retinal inflammation.

Redox Biol. 2023 Aug 06. pii: S2213-2317(23)00241-0. [Epub ahead of print]65 102840

Comparative mechanistic study of RPE cell death induced by different oxidative stresses.

Yao Tong, Yinga Wu, Jing Ma, Masataka Ikeda, Tomomi Ide, Courtney T Griffin, Xi-Qin Ding, Shusheng Wang.

  Oxidative stress is hypothesized to drive the progression of age-related macular degeneration (AMD). Retinal pigment epithelial (RPE) cell layer is important for supporting the function of retina and is particularly susceptible to oxidative stress-induced cell death. How RPE cells die in AMD, especially in geographic atrophy (GA), a late stage of dry AMD, is still controversial. The goal of this study is to compare the features and mechanisms of RPE cell death induced by different oxidative stresses, to identify potential universal therapeutic targets for GA. RPE cell death was induced both in vitro and ex vivo by 4-Hydroxynonenal (4-HNE), a major product of lipid peroxidation, sodium iodate (NaIO3) that has been widely used to model RPE cell death in dry AMD, a ferroptosis inducer RAS-selective lethal 3 (RSL3) or a necroptosis inducer shikonin. We found that RPE necroptosis and ferroptosis show common and distinct features. Common features include receptor-interacting protein kinase (RIPK)1/RIPK3 activation and lipid reactive oxygen species (ROS) accumulation, although lipid ROS accumulation is much milder during necroptosis. This supports cross talk between RPE ferroptosis and necroptosis pathways and is consistent with the rescue of RPE necroptosis and ferroptosis by RIPK1 inhibitor Necrostatin-1 (Nec-1) or in Ripk3-/- RPE explants. Distinct feature includes activated mixed lineage kinase domain like pseudokinase (MLKL) that is translocated to the cell membrane during necroptosis, which is not happening in ferroptosis. This is consistent with the failure to rescue RPE ferroptosis by MLKL inhibitor necrosulfonamide (NSA) or in Mlkl-/- RPE explants. Using this framework, we found that 4-HNE and NaIO3 induced RPE cell death likely through necroptosis based on the molecular features and the rescuing effect by multiple inhibitors. Our studies suggest that multiple markers and inhibitors are required to distinguish RPE necroptosis and ferroptosis, and that necroptosis inhibitor Nec-1 could be a potential therapeutic compound for GA since it inhibits RIPK1/RIPK3 activation and lipid ROS accumulation occurred in both necroptosis and ferroptosis pathways.

Keywords:  Age-related macular degeneration; Ferroptosis; Geographic atrophy; Necroptosis; Oxidative stress; RPE cell death; ex vivo model

DOI:  https://doi.org/10.1016/j.redox.2023.102840
Cells. 2023 Jul 30. pii: 1969. [Epub ahead of print]12(15):

The Role of Mitophagy in Glaucomatous Neurodegeneration.

Dimitrios Stavropoulos, Manjot K Grewal, Bledi Petriti, Kai-Yin Chau, Christopher J Hammond, David F Garway-Heath, Gerassimos Lascaratos.

  This review aims to provide a better understanding of the emerging role of mitophagy in glaucomatous neurodegeneration, which is the primary cause of irreversible blindness worldwide. Increasing evidence from genetic and other experimental studies suggests that mitophagy-related genes are implicated in the pathogenesis of glaucoma in various populations. The association between polymorphisms in these genes and increased risk of glaucoma is presented. Reduction in intraocular pressure (IOP) is currently the only modifiable risk factor for glaucoma, while clinical trials highlight the inadequacy of IOP-lowering therapeutic approaches to prevent sight loss in many glaucoma patients. Mitochondrial dysfunction is thought to increase the susceptibility of retinal ganglion cells (RGCs) to other risk factors and is implicated in glaucomatous degeneration. Mitophagy holds a vital role in mitochondrial quality control processes, and the current review explores the mitophagy-related pathways which may be linked to glaucoma and their therapeutic potential.

Keywords:  genetics; glaucoma; glaucomatous neurodegeneration; mitochondria; mitochondrial dysfunction; mitophagy; primary open-angle glaucoma

DOI:  https://doi.org/10.3390/cells12151969
Int Ophthalmol. 2023 Aug 10.

Neuroprotective effects of idebenone on hydrogen peroxide-induced oxidative damage in retinal ganglion cells-5.

Yuping Wang, Jing Wang, Xi Zhang, Yifan Feng, Yuanzhi Yuan.

   PURPOSE: To investigate the neuroprotective effect of idebenone against hydrogen peroxide (H2O2)-induced oxidative damage in retinal ganglion cells-5 (RGC-5 cells).
METHODS: RGC-5 cells were pre-treated with various idebenone concentrations (5, 10, and 20 µM) for 12 h and were then subjected to 300 µM H2O2 for a further 12 h. Apoptosis in RGC-5 was measured by flow cytometry. The changes of mitochondrial membrane potential (MMP) were detected by JC-1 staining. Autophagy in RGC-5 cells was observed by transmission electron microscopy. Western blots were used to measure the expression of autophagy-related protein light chain 3 (LC3), Beclin-1, and the release of Cytochrome c (Cyt-c).
RESULTS: Flow cytometry showed that the apoptosis rates in the normal control group, H2O2 group, and idebenone groups were 6.48 ± 0.55%, 27.3 ± 0.51%, 22.8 ± 0.52%, 15.45 ± 0.81%, and 12.59 ± 0.58%, respectively (F = 559.7, P < 0.0001). After incubation with H2O2, the number of autophagosomes increased significantly, whereas it was decreased in the idebenone groups. After incubation of RGC-5 cells with H2O2, MMP levels were significantly decreased, while idebenone could prevent the decrease in MMP levels. Compared with that in the normal control group, LC3 II/I, the expression levels of Beclin-1 and Cyt-c were increased significantly in the H2O2 group (P < 0.05). Compared with that in the H2O2 group, LC3 II/I, the expression of Beclin-1 and Cyt-c was significantly decreased in idebenone groups (P < 0.05).
CONCLUSIONS: Idebenone protects RGC-5 cells against H2O2-induced oxidative damage by reducing mitochondrial damage and autophagic activity.

Keywords:  Apoptosis; Autophagy; Glaucomatous optic neuropathy; Idebenone; Oxidative stress

DOI:  https://doi.org/10.1007/s10792-023-02831-x
Nutrients. 2023 Jul 29. pii: 3375. [Epub ahead of print]15(15):

Redox Regulation in Age-Related Cataracts: Roles for Glutathione, Vitamin C, and the NRF2 Signaling Pathway.

Eloy Bejarano, Jasper Weinberg, Madison Clark, Allen Taylor, Sheldon Rowan, Elizabeth A Whitcomb.

  Age is the biggest risk factor for cataracts, and aberrant oxidative modifications are correlated with age-related cataracts, suggesting that proper redox regulation is important for lens clarity. The lens has very high levels of antioxidants, including ascorbate and glutathione that aid in keeping the lens clear, at least in young animals and humans. We summarize current functional and genetic data supporting the hypothesis that impaired regulation of oxidative stress leads to redox dysregulation and cataract. We will focus on the essential endogenous antioxidant glutathione and the exogenous antioxidant vitamin C/ascorbate. Additionally, gene expression in response to oxidative stress is regulated in part by the transcription factor NRF2 (nuclear factor erythroid 2-related factor 2 [NFE2L2]), thus we will summarize our data regarding cataracts in Nrf2-/- mice. In this work, we discuss the function and integration of these capacities with the objective of maintaining lens clarity.

Keywords:  NRF2; ascorbate/vitamin C; glutathione; lens; redox

DOI:  https://doi.org/10.3390/nu15153375
Front Mol Neurosci. 2023 ;16 1149024

Purification of retinal ganglion cells using low-pressure flow cytometry.

Kiran J Mcloughlin, Afnan M Aladdad, Andrew J Payne, Anna I Boda, Sayra Nieto-Gomez, Karl E Kador.

  Purified Retinal Ganglion Cells (RGCs) for in vitro study have been a valuable tool in the study of neural regeneration and in the development of therapies to treat glaucoma. Traditionally, RGCs have been isolated from early postnatal rats and mice, and more recently from human in vitro derived retinal organoids using a two-step immunopanning technique based upon the expression of Thy-1. This technique, however, limits the time periods from which RGCs can be isolated, missing the earliest born RGCs at which time the greatest stage of axon growth occurs, as well as being limited in its use with models of retinal degeneration as Thy-1 is downregulated following injury. While fluorescence associated cell sorting (FACS) in combination with new optogenetically labeled RGCs would be able to overcome this limitation, the use of traditional FACS sorters has been limited to genomic and proteomic studies, as RGCs have little to no survival post-sorting. Here we describe a new method for RGC isolation utilizing a combined immunopanning-fluorescence associated cell sorting (IP-FACS) protocol that initially depletes macrophages and photoreceptors, using immunopanning to enrich for RGCs before using low-pressure FACS to isolate these cells. We demonstrate that RGCs isolated via IP-FACS when compared to RGCs isolated via immunopanning at the same age have similar purity as measured by antibody staining and qRT-PCR; survival as measured by live dead staining; neurite outgrowth; and electrophysiological properties as measured by calcium release response to glutamate. Finally, we demonstrate the ability to isolate RGCs from early embryonic mice prior to the expression of Thy-1 using Brn3b-eGFP optogenetically labeled cells. This method provides a new approach for the isolation of RGCs for the study of early developed RGCs, the study of RGC subtypes and the isolation of RGCs for cell transplantation studies.

Keywords:  FACS (fluorescence-activated cell sorting); cell isolation; immunopanning; neurite outgowth; retinal ganglion cell (RGC)

DOI:  https://doi.org/10.3389/fnmol.2023.1149024
Invest Ophthalmol Vis Sci. 2023 08 01. 64(11): 10

Impaired Lysosome Reformation in Chloroquine-Treated Retinal Pigment Epithelial Cells.

M Helena Cardoso, Michael J Hall, Thomas Burgoyne, Pedro Fale, Tina Storm, Cristina Escrevente, Pedro Antas, Miguel C Seabra, Clare E Futter.

Purpose: To model the in vivo effects of chloroquine on the retinal pigment epithelium in experimentally tractable cell culture systems and determine the effects of mild chloroquine treatment on lysosome function and turnover.
Methods: Effects of low-dose chloroquine treatment on lysosomal function and accessibility to newly endocytosed cargo were investigated in primary and embryonic stem cell-derived RPE cells and ARPE19 cells using fluorescence and electron microscopy of fluorescent and gold-labeled probes. Lysosomal protein expression and accumulation were measured by quantitative PCR and Western blotting.
Results: Initial chloroquine-induced lysosome neutralization was followed by partial recovery, lysosomal expansion, and accumulation of undegraded endocytic, phagocytic, and autophagic cargo and inhibition of cathepsin D processing. Accumulation of enlarged lysosomes was accompanied by a gradual loss of accessibility of these structures to the endocytic pathway, implying impaired lysosome reformation. Chloroquine-induced accumulation of pro-cathepsin D, as well as the lysosomal membrane protein, LAMP1, was reproduced by treatment with protease inhibitors and preceded changes in lysosomal gene expression.
Conclusions: Low-dose chloroquine treatment inhibits lysosome reformation, causing a gradual depletion of lysosomes able to interact with cargo-carrying vacuoles and degrade their content. The resulting accumulation of newly synthesized pro-cathepsin D and LAMP1 reflects inhibition of normal turnover of lysosomal constituents and possibly lysosomes themselves. A better understanding of the mechanisms underlying lysosome reformation may reveal new targets for the treatment of chloroquine-induced retinopathy.

DOI: https://doi.org/10.1167/iovs.64.11.10
Immunol Rev. 2023 Aug 09.

Clearance phagocytosis by the retinal pigment epithelial during photoreceptor outer segment renewal: Molecular mechanisms and relation to retinal inflammation.

Stephanie A Lieffrig, Gavin Gyimesi, Yingyu Mao, Silvia C Finnemann.

  Mammalian photoreceptor outer segment renewal is a highly coordinated process that hinges on timed cell signaling between photoreceptor neurons and the adjacent retinal pigment epithelial (RPE). It is a strictly rhythmic, synchronized process that underlies in part circadian regulation. We highlight findings from recently developed methods that quantify distinct phases of outer segment renewal in retinal tissue. At light onset, outer segments expose the conserved "eat-me" signal phosphatidylserine exclusively at their distal, most aged tip. A coordinated two-receptor efferocytosis process follows, in which ligands bridge outer segment phosphatidylserine with the RPE receptors αvβ5 integrin, inducing cytosolic signaling toward Rac1 and focal adhesion kinase/MERTK, and with MERTK directly, additionally inhibiting RhoA/ROCK and thus enabling F-actin dynamics favoring outer segment fragment engulfment. Photoreceptors and RPE persist for life with each RPE cell in the eye servicing dozens of overlying photoreceptors. Thus, RPE cells phagocytose more often and process more material than any other cell type. Mutant mice with impaired outer segment renewal largely retain functional photoreceptors and retinal integrity. However, when anti-inflammatory signaling in the RPE via MERTK or the related TYRO3 is lacking, catastrophic inflammation leads to immune cell infiltration that swiftly destroys the retina causing blindness.

Keywords:  cell signaling; circadian rhythm; phagocytosis; photoreceptors; renewal; retinal pigment epithelial

DOI:  https://doi.org/10.1111/imr.13264