bims-mideyd 2024-08-18 papers

bims-mideyd

Biomed News

on Mitochondrial dysfunction in eye diseases

Issue of 2024–08–18
six papers selected by
Rajalekshmy “Raji” Shyam, Indiana University Bloomington

The Scavenging Activity of Coenzyme Q10 Plus a Nutritional Complex on Human Retinal Pigment Epithelial Cells.
Inducible RPE-specific GPX4 knockout causes oxidative stress and retinal degeneration with features of age-related macular degeneration.
Dielectrophoretic characterization of peroxidized retinal pigment epithelial cells as a model of age-related macular degeneration.
Recycling the recyclers: lysophagy emerges as a new pharmacological target for retinal degeneration.
The significance of upper glycolytic components in regulating retinal pigment epithelial cellular behavior.
PDK4-mediated metabolic reprogramming is a potential therapeutic target for neovascular age-related macular degeneration.

Int J Mol Sci. 2024 Jul 24. pii: 8070. [Epub ahead of print]25(15):

The Scavenging Activity of Coenzyme Q10 Plus a Nutritional Complex on Human Retinal Pigment Epithelial Cells.

Maria Hernandez, Sergio Recalde, Jaione Bezunartea, Maite Moreno-Orduña, Idoia Belza, Ainara Chas-Prat, Elena Perugini, Alfredo Garcia-Layana, Patricia Fernández-Robredo.

  Age-related macular degeneration (AMD) and diabetic retinopathy (DR) are common retinal diseases responsible for most blindness in working-age and elderly populations. Oxidative stress and mitochondrial dysfunction play roles in these pathogenesis, and new therapies counteracting these contributors could be of great interest. Some molecules, like coenzyme Q10 (CoQ10), are considered beneficial to maintain mitochondrial homeostasis and contribute to the prevention of cellular apoptosis. We investigated the impact of adding CoQ10 (Q) to a nutritional antioxidant complex (Nutrof Total®; N) on the mitochondrial status and apoptosis in an in vitro hydrogen peroxide (H2O2)-induced oxidative stress model in human retinal pigment epithelium (RPE) cells. H2O2 significantly increased 8-OHdG levels (p < 0.05), caspase-3 (p < 0.0001) and TUNEL intensity (p < 0.01), and RANTES (p < 0.05), caspase-1 (p < 0.05), superoxide (p < 0.05), and DRP-1 (p < 0.05) levels, and also decreased IL1β, SOD2, and CAT gene expression (p < 0.05) vs. control. Remarkably, Q showed a significant recovery in IL1β gene expression, TUNEL, TNFα, caspase-1, and JC-1 (p < 0.05) vs. H2O2, and NQ showed a synergist effect in caspase-3 (p < 0.01), TUNEL (p < 0.0001), mtDNA, and DRP-1 (p < 0.05). Our results showed that CoQ10 supplementation is effective in restoring/preventing apoptosis and mitochondrial stress-related damage, suggesting that it could be a valid strategy in degenerative processes such as AMD or DR.

Keywords:  ARPE-19; DRP-1; age-related macular degeneration (AMD); caspase-3; coenzyme Q10; diabetic retinopathy (DR); mitochondrial stress; oxidative stress

DOI:  https://doi.org/10.3390/ijms25158070
Exp Eye Res. 2024 Aug 10. pii: S0014-4835(24)00249-5. [Epub ahead of print]247 110028

Inducible RPE-specific GPX4 knockout causes oxidative stress and retinal degeneration with features of age-related macular degeneration.

Alaina M Wojciechowski, Brent A Bell, Ying Song, Brandon D Anderson, Alexa Conomikes, Cecilia Petruconis, Joshua L Dunaief.

  Age-related macular degeneration (AMD) is one of the leading causes of vision loss in the elderly. This disease involves oxidative stress burden in the retina leading to death of retinal pigment epithelial (RPE) cells and photoreceptors. The retina is susceptible to oxidative stress, in part due to high metabolic activity and high concentration of polyunsaturated fatty acids that undergo lipid peroxidation chain reactions. Antioxidant enzymes exist in the retina to combat this stress, including glutathione peroxidase 4 (GPX4). GPX4 specifically reduces oxidized lipids, protecting against lipid peroxidation-induced oxidative stress, which is noted in dry AMD. We hypothesize that Gpx4 knockout within the RPE will result in an environment of chronic oxidative stress yielding degeneration akin to AMD. C57BL/6J mice with a floxed Gpx4 gene were mated with Rpe65Cre/ER mice. Offspring containing Rpe65Cre ± alleles and either Gpx4 WT or Gpx4 fl/fl alleles were administered tamoxifen to induce Gpx4 knockout in Gpx4 fl/fl mice. At sequential timepoints, retinal phenotypes were assessed via in vivo imaging utilizing confocal scanning laser ophthalmoscopy and optical coherence tomography (OCT), and visual function was probed by electroretinography. Retinas were studied post-mortem by immunohistochemical analyses, electron microscopy, plastic sectioning, and quantitative polymerase chain reaction and Western analyses. The RPE-specific Gpx4 knockout model was validated via Western analysis indicating diminished GPX4 protein only within the RPE and not the neural retina. Following Gpx4 knockout, RPE cells became dysfunctional and died, with significant cell loss occurring 2 weeks post-knockout. Progressive thinning of the photoreceptor layer followed RPE degeneration and was accompanied by loss of visual function. OCT and light microscopy showed hyperreflective foci and enlarged, pigmented cells in and above the RPE layer. Electron microscopy revealed decreased mitochondrial cristae and loss of basal and apical RPE ultrastructure. Finally, there was increased carboxyethylpyrrole staining, indicating oxidation of docosahexaenoic acid, and increased levels of mRNAs encoding oxidative stress-associated genes in the RPE and photoreceptors. Overall, we show that RPE-localized GPX4 is necessary for the health of the RPE and outer retina, and that knockout recapitulates phenotypes of dry AMD.

Keywords:  AMD; GPX4; Lipid peroxidation; Oxidative stress; Retina

DOI:  https://doi.org/10.1016/j.exer.2024.110028
BMC Ophthalmol. 2024 Aug 13. 24(1): 340

Dielectrophoretic characterization of peroxidized retinal pigment epithelial cells as a model of age-related macular degeneration.

Dharm Singh Yadav, Ioan Tivig, Tudor Savopol, Mihaela G Moisescu.

   BACKGROUND: Age-related macular degeneration (AMD) is a prevalent ocular pathology affecting mostly the elderly population. AMD is characterized by a progressive retinal pigment epithelial (RPE) cell degeneration, mainly caused by an impaired antioxidative defense. One of the AMD therapeutic procedures involves injecting healthy RPE cells into the subretinal space, necessitating pure, healthy RPE cell suspensions. This study aims to electrically characterize RPE cells to demonstrate a possibility using simulations to separate healthy RPE cells from a mixture of healthy/oxidized cells by dielectrophoresis.
METHODS: BPEI-1 rat RPE cells were exposed to hydrogen peroxide to create an in-vitro AMD cellular model. Cell viability was evaluated using various methods, including microscopic imaging, impedance-based real-time cell analysis, and the MTS assay. Healthy and oxidized cells were characterized by recording their dielectrophoretic spectra, and electric cell parameters (crossover frequency, membrane conductivity and permittivity, and cytoplasm conductivity) were computed. A COMSOL simulation was performed on a theoretical microfluidic-based dielectrophoretic separation chip using these parameters.
RESULTS: Increasing the hydrogen peroxide concentration shifted the first crossover frequency toward lower values, and the cell membrane permittivity progressively increased. These changes were attributed to progressive membrane peroxidation, as they were diminished when measured on cells treated with the antioxidant N-acetylcysteine. The changes in the crossover frequency were sufficient for the efficient separation of healthy cells, as demonstrated by simulations.
CONCLUSIONS: The study demonstrates that dielectrophoresis can be used to separate healthy RPE cells from oxidized ones based on their electrical properties. This method could be a viable approach for obtaining pure, healthy RPE cell suspensions for AMD therapeutic procedures.

Keywords:  Age-related macular degeneration; Cell purification simulations; Dielectrophoresis; Lipid peroxidation; Membrane permittivity; Retinal pigment epithelium

DOI:  https://doi.org/10.1186/s12886-024-03617-0
Autophagy. 2024 Aug 12.

Recycling the recyclers: lysophagy emerges as a new pharmacological target for retinal degeneration.

Juan Ignacio Jiménez-Loygorri, Patricia Boya.

  Dysregulated macroautophagy/autophagy is one of the hallmarks of aging and has also been linked to higher incidence of several age-associated diseases such as age-related macular degeneration (AMD). The main cell type affected in AMD is the retinal pigment epithelium (RPE), and this disease can lead to central vision loss. Despite affecting around 8.7% of the population between 45-85 years, its etiopathogenesis remains unknown. In our recent manuscript using the pharmacological sodium iodate (SI) model of AMD we identified severe lysosomal membrane permeabilization (LMP) in the RPE, that leads to autophagy flux blockage and proteostasis defects. Treatment with the natural compound urolithin A (UA) reduces RPE cell death and alleviates vision loss, concurrent with full autophagy restoration. While UA was initially described as a specific mitophagy inducer, we now show that it is also able to promote SQSTM1/p62-dependent lysophagy in the context of lysosomal damage and LMP. Genetic downregulation of SQSTM1/p62 fully abolishes the effect of UA on lysophagy while mitophagy stimulation remains unaffected. In summary, these findings highlight the wide range of pathways modulated by UA and its potential implementation in the management of AMD and other diseases involving lysosomal damage.

Keywords:  AMD; RPE; autophagy; lysosomal membrane permeabilization; lysosome

DOI:  https://doi.org/10.1080/15548627.2024.2391726
Sci Rep. 2024 08 14. 14(1): 18862

The significance of upper glycolytic components in regulating retinal pigment epithelial cellular behavior.

Armaan Naghdi, Nicole Oska, Thangal Yumnamcha, Shaimaa Eltanani, Mohamed Shawky, Rao Me, Ahmed S Ibrahim.

  Cell adhesion to the extracellular matrix and its natural outcome of cell spreading, along with the maintenance of barrier activity, are essential behaviors of epithelial cells, including retinal pigment epithelium (RPE). Disruptions in these characteristics can result in severe vision-threatening diseases such as diabetic macular edema and age-related macular degeneration. However, the precise mechanisms underlying how RPE cells regulate their barrier integrity and cell spreading are not fully understood. This study aims to elucidate the relative importance of upper glycolytic components in governing these cellular behaviors of RPE cells. Electric Cell-Substrate Impedance Sensing (ECIS) technology was utilized to assess in real-time the effects of targeting various upper glycolytic enzymes on RPE barrier function and cell spreading by measuring cell resistance and capacitance, respectively. Specific inhibitors used included WZB117 for Glut1 inhibition, Lonidamine for Hexokinase inhibition, PFK158 for PFKFB3/PFK axis inhibition, and TDZD-8 for Aldolase inhibition. Additionally, the viability of RPE cells was evaluated using a lactate dehydrogenase (LDH) cytotoxicity assay. The most significant decrease in electrical resistance and increase in capacitance of RPE cells were observed due to dose-dependent inhibition of Glut1 using WZB117, as well as Aldolase inhibition with TDZD-8. LDH level analysis at 24-72 h post-treatment with WZB117 (1 and 10 μM) or TDZD-8 (1 μM) showed no significant difference compared to the control, indicating that the disruption of RPE functionality was not attributed to cell death. Lastly, inhibition of other upper glycolytic components, including PFKFB3/PFK with PFK158 or Hexokinase with Lonidamine, did not significantly affect RPE cell behavior. This study provides insights into the varied roles of upper glycolytic components in regulating the functionality of RPE cells. Specifically, it highlights the critical roles of Glut1 and Aldolase in preserving barrier integrity and promoting RPE cell adhesion and spreading. Such understanding will guide the development of safe interventions to treat RPE cell dysfunction in various retinal disorders.

Keywords:  Age-related macular degeneration (AMD); Barrier integrity; Capacitance; Cell adhesion; Cell spreading; Electric cell-substrate impedance sensing (ECIS); Glycolysis; Resistance

DOI:  https://doi.org/10.1038/s41598-024-68343-5
Cell Death Dis. 2024 Aug 09. 15(8): 582

PDK4-mediated metabolic reprogramming is a potential therapeutic target for neovascular age-related macular degeneration.

Juhee Kim, Yujin Jeon, Jinyoung Son, Haushabhau S Pagire, Suvarna H Pagire, Jin Hee Ahn, Akiyoshi Uemura, In-Kyu Lee, Sungmi Park, Dong Ho Park.

Age-related macular degeneration (AMD) causes severe blindness in the elderly due to choroidal neovascularization (CNV), which results from the dysfunction of the retinal pigment epithelium (RPE). While normal RPE depends exclusively on mitochondrial oxidative phosphorylation for energy production, the inflammatory conditions associated with metabolic reprogramming of the RPE play a pivotal role in CNV. Although mitochondrial pyruvate dehydrogenase kinase (PDK) is a central node of energy metabolism, its role in the development of CNV in neovascular AMD has not been investigated. In the present study, we used a laser-induced CNV mouse model to evaluate the effects of Pdk4 gene ablation and treatment with pan-PDK or specific PDK4 inhibitors on fluorescein angiography and CNV lesion area. Among PDK isoforms, only PDK4 was upregulated in the RPE of laser-induced CNV mice, and Pdk4 gene ablation attenuated CNV. Next, we evaluated mitochondrial changes mediated by PDK1-4 inhibition using siRNA or PDK inhibitors in inflammatory cytokine mixture (ICM)-treated primary human RPE (hRPE) cells. PDK4 silencing only in ICM-treated hRPE cells restored mitochondrial respiration and reduced inflammatory cytokine secretion. Likewise, GM10395, a specific PDK4 inhibitor, restored oxidative phosphorylation and decreased ICM-induced upregulation of inflammatory cytokine secretion. In a laser-induced CNV mouse model, GM10395 significantly alleviated CNV. Taken together, we demonstrate that specific PDK4 inhibition could be a therapeutic strategy for neovascular AMD by preventing mitochondrial metabolic reprogramming in the RPE under inflammatory conditions.

DOI: https://doi.org/10.1038/s41419-024-06968-0