bims-mideyd 2023-08-27 papers

bims-mideyd

Biomed News

on Mitochondrial dysfunction in eye diseases

Issue of 2023‒08‒27
eleven papers selected by
Raji Shyam, Indiana University Bloomington

Exogenous CFH Modulates Levels of Pro-Inflammatory Mediators to Prevent Oxidative Damage of Retinal Pigment Epithelial Cells with the At-Risk CFH Y402H Variant.
Endogenous Galectin-1 Modulates Cell Biological Properties of Immortalized Retinal Pigment Epithelial Cells In Vitro.
Selenium Protects ARPE-19 and ACBRI 181 Cells against High Glucose-Induced Oxidative Stress.
The potential for mitochondrial therapeutics in the treatment of primary open-angle glaucoma: a review.
Loss of Prom1 impairs autophagy and promotes epithelial-mesenchymal transition in mouse retinal pigment epithelial cells.
The AKT2/SIRT5/TFEB pathway as a potential therapeutic target in atrophic AMD.
Autophagy deficiency protects against ocular hypertension and neurodegeneration in experimental and spontanous glaucoma mouse models.
The evolution of selective autophagy as a mechanism of oxidative stress response: The evolutionarily acquired ability of selective autophagy receptors to respond to oxidative stress is beneficial for human longevity.
P36-A151 Primary human RPE cells as individual starting material for non-viral gene transfer of therapeutically active factors.
Comparison between sodium iodate and lipid peroxide murine models of age-related macular degeneration for drug evaluation-a narrative review.
Comprehensive Analysis of Photoreceptor Outer Segments: Flow Cytometry Characterization and Stress-Driven Impact on Retinal Pigment Epithelium Phagocytosis.

Antioxidants (Basel). 2023 Jul 31. pii: 1540. [Epub ahead of print]12(8):

Exogenous CFH Modulates Levels of Pro-Inflammatory Mediators to Prevent Oxidative Damage of Retinal Pigment Epithelial Cells with the At-Risk CFH Y402H Variant.

Henry Velazquez-Soto, Sergio Groman-Lupa, Marisa Cruz-Aguilar, Alberto L Salazar, Juan C Zenteno, Maria C Jimenez-Martinez.

  Age-related macular degeneration (AMD) is a complex, progressive degenerative retinal disease. Retinal pigment epithelial (RPE) cells play an important role in the immune defense of the eye and their dysfunction leads to the progressive irreversible degeneration of photoreceptors. Genetic factors, chronic inflammation, and oxidative stress have been implicated in AMD pathogenesis. Oxidative stress causes RPE injury, resulting in a chronic inflammatory response and cell death. The Y402H polymorphism in the complement factor H (CFH) protein is an important risk factor for AMD. However, the functional significance of CFH Y402H polymorphism remains unclear. In the present study, we investigated the role of CFH in the pro-inflammatory response using an in vitro model of oxidative stress in the RPE with the at-risk CFH Y402H variant. ARPE-19 cells with the at-risk CFH Y402H variant were highly susceptible to damage caused by oxidative stress, with increased levels of inflammatory mediators and pro-apoptotic factors that lead to cell death. Pretreatment of the ARPE-19 cell cultures with exogenous CFH prior to the induction of oxidative stress prevented damage and cell death. This protective effect may be related to the negative regulation of pro-inflammatory cytokines. CFH contributes to cell homeostasis and is required to modulate the pro-inflammatory cytokine response under oxidative stress in the ARPE-19 cells with the at-risk CFH Y402H variant.

Keywords:  AMD; CFH; RPE; cell death; cytokines; oxidative stress

DOI:  https://doi.org/10.3390/antiox12081540
Int J Mol Sci. 2023 Aug 10. pii: 12635. [Epub ahead of print]24(16):

Endogenous Galectin-1 Modulates Cell Biological Properties of Immortalized Retinal Pigment Epithelial Cells In Vitro.

Caspar Liesenhoff, Simon Martin Paulus, Caroline Havertz, Arie Geerlof, Siegfried Priglinger, Claudia Sybille Priglinger, Andreas Ohlmann.

  In the eye, an increase in galectin-1 is associated with various chorioretinal diseases, in which retinal pigment epithelium (RPE) cells play a crucial role in disease development and progression. Since little is known about the function of endogenous galectin-1 in these cells, we developed a galectin-1-deficient immortalized RPE cell line (ARPE-19-LGALS1-/-) using a sgRNA/Cas9 all-in-one expression vector and investigated its cell biological properties. Galectin-1 deficiency was confirmed by Western blot analysis and immunocytochemistry. Cell viability and proliferation were significantly decreased in ARPE-19-LGALS1-/- cells when compared to wild-type controls. Further on, an increased attachment of galectin-1-deficient RPE cells was observed by cell adhesion assay when compared to control cells. The diminished viability and proliferation, as well as the enhanced adhesion of galectin-1-deficient ARPE-19 cells, could be blocked, at least in part, by the additional treatment with human recombinant galectin-1. In addition, a significantly reduced migration was detected in ARPE-19-LGALS1-/- cells. In comparison to control cells, galectin-1-deficient RPE cells had enhanced expression of sm-α-actin and N-cadherin, whereas expression of E-cadherin showed no significant alteration. Finally, a compensatory expression of galectin-8 mRNA was observed in ARPE-19-LGALS1-/- cells. In conclusion, in RPE cells, endogenous galectin-1 has crucial functions for various cell biological processes, including viability, proliferation, migration, adherence, and retaining the epithelial phenotype.

Keywords:  ARPE-19; cell attachment; cell proliferation; cell viability; compensatory gene expression; epithelial–mesenchymal transition; galectin-1; galectin-1 deficiency; galectin-1 knockout; retinal pigment epithelium cells

DOI:  https://doi.org/10.3390/ijms241612635
Molecules. 2023 Aug 09. pii: 5961. [Epub ahead of print]28(16):

Selenium Protects ARPE-19 and ACBRI 181 Cells against High Glucose-Induced Oxidative Stress.

Handan Bardak, Abdülhadi Cihangir Uğuz, Yavuz Bardak, Javier Rocha-Pimienta, Jonathan Delgado-Adámez, Javier Espino.

  Diabetic retinopathy (DR), a complication of diabetes mellitus (DM), can cause severe visual loss. The retinal pigment epithelium (RPE) plays a crucial role in retinal physiology but is vulnerable to oxidative damage. We investigated the protective effects of selenium (Se) on retinal pigment epithelium (ARPE-19) and primary human retinal microvascular endothelial (ACBRI 181) cells against high glucose (HG)-induced oxidative stress and apoptotic cascade. To achieve this objective, we utilized varying concentrations of D-glucose (ranging from 5 to 80 mM) to induce the HG model. HG-induced oxidative stress in ARPE-19 and ACBRI 181 cells and the apoptotic cascade were evaluated by determining Ca2+ overload, mitochondrial membrane depolarization, caspase-3/-9 activation, intracellular reactive oxygen species (ROS), lipid peroxidation (LP), glutathione (GSH), glutathione peroxidase (GSH-Px), vascular endothelial growth factor (VEGF) and apoptosis levels. A cell viability assay utilizing MTT was conducted to ascertain the optimal concentration of Se to be employed. The quantification of MTT, ROS, VEGF levels, and caspase-3 and -9 activation was accomplished using a plate reader. To quantitatively assess LP and GSH levels, GSH-Px activities were utilized by spectrophotometer and apoptosis, mitochondrial membrane depolarization, and the release of Ca2+ from intracellular stores were evaluated by spectrofluorometer. Our investigation revealed a significant augmentation in oxidative stress induced by HG, leading to cellular damage through modulation of mitochondrial membrane potential, ROS levels, and intracellular Ca2+ release. Incubation with Se resulted in a notable reduction in ROS production induced by HG, as well as a reduction in apoptosis and the activation of caspase-3 and -9. Additionally, Se incubation led to decreased levels of VEGF and LP while concurrently increasing levels of GSH and GSH-Px. The findings from this study strongly suggest that Se exerts a protective effect on ARPE-19 and ACBRI 181 cells against HG-induced oxidative stress and apoptosis. This protective mechanism is partially mediated through the intracellular Ca2+ signaling pathway.

Keywords:  ARPE-19 cells; high glucose; oxidative stress; selenium

DOI:  https://doi.org/10.3390/molecules28165961
Front Physiol. 2023 ;14 1184060

The potential for mitochondrial therapeutics in the treatment of primary open-angle glaucoma: a review.

Grace Kuang, Mina Halimitabrizi, Amy-Ann Edziah, Rebecca Salowe, Joan M O'Brien.

  Glaucoma, an age-related neurodegenerative disease, is characterized by the death of retinal ganglion cells (RGCs) and the corresponding loss of visual fields. This disease is the leading cause of irreversible blindness worldwide, making early diagnosis and effective treatment paramount. The pathophysiology of primary open-angle glaucoma (POAG), the most common form of the disease, remains poorly understood. Current available treatments, which target elevated intraocular pressure (IOP), are not effective at slowing disease progression in approximately 30% of patients. There is a great need to identify and study treatment options that target other disease mechanisms and aid in neuroprotection for POAG. Increasingly, the role of mitochondrial injury in the development of POAG has become an emphasized area of research interest. Disruption in the function of mitochondria has been linked to problems with neurodevelopment and systemic diseases. Recent studies have shown an association between RGC death and damage to the cells' mitochondria. In particular, oxidative stress and disrupted oxidative phosphorylation dynamics have been linked to increased susceptibility of RGC mitochondria to secondary mechanical injury. Several mitochondria-targeted treatments for POAG have been suggested, including physical exercise, diet and nutrition, antioxidant supplementation, stem cell therapy, hypoxia exposure, gene therapy, mitochondrial transplantation, and light therapy. Studies have shown that mitochondrial therapeutics may have the potential to slow the progression of POAG by protecting against mitochondrial decline associated with age, genetic susceptibility, and other pathology. Further, these therapeutics may potentially target already present neuronal damage and symptom manifestations. In this review, the authors outline potential mitochondria-targeted treatment strategies and discuss their utility for use in POAG.

Keywords:  glaucoma; mitochondrial dysfunction; mitochondrial therapeutics; neurodegeneration; oxidative Stress

DOI:  https://doi.org/10.3389/fphys.2023.1184060
J Cell Physiol. 2023 Aug 23.

Loss of Prom1 impairs autophagy and promotes epithelial-mesenchymal transition in mouse retinal pigment epithelial cells.

Sujoy Bhattacharya, Jinggang Yin, Weihong Huo, Edward Chaum.

  Mutations in the Prominin-1 (Prom1) gene disrupt photoreceptor disk morphogenesis, leading to macular dystrophies. We have shown that human retinal pigment epithelial (RPE) homeostasis is under the control of Prom1-dependent autophagy, demonstrating that Prom1 plays different roles in the photoreceptors and RPE. It is unclear if retinal and macular degeneration caused by the loss of Prom1 function is a cell-autonomous feature of the RPE or a generalized disease of photoreceptor degeneration. In this study, we investigated whether Prom1 is required for mouse RPE (mRPE) autophagy and phagocytosis, which are cellular processes essential for photoreceptor survival. We found that Prom1-KO decreases autophagy flux, activates mTORC1, and concomitantly decreases transcription factor EB (TFEB) and Cathepsin-D activities in mRPE cells. In addition, Prom1-KO reduces the clearance of bovine photoreceptor outer segments in mRPE cells due to increased mTORC1 and reduced TFEB activities. Dysfunction of Prom1-dependent autophagy correlates with both a decrease in ZO-1 and E-cadherin and a concomitant increase in Vimentin, SNAI1, and ZEB1 levels, consistent with induction of epithelial-mesenchymal transition (EMT) in Prom1-KO mRPE cells. Our results demonstrate that Prom1-mTORC1-TFEB signaling is a central driver of cell-autonomous mRPE homeostasis. We show that Prom1-KO in mRPE leads to RPE defects similar to that seen in atrophic age-related macular degeneration and opens new avenues of investigation targeting Prom1 in retinal degenerative diseases.

Keywords:  Stargardt-like macular dystrophy; TFEB; age-related macular degeneration; lysosomal activity; mTORC1; phagocytosis

DOI:  https://doi.org/10.1002/jcp.31094
bioRxiv. 2023 Aug 09. pii: 2023.08.08.552343. [Epub ahead of print]

The AKT2/SIRT5/TFEB pathway as a potential therapeutic target in atrophic AMD.

Sayan Ghosh, Ruchi Sharma, Sridhar Bammidi, Victoria Koontz, Mihir Nemani, Meysam Yazdankhah, Katarzyna M Kedziora, Callen T Wallace, Cheng Yu-Wei, Jonathan Franks, Devika Bose, Dhivyaa Rajasundaram, Stacey Hose, José-Alain Sahel, Rosa Puertollano, Toren Finkel, J Samuel Zigler, Yuri Sergeev, Simon C Watkins, Eric S Goetzman, Miguel Flores-Bellver, Kai Kaarniranta, Akrit Sodhi, Kapil Bharti, James T Handa, Debasish Sinha.

Age-related macular degeneration (AMD), the leading cause of geriatric blindness, is a multi-factorial disease with retinal-pigmented epithelial (RPE) cell dysfunction as a central pathogenic driver. With RPE degeneration, lysosomal function is a core process that is disrupted. Transcription factors EB/E3 (TFEB/E3) tightly control lysosomal function; their disruption can cause aging disorders, such as AMD. Here, we show that induced pluripotent stem cells (iPSC)-derived RPE cells with the complement factor H variant [ CFH (Y402H)] have increased AKT2, which impairs TFEB/TFE3 nuclear translocation and lysosomal function. Increased AKT2 can inhibit PGC1α, which downregulates SIRT5, an AKT2 binding partner. SIRT5 and AKT2 co-regulate each other, thereby modulating TFEB-dependent lysosomal function in the RPE. Failure of the AKT2/SIRT5/TFEB pathway in the RPE induced abnormalities in the autophagy-lysosome cellular axis by upregulating secretory autophagy, thereby releasing a plethora of factors that likely contribute to drusen formation, a hallmark of AMD. Finally, overexpressing AKT2 in RPE cells in mice led to an AMD-like phenotype. Thus, targeting the AKT2/SIRT5/TFEB pathway could be a potential therapy for atrophic AMD.

DOI: https://doi.org/10.1101/2023.08.08.552343
Cell Death Dis. 2023 Aug 24. 14(8): 554

Autophagy deficiency protects against ocular hypertension and neurodegeneration in experimental and spontanous glaucoma mouse models.

Angela Dixon, Myoung Sup Shim, April Nettesheim, Aislyn Coyne, Chien-Chia Su, Haiyan Gong, Paloma B Liton.

Glaucoma is a group of diseases that leads to chronic degeneration of retinal ganglion cell (RGC) axons and progressive loss of RGCs, resulting in vision loss. While aging and elevated intraocular pressure (IOP) have been identified as the main contributing factors to glaucoma, the molecular mechanisms and signaling pathways triggering RGC death and axonal degeneration are not fully understood. Previous studies in our laboratory found that overactivation of autophagy in DBA/2J::GFP-LC3 mice led to RGC death and optic nerve degeneration with glaucomatous IOP elevation. We found similar findings in aging GFP-LC3 mice subjected to chronic IOP elevation. Here, we further investigated the impact of autophagy deficiency on autophagy-deficient DBA/2J-Atg4bko and DBA/2J-Atg4b+/- mice, generated in our laboratory via CRISPR/Cas9 technology; as well as in Atg4bko mice subjected to the experimental TGFβ2 chronic ocular hypertensive model. Our data shows that, in contrast to DBA/2J and DBA/2J-Atg4b+/- littermates, DBA/2J-Atg4bko mice do not develop glaucomatous IOP elevation. Atg4b deficiency also protected against glaucomatous IOP elevation in the experimental TGFβ2 chronic ocular hypertensive model. Atg4 deletion did not compromise RGC or optic nerve survival in Atg4bko mice. Moreover, our results indicate a protective role of autophagy deficiency against RGC death and ON atrophy in the hypertensive DBA/2J-Atg4b+/- mice. Together, our data suggests a pathogenic role of autophagy activation in ocular hypertension and glaucoma.

DOI: https://doi.org/10.1038/s41419-023-06086-3
Bioessays. 2023 Aug 21. e2300076

The evolution of selective autophagy as a mechanism of oxidative stress response: The evolutionarily acquired ability of selective autophagy receptors to respond to oxidative stress is beneficial for human longevity.

Joshua Ratliffe, Tetsushi Kataura, Elsje G Otten, Viktor I Korolchuk.

  Ageing is associated with a decline in autophagy and elevated reactive oxygen species (ROS), which can breach the capacity of antioxidant systems. Resulting oxidative stress can cause further cellular damage, including DNA breaks and protein misfolding. This poses a challenge for longevous organisms, including humans. In this review, we hypothesise that in the course of human evolution selective autophagy receptors (SARs) acquired the ability to sense and respond to localised oxidative stress. We posit that in the vicinity of protein aggregates and dysfunctional mitochondria oxidation of key cysteine residues in SARs induces their oligomerisation which initiates autophagy. The degradation of damaged cellular components thus could reduce ROS production and restore redox homeostasis. This evolutionarily acquired function of SARs may represent one of the biological adaptations that contributed to longer lifespan. Inversely, loss of this mechanism can lead to age-related diseases associated with impaired autophagy and oxidative stress.

Keywords:  ageing; aggrephagy; autophagy; mitophagy; neurodegeneration; oxidative stress; selective autophagy receptors

DOI:  https://doi.org/10.1002/bies.202300076
BMJ Open Ophthalmol. 2023 08;8(Suppl 2): A15

P36-A151 Primary human RPE cells as individual starting material for non-viral gene transfer of therapeutically active factors.

Sandra Johnen, Walter Peter, Thumann Gabriele.

PURPOSE: Our aging society leads to an increasing incidence of neurodegenerative diseases. To date, the development of defined therapies has been hampered because the pathological mechanisms are poorly understood. Cell-based additive gene therapies to enhance the expression of protective factors are considered a promising modality for the treatment of neurodegenerative diseases, such as agerelated macular degeneration (AMD). We have developed a method to stably overexpress the genes encoding pigment epithelium-derived factor (PEDF) and brain-derived neurotrophic factor (BDNF) into the genome of primary human retinal pigment epithelial (RPE) cells by electroporation using the Sleeping Beauty (SB) transposon system. BDNF is the most abundant neurotrophin in the central nervous system. PEDF is a multifunctional protein with anti-angiogenic and neurotrophic properties.METHODS: Primary RPE cells were isolated from various human donor eyes and maintained individually in culture. After reaching confluence, RPE cells were trypsinized and co-transfected in suspension with two plasmids encoding SB100X transposase and the transposon carrying a PEDF and BDNF transcription cassette, respectively. The results of transfection were evaluated by different methods including microscopy, immunoblotting, ELISA, and quantitative PCR (qPCR).
RESULTS: Seeding of sufficient numbers of primary human RPE cells allows cultivation and growth into an integrated monolayer of pigmented, hexagonally shaped cells, independent of the donor age (65.3 ± 9.94 a, min: 49 a, max: 83 a, n = 12), post-mortem time of isolation (37.3 ± 17.0 h, min: 16 h, max: 68 h), and cultivation time (27.6 ± 14.1 d, min: 13 d, max: 61 d). Successful transfection was demonstrated in experiments performed independently. Applied electrical pulses had no negative effects on cell morphology. Gene expression of PEDF and BDNF was significantly increased compared with non-transfected control cells. Secretion of recombinant PEDF and BDNF proteins was also significantly elevated and remained stable over time. CONCLUSION: The studies using primary human RPE cells are an important step in the development of a cell-based PEDF or BDNF gene therapy that could be applied as an advanced therapy medicinal product to treat AMD or other degenerative retinal diseases.

DOI: https://doi.org/10.1136/bmjophth-2023-EEBA.35
Ann Eye Sci. 2022 Mar 15. pii: 8. [Epub ahead of print]7

Comparison between sodium iodate and lipid peroxide murine models of age-related macular degeneration for drug evaluation-a narrative review.

Soo-Young Kim, Haohua Qian.

  Objective: In this review, non-transgenic models of age-related macular degeneration (AMD) are discussed, with focuses on murine retinal degeneration induced by sodium iodate and lipid peroxide (HpODE) as preclinical study platforms.Background: AMD is the most common cause of vision loss in a world with an increasingly aging population. The major phenotypes of early and intermediate AMD are increased drusen and autofluorescence, Müller glia activation, infiltrated subretinal microglia and inward moving retinal pigment epithelium cells. Intermediate AMD may progress to advanced AMD, characterized by geography atrophy and/or choroidal neovascularization. Various transgenic and non-transgenic animal models related to retinal degeneration have been generated to investigate AMD pathogenesis and pathobiology, and have been widely used as potential therapeutic evaluation platforms.
Methods: Two retinal degeneration murine models induced by sodium iodate and HpODE are described. Distinct pathological features and procedures of these two models are compared. In addition, practical protocol and material preparation and assessment methods are elaborated.
Conclusion: Retina degeneration induced by sodium iodate and HpODE in mouse eye resembles many clinical aspects of human AMD and complimentary to the existent other animal models. However, standardization of procedure and assessment protocols is needed for preclinical studies. Further studies of HpODE on different routes, doses and species will be valuable for the future extensive use. Despite many merits of murine studies, differences between murine and human should be always considered.

Keywords:  Microglia; Photoreceptor; Retina degeneration; Retinal pigment epithelium

DOI:  https://doi.org/10.21037/aes-21-25
Int J Mol Sci. 2023 Aug 17. pii: 12889. [Epub ahead of print]24(16):

Comprehensive Analysis of Photoreceptor Outer Segments: Flow Cytometry Characterization and Stress-Driven Impact on Retinal Pigment Epithelium Phagocytosis.

Haoqian Liang, Qiang Wu, Xinzheng Victor Guo, Linda Chan, Tin Mao, Cinzia Stella, Axel Guilbaud, Julien Camperi.

  Phagocytosis is one of the key functions of retinal pigment epithelium (RPE) cells, which maintain photoreceptor health by removing photoreceptor outer segments (POSs) that are regularly shed. A deficiency in RPE function to phagocytose POSs may lead to vision loss in inherited retinal diseases and eventually to age-related macular degeneration (AMD) with geographic atrophy. Significant progress has been made in the field of cell replacement therapy for AMD using stem-cell-derived RPE. To test their function, RPE cells are incubated with purified bovine POSs for the demonstration of efficient binding, internalization, and digestion of POSs. Here, we present an image-based method to measure phagocytosis activity by using POSs labeled with a pH-sensitive fluorescent dye, which has low fluorescence at neutral pH outside of the cell and high fluorescence at low pH inside the phagosome. Further, we introduce a unique flow-cytometry-based method for the characterization of POSs by measuring specific markers for POSs such as rhodopsin and opsin. Using this method, we demonstrated a comparable quality of several bovine POS isolation batches and a reliable assessment of POS quality on RPE phagocytosis assay performance when subjected to different stress conditions. This work provides new tools to characterize POSs and insight into RPE phagocytosis assay development for the functional evaluation of RPE cells in the field of cell replacement therapy.

Keywords:  flow cytometry; fluorescence scanning; phagocytosis assay; photoreceptor outer segments; retinal pigment epithelium cells

DOI:  https://doi.org/10.3390/ijms241612889