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19 pages, 3267 KiB  
Article
Human Retinal Organoid Model of Ocular Toxoplasmosis
by Liam M. Ashander, Grace E. Lidgerwood, Amanda L. Lumsden, João M. Furtado, Alice Pébay and Justine R. Smith
Pathogens 2025, 14(3), 286; https://doi.org/10.3390/pathogens14030286 - 14 Mar 2025
Viewed by 910
Abstract
The health burden of ocular toxoplasmosis is substantial, and there is an unmet need for safe and curative anti-microbial drugs. One major barrier to research on new therapeutics is the lack of in vitro human-based models beyond two-dimensional cultured cells and tissue explants. [...] Read more.
The health burden of ocular toxoplasmosis is substantial, and there is an unmet need for safe and curative anti-microbial drugs. One major barrier to research on new therapeutics is the lack of in vitro human-based models beyond two-dimensional cultured cells and tissue explants. We aimed to address this research gap by establishing a human retinal organoid model of ocular toxoplasmosis. Retinal organoids, generated from human induced pluripotent stem cells and grown to two stages of organization, were incubated with a suspension of live or heat-killed GT-1 strain T. gondii tachyzoites, or medium without tachyzoites. Both developing (1 month post-isolation) and matured (6 months post-isolation) organoids were susceptible to infection. Spread of live parasites from the margin to the entire organoid over 1 week was indicated by immunolabelling for T. gondii surface antigen 1. This progression was accompanied by changes in the levels of selected tachyzoite transcripts—SAG1, GRA6, and ROP16—and human cytokine transcripts—CCL2, CXCL8, CXCL10, and IL6—in infected versus control conditions. Our human retinal organoid model of ocular toxoplasmosis offers the opportunity for many future lines of study, including tachyzoite interactions with retinal cell populations and leukocyte subsets, parasite stage progression, and disease processes of different T. gondii strains, as well as drug testing. Full article
(This article belongs to the Special Issue Parasitic Diseases in the Contemporary World)
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11 pages, 2296 KiB  
Communication
SVM-Based Optical Detection of Retinal Ganglion Cell Apoptosis
by Mukhit Kulmaganbetov, Ryan Bevan, Andrew Want, Nantheera Anantrasirichai, Alin Achim, Julie Albon and James Morgan
Photonics 2025, 12(2), 128; https://doi.org/10.3390/photonics12020128 - 31 Jan 2025
Cited by 1 | Viewed by 822
Abstract
Background: Retinal ganglion cell (RGC) loss is crucial in eye diseases like glaucoma. Axon damage and dendritic degeneration precede cell death, detectable within optical coherence tomography (OCT) resolution, indicating their correlation with neuronal degeneration. The purpose of this study is to evaluate [...] Read more.
Background: Retinal ganglion cell (RGC) loss is crucial in eye diseases like glaucoma. Axon damage and dendritic degeneration precede cell death, detectable within optical coherence tomography (OCT) resolution, indicating their correlation with neuronal degeneration. The purpose of this study is to evaluate the optical changes of early retinal degeneration. Methods: The detection of optical changes in the axotomised retinal explants was completed in six C57BL/6J mice. OCT images were acquired up to 120 min from enucleation. A grey-level co-occurrence-based texture analysis was performed on the inner plexiform layer (IPL) to monitor changes in the optical speckles using a principal component analysis (PCA) and a support vector machine (SVM). In parallel tests, retinal transparency was confirmed by a comparison of the modulation transfer functions (MTFs) at 0 and 120 min. Results: Quantitative confirmation by analysis of the MTFs confirmed the non-degradation of optical transparency during the imaging period: MTF (fx) = 0.267 ± 0.02. Textural features in the IPL could discriminate between the optical signals of RGC degeneration. The mean accuracy of the SVM classification was 86.3%; discrimination was not enhanced by the combination of the SVM and PCA (81.9%). Conclusions: Optical changes in the IPL can be detected using OCT following RGC axotomy. High-resolution OCT can provide an index of retinal neuronal integrity and its degeneration. Full article
(This article belongs to the Special Issue Biomedical Optics:Imaging, Sensing and Therapy)
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20 pages, 2971 KiB  
Article
Intravitreal Metformin Protects Against Choroidal Neovascularization and Light-Induced Retinal Degeneration
by Jason F. Xiao, Wendy Luo, Amir Mani, Hugo Barba, Aniruddhsingh Solanki, Steven Droho, Jeremy A. Lavine and Dimitra Skondra
Int. J. Mol. Sci. 2024, 25(21), 11357; https://doi.org/10.3390/ijms252111357 - 22 Oct 2024
Cited by 3 | Viewed by 4549
Abstract
Neovascular age-related macular degeneration (nAMD), a leading cause of blindness in older adults, presents a challenging pathophysiology involving choroidal neovascularization (CNV) and retinal degeneration. Current treatments relying on intravitreal (IVT) administration of anti-angiogenic agents are costly and of moderate effectiveness. Metformin, the common [...] Read more.
Neovascular age-related macular degeneration (nAMD), a leading cause of blindness in older adults, presents a challenging pathophysiology involving choroidal neovascularization (CNV) and retinal degeneration. Current treatments relying on intravitreal (IVT) administration of anti-angiogenic agents are costly and of moderate effectiveness. Metformin, the common anti-diabetic drug, has been associated with decreased odds of developing AMD. Studies have shown that metformin can mitigate cellular aging, neoangiogenesis, and inflammation across multiple diseases. This preclinical study assessed metformin’s impact on vessel growth using choroidal explants before exploring IVT metformin’s effects on laser-induced CNV and light-induced retinal degeneration in C57BL/6J and BALB/cJ mice, respectively. Metformin reduced new vessel growth in choroidal explants in a dose-dependent relationship. Following laser induction, IVT metformin suppressed CNV and decreased peripheral infiltration of IBA1+ macrophages/microglia. Furthermore, IVT metformin protected against retinal thinning in response to light-induced degeneration. IVT metformin downregulated genes in the choroid and retinal pigment epithelium which are associated with angiogenesis and inflammation, two key processes that drive nAMD progression. These findings underscore metformin’s capacity as an anti-angiogenic and neuroprotective agent, demonstrating this drug’s potential as an accessible option to help manage nAMD. Full article
(This article belongs to the Special Issue Age-Related Macular Degeneration and Diabetic Retinopathy)
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29 pages, 1074 KiB  
Review
A Comparative Analysis of Models for AAV-Mediated Gene Therapy for Inherited Retinal Diseases
by Almaqdad Alsalloum, Ekaterina Gornostal, Natalia Mingaleva, Roman Pavlov, Ekaterina Kuznetsova, Ekaterina Antonova, Aygun Nadzhafova, Daria Kolotova, Vitaly Kadyshev, Olga Mityaeva and Pavel Volchkov
Cells 2024, 13(20), 1706; https://doi.org/10.3390/cells13201706 - 15 Oct 2024
Cited by 4 | Viewed by 3771
Abstract
Inherited retinal diseases (IRDs) represent a diverse group of genetic disorders leading to progressive degeneration of the retina due to mutations in over 280 genes. This review focuses on the various methodologies for the preclinical characterization and evaluation of adeno-associated virus (AAV)-mediated gene [...] Read more.
Inherited retinal diseases (IRDs) represent a diverse group of genetic disorders leading to progressive degeneration of the retina due to mutations in over 280 genes. This review focuses on the various methodologies for the preclinical characterization and evaluation of adeno-associated virus (AAV)-mediated gene therapy as a potential treatment option for IRDs, particularly focusing on gene therapies targeting mutations, such as those in the RPE65 and FAM161A genes. AAV vectors, such as AAV2 and AAV5, have been utilized to deliver therapeutic genes, showing promise in preserving vision and enhancing photoreceptor function in animal models. Despite their advantages—including high production efficiency, low pathogenicity, and minimal immunogenicity—AAV-mediated therapies face limitations such as immune responses beyond the retina, vector size constraints, and challenges in large-scale manufacturing. This review systematically compares different experimental models used to investigate AAV-mediated therapies, such as mouse models, human retinal explants (HREs), and induced pluripotent stem cell (iPSC)-derived retinal organoids. Mouse models are advantageous for genetic manipulation and detailed investigations of disease mechanisms; however, anatomical differences between mice and humans may limit the translational applicability of results. HREs offer valuable insights into human retinal pathophysiology but face challenges such as tissue degradation and lack of systemic physiological effects. Retinal organoids, on the other hand, provide a robust platform that closely mimics human retinal development, thereby enabling more comprehensive studies on disease mechanisms and therapeutic strategies, including AAV-based interventions. Specific outcomes targeted in these studies include vision preservation and functional improvements of retinas damaged by genetic mutations. This review highlights the strengths and weaknesses of each experimental model and advocates for their combined use in developing targeted gene therapies for IRDs. As research advances, optimizing AAV vector design and delivery methods will be critical for enhancing therapeutic efficacy and improving clinical outcomes for patients with IRDs. Full article
(This article belongs to the Special Issue Organoids as an Experimental Tool)
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16 pages, 3908 KiB  
Article
A New Ex Vivo Model Based on Mouse Retinal Explants for the Study of Ocular Toxoplasmosis
by Veronica Rodriguez Fernandez, Rosario Amato, Simona Piaggi, Barbara Pinto, Giovanni Casini and Fabrizio Bruschi
Pathogens 2024, 13(8), 701; https://doi.org/10.3390/pathogens13080701 - 19 Aug 2024
Viewed by 1597
Abstract
Ocular toxoplasmosis is the most prevalent clinical manifestation of T. gondii infection, which causes irreversible retinal damage. Different experimental models have been developed to study this pathology. In the present study, a new, ex vivo model is proposed to contribute to the elucidation [...] Read more.
Ocular toxoplasmosis is the most prevalent clinical manifestation of T. gondii infection, which causes irreversible retinal damage. Different experimental models have been developed to study this pathology. In the present study, a new, ex vivo model is proposed to contribute to the elucidation of disease mechanisms and to possible therapeutic solutions. Ex-vivo retinal explants, prepared from mouse retinas following established protocols, were incubated with T. gondii tachyzoites maintained in Vero cells. At different times, starting at 12 h up to 10 days of incubation, the explants were analyzed with immunofluorescence and Western blot to investigate their responses to parasite infection. T. gondii invasion of the retinal thickness was evident after 3 days in culture, where parasites could be detected around retinal cell nuclei. This was paralleled by putative cyst formation and microglial activation. At the same time, an evident increase in inflammatory and oxidative stress markers was detected in infected explants compared to controls. Cell death also appeared to occur in retinal explants after 3 days of T. gondii infection, and it was characterized by increased necroptotic but not apoptotic markers. The proposed model recapitulates the main characteristics of T. gondii retinal infection within 3 days of incubation and, therefore, allows for studying the very early events of the process. In addition, it requires only a limited number of animals and offers easy manipulation and accessibility for setting up different experimental conditions and assessing the effects of putative drugs for therapy. Full article
(This article belongs to the Section Parasitic Pathogens)
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19 pages, 2721 KiB  
Article
Nogo Receptor Antagonist LOTUS Promotes Neurite Outgrowth through Its Interaction with Teneurin-4
by Yuji Kurihara, Yuki Kawaguchi, Yuki Ohta, Nana Kawasaki, Yuki Fujita and Kohtaro Takei
Cells 2024, 13(16), 1369; https://doi.org/10.3390/cells13161369 - 17 Aug 2024
Viewed by 1432
Abstract
Neurite outgrowth is a crucial process for organizing neuronal circuits in neuronal development and regeneration after injury. Regenerative failure in the adult mammalian central nervous system (CNS) is attributed to axonal growth inhibitors such as the Nogo protein that commonly binds to Nogo [...] Read more.
Neurite outgrowth is a crucial process for organizing neuronal circuits in neuronal development and regeneration after injury. Regenerative failure in the adult mammalian central nervous system (CNS) is attributed to axonal growth inhibitors such as the Nogo protein that commonly binds to Nogo receptor-1 (NgR1). We previously reported that lateral olfactory tract usher substance (LOTUS) functions as an endogenous antagonist for NgR1 in forming neuronal circuits in the developing brain and improving axonal regeneration in the adult injured CNS. However, another molecular and cellular function of LOTUS remains unknown. In this study, we found that cultured retinal explant neurons extend their neurites on the LOTUS-coating substrate. This action was also observed in cultured retinal explant neurons derived from Ngr1-deficient mouse embryos, indicating that the promoting action of LOTUS on neurite outgrowth may be mediated by unidentified LOTUS-binding protein(s). We therefore screened the binding partner(s) of LOTUS by using a liquid chromatography-tandem mass spectrometry (LC-MS/MS). LC-MS/MS analysis and pull-down assay showed that LOTUS interacts with Teneurin-4 (Ten-4), a cell adhesion molecule. RNAi knockdown of Ten-4 inhibited neurite outgrowth on the LOTUS substrate in retinoic acid (RA)-treated Neuro2A cells. Furthermore, a soluble form of Ten-4 attenuates the promoting action on neurite outgrowth in cultured retinal explant neurons on the LOTUS substrate. These results suggest that LOTUS promotes neurite outgrowth by interacting with Ten-4. Our findings may provide a new molecular mechanism of LOTUS to contribute to neuronal circuit formation in development and to enhance axonal regeneration after CNS injury. Full article
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19 pages, 5473 KiB  
Article
Arylphthalide Delays Diabetic Retinopathy via Immunomodulating the Early Inflammatory Response in an Animal Model of Type 1 Diabetes Mellitus
by Francisco Martín-Loro, Fátima Cano-Cano, María J. Ortega, Belén Cuevas, Laura Gómez-Jaramillo, María del Carmen González-Montelongo, Jan Cedric Freisenhausen, Almudena Lara-Barea, Antonio Campos-Caro, Eva Zubía, Manuel Aguilar-Diosdado and Ana I. Arroba
Int. J. Mol. Sci. 2024, 25(15), 8440; https://doi.org/10.3390/ijms25158440 - 2 Aug 2024
Cited by 2 | Viewed by 1852
Abstract
Diabetic retinopathy (DR) is one of the most prevalent secondary complications associated with diabetes. Specifically, Type 1 Diabetes Mellitus (T1D) has an immune component that may determine the evolution of DR by compromising the immune response of the retina, which is mediated by [...] Read more.
Diabetic retinopathy (DR) is one of the most prevalent secondary complications associated with diabetes. Specifically, Type 1 Diabetes Mellitus (T1D) has an immune component that may determine the evolution of DR by compromising the immune response of the retina, which is mediated by microglia. In the early stages of DR, the permeabilization of the blood–retinal barrier allows immune cells from the peripheral system to interact with the retinal immune system. The use of new bioactive molecules, such as 3-(2,4-dihydroxyphenyl)phthalide (M9), with powerful anti-inflammatory activity, might represent an advance in the treatment of diseases like DR by targeting the immune systems responsible for its onset and progression. Our research aimed to investigate the molecular mechanisms involved in the interaction of specific cells of the innate immune system during the progression of DR and the reduction in inflammatory processes contributing to the pathology. In vitro studies were conducted exposing Bv.2 microglial and Raw264.7 macrophage cells to proinflammatory stimuli for 24 h, in the presence or absence of M9. Ex vivo and in vivo approaches were performed in BB rats, an animal model for T1D. Retinal explants from BB rats were cultured with M9. Retinas from BB rats treated for 15 days with M9 via intraperitoneal injection were analyzed to determine survival, cellular signaling, and inflammatory markers using qPCR, Western blot, or immunofluorescence approaches. Retinal structure images were acquired via Spectral-Domain–Optical Coherence Tomography (SD-OCT). Our results show that the treatment with M9 significantly reduces inflammatory processes in in vitro, ex vivo, and in vivo models of DR. M9 works by inhibiting the proinflammatory responses during DR progression mainly affecting immune cell responses. It also induces an anti-inflammatory response, primarily mediated by microglial cells, leading to the synthesis of Arginase-1 and Hemeoxygenase-1(HO-1). Ultimately, in vivo administration of M9 preserves the retinal integrity from the degeneration associated with DR progression. Our findings demonstrate a specific interaction between both retinal and systemic immune cells in the progression of DR, with a differential response to treatment, mainly driven by microglia in the anti-inflammatory action. In vivo treatment with M9 induces a switch in immune cell phenotypes and functions that contributes to delaying the DR progression, positioning microglial cells as a new and specific therapeutic target in DR. Full article
(This article belongs to the Special Issue Advances in the Pathophysiology and Treatment of Diabetic Retinopathy)
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16 pages, 3036 KiB  
Article
Protection against Oxidative Stress by Coenzyme Q10 in a Porcine Retinal Degeneration Model
by Leonie Deppe, Ana M. Mueller-Buehl, Teresa Tsai, Carl Erb, H. Burkhard Dick and Stephanie C. Joachim
J. Pers. Med. 2024, 14(4), 437; https://doi.org/10.3390/jpm14040437 - 22 Apr 2024
Cited by 4 | Viewed by 2363
Abstract
Oxidative stress plays an important role in neurodegenerative diseases, including glaucoma. Therefore, we analyzed if the antioxidant coenzyme Q10 (CoQ10), which is also commercially available, can prevent retinal degeneration induced by hydrogen peroxide (H2O2) in a porcine organ culture [...] Read more.
Oxidative stress plays an important role in neurodegenerative diseases, including glaucoma. Therefore, we analyzed if the antioxidant coenzyme Q10 (CoQ10), which is also commercially available, can prevent retinal degeneration induced by hydrogen peroxide (H2O2) in a porcine organ culture model. Retinal explants were cultivated for eight days, and H2O2 (500 µM, 3 h) induced the oxidative damage. CoQ10 therapy was applied (700 µM, 48 h). Retinal ganglion cells (RGCs) and microglia were examined immunohistologically in all groups (control, H2O2, H2O2 + CoQ10). Cellular, oxidative, and inflammatory genes were quantified via RT-qPCR. Strong RGC loss was observed with H2O2 (p ≤ 0.001). CoQ10 elicited RGC protection compared to the damaged group at a histological (p ≤ 0.001) and mRNA level. We detected more microglia cells with H2O2, but CoQ10 reduced this effect (p = 0.004). Cellular protection genes (NRF2) against oxidative stress were stimulated by CoQ10 (p ≤ 0.001). Furthermore, mitochondrial oxidative stress (SOD2) increased through H2O2 (p = 0.038), and CoQ10 reduced it to control level. Our novel results indicate neuroprotection via CoQ10 in porcine retina organ cultures. In particular, CoQ10 appears to protect RGCs by potentially inhibiting apoptosis-related pathways, activating intracellular protection and reducing mitochondrial stress. Full article
(This article belongs to the Special Issue Glaucoma Management in the Era of Personalized Medicine)
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24 pages, 3915 KiB  
Article
Integrative Kinase Activity Profiling and Phosphoproteomics of rd10 Mouse Retina during cGMP-Dependent Retinal Degeneration
by Akanksha Roy, Jiaming Zhou, Merijn Nolet, Charlotte Welinder, Yu Zhu, François Paquet-Durand, John Groten, Tushar Tomar and Per Ekström
Int. J. Mol. Sci. 2024, 25(6), 3446; https://doi.org/10.3390/ijms25063446 - 19 Mar 2024
Cited by 3 | Viewed by 2182
Abstract
Inherited retinal degenerative diseases (IRDs) are a group of rare diseases that lead to a progressive loss of photoreceptor cells and, ultimately, blindness. The overactivation of cGMP-dependent protein kinase G (PKG), one of the key effectors of cGMP-signaling, was previously found to be [...] Read more.
Inherited retinal degenerative diseases (IRDs) are a group of rare diseases that lead to a progressive loss of photoreceptor cells and, ultimately, blindness. The overactivation of cGMP-dependent protein kinase G (PKG), one of the key effectors of cGMP-signaling, was previously found to be involved in photoreceptor cell death and was studied in murine IRD models to elucidate the pathophysiology of retinal degeneration. However, PKG is a serine/threonine kinase (STK) with several hundred potential phosphorylation targets and, so far, little is known about the specificity of the target interaction and downstream effects of PKG activation. Here, we carried out both the kinome activity and phosphoproteomic profiling of organotypic retinal explant cultures derived from the rd10 mouse model for IRD. After treating the explants with the PKG inhibitor CN03, an overall decrease in peptide phosphorylation was observed, with the most significant decrease occurring in seven peptides, including those from the known PKG substrate cyclic-AMP-response-element-binding CREB, but also Ca2+/calmodulin-dependent kinase (CaMK) peptides and TOP2A. The phosphoproteomic data, in turn, revealed proteins with decreased phosphorylation, as well as proteins with increased phosphorylation. The integration of both datasets identified common biological networks altered by PKG inhibition, which included kinases predominantly from the so-called AGC and CaMK families of kinases (e.g., PKG1, PKG2, PKA, CaMKs, RSKs, and AKTs). A pathway analysis confirmed the role of CREB, Calmodulin, mitogen-activated protein kinase (MAPK) and CREB modulation. Among the peptides and pathways that showed reduced phosphorylation activity, the substrates CREB, CaMK2, and CaMK4 were validated for their retinal localization and activity, using immunostaining and immunoblotting in the rd10 retina. In summary, the integrative analysis of the kinome activity and phosphoproteomic data revealed both known and novel PKG substrates in a murine IRD model. This data establishes a basis for an improved understanding of the biological pathways involved in cGMP-mediated photoreceptor degeneration. Moreover, validated PKG targets like CREB and CaMKs merit exploration as novel (surrogate) biomarkers to determine the effects of a clinical PKG-targeted treatment for IRDs. Full article
(This article belongs to the Special Issue Retinal Diseases: From Molecular Pathology to Therapies)
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17 pages, 3031 KiB  
Article
Sustained Extracellular Electrical Stimulation Modulates the Permeability of Gap Junctions in rd1 Mouse Retina with Photoreceptor Degeneration
by Sophie Stürmer, Sylvia Bolz, Eberhart Zrenner, Marius Ueffing and Wadood Haq
Int. J. Mol. Sci. 2024, 25(3), 1616; https://doi.org/10.3390/ijms25031616 - 28 Jan 2024
Viewed by 2015
Abstract
Neurons build vast gap junction-coupled networks (GJ-nets) that are permeable to ions or small molecules, enabling lateral signaling. Herein, we investigate (1) the effect of blinding diseases on GJ-nets in mouse retinas and (2) the impact of electrical stimulation on GJ permeability. GJ [...] Read more.
Neurons build vast gap junction-coupled networks (GJ-nets) that are permeable to ions or small molecules, enabling lateral signaling. Herein, we investigate (1) the effect of blinding diseases on GJ-nets in mouse retinas and (2) the impact of electrical stimulation on GJ permeability. GJ permeability was traced in the acute retinal explants of blind retinal degeneration 1 (rd1) mice using the GJ tracer neurobiotin. The tracer was introduced via the edge cut method into the GJ-net, and its spread was visualized in histological preparations (fluorescent tagged) using microscopy. Sustained stimulation was applied to modulate GJ permeability using a single large electrode. Our findings are: (1) The blind rd1 retinas displayed extensive intercellular coupling via open GJs. Three GJ-nets were identified: horizontal, amacrine, and ganglion cell networks. (2) Sustained stimulation significantly diminished the tracer spread through the GJs in all the cell layers, as occurs with pharmaceutical inhibition with carbenoxolone. We concluded that the GJ-nets of rd1 retinas remain coupled and functional after blinding disease and that their permeability is regulatable by sustained stimulation. These findings are essential for understanding molecular signaling in diseases over coupled networks and therapeutic approaches using electrical implants, such as eliciting visual sensations or suppressing cortical seizures. Full article
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21 pages, 83361 KiB  
Article
Subconfluent ARPE-19 Cells Display Mesenchymal Cell-State Characteristics and Behave like Fibroblasts, Rather Than Epithelial Cells, in Experimental HCMV Infection Studies
by Preethi Golconda, Mariana Andrade-Medina and Adam Oberstein
Viruses 2024, 16(1), 49; https://doi.org/10.3390/v16010049 - 28 Dec 2023
Cited by 5 | Viewed by 2670
Abstract
Human cytomegalovirus (HCMV) has a broad cellular tropism and epithelial cells are important physiological targets during infection. The retinal pigment epithelial cell line ARPE-19 has been used to model HCMV infection in epithelial cells for decades and remains a commonly used cell type [...] Read more.
Human cytomegalovirus (HCMV) has a broad cellular tropism and epithelial cells are important physiological targets during infection. The retinal pigment epithelial cell line ARPE-19 has been used to model HCMV infection in epithelial cells for decades and remains a commonly used cell type for studying viral entry, replication, and the cellular response to infection. We previously found that ARPE-19 cells, despite being derived from an epithelial cell explant, express extremely low levels of canonical epithelial proteins, such as E-cadherin and EpCAM. Here, we perform comparative studies of ARPE-19 and additional epithelial cell lines with strong epithelial characteristics. We find that ARPE-19 cells cultured under subconfluent conditions resemble mesenchymal fibroblasts, rather than epithelial cells; this is consistent with previous studies showing that ARPE-19 cultures require extended periods of high confluency culture to maintain epithelial characteristics. By reanalyzing public gene expression data and using machine learning, we find evidence that ARPE-19 cultures maintained across many labs exhibit mesenchymal characteristics and that the majority of studies employing ARPE-19 use them in a mesenchymal state. Lastly, by performing experimental HCMV infections across mesenchymal and epithelial cell lines, we find that ARPE-19 cells behave like mesenchymal fibroblasts, producing logarithmic yields of cell-free infectious progeny, while cell lines with strong epithelial character exhibit an atypical infectious cycle and naturally restrict the production of cell-free progeny. Our work highlights important characteristics of the ARPE-19 cell line and suggests that subconfluent ARPE-19 cells may not be optimal for modeling epithelial infection with HCMV or other human viruses. It also suggests that HCMV biosynthesis and/or spread may occur quite differently in epithelial cells compared to mesenchymal cells. These differences could contribute to viral persistence or pathogenesis in epithelial tissues. Full article
(This article belongs to the Special Issue 65-Year Anniversary of the Discovery of Cytomegalovirus)
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21 pages, 5906 KiB  
Article
The PKG Inhibitor CN238 Affords Functional Protection of Photoreceptors and Ganglion Cells against Retinal Degeneration
by Arianna Tolone, Wadood Haq, Alexandra Fachinger, Akanksha Roy, Sandeep Kesh, Andreas Rentsch, Sophie Wucherpfennig, Yu Zhu, John Groten, Frank Schwede, Tushar Tomar, Friedrich W. Herberg, Vasilica Nache and François Paquet-Durand
Int. J. Mol. Sci. 2023, 24(20), 15277; https://doi.org/10.3390/ijms242015277 - 17 Oct 2023
Cited by 8 | Viewed by 3060
Abstract
Hereditary retinal degeneration (RD) is often associated with excessive cGMP signalling in photoreceptors. Previous research has shown that inhibition of cGMP-dependent protein kinase G (PKG) can reduce photoreceptor loss in two different RD animal models. In this study, we identified a PKG inhibitor, [...] Read more.
Hereditary retinal degeneration (RD) is often associated with excessive cGMP signalling in photoreceptors. Previous research has shown that inhibition of cGMP-dependent protein kinase G (PKG) can reduce photoreceptor loss in two different RD animal models. In this study, we identified a PKG inhibitor, the cGMP analogue CN238, which preserved photoreceptor viability and functionality in rd1 and rd10 mutant mice. Surprisingly, in explanted retinae, CN238 also protected retinal ganglion cells from axotomy-induced retrograde degeneration and preserved their functionality. Furthermore, kinase activity-dependent protein phosphorylation of the PKG target Kv1.6 was reduced in CN238-treated rd10 retinal explants. Ca2+-imaging on rd10 acute retinal explants revealed delayed retinal ganglion cell repolarization with CN238 treatment, suggesting a PKG-dependent modulation of Kv1-channels. Together, these results highlight the strong neuroprotective capacity of PKG inhibitors for both photoreceptors and retinal ganglion cells, illustrating their broad potential for the treatment of retinal diseases and possibly neurodegenerative diseases in general. Full article
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19 pages, 2780 KiB  
Article
Artificial Vision: The High-Frequency Electrical Stimulation of the Blind Mouse Retina Decay Spike Generation and Electrogenically Clamped Intracellular Ca2+ at Elevated Levels
by Lucia Peiroten, Eberhart Zrenner and Wadood Haq
Bioengineering 2023, 10(10), 1208; https://doi.org/10.3390/bioengineering10101208 - 16 Oct 2023
Cited by 4 | Viewed by 2263
Abstract
Background: The electrical stimulation (stim) of retinal neurons enables blind patients to experience limited artificial vision. A rapid response outage of the stimulated ganglion cells (GCs) allows for a low visual sensation rate. Hence, to elucidate the underlying mechanism, we investigated different stim [...] Read more.
Background: The electrical stimulation (stim) of retinal neurons enables blind patients to experience limited artificial vision. A rapid response outage of the stimulated ganglion cells (GCs) allows for a low visual sensation rate. Hence, to elucidate the underlying mechanism, we investigated different stim parameters and the role of the neuromodulator calcium (Ca2+). Methods: Subretinal stim was applied on retinal explants (blind rd1 mouse) using multielectrode arrays (MEAs) or single metal electrodes, and the GC activity was recorded using Ca2+ imaging or MEA, respectively. Stim parameters, including voltage, phase polarity, and frequency, were investigated using specific blockers. Results: At lower stim frequencies (<5 Hz), GCs responded synaptically according to the stim pulses (stim: biphasic, cathodic-first, −1.6/+1.5 V). In contrast, higher stim frequencies (≥5 Hz) also activated GCs directly and induced a rapid GC spike response outage (<500 ms, MEA recordings), while in Ca2+ imaging at the same frequencies, increased intracellular Ca2+ levels were observed. Conclusions: Our study elucidated the mechanisms involved in stim-dependent GC spike response outage: sustained high-frequency stim-induced spike outage, accompanied by electrogenically clamped intracellular Ca2+ levels at elevated levels. These findings will guide future studies optimizing stim paradigms for electrical implant applications for interfacing neurons. Full article
(This article belongs to the Special Issue Pathophysiology and Translational Research of Retinal Diseases)
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17 pages, 3787 KiB  
Article
NMOSD IgG Impact Retinal Cells in Murine Retinal Explants
by Hannah Nora Wolf, Veronika Ehinger, Larissa Guempelein, Pratiti Banerjee, Tania Kuempfel, Joachim Havla and Diana Pauly
Curr. Issues Mol. Biol. 2023, 45(9), 7319-7335; https://doi.org/10.3390/cimb45090463 - 7 Sep 2023
Cited by 1 | Viewed by 1971
Abstract
Neuromyelitis optica spectrum disorders (NMOSD) are chronic inflammatory diseases of the central nervous system, characterized by autoantibodies against aquaporin-4. The symptoms primarily involve severe optic neuritis and longitudinally extensive transverse myelitis. Although the disease progression is typically relapse-dependent, recent studies revealed retinal neuroaxonal [...] Read more.
Neuromyelitis optica spectrum disorders (NMOSD) are chronic inflammatory diseases of the central nervous system, characterized by autoantibodies against aquaporin-4. The symptoms primarily involve severe optic neuritis and longitudinally extensive transverse myelitis. Although the disease progression is typically relapse-dependent, recent studies revealed retinal neuroaxonal degeneration unrelated to relapse activity, potentially due to anti-aquaporin-4-positive antibodies interacting with retinal glial cells such as Müller cells. In this exploratory study, we analysed the response of mouse retinal explants to NMOSD immunoglobulins (IgG). Mouse retinal explants were treated with purified IgG from patient or control sera for one and three days. We characterized tissue response patterns through morphological changes, chemokine secretion, and complement expression. Mouse retinal explants exhibited a basic proinflammatory response ex vivo, modified by IgG addition. NMOSD IgG, unlike control IgG, increased gliosis and decreased chemokine release (CCL2, CCL3, CCL4, and CXCL-10). Complement component expression by retinal cells remained unaltered by either IgG fraction. We conclude that human NMOSD IgG can possibly bind in the mouse retina, altering the local cellular environment. This intraretinal stress may contribute to retinal degeneration independent of relapse activity in NMOSD, suggesting a primary retinopathy. Full article
(This article belongs to the Special Issue Molecular Mechanism and Regulation in Neuroinflammation)
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23 pages, 4939 KiB  
Article
Glaucoma-Associated CDR1 Peptide Promotes RGC Survival in Retinal Explants through Molecular Interaction with Acidic Leucine Rich Nuclear Phosphoprotein 32A (ANP32A)
by Carsten Schmelter, Kristian Nzogang Fomo, Alina Brueck, Natarajan Perumal, Sascha D. Markowitsch, Gokul Govind, Thomas Speck, Norbert Pfeiffer and Franz H. Grus
Biomolecules 2023, 13(7), 1161; https://doi.org/10.3390/biom13071161 - 22 Jul 2023
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Abstract
Glaucoma is a complex, multifactorial optic neuropathy mainly characterized by the progressive loss of retinal ganglion cells (RGCs) and their axons, resulting in a decline of visual function. The pathogenic molecular mechanism of glaucoma is still not well understood, and therapeutic strategies specifically [...] Read more.
Glaucoma is a complex, multifactorial optic neuropathy mainly characterized by the progressive loss of retinal ganglion cells (RGCs) and their axons, resulting in a decline of visual function. The pathogenic molecular mechanism of glaucoma is still not well understood, and therapeutic strategies specifically addressing the neurodegenerative component of this ocular disease are urgently needed. Novel immunotherapeutics might overcome this problem by targeting specific molecular structures in the retina and providing direct neuroprotection via different modes of action. Within the scope of this research, the present study showed for the first time beneficial effects of the synthetic CDR1 peptide SCTGTSSDVGGYNYVSWYQ on the viability of RGCs ex vivo in a concentration-dependent manner compared to untreated control explants (CTRL, 50 µg/mL: p < 0.05 and 100 µg/mL: p < 0.001). Thereby, this specific peptide was identified first as a potential biomarker candidate in the serum of glaucoma patients and was significantly lower expressed in systemic IgG molecules compared to healthy control subjects. Furthermore, MS-based co-immunoprecipitation experiments confirmed the specific interaction of synthetic CDR1 with retinal acidic leucine-rich nuclear phosphoprotein 32A (ANP32A; p < 0.001 and log2 fold change > 3), which is a highly expressed protein in neurological tissues with multifactorial biological functions. In silico binding prediction analysis revealed the N-terminal leucine-rich repeat (LRR) domain of ANP32A as a significant binding site for synthetic CDR1, which was previously reported as an important docking site for protein-protein interactions (PPI). In accordance with these findings, quantitative proteomic analysis of the retinae ± CDR1 treatment resulted in the identification of 25 protein markers, which were significantly differentially distributed between both experimental groups (CTRL and CDR1, p < 0.05). Particularly, acetyl-CoA biosynthesis I-related enzymes (e.g., DLAT and PDHA1), as well as cytoskeleton-regulating proteins (e.g., MSN), were highly expressed by synthetic CDR1 treatment in the retina; on the contrary, direct ANP32A-interacting proteins (e.g., NME1 and PPP2R4), as well as neurodegenerative-related markers (e.g., CEND1), were identified with significant lower abundancy in the CDR1-treated retinae compared to CTRL. Furthermore, retinal protein phosphorylation and histone acetylation were also affected by synthetic CDR1, which are both partially controlled by ANP32A. In conclusion, the synthetic CDR1 peptide provides a great translational potential for the treatment of glaucoma in the future by eliciting its neuroprotective mechanism via specific interaction with ANP32A’s N terminal LRR domain. Full article
(This article belongs to the Special Issue Functional Peptides and Their Interactions)
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