R28 Cells Research Articles

The role of nicotinamide riboside in the preservation of retinal ganglion cells using an in vitro glutamate-induced excitotoxicity model

Delaying or preventing the loss of retinal ganglion cells (RGCs) in glaucoma is needed for vision preservation. Glutamate-mediated neurotoxicity arises from the excessive stimulation of N-methyl-D-aspartate membrane receptors by glutamate. This overstimulation, occurring specifically in RGCs, triggers a progressive deterioration of the optic nerve that ultimately leads to the vision loss in glaucoma. Our previous investigation demonstrated that nicotinamide riboside (NR) effectively preserved RGCs in multiple mouse models of glaucoma. To investigate the precise role of NR concerning RGCs which remains uncertain, a glutamate-induced excitotoxicity RGCs damage model was established using R28 cells in this study. Results showed that NR treatment could not only prevent the decrease in cell viability but also effectively inhibit the apoptosis of R28 cells induced by glutamate, as proven by flow cytometry and expression of key pro-apoptotic proteins. Additionally, it significantly attenuated oxidative stress induced by glutamate, as evaluated by the production of inflammatory factors, reactive oxygen species (ROS) and mitochondrial ROS (mtROS). Furthermore, NR elevated the intracellular nicotinamide adenine dinucleotide (NAD+) levels in R28 cells. Lastly, we used RNA-seq to reveal the underlying mechanism of NR protection. Combining the results of RNA-seq and Western blot, we found that NR also restored the decreased protein expression of sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor-gamma coactivator (PGC1α) induced by glutamate. These findings strongly indicated that NR exhibits a protective effect against R28 cell apoptosis in a glutamate-induced excitotoxicity RGCs damage model. This protective effect is likely mediated through the activation of the SIRT1/PGC1α pathway, achieved by increasing intracellular NAD + levels.

Intravitreal indocyanine green is toxic to the retinal cells

BackgroundIndocyanine green (ICG) is widely used to stain the epiretinal membranes and internal limiting membranes during the pars plana vitrectomy (PPV). This study aims to evaluate the effect of ICG on rat retinas and various retinal cell lines, including ARPE-19 cells, rMC-1 cells, BV2 cells, HRMECs and R28 cells. MethodsICG solutions were prepared and diluted with glucose solution (GS) according to the standard clinical protocols. The retinal cell lines, including ARPE-19 cells, rMC-1 cells, BV2 cells, HRMECs and R28 cells, were treated with the following solutions: normal glucose (NG, 5 mM), GS-1 (92.5 mM glucose), GS-2 (185.02 mM glucose), ICG-1 (92.5 mM glucose + 0.43 mM ICG), or ICG-2 (185.02 mM glucose + 0.86 mM ICG) for durations of 15 or 30 min. In vivo, the right eyes of the rats were intravitreally injected with ICG-1 or ICG-2 (2 μL), while the left eyes were intravitreally injected with GS-1 or GS-2, served as the osmotic controls, for 30 min or 60 min. The rats intravitreally injected with an equivalent volume of NG or 1x phosphate-buffered saline (1x PBS) were served as the normal control or vehicle control. The cell viability was measured with the Cell Counting Kit-8 (CCK-8), while the cell death in retinal cryosections was detected with the TUNEL assay. ResultsThe viabilities of the different retinal cell lines involved in this study were significantly reduced by both ICG-1 and ICG-2 treatments at both time points, with ICG-2 resulting in lower cell viability compared to the NG group and the osmotic control group. Additionally, GS-2 treatment also exhibited a decrease in retinal cell viabilities in vitro. To further confirm these results, intravitreal injection of ICG induced more apoptotic cell death in rat retinas as evidenced by the TUNEL assay. ConclusionsThe exposure of ICG leads to an augmented retinal cell death, which is directly proportional to the concentration and duration of exposure, both in vivo and in vitro. Caution should be exercised during vitrectomy procedures involving ICG administration during clinical practice. It is recommended to advocate for lower concentrations of ICG with reduced exposure time during ocular surgeries.

Self-Assembly Hypoxic and ROS Dual Response Nano Prodrug as a New Therapeutic Approach for Glaucoma Treatments.

Glaucoma is an irreversible blinding eye disease characterized by retinal ganglion cell (RGC) death.Previous studies have demonstrated that protecting mitochondria and activating the CaMKII/CREB signaling pathway can effectively protect RGC and axon. However, currently treatments are often unsatisfactory, and the pathogenesis of glaucoma requires further elucidation. In this study, a ROS-responsive dual drug conjugate (OLN monomer) is first designed that simultaneously bonds nicotinamide and oleic acid. The conjugate self-assembled into nanoparticles (uhOLN-NPs) through the aggregation of multiple micelles and possesses ROS scavenging capability. Then, a polymer with a hypoxic response function is designed, which encapsulates uhOLN-NPs to form nanoparticles with hypoxic and ROS responses (HOLN-NPs). Under hypoxia in RGCs, the azo bond of HOLN-NPs breaks and releases uhOLN-NPs. Meanwhile, under high ROS conditions, the thioketone bond broke, leading to the dissociation of nano-prodrug. The released nicotinamide and oleic acid co-scavenge ROS and activate the CaMKII/CREB pathway, protecting mitochondria in RGCs. HOLN-NPs exhibit a significantly superior protective effect on R28 cells in glutamate models of glaucoma. The accumulation of HOLN-NPs in retinal RGCs lead to significant inhibition of RGC apoptosis and axonal damage in vivo. Notably, HOLN-NPs provide a new therapeutic approach for patients with neurodegenerative disease.

Pigment Epithelium-Derived Factor Binding to VEGFR-1 (Flt-1) Increases the Survival of Retinal Neurons.

The purpose of this study was to examine possible involvement of vascular endothelial growth factor (VEGF) receptor (VEGFR)-1/Flt-1 in pigment epithelium-derived factor (PEDF)-promoted survival of retinal neurons. Survival of growth factor-deprived retinal ganglion cells (RGCs) and R28 cells and activation of ERK-1/-2 MAP kinases were assessed in the presence of PEDF, placental growth factor (PlGF), and VEGF using cell cultures, viability assays and quantitation of ERK-1/-2 phosphorylation. VEGFR-1/Flt-1 expression was determined using quantitative PCR (qPCR) and Western blotting. VEGFR-1/Flt-1 was knocked down in R28 cells by small interfering RNA (siRNA). Binding of a PEDF-IgG Fc fusion protein (PEDF-Fc) to retinal neurons, immobilized VEGFR-1/Flt-1 and VEGFR-1/Flt-1-derived peptides was studied using binding assays and peptide scanning. PEDF in combination with PlGF stimulated increased cell survival and ERK-1/-2 MAP kinase activation compared to effects of either factor alone. VEGFR-1/Flt-1 expression in RGCs and R28 cells was significantly upregulated by hypoxia, VEGF, and PEDF. VEGFR-1/Flt-1 ligands (VEGF and PlGF) or soluble VEGFR-1 (sflt-1) competed with PEDF-Fc for binding to R28 cells. Depleting R28 cells of VEGFR-1/Flt-1 resulted in reduced PEDF-Fc binding when comparing VEGFR-1/Flt-1 siRNA- and control siRNA-treated cells. PEDF-Fc interacted with immobilized sflt-1, which was specifically blocked by VEGF and PlGF. PEDF-Fc binding sites were mapped to VEGFR-1/Flt-1 extracellular domains D3 and D4. Peptides corresponding to D3 and D4 specifically inhibited PEDF-Fc binding to R28 cells. These peptides and sflt-1 significantly inhibited PEDF-promoted survival of R28 cells. These results suggest that PEDF can target VEGFR-1/Flt-1 and this interaction plays a significant role in PEDF-mediated neuroprotection in the retina.

Neuroretinal Cell Culture Model as a Tool for the Development of New Therapeutic Approaches for Oxidative Stress-Induced Ocular Diseases, with a Focus on Glaucoma.

Glaucoma is a heterogeneous group of optic neuropathies characterized by a progressive degeneration of the retinal ganglion cells (RGCs), leading to irreversible vision loss. Nowadays, the traditional therapeutic approach to glaucoma consists of lowering the intraocular pressure (IOP), which does not address the neurodegenerative features of the disease. Besides animal models of glaucoma, there is a considerable need for in vitro experimental models to propose new therapeutic strategies for this ocular disease. In this study, we elucidated the pathological mechanisms leading to neuroretinal R28 cell death after exposure to glutamate and hydrogen peroxide (H2O2) in order to develop new therapeutic approaches for oxidative stress-induced retinal diseases, including glaucoma. We were able to show that glutamate and H2O2 can induce a decrease in R28 cell viability in a concentration-dependent manner. A cell viability of about 42% was found after exposure to 3 mM of glutamate and about 56% after exposure to 100 µM of H2O2 (n = 4). Label-free quantitative mass spectrometry analysis revealed differential alterations of 193 and 311 proteins in R28 cells exposed to 3 mM of glutamate and 100 µM of H2O2, respectively (FDR < 1%; p < 0.05). Bioinformatics analysis indicated that the protein changes were associated with the dysregulation of signaling pathways, which was similar to those observed in glaucoma. Thus, the proteomic alteration induced by glutamate was associated with the inhibition of the PI3K/AKT signaling pathway. On the other hand, H2O2-induced toxicity in R28 cells was linked to the activation of apoptosis signaling and the inhibition of the mTOR and ERK/MAPK signaling pathways. Furthermore, the data show a similarity in the inhibition of the EIF2 and AMPK signaling pathways and the activation of the sumoylation and WNT/β-catenin signaling pathways in both groups. Our findings suggest that the exposure of R28 cells to glutamate and H2O2 could induce glaucoma-like neurodegenerative features and potentially provide a suitable tool for the development of new therapeutic strategies for retinal diseases.

Geniposide Attenuates Oxygen-glucose Deprivation/Recovery-induced Retinal Ganglion Cell Injury via Akt/Nrf-2 Signaling Pathway.

Glaucoma is an eye disease. Its pathological process involves retinal ischemia-reperfusion (I/R), which causes irreversible blindness in patients. Geniposide (Gen), a bioactive iridoid glycoside extracted from the fruit of gardenia, exhibits many biological effects, such as anti-oxidative stress, anti-inflammation, anti-apoptosis, anti-endoplasmic reticulum stress, and anti-thrombotic effects. However, its therapeutic potential for the retinal I/R injury remains unclear. This study investigated the protective effect of Gen against I/R injury by inhibiting abnormal reactive oxygen species (ROS) and retinal neuron apoptosis. We used oxygen-glucose deprivation/reoxygenation (OGD/R) to induce R28 cells to mimic the pathological process of I/R in glaucoma. We conducted CCK-8 analysis and TUNEL staining to examine cell proliferation and apoptosis in glaucoma. Western blotting was used to assay the expressions of apoptosis and Akt/Nrf-2 pathway-related proteins. The production of ROS was detected by using the corresponding kit. Cell viability decreased, whereas TUNEL staining-positive cells and ROS production increased after the OGD/R injury. The contents of cleaved caspase-3 and Bax/Bcl-2 increased after the OGD/R injury. Treatment with 200 μM of Gen effectively improved the cell viability and suppressed cell apoptosis and ROS production. In addition, Gen could significantly promote the activation of the Akt/Nrf-2 signaling pathway in R28 cells, which was blocked by the inhibition of Akt/Nrf-2. We in vivo verified the neuroprotective effect of Gen by establishing an acute high intraocular pressure (aHIOP) model and obtained similar results to those of the in vitro experimental results. Hence, it can be suggested that Gen provides neuroprotection against the OGD/R-induced injury of R28 cells by activating the Akt/Nrf-2 signaling pathway, which is beneficial for the clinical treatment of glaucoma.

Neuroprotective and anti-inflammatory effects of eicosane on glutamate and NMDA-induced retinal ganglion cell injury.

To investigate the protective effects, antioxidant potential, and anti-inflammatory mechanisms of eicosane on glutamate-induced cell damage and on N-methyl-D-aspartate (NMDA)-induced retinal ganglion cell (RGC) injury in a mouse model of glaucoma. The protective effects of eicosane on the rat R28 retinal precursor cell line were assessed using cell counting kit-8 assays and Hoechst-propidium iodide staining. Intracellular reactive oxygen species (ROS) production was measured using the fluorescent probe 2'-7'-dichlorofluorescin diacetate and flow cytometry. The protective role of eicosane on NMDA-induced RGC injury in a mouse glaucoma model was determined by immunostaining of frozen sections of retina. The effects of eicosane on the metabolome of the retina in mice with NMDA-induced RGC damage were evaluated by liquid chromatography-mass spectroscopy (LC-MS) and untargeted metabolomics analyses. Eicosane treatment significantly attenuated glutamate-induced damage to R28 cells in vitro. Eicosane also protected RGCs against NMDA-induced injury in a mouse glaucoma model. Untargeted metabolomics analyses showed that eicosane increased multiple metabolites, including L-arginine and L-carnitine, in the retina. Eicosane has protective effects, antioxidant potential, and anti-inflammatory properties in an in vitro model of glutamate-induced cell damage and in an in vivo model of NMDA-induced RGC injury in mouse glaucoma through modulation of L-arginine and/or L-carnitine metabolism.

Neuroprotective and Anti-Inflammatory Activities of Hybrid Small-Molecule SA-10 in Ischemia/Reperfusion-Induced Retinal Neuronal Injury Models.

To induce I/R injury and inflammation, we subjected R28 and primary microglial cells to oxygen glucose deprivation (OGD) for 6 h in vitro or treated these cells with a cocktail of TNF-α, IL-1β and IFN-γ for 1 h, followed by the addition of SA-10 (10 µM). Inhibition of microglial activation, ROS scavenging, cytoprotective and anti-inflammatory activities were measured. In vivo I/R-injured mouse retinas were treated with either PBS or SA-10 (2%) intravitreally, and pattern electroretinogram (ERG), spectral-domain optical coherence tomography, flash ERG and retinal immunocytochemistry were performed. SA-10 significantly inhibited microglial activation and inflammation in vitro. Compared to the control, the compound SA-10 significantly attenuated cell death in both microglia (43% vs. 13%) and R28 cells (52% vs. 17%), decreased ROS (38% vs. 68%) production in retinal microglia cells, preserved neural retinal function and increased SOD1 in mouse eyes. SA-10 is protective to retinal neurons by decreasing oxidative stress and inflammatory cytokines.

Ripa-56 protects retinal ganglion cells in glutamate-induced retinal excitotoxic model of glaucoma

Glaucoma is a prevalent cause of blindness globally, characterized by the progressive degeneration of retinal ganglion cells (RGCs). Among various factors, glutamate excitotoxicity stands out as a significant contributor of RGCs loss in glaucoma. Our study focused on Ripa-56 and its protective effect against NMDA-induced retinal damage in mice, aiming to delve into the potential underlying mechanism. The R28 cells were categorized into four groups: glutamate (Glu), Glu + Ripa-56, Ripa-56 and Control group. After 24 h of treatment, cell death was assessed by PI / Hoechst staining. Mitochondrial membrane potential changes, apoptosis and reactive oxygen species (ROS) production were analyzed using flow cytometry. The alterations in the expression of RIP-1, p-MLKL, Bcl-2, BAX, Caspase-3, Gpx4 and SLC7A11 were examined using western blot analysis. C57BL/6j mice were randomly divided into NMDA, NMDA + Ripa-56, Ripa-56 and control groups. Histological changes in the retina were evaluated using hematoxylin and eosin (H&E) staining. RGCs survival and the protein expression changes of RIP-1, Caspase-3, Bcl-2, Gpx4 and SLC7A11 were observed using immunofluorescence. Ripa-56 exhibited a significant reduction in the levels of RIP-1, p-MLKL, Caspase-3, and BAX induced by glutamate, while promoting the expression of Bcl-2, Gpx-4, and SLC7A1 in the Ripa-56-treated group. In our study, using an NMDA-induced normal tension glaucoma mice model, we employed immunofluorescence and H&E staining to observe that Ripa-56 treatment effectively ameliorated retinal ganglion cell loss, mitigating the decrease in retinal ganglion cell layer and bipolar cell layer thickness caused by NMDA. In this study, we have observed that Ripa-56 possesses remarkable anti- necroptotic, anti-apoptotic and anti-ferroptosis properties. It demonstrates the ability to combat not only glutamate-induced excitotoxicity in R28 cells, but also NMDA-induced retinal excitotoxicity in mice. Therefore, Ripa-56 could be used as a potential retinal protective agent.

Role of Interleukin-21 in retinal ischemia-reperfusion injury: Unveiling the impact on retinal ganglion cell apoptosis

BackgroundRetinal ischemia–reperfusion (I/R) serves as a significant contributor to ocular diseases, triggering a cascade of pathological processes. The interplay between neuroinflammation and the apoptosis of retinal ganglion cell (RGC) is a well-explored aspect of retinal I/R-induced tissue damage. Within this intricate landscape, the inflammatory cytokine Interleukin-21 (IL21) emerges as a potent mediator of neuroinflammation with known detrimental effects on neuronal integrity. However, its specific impact on RGC apoptosis in the context of retinal I/R has remains to be uncovered. This study aims to unravel the potential anti-apoptotic effects of IL21 siRNA on RGC, shedding light on the neuroprotection of retinal I/R. MethodsSprague-Dawley (SD) rats underwent a controlled elevation of intraocular pressure (IOP) to 110 mmHg for 60 min to simulate retinal I/R conditions. To explore the influence of IL21 on RGC apoptosis and its underlying molecular mechanisms, a comprehensive array of techniques such immunohistochemistry, immunofluorescence, TUNEL, Hematoxylin-eosin (H&E), immunoblotting, and qRT-PCR were carried out. ResultsThe landscape of retinal I/R injury revealed an increase in the expression of IL21, reaching its peak at 72 h. Notably, IL21 markedly induced RGC apoptosis within the retinal I/R milieu. The introduction of IL21 siRNA showed promising outcomes, manifesting as an amelioration of neurological function deficits, a reduction in RGC loss, and an increase in the thickness of the inner retinal layer at the 72-hour reperfusion. Additionally, IL21 siRNA demonstrated its ability to hinder the release of proteins associated with apoptosis via the JAK/STAT signaling pathway. In the in vitro setting, IL21 siRNA efficiently reduced R28 cell apoptosis by suppressing the production of proteins associated with apoptosis by regulating the JAK/STAT signaling pathway. ConclusionsThis study provides evidence for the pathogenic role of IL21 in retinal I/R. The findings underscore IL21 siRNA as a promising therapeutic target for ischemic retinal injury. Its efficacy lies in its ability to mitigate RGC apoptosis by suppressing the JAK/STAT signaling pathway. These findings not only enhance our comprehension of retinal I/R pathology but also suggests IL21 siRNA as a potential transformative factor in the development of targeted therapies for ischemic retinal injuries.

P38 MAPK inhibitor SB202190 suppresses ferroptosis in the glutamate-induced retinal excitotoxicity glaucoma model.

JOURNAL/nrgr/04.03/01300535-202410000-00031/figure1/v/2024-02-06T055622Z/r/image-tiff Glutamate excitotoxicity has been shown to play an important role in glaucoma, and glutamate can induce ferroptosis. The p38 mitogen-activated protein kinase (MAPK) pathway inhibitor SB202190 has a potential ability to suppress ferroptosis, and its downstream targets, such as p53, have been shown to be associated with ferroptosis. However, whether ferroptosis also occurs in retinal ganglion cells in response to glutamate excitotoxicity and whether inhibition of ferroptosis reduces the loss of retinal ganglion cells induced by glutamate excitotoxicity remain unclear. This study investigated ferroptosis in a glutamate-induced glaucoma rat model and explored the effects and molecular mechanisms of SB202190 on retinal ganglion cells. A glutamate-induced excitotoxicity model in R28 cells and an N-methyl-D-aspartate-induced glaucoma model in rats were used. In vitro experiments showed that glutamate induced the accumulation of iron and lipid peroxide and morphological changes of mitochondria in R28 cells, and SB202190 inhibited these changes. Glutamate induced the levels of p-p38 MAPK/p38 MAPK and SAT1 and decreased the expression levels of ferritin light chain, SLC7A11, and GPX4. SB202190 inhibited the expression of iron death-related proteins induced by glutamate. In vivo experiments showed that SB202190 attenuated N-methyl-D-aspartate-induced damage to rat retinal ganglion cells and improved visual function. These results suggest that SB202190 can inhibit ferroptosis and protect retinal ganglion cells by regulating ferritin light chain, SAT1, and SLC7A11/Gpx4 pathways and may represent a potential retina protectant.

Calbindin 2-specific deletion of arginase 2 preserves visual function after optic nerve crush

We previously found that global deletion of the mitochondrial enzyme arginase 2 (A2) limits optic nerve crush (ONC)-induced neuronal death. Herein, we examined the cell-specific role of A2 in this pathology by studies using wild type (WT), neuronal-specific calbindin 2 A2 KO (Calb2cre/+ A2 f/f), myeloid-specific A2 KO (LysMcre/+ A2f/f), endothelial-specific A2 KO (Cdh5cre/+ A2f/f), and floxed controls. We also examined the impact of A2 overexpression on mitochondrial function in retinal neuronal R28 cells. Immunolabeling showed increased A2 expression in ganglion cell layer (GCL) neurons of WT mice within 6 h-post injury and inner retinal neurons after 7 days. Calb2 A2 KO mice showed improved neuronal survival, decreased TUNEL-positive neurons, and improved retinal function compared to floxed littermates. Neuronal loss was unchanged by A2 deletion in myeloid or endothelial cells. We also found increased expression of neurotrophins (BDNF, FGF2) and improved survival signaling (pAKT, pERK1/2) in Calb2 A2 KO retinas within 24-hour post-ONC along with suppression of inflammatory mediators (IL1β, TNFα, IL6, and iNOS) and apoptotic markers (cleavage of caspase3 and PARP). ONC increased GFAP and Iba1 immunostaining in floxed controls, and Calb2 A2 KO dampened this effect. Overexpression of A2 in R28 cells increased Drp1 expression, and decreased mitochondrial respiration, whereas ABH-induced inhibition of A2 decreased Drp1 expression and improved mitochondrial respiration. Finally, A2 overexpression or excitotoxic treatment with glutamate significantly impaired mitochondrial function in R28 cells as shown by significant reductions in basal respiration, maximal respiration, and ATP production. Further, glutamate treatment of A2 overexpressing cells did not induce further deterioration in their mitochondrial function, indicating that A2 overexpression or glutamate insult induce comparable alterations in mitochondrial function. Our data indicate that neuronal A2 expression is neurotoxic after injury, and A2 deletion in Calb2 expressing neurons limits ONC-induced retinal neurodegeneration and improves visual function.

Unveiling Neuroprotection and Regeneration Mechanisms in Optic Nerve Injury: Insight from Neural Progenitor Cell Therapy with Focus on Vps35 and Syntaxin12.

Axonal degeneration resulting from optic nerve damage can lead to the progressive death of retinal ganglion cells (RGCs), culminating in irreversible vision loss. We contrasted two methods for inducing optic nerve damage: optic nerve compression (ONCo) and optic nerve crush (ONCr). These were assessed for their respective merits in simulating traumatic optic neuropathies and neurodegeneration. We also administered neural progenitor cells (NPCs) into the subtenon space to validate their potential in mitigating optic nerve damage. Our findings indicate that both ONCo and ONCr successfully induced optic nerve damage, as shown by increases in ischemia and expression of genes linked to neuronal regeneration. Post NPC injection, recovery in the expression of neuronal regeneration-related genes was more pronounced in the ONCo model than in the ONCr model, while inflammation-related gene expression saw a better recovery in ONCr. In addition, the proteomic analysis of R28 cells in hypoxic conditions identified Vps35 and Syntaxin12 genes. Vps35 preserved the mitochondrial function in ONCo, while Syntaxin12 appeared to restrain inflammation via the Wnt/β-catenin signaling pathway in ONCr. NPCs managed to restore damaged RGCs by elevating neuroprotection factors and controlling inflammation through mitochondrial homeostasis and Wnt/β-catenin signaling in hypoxia-injured R28 cells and in both animal models. Our results suggest that ischemic injury and crush injury cause optic nerve damage via different mechanisms, which can be effectively simulated using ONCo and ONCr, respectively. Moreover, cell-based therapies such as NPCs may offer promising avenues for treating various optic neuropathies, including ischemic and crush injuries.

Metformin protects against retinal ischemia/reperfusion injury through AMPK-mediated mitochondrial fusion

Retinal ischemia/reperfusion (I/R) injury is a common pathological process responsible for cellular damage in glaucoma, diabetic retinopathy and hypertensive retinopathy. Metformin is a biguanide drug that exerts strong effects on multiple diseases. This study aims to evaluate the protective effect of metformin against retinal I/R injury and its underlying mechanism. I/R induced reduction in retina thickness and cell number in ganglion cell layer, and metformin alleviated I/R-induced retinal injury. Both retinal I/R and simulated ischemia/reperfusion (SIR) in R28 cells down-regulated expression of mitochondrial fusion protein Mfn2 and OPA1, which led to mitochondrial fission. Metformin also alleviated damage in R28 cells, and reversed the alteration in Mfn2 and OPA1, mitochondrial fission and mitochondrial membrane potential (MMP) disruption-induced by I/R or SIR as well. Intriguingly, inhibition of AMPK by compound C or siRNA prevented metformin-mediated up-regulation of Mfn2 and OPA1. Compound C and knockdown of Mfn2 or OPA1 dramatically alleviated the protective effect of metformin against intracellular ROS generation, MMP disruption, mitochondrial fission and loss of RGCs in ganglion cell layer induced by SIR or I/R. Moreover, scavenging mitochondrial ROS (mito-ROS) by mito-TEMPO exerted the similar protection against I/R-induced retinal injury or SIR-induced damage in R28 cells as metformin. Our data show for the first time that metformin protects against retinal I/R injury through AMPK-mediated mitochondrial fusion and the decreased mito-ROS generation. These findings might also repurpose metformin as a therapeutic agent for retinal I/R injury.

Integrated analysis of long non-coding RNAs and mRNAs associated with glaucoma in vitro.

In recent years, the biological functions and important roles of long non-coding RNAs (lncRNAs) have been widely reported in many diseases. Although glaucoma is the leading cause of blindness worldwide, the specific mechanisms of lncRNAs in the pathogenesis and progression of glaucoma remain unclear. Our research aims to elucidate the differentially expressed lncRNAs and mRNAs in glaucoma and to provide a basis for further exploration of the specific mechanism of action of lncRNAs in the progression of glaucoma. We performed RNA sequencing on samples from a pressurized model of R28 cells and performed bioinformatics analyses on the sequencing results. The expression consistency of lncRNAs in clinical samples from patients with glaucoma or cataracts was detected using real-time quantitative polymerase chain reaction (RT-qPCR). RNA sequencing results showed that lncRNAs in cluster 5 were upregulated with increasing stress after typing all significantly altered lncRNAs using k-means in a cellular stress model. KEGG analysis indicated that they were associated with neurodegenerative diseases. Differentially expressed lncRNAs were verified by RT-qPCR, and the lncRNA expression levels of AC120246.2 and XLOC_006247 were significantly higher in the aqueous humor (AH) of patients with glaucoma than in those with cataracts. For LOC102551819, there was almost no expression in the AH and trabecular meshwork in patients with glaucoma but high expression was observed in the iris. Our research proposes potential diagnostic or intervention targets for clinical applications as well as a theoretical basis for more in-depth research on the function of lncRNAs in glaucoma.

DTX3L induced NLRP3 ubiquitination inhibit R28 cell pyroptosis in OGD/R injury

Ischemia/reperfusion (I/R) injury is one of the most common etiologies in many diseases. Retinal I/R leads to cytokine storm, resulting in tissue damage and cell death. Pyroptosis, a novel type of regulated cell death, occurs after cellular I/R injury. In this study, we established an oxygen glucose deprivation (OGD/R) cellular model (R28) to simulate retinal I/R injury. We conducted an LDH assay, and EthD-III and PI staining procedures to confirm pyroptosis. Mass spectrometry and bioinformatics analysis were used to identify the possible proteins interacting with NLRP3. Co-IP and various molecular biology techniques were used to investigate the possible modes regulating NLRP3 by DTX3L. EthD-III, PI staining and LDH assays demonstrated pyroptosis induced by OGD/R injury, mediated via NLRP3 pathway. Mass spectrometry and bioinformatics analysis screened out three candidate proteins interacting with NLRP3, and further Co-IP experiment indicated that DTX-3L may interact with NLRP3 to regulate its protein levels after injury. Co-IP experiments and various molecular biology methods demonstrated that DTX3L ubiquitinates NLRP3 resulting in pyroptosis after R28 OGD/R injury. Further, NLRP3 LRR and DTX3L RING domains interact with each other. Our study demonstrated that DTX3L may ubiquitinate NLRP3 to regulate OGD/R-induced pyroptosis globally in R28 cells.

Inhibition of mitochondrial VDAC1 oligomerization alleviates apoptosis and necroptosis of retinal neurons following OGD/R injury

Ischemia-reperfusion (I/R) injury is a common pathological mechanism in many retinal diseases, which can lead to cell death via mitochondrial dysfunction. Voltage-dependent anion channel 1 (VDAC1), which is mainly located in the outer mitochondrial membrane, is the gatekeeper of mitochondria. The permeability of mitochondrial membrane can be regulated by controlling the oligomerization of VDAC1. However, the functional mechanism of VDAC1 in retinal I/R injury was unclear. Our results demonstrate that oxygen-glucose deprivation and re-oxygenation (OGD/R) injury leads to apoptosis, necroptosis, and mitochondrial dysfunction of R28 cells. The OGD/R injury increases the levels of VDAC1 oligomerization. Inhibition of VDAC1 oligomerization by VBIT-12 rescued mitochondrial dysfunction by OGD/R and also reduced apoptosis/necroptosis of R28 cells. In vivo, the use of VBIT-12 significantly reduced aHIOP-induced neuronal death (apoptosis/necroptosis) in the rat retina. Our findings indicate that VDAC1 oligomers may open and enlarge mitochondrial membrane pores during OGD/R injury, leading to the release of death-related factors in mitochondria, resulting in apoptosis and necroptosis. This study provides a potential therapeutic strategy against ocular diseases caused by I/R injury.

Mitochondria-associated endoplasmic reticulum membranes dysfunction contributes to PARP-1-dependent cell death under oxidative stress in retinal precursor cells.

Persistent poly (ADP-ribose) polymerase 1 (PARP-1) activation has proven detrimental and can lead to PARP-1-dependent cell death. Mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) serve as essential hubs for many biological pathways, such as autophagy and mitochondria fission and fusion. This study aimed to alleviate the effects of hydrogen peroxide (H2 O2 )-induced persistent PARP-1 activation and MAM dysregulation by the usage of a PARP-1 inhibitor. Results showed that receptor-interacting protein kinase (RIPK) 1 inhibitor (necrostatin-1) and PARP-1 inhibitor (olaparib) protected retinal precursor cells from H2 O2 -induced death, while a pan-caspase inhibitor (Z-VAD-FMK) failed to protect R28 cells. Olaparib also alleviated H2 O2 -induced MAM dysregulation, as evidenced by decreased VDAC1/ITPR3 interactions and reduced mitochondrial membrane potential collapse. Additionally, olaparib also inhibited H2 O2 -induced autophagy. Inhibiting autophagic flux increased MAM signaling under both normal and oxidative conditions. Furthermore, H2 O2 treatment caused a reduction in the protein level of mitofusin-2 (MFN2) in a dose- and time-dependent manner. Mfn2 knockdown was found to further magnify MAM dysregulation and mitochondrial dysfunction under normal and oxidative conditions. Mfn2 overexpression surprisingly enhanced H2 O2 -induced MAM signaling and failed to rescue H2 O2 -induced mitochondrial dysfunction. These results indicate that MAMs probably serve as a membrane source for oxidative stress-associated autophagy. MAM dysregulation also contributed to H2 O2 -induced PARP-1-dependent cell death. However, more studies are required to decipher the link between the modulation of Mfn2 expression, changes in MAM integrity, and alterations in mitochondrial performances.

Autophagy and senescence of rat retinal precursor cells under high glucose.

Diabetic retinopathy (DR) is a common diabetic ocular disease characterized by retinal ganglion cell (RGC) changes. An abnormal environment, hyperglycemia, may progressively alter the structure and function of RGCs, which is a primary pathological feature of retinal neurodegeneration in DR. Accumulated studies confirmed autophagy and senescence play a vital role in DR; however, the underlying mechanisms need to be clarified. This study included the microarray expression profiling dataset GSE60436 from Gene Expression Omnibus (GEO) to conduct the bioinformatics analysis. The R software was used to identify autophagy-related genes (ARGs) that were differentially expressed in fibrovascular membranes (FVMs) and normal retinas. Co-expression and tissue-specific expression were elicited for the filtered genes. The genes were then analyzed by ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and Gene Set Enrichment Analysis (GSEA). R28 cells were cultured with high glucose, detected by reverse transcription-quantitative (RT-qPCR) and stained by apoptosis kit. In the retina, 31 differentially expressed ARGs (24 up-regulated genes) were discovered and enriched. The enrichment results revealed that differentially expressed ARGs were significantly enriched in autophagy, apoptosis, aging, and neural function. Four hub genes (i.e., TP53, CASP1, CCL2, and CASP1) were significantly up-regulated. Upregulation of cellular autophagy and apoptosis level was detected in the hyperglycemia model in vitro. Our results provide evidence for the autophagy and cellular senescence mechanisms involved in retinal hyperglycemia injury, and the protective function of autophagy is limited. Further study may favour understanding the disease progression and neuroprotection of DR.

Adipose mesenchymal stem cell-derived extracellular vesicles reduce glutamate-induced excitotoxicity in the retina.

Adipose mesenchymal stem cells (ADSCs) have protective effects against glutamate-induced excitotoxicity, but ADSCs are limited in use for treatment of optic nerve injury. Studies have shown that the extracellular vesicles (EVs) secreted by ADSCs (ADSC-EVs) not only have the function of ADSCs, but also have unique advantages including non-immunogenicity, low probability of abnormal growth, and easy access to target cells. In the present study, we showed that intravitreal injection of ADSC-EVs substantially reduced glutamate-induced damage to retinal morphology and electroretinography. In addition, R28 cell pretreatment with ADSC-EVs before injury inhibited glutamate-induced overload of intracellular calcium, downregulation of α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor (AMPAR) subunit GluA2, and phosphorylation of GluA2 and protein kinase C alpha in vitro. A protein kinase C alpha agonist, 12-O-tetradecanoylphorbol 13-acetate, inhibited the neuroprotective effects of ADSC-EVs on glutamate-induced R28 cells. These findings suggest that ADSC-EVs ameliorate glutamate-induced excitotoxicity in the retina through inhibiting protein kinase C alpha activation.