Viability Of Retinal Ganglion Cells Research Articles

L-Serine Protects Murine Retinal Ganglion Cells from Oxidative Stress via Modulation of Mitochondrial Dysfunction

Purpose This study aimed to investigate the effects of l-serine on mitochondrial dysfunction in retinal ganglion cells after exposure to H2O2-induced oxidative stress. Methods Retinal ganglion cells obtained from C57BL6 mice (postnatal days 1–4) were purified and cultured. A cell viability assay was performed following exposure to H2O2-induced oxidative stress to assess the cytoprotective effects of l-serine on retinal ganglion cells. Flow cytometry with CellROX Deep Red and MitoSOX dyes was performed to analyze the cytoplasmic and mitochondrial reactive oxygen species levels, respectively. Staining with the fluorescent probe JC-1 was used to detect changes in the mitochondrial membrane potential. The oxygen consumption rate and Bioenergetic Health Index were used to evaluate mitochondrial respiration. Results H2O2 treatment was found to induce mitochondrial dysfunction in retinal ganglion cells. Pretreatment with l-serine prevented cytotoxicity and significantly increased the viability of retinal ganglion cells following exposure to H2O2-induced oxidative stress (p < .05). l-Serine alleviated reactive oxygen species production in retinal ganglion cells following exposure to H2O2-induced oxidative (p < .05). Further, it successfully mitigated H2O2-induced mitochondrial depolarization in retinal ganglion cells (p < .05) and significantly increased the oxygen consumption rate and Bioenergetic Health Index in retinal ganglion cells following exposure to H2O2-induced oxidative stress (p < .05). Conclusion Pretreatment with l-serine protected retinal ganglion cells from H2O2-induced oxidative stress by improving mitochondrial function. The findings of the present study suggest that l-serine is a potential candidate for treatment of reactive oxygen species-related ocular diseases such as mitochondrial optic neuropathies.

Molecular mechanism of high glucose-induced mitochondrial DNA damage in retinal ganglion cells.

Mitochondrial DNA damage in retinal ganglion cells (RGCs) may be closely related to lesions of glaucoma. RGCs were cultured with different concentrations of glucose and grouped into 3 groups, namely normal control (NC) group, Low-Glu group, and High-Glu group. Cell viability was measured with cell counting kit-8, and cell apoptosis was measured using flow cytometry. The DNA damage was measured with comet assay, and the morphological changes of damaged mitochondria in RGCs were observed using TEM. Western blot analyzed the expression of MRE11, RAD50, and NBS1 protein. Cell viability of RGCs in Low-Glu and High-Glu groups were lower than that of NC group in 48 and 96 h. The cell apoptosis in NC group was 4.9%, the Low-Glu group was 12.2% and High-Glu group was 24.4%. The comet imaging showed that NC cells did not have tailings, but the low-Glu and high-Glu group cells had tailings, indicating that the DNA of RGCs had been damaged. TEM, mitochondrial membrane potential, ROS, mitochondrial oxygen consumption, and ATP content detection results showed that RGCs cultured with high glucose occurred mitochondrial morphology changes and dysfunction. MRE11, RAD50, and NBS1 protein expression associated with DNA damage repair pathway in High-Glu group declined compared with Low-Glu group. Mitochondrial DNA damage caused by high glucose will result in apoptosis of retinal ganglion cells in glaucoma.

Rutin promotes M2 phenotype microglia polarization by suppressing the JAK/STAT3 signaling to protect against retinal ischemia-reperfusion injury.

We aimed to investigate the neuroprotective effect of rutin on retinal ganglion cells (RGCs) under ischemia-reperfusion (I/R) conditions and the underlying mechanisms involving microglia polarization and JAK/STAT3 signaling. RGCs isolated from C57/Bl6 mice were co-cultured with BV2 microglial cells under normal or in vitro oxygen-glucose deprivation and reoxygenation (OGD/R) conditions. Rutin's effects were evaluated by assessing cell viability, apoptosis rates, cytokine levels, microglial polarization markers and JAK/STAT3 phosphorylation levels. The specific target is confirmed through the inhibitory effect of rutin on the respectively activated signaling factors. Furthermore, molecular docking analyses elucidated rutin-JAK1 interactions. OGD/R conditions significantly reduced RGC viability, exacerbated by BV2 co-culture. However, both 1 μM and 5 μM rutin treatment dose-dependently enhanced RGC viability, reduced apoptosis, and suppressed pro-inflammatory cytokine levels. Western blot analysis indicated that rutin promoted the M2 microglial phenotype and suppressed JAK/STAT3 signaling. Notably, rutin selectively inhibited JAK1 phosphorylation without affecting STAT3. Molecular docking highlighted potential interaction sites between rutin and specific JAK1 pseudokinase domain. Rutin exerts neuroprotective effects against retinal I/R injury by promoting M2 microglial polarization, potentially through the selective inhibition of JAK1 phosphorylation within the JAK/STAT3 signaling pathway. These findings provide a foundation for the therapeutic potential of rutin in retinal I/R injuries.

The Inflammasome-Dependent Dysfunction and Death of Retinal Ganglion Cells after Repetitive Intraocular Pressure Spikes.

The dysfunction and selective loss of retinal ganglion cells (RGCs) is a known cause of vision loss in glaucoma and other neuropathies, where ocular hypertension (OHT) is the major risk factor. We investigated the impact of transient non-ischemic OHT spikes (spOHT) on RGC function and viability in vivo to identify cellular pathways linking low-grade repetitive mechanical stress to RGC pathology. We found that repetitive spOHT had an unexpectedly high impact on intraocular homeostasis and RGC viability, while exposure to steady OHT (stOHT) of a similar intensity and duration failed to induce pathology. The repetitive spOHT induced the rapid activation of the inflammasome, marked by the upregulation of NLRP1, NLRP3, AIM2, caspases -1, -3/7, -8, and Gasdermin D (GSDMD), and the release of interleukin-1β (IL-1β) and other cytokines into the vitreous. Similar effects were also detected after 5 weeks of exposure to chronic OHT in an induced glaucoma model. The onset of these immune responses in both spOHT and glaucoma models preceded a 50% deficit in pattern electroretinogram (PERG) amplitude and a significant loss of RGCs 7 days post-injury. The inactivation of inflammasome complexes in Nlrp1-/-, Casp1-/-, and GsdmD-/- knockout animals significantly suppressed the spOHT-induced inflammatory response and protected RGCs. Our results demonstrate that mechanical stress produced by acute repetitive spOHT or chronic OHT is mechanistically linked to inflammasome activation, which leads to RGC dysfunction and death.

MicroRNA-93 promotes the pathogenesis of glaucoma by inhibiting matrix metalloproteinases as well as up-regulating extracellular matrix and Rho/ROCK signaling pathways

ObjectiveTo investigate the effect and potential molecular mechanism of microRNA-93 (miR-93) on retinal ganglion cells (RGCs) apoptosis as well as retinal damage in acute glaucoma mice. MethodsRGCs apoptosis were induced by oxygen-glucose deprivation and reperfusion (OGD/R). The pro-apoptotic effect of miR-93 was evaluated by transfecting miR-93 mimics or miR-93 inhibitor into OGD/R-induced RGCs. The viability and apoptosis of RGCs were determined by MTT assay and flow cytometry. Mouse model of acute glaucoma were successfully induced via high intraocular pressure (IOP), and then these model animals were randomly divided into vehicle group, miR-93 mimics group or miR-93 inhibitor group (n = 10), using healthy mice as normal control. Histopathologic changes of retinal tissue were evaluated by Hematoxylin and Eosin (H&E) staining method. Moreover, cell counts of retinal ganglion cell layer and mean thickness of different layers were also determined. Quantitative real-time PCR (qPCR) and western blotting analysis were used to detect the mRNA and protein expression levels of extracellular matrix (ECM), matrix metalloproteinases (MMPs) and Rho/ROCK signaling pathway. ResultsmiR-93 mimics significantly decreased or promoted the viability and apoptosis of OGD/R-induced RGCs, respectively. In addition, miR-93 mimics significantly exacerbated the degree of retinal tissue damage in mice with acute glaucoma, which was accompanied by a decrease in the number of ganglion cell layer (GCL) cells and the thickness of different tissue layers. Moreover, miR-93 mimics significantly increased IOP in mice with acute glaucoma. Significantly, miR-93 inhibitors significantly reversed the above changes. In addition, results of Western blot analysis showed that miR-93 mimics increased and decreased the expression of ECM-associated and MMP-associated proteins, respectively, by activating the Rho/ROCK signaling pathway. In contrast, miR-93 significantly decreased and increased the expression of ECM-associated and MMP-associated proteins, and suppressed the expression of Rho/ROCK signaling pathway-related proteins. ConclusionmiR-93 can promote the development of glaucoma by activating Rho/ROCK signaling pathway to mediate the accumulation of ECM-related proteins as well as the down-regulation of MMP-related proteins.

Pyrroloquinoline quinone drives ATP synthesis in vitro and in vivo and provides retinal ganglion cell neuroprotection

Retinal ganglion cells are highly metabolically active requiring strictly regulated metabolism and functional mitochondria to keep ATP levels in physiological range. Imbalances in metabolism and mitochondrial mechanisms can be sufficient to induce a depletion of ATP, thus altering retinal ganglion cell viability and increasing cell susceptibility to death under stress. Altered metabolism and mitochondrial abnormalities have been demonstrated early in many optic neuropathies, including glaucoma, autosomal dominant optic atrophy, and Leber hereditary optic neuropathy. Pyrroloquinoline quinone (PQQ) is a quinone cofactor and is reported to have numerous effects on cellular and mitochondrial metabolism. However, the reported effects are highly context-dependent, indicating the need to study the mechanism of PQQ in specific systems. We investigated whether PQQ had a neuroprotective effect under different retinal ganglion cell stresses and assessed the effect of PQQ on metabolic and mitochondrial processes in cortical neuron and retinal ganglion cell specific contexts. We demonstrated that PQQ is neuroprotective in two models of retinal ganglion cell degeneration. We identified an increased ATP content in healthy retinal ganglion cell-related contexts both in in vitro and in vivo models. Although PQQ administration resulted in a moderate effect on mitochondrial biogenesis and content, a metabolic variation in non-diseased retinal ganglion cell-related tissues was identified after PQQ treatment. These results suggest the potential of PQQ as a novel neuroprotectant against retinal ganglion cell death.

Glaucoma-Associated CDR1 Peptide Promotes RGC Survival in Retinal Explants through Molecular Interaction with Acidic Leucine Rich Nuclear Phosphoprotein 32A (ANP32A).

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.

Urine-derived mesenchymal stem cells-derived exosomes enhances survival and proliferation of aging retinal ganglion cells

ObjectivesThis study was designed to investigate to test the effect of exosomes from urine-derived mesenchymal stem cells (USCs) on the survival and viability of aging retinal ganglion cells (RGCs), and explored the preliminary related mechanisms.MethodsPrimary USCs were cultured and identified by immunofluorescence staining. Aging RGCs models were established by D-galactose treatment and identified by β-Galactosidase staining. After treatment with USCs conditioned medium (with USCs removal), flow cytometry was performed to examine the apoptosis and cell cycle of RGCs. Cell viability of RGCs was detected by Cell-counting Kit 8 (CCK8) assay. Moreover, gene sequencing and bioinformatics analysis were applied to analyze the genetic variation after medium treatment in RGCs along with the biological functions of differentially expressed genes (DEGs).ResultsThe number of apoptotic aging RGCs was significantly reduced in USCs medium-treated RGCs. Besides, USCs-derived exosomes exert significant promotion on the cell viability and proliferation of aging RGCs. Further, sequencing data analyzed and identified DEGs expressed in aging RGCs and aging RGCs treated with USCs conditioned medium. The sequencing outcomes demonstrated 117 upregulated genes and 186 downregulated genes in normal RGCs group vs aging RGCs group, 137 upregulated ones and 517 downregulated ones in aging RGCs group vs aging RGCs + USCs medium group. These DEGs involves in numerous positive molecular activities to promote the recovery of RGCs function.ConclusionsCollectively, the therapeutic potentials of USCs-derived exosomes include suppression on cell apoptosis, enhancement on cell viability and proliferation of aging RGCs. The underlying mechanism involves multiple genetic variation and changes of transduction signaling pathways.

Improving adeno-associated viral (AAV) vector-mediated transgene expression in retinal ganglion cells: comparison of five promoters

Recombinant adeno-associated viral vectors (AAVs) are an effective system for gene transfer. AAV serotype 2 (AAV2) is commonly used to deliver transgenes to retinal ganglion cells (RGCs) via intravitreal injection. The AAV serotype however is not the only factor contributing to the effectiveness of gene therapies. Promoters influence the strength and cell-selectivity of transgene expression. This study compares five promoters designed to maximise AAV2 cargo space for gene delivery: chicken β-actin (CBA), cytomegalovirus (CMV), short CMV early enhancer/chicken β-actin/short β-globulin intron (sCAG), mouse phosphoglycerate kinase (PGK), and human synapsin (SYN). The promoters driving enhanced green fluorescent protein (eGFP) were examined in adult C57BL/6J mice eyes and tissues of the visual system. eGFP expression was strongest in the retina, optic nerves and brain when driven by the sCAG and SYN promoters. CBA, CMV, and PGK had moderate expression by comparison. The SYN promoter had almost exclusive transgene expression in RGCs. The PGK promoter had predominant expression in both RGCs and AII amacrine cells. The ubiquitous CBA, CMV, and sCAG promoters expressed eGFP in a variety of cell types across multiple retinal layers including Müller glia and astrocytes. We also found that these promoters could transduce human retina ex vivo, although expression was predominantly in glial cells due to low RGC viability. Taken together, this promoter comparison study contributes to optimising AAV-mediated transduction in the retina, and could be valuable for research in ocular disorders, particularly those with large or complex genetic cargos.

Investigating the potential neuroprotective effect of semaglutide in the mouse retina

AbstractPurpose: To investigate the potential locally mediated neuroprotective effect of the glucagon‐like peptide 1 receptor agonist, semaglutide, in the mouse retina, including an assessment of the underlying mechanisms of action.Methods: The study was conducted using surgically isolated retinas from female C57BL/6jRj mice as ex vivo model system. Retinas were exposed to different concentrations (10–100 nM) of semaglutide for different exposure durations (2–48 h). Retinal explant viability was assessed employing the lactate dehydrogenase release assay. The number of retinal ganglion cells (RGCs) was quantified employing immunohistochemistry. The acute and intermediate effect of semaglutide on the ex vivo retinal glucose metabolism under euglycemic conditions was assessed through metabolic mapping employing [U‐13C]glucose, followed by relative analyte quantification employing gas chromatography–mass spectrometry. The influence of semaglutide on the retinal mitochondrial function was assessed by employing the Seahorse assay on isolated retinal mitochondria.Results: Semaglutide exerted no effect on the retinal explant or RGC viability. Semaglutide may acutely potentiate the retinal ex vivo glucose metabolism/mitochondrial function indicated by statistically significant glucose hypermetabolism (increased 13C‐enrichment) of certain tricarboxylic acid intermediates (citrate and α‐ketoglutarate) relative to the control. Results on the effect of semaglutide on the mitochondrial function/oxygen consumption rate are not yet available.Conclusions: Under the experimental conditions, semaglutide did not influence the retinal explant or RGC‐specific viability. Semaglutide may acutely potentiate the retinal ex vivo glucose metabolism by enhancing the mitochondrial function. Additional studies are needed for clarification.

Novel insights into neuronal and vascular damage glaucoma

AbstractDespite decades of clinical and basic research, we still do not understand the factors that cause retinal ganglion cell death and loss of vision in glaucoma patients. This keynote lecture will provide insights into new mechanisms that lead to early neuronal and neurovascular dysregulation in glaucoma. Several pathological changes associated with retinal ganglion cell damage will be discussed including dendritic retraction and synapse loss, metabolic deficits, and loss of neurovascular coupling. The role of pericytes in retinal capillary function will be presented, including newly identified inter‐pericyte tunnelling nanotubes, and their impact on retinal ganglion cell viability. These new data will highlight therapeutic opportunities to alleviate key pathological processes and restore visual function in glaucoma and other optic neuropathies.

Viability of mitochondria-labeled retinal ganglion cells in organotypic retinal explant cultures by two methods

Retinal explant cultures provide a valuable system to study retinal function in vitro. This study established a new retinal explant culture method to prolong the survival of retinal ganglion cells (RGCs). Explants were prepared in two different ways: with or without optic nerve. Retinas from newborn mice that had received an injection of MitoTracker Red into the contralateral superior colliculus to label axonal mitochondria were cultured as organotypic culture for 7 days in vitro. At several time points during the culture, viability of RGCs was assessed by multi-electrode array recording, and morphology by immunohistochemical methods. During the culture, the thickness of the retinal tissue in both groups gradually decreased, however, the structure of the layers of the retina could be identified. Massive apoptosis in the retinal ganglion cell layer (GCL) appeared on the first day of culture, thereafter the number of apoptotic cells decreased. Glial activation was observed throughout the culture, and there was no difference in morphology between the two groups. RGCs loss was exacerbated on 3rdday of culture, and RGCs loss in retinal explants with preserved optic nerve was significantly lower than in retinas that did not preserve the optic nerve. More and longer-lasting mitochondrial signals were observed in the injured area of the optic nerve-preserving explants. Retinal explants provide an invaluable tool for studying retinal function and developing treatments for ocular diseases. The optic nerve-preserving culture helps preserve the integrity of RGCs. The higher number of mitochondria in the nerve-preserving cultures may help maintain viability of RGCs.

Pregabalin Mediates Retinal Ganglion Cell Survival From Retinal Ischemia/Reperfusion Injury Via the Akt/GSK3β/β-Catenin Signaling Pathway.

Progressive retinal ganglion cell (RGC) loss induced by retinal ischemia/reperfusion (RIR) injury leads to irreversible visual impairment. Pregabalin (PGB) is a promising drug for neurodegenerative diseases. However, with regard to RGC survival, its specific role and exact mechanism after RIR injury remain unclear. In this study, we sought to investigate whether PGB could protect RGCs from mitochondria-related apoptosis induced by RIR and explore the possible mechanisms. C57BL/6J mice and primary RGCs were pretreated with PGB prior to ischemia/reperfusion modeling. The retinal structure and cell morphology were assessed by immunochemical assays and optical coherence tomography. CCK8 was used to assay cell viability, and an electroretinogram was performed to detect RGC function. Mitochondrial damage was assessed by a reactive oxygen species (ROS) assay kit and transmission electron microscopy. Western blot and immunofluorescence assays quantified the expression of proteins associated with the Akt/GSK3β/β-catenin pathway. Treatment with PGB increased the viability of RGCs in vitro. Consistently, PGB preserved the normal thickness of the retina, upregulated Bcl-2, reduced the ratio of cleaved caspase-3/caspase-3 and the expression of Bax in vivo. Meanwhile, PGB improved mitochondrial structure and prevented excessive ROS production. Moreover, PGB restored the amplitudes of oscillatory potentials and photopic negative responses following RIR. The mechanisms underlying its neuroprotective effects were attributed to upregulation of the Akt/GSK3β/β-catenin pathway. However, PGB-mediated neuroprotection was suppressed when using MK2206 (an Akt inhibitor), whereas it was preserved when treated with TWS119 (a GSK3β inhibitor). PGB exerts a protective effect against RGC apoptosis induced by RIR injury, mediated by the Akt/GSK3β/β-catenin pathway.

Synthetic antibody-derived immunopeptide provides neuroprotection in glaucoma through molecular interaction with retinal protein histone H3.1.

Glaucoma is a group of optic neuropathies characterized by the progressive degeneration of retinal ganglion cells (RGCs) as well as their axons leading to irreversible loss of sight. Medical management of the intraocular pressure (IOP) still represents the gold standard in glaucoma therapy, which only manages a single risk factor and does not directly address the neurodegenerative component of this eye disease. Recently, our group showed that antibody-derived immunopeptides (encoding complementarity-determining regions, CDRs) provide attractive glaucoma medication candidates and directly interfere its pathogenic mechanisms by different modes of action. In accordance with these findings, the present study showed the synthetic complementary-determining region 2 (CDR2) peptide (INSDGSSTSYADSVK) significantly increased RGC viability in vitro in a concentration-dependent manner (p < 0.05 using a CDR2 concentration of 50 μg/mL). Employing state-of the-art immunoprecipitation experiments, we confirmed that synthetic CDR2 exhibited a high affinity toward the retinal target protein histone H3.1 (HIST1H3A) (p < 0.001 and log2-fold change > 3). Furthermore, molecular dynamics (MD) simulations along with virtual docking analyses predicted potential CDR2-specific binding regions of HIST1H3A, which might represent essential post-translational modification (PTM) sites for epigenetic regulations. Quantitative mass spectrometry (MS) analysis of retinas demonstrated 39 proteins significantly affected by CDR2 treatment (p < 0.05). An up-regulation of proteins involved in the energy production (e.g., ATP5F1B and MT-CO2) as well as the regulatory ubiquitin proteasome system (e.g., PSMC5) was induced by the synthetic CDR2 peptide. On the other hand, CDR2 reduced metabolic key enzymes (e.g., DDAH1 and MAOB) as well as ER stress-related proteins (e.g., SEC22B and VCP) and these data were partially confirmed by microarray technology. Our outcome measurements indicate that specific protein-peptide interactions influence the regulatory epigenetic function of HIST1H3A promoting the neuroprotective mechanism on RGCs in vitro. In addition to IOP management, such synthetic peptides as CDR2 might serve as a synergistic immunotherapy for glaucoma in the future.

Tongqiao Mingmu formula alleviates retinal ganglion cell autophagy through PI3K/AKT/mTOR pathway.

Glaucoma is a severe blindness-causing optic nerve disease characterized by a loss of retinal ganglion cells (RGCs). Previous studies have shown that the Tongqiao Mingmu (TQMM) formula can reduce retinal and optic nerve damage, but its mechanism of action requires further elucidation. In this study, an RGC injury model was prepared using glutamate and then treated with serum-containing drug from the TQMM formula (hereafter called "TQMM formula serum"). In the glutamate-induced RGC injury model, cell viability decreased with an increase in glutamate concentration, whereas the expression of autophagy-related biomarkers LC3 and Belicin-1 increased. An adenovirus transfection experiment revealed that glutamate markedly promoted autophagic flux in RGCs. Notably, TQMM formula serum inhibited the expression of autophagy-related biomarkers, reduced autophagy flux, and reversed the damage caused by glutamate to RGCs. Furthermore, the PI3K inhibitor LY294002 was used to intervene in the RGC autophagy model and was found to suppress the PI3K/AKT/mTOR pathway and enhance RGC autophagy. However, TQMM formula serum could generate an opposite effect and upregulate the expressions of the PI3K/AKT/mTOR pathway genes and proteins. In conclusion, the TQMM formula can prevent glutamate-induced autophagy in RGCs, possibly by activating the PI3K/AKT/mTOR pathway and reducing the expression of autophagy-related biomarkers LC3 and Belicin-1 to attenuate autophagy and maintain RGC viability.

MTORC1 regulates high levels of protein synthesis in retinal ganglion cells of adult mice

Mechanistic target of rapamycin (mTOR) and mTOR complex 1 (mTORC1), linchpins of the nutrient sensing and protein synthesis pathways, are present at relatively high levels in the ganglion cell layer (GCL) and retinal ganglion cells (RGCs) of rodent and human retinas. However, the role of mTORCs in the control of protein synthesis in RGC is unknown. Here, we applied the SUrface SEnsing of Translation (SUnSET) method of nascent protein labeling to localize and quantify protein synthesis in the retinas of adult mice. We also used intravitreal injection of an adeno-associated virus 2 vector encoding Cre recombinase in the eyes of mtor- or rptor-floxed mice to conditionally knockout either both mTORCs or only mTORC1, respectively, in cells within the GCL. A novel vector encoding an inactive Cre mutant (CreΔC) served as control. We found that retinal protein synthesis was highest in the GCL, particularly in RGC. Negation of both complexes or only mTORC1 significantly reduced protein synthesis in RGC. In addition, loss of mTORC1 function caused a significant reduction in the pan-RGC marker, RNA-binding protein with multiple splicing, with little decrease of the total number of cells in the RGC layer, even at 25 weeks after adeno-associated virus-Cre injection. These findings reveal that mTORC1 signaling is necessary for maintaining the high rate of protein synthesis in RGCs of adult rodents, but it may not be essential to maintain RGC viability. These findings may also be relevant to understanding the pathophysiology of RGC disorders, including glaucoma, diabetic retinopathy, and optic neuropathies.

Acteoside attenuates hydrogen peroxide-induced injury of retinal ganglion cells via the CASC2/miR-155/mTOR axis.

BackgroundLoss of retinal ganglion cells (RGCs), which eventually leads to optic nerve atrophy and vision loss, is the main cause of glaucoma and traumatic optic neuropathy. Acteoside is the effective component of Yunnan Kudingcha, which has been reported to exert neuroprotective effects and protects RGCs from injury. However, the underlying mechanisms of acteoside in RGC injury remain largely elusive.MethodsHuman RGCs was treated with hydrogen peroxide (H2O2). The expression of miR-155 and lncRNA CASC2 in RGC-5 cells was measured by RT-qPCR. The viability of RGCs was determined by the MTT assay. Flow cytometry and TUNEL staining were used to detect cell apoptosis. The malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were determined using ELISA kits. The mTOR and autophagic proteins were measured by western blot.ResultsWe identified the expression of miR-155 was upregulated in H2O2-treated RGCs, and enhanced miR-155 promoted RGC autophagy and apoptosis. Acteoside administration reduced miR-155 expression and abolished miR-155-mediated RGC injury. The expression of CASC2 was decreased in H2O2-treated RGCs. Acteoside administration could increase CASC2 expression and CASC2 overexpression reverses the effect of miR-155 overexpression on acteoside treatment-RGCs. Mechanistically, we discovered that highly expressed miR-155 promoted RGC autophagy and apoptosis via the mTOR pathway. In addition, acteoside attenuated RGC autophagy and apoptosis via the miR-155/mTOR axis. Together, these results identify a mechanism by which acteoside attenuates H2O2-induced RGC apoptosis and autophagy via the CASC2/miR-155/mTOR axis.ConclusionsActeoside protects RGC-5 cells against H2O2-induced cell injury via the CASC2/miR-155/mTOR axis. These results provide new insights for early medical interventions in patients with glaucoma.

MicroRNA-145-5p targeting of TRIM2 mediates the apoptosis of retinal ganglion cells via the PI3K/AKT signaling pathway in glaucoma.

There is accumulating evidence to suggest that microRNAs (miRNAs) are associated with the progressive optic neuropathy including glaucoma. Apoptosis of retinal ganglion cells (RGCs) is a hallmark of glaucoma. The present study focused on the effects of miR-145-5p on RGC apoptosis in glaucoma. We established a glaucoma rat model by intraocular injection of N-methyl-d-aspartic acid (NMDA). RGCs were isolated from newborn rats and treated with NMDA. Hematoxylin and eosin staining was performed to detect morphological changes in the retinas of rats. The expression of miR-145-5p and tripartite motif-containing 2 (TRIM2) in RGCs was measured by RT-qPCR. The viability of RGCs was measured by MTT assay. Flow cytometry analysis and TUNEL assays were conducted to assess the apoptosis of RGCs. The interaction between miR-145-5p and TRIM2 was investigated using a luciferase reporter assay. Rats injected with NMDA showed a thinner ganglion cell layer (GCL) and inner plexiform layer (IPL) as well as increased expression of miR-145-5p. Silencing of miR-145-5p significantly increased the GCL and IPL in the glaucoma rat model. Moreover, miR-145-5p expression was upregulated in RGCs ex vivo in response to NMDA. Silencing of miR-145-5p promoted cell viability and suppressed apoptosis in NMDA-treated RGCs. Mechanistically, miR-145-5p targeted the TRIM2 3' untranslated region to suppress its expression. TRIM2 was upregulated in NMDA-treated RGCs and protected RGCs against NMDA-induced apoptosis. Furthermore, miR-145-5p suppressed the PI3K/AKT pathway by downregulating TRIM2 in NMDA-treated RGCs. Suppression of miR-145-5p inhibited the apoptosis of RGCs via TRIM2-mediated activation of the PI3K/AKT signaling pathway in NMDA-induced glaucoma.

Carbon Nanotube Polymer Scaffolds as a Conductive Alternative for the Construction of Retinal Sheet Tissue.

With the great success of graphene in the biomedical field, carbon nanotubes have attracted increasing attention for different applications in ophthalmology. Here, we report a novel retinal sheet composed of carbon nanotubes (CNTs) and poly(lactic-co-glycolic acid) (PLGA) that can enhance retinal cell therapy. By tuning our CNTs to regulate the mechanical characteristics of retina sheets, we were able to improve the in vitro viability of retinal ganglion cells derived from human-induced pluripotent stem cells incorporated into CNTs. Engrafted retinal ganglion cells displayed signs of regenerating processes along the optic nerve. Compared with PLGA scaffolds, CNT-PLGA retinal sheet tissue has excellent electrical conductivity, biocompatibility, and biodegradation. This new biomaterial offers new insight into retinal injury, repair, and regeneration.

Age Related Response of Neonatal Rat Retinal Ganglion Cells to Reduced TrkB Signaling in vitro and in vivo.

During development of retinofugal pathways there is naturally occurring cell death of at least 50% of retinal ganglion cells (RGCs). In rats, RGC death occurs over a protracted pre- and early postnatal period, the timing linked to the onset of axonal ingrowth into central visual targets. Gene expression studies suggest that developing RGCs switch from local to target-derived neurotrophic support during this innervation phase. Here we investigated, in vitro and in vivo, how RGC birthdate affects the timing of the transition from intra-retinal to target-derived neurotrophin dependence. RGCs were pre-labeled with 5-Bromo-2′-Deoxyuridine (BrdU) at embryonic (E) day 15 or 18. For in vitro studies, RGCs were purified from postnatal day 1 (P1) rat pups and cultured with or without: (i) brain derived neurotrophic factor (BDNF), (ii) blocking antibodies to BDNF and neurotrophin 4/5 (NT-4/5), or (iii) a tropomyosin receptor kinase B fusion protein (TrkB-Fc). RGC viability was quantified 24 and 48 h after plating. By 48 h, the survival of purified βIII-tubulin immunopositive E15 but not E18 RGCs was dependent on addition of BDNF to the culture medium. For E18 RGCs, in the absence of exogenous BDNF, addition of blocking antibodies or TrkB-Fc reduced RGC viability at both 24 and 48 h by 25–40%. While this decrease was not significant due to high variance, importantly, each blocking method also consistently reduced complex process expression in surviving RGCs. In vivo, survival of BrdU and Brn3a co-labeled E15 or E18 RGCs was quantified in rats 24 h after P1 or P5 injection into the eye or contralateral superior colliculus (SC) of BDNF and NT-4/5 antibodies, or serum vehicle. The density of E15 RGCs 24 h after P1 or P5 injection of blocking antibodies was reduced after SC but not intraretinal injection. Antibody injections into either site had little obvious impact on viability of the substantially smaller population of E18 RGCs. In summary, most early postnatal RGC death in the rat involves the elimination of early-born RGCs with their survival primarily dependent upon the availability of target derived BDNF during this time. In contrast, late-born RGC survival may be influenced by additional factors, suggesting an association between RGC birthdate and developmental death mechanisms.