GSH-Independent Induction of ER Stress during Hypoglycaemia in the Retinal Cells of Mice.
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ABSTRACT:
SUBMITTER: Fresia D
PROVIDER: S-EPMC8201117 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Hyperhomocysteinemia is implicated in retinal neurovascular diseases including arterial occlusive disease, venous occlusive disease and pseudoexfoliation glaucoma. The mechanism for these diseases is not known. Here we used hyperhomocysteinemic mice lacking the gene encoding cystathionine-beta-synthase (cbs-/-) to examine whether ER stress could be a mechanism for the retinal neurovasculopathy reported in these mice. Retinas of cbs+/+ and cbs-/- mice (age: 3-5 wks) were used to investigate the expression of ER stress genes (BiP/GRP78, Perk, Atf6, Atf4, Ire1?, Chop) and the proteins they encode. The levels of poly(ADP-ribose) polymerase (PARP) and cleaved cysteine-aspartic proteases-3 (caspase-3), proteins known to be involved in apoptosis, were also examined. Quantitative reverse transcription polymerase chain reaction and western blotting revealed an increase in BiP/GRP78 and PERK in retinas of cbs-/- mice compared with cbs+/+ mice. There was an elevation of CCAAT-enhancer-binding protein homologous protein (CHOP) in retinal cryosections of cbs-/- mice indicating apoptosis, which was confirmed by increased levels of PARP and cleaved caspase-3. The data suggest that the genes and proteins that are major players in the ER stress pathway, particularly the PERK pathway, are upregulated in retinas of cbs-/- mice. The data support a role for ER stress in the pathophysiology associated with the hyperhomocysteinemia-linked retinal disease.
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Project description:Age-related macular degeneration (AMD) is a degenerative disease of the retina and the leading cause of blindness in the elderly. Retinal pigment epithelial (RPE) cell death and the resultant photoreceptor apoptosis are characteristic of late-stage dry AMD, especially geographic atrophy (GA). Although oxidative stress and inflammation have been associated with GA, the nature and underlying mechanism for RPE cell death remains controversial, which hinders the development of targeted therapy for dry AMD. The purpose of this study is to systematically dissect the mechanism of RPE cell death induced by oxidative stress. Our results show that characteristic features of apoptosis, including DNA fragmentation, caspase 3 activation, chromatin condensation and apoptotic body formation, were not observed during RPE cell death induced by either hydrogen peroxide or tert-Butyl hydroperoxide. Instead, this kind of cell death can be prevented by RIP kinase inhibitors necrostatins but not caspase inhibitor z-VAD, suggesting necrotic feature of RPE cell death. Moreover, ATP depletion, receptor interacting protein kinase 3 (RIPK3) aggregation, nuclear and plasma membrane leakage and breakdown, which are the cardinal features of necrosis, were observed in RPE cells upon oxidative stress. Silencing of RIPK3, a key protein in necrosis, largely prevented oxidative stress-induced RPE death. The necrotic nature of RPE death is consistent with the release of nuclear protein high mobility group protein B1 into the cytoplasm and cell medium, which induces the expression of inflammatory gene TNF? in healthy RPE and THP-1 cells. Interestingly, features of pyroptosis or autophagy were not observed in oxidative stress-treated RPE cells. Our results unequivocally show that necrosis, but not apoptosis, is a major type of cell death in RPE cells in response to oxidative stress. This suggests that preventing oxidative stress-induced necrotic RPE death may be a viable approach for late-stage dry AMD.
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Project description:BackgroundWe aimed to determine sphingolipid levels and examine apoptotic pathways in human retinal pigment epithelial cells (ARPE-19) undergoing endoplasmic reticulum (ER) stress.MethodsCells were treated with tunicamycin (TM) to induce ER stress and tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor, was administered to decrease cytotoxicity. Cell viability was measured by MTT assay. Levels of C16-C24 sphingomyelins (SM) and C16-C24 ceramides (CERs) were determined by LC-MS/MS. Glucose-regulated protein 78-kd (GRP78) and nuclear factor kappa-b subunit 1 (NFκB1) gene expressions were evaluated by quantitative PCR analysis, while GRP 78, NF-κB p65, cleaved caspase-3 and caspase-12 protein levels were assesed by immunofluorescence. Ceramide-1-phosphate (C1P) levels were determined by immunoassay, while caspase -3 and -12 activity in cell lysates were measured via a fluorometric method.ResultsInduction of ER stress in TM treated groups were confirmed by significantly increased mRNA and protein levels of GRP78. TM significantly decreased cell viability compared to controls. Treatment with TUDCA along with TM significantly increased cell viability compared to the TM group. A significant increase was observed in C22-C24 CERs, C1P, caspase-3, caspase-12, NFκB1 mRNA and NF-κB p65 protein levels in cells treated with TM compared to controls. Administration of TUDCA lead to a partial decrease in GRP78 expression, NFκB1 mRNA, NF-κB p65 protein, C22-C24 CERs and C1P levels along with a decrease in caspase-3 and -12 activity.ConclusionsThe results of this study reveal the presence of increased long chain CERs, C1P and apoptotic markers in retinal cells undergoing ER stress.
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