Oxidative stress induces lysosomal membrane permeabilization and ceramide accumulation in retinal pigment epithelial cells
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ABSTRACT:
PROVIDER: PRJNA994552 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR25268744.fastq.gz | Fastqsanger.gz | |||
| SRR25268745.fastq.gz | Fastqsanger.gz | |||
| SRR25268746.fastq.gz | Fastqsanger.gz | |||
| SRR25268747.fastq.gz | Fastqsanger.gz | |||
| SRR25268748.fastq.gz | Fastqsanger.gz |
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Project description:Oxidative stress has been implicated in the pathogenesis of age-related macular degeneration, the leading cause of blindness in older adults, with retinal pigment epithelium (RPE) cells playing a key role. To better understand the cytotoxic mechanisms underlying oxidative stress, we used a mouse model of iron overload, as iron can catalyze reactive oxygen species formation in the RPE. In a liver-specific Hepc (Hamp) knockout murine model of systemic iron overload, RPE cells accumulated lipid peroxidation adducts and lysosomes, developed progressive hypertrophy, and underwent cell death. Proteomic and lipidomic analyses revealed accumulation of lysosomal proteins, ceramide biosynthetic enzymes, and ceramides. The proteolytic enzyme cathepsin D had impaired maturation. A large proportion of lysosomes were galectin-3 positive, suggesting cytotoxic lysosomal membrane permeabilization. RNA sequencing was performed and did not show evidence of upregulation of CLEAR network genes, suggesting that the lysosomal accumulation was due to decreased lysosomal turnover and not increased lysosomal biogenesis. Collectively, these results demonstrate that iron overload induces lysosomal accumulation and impairs lysosomal function, likely owing to iron-induced lipid peroxides that can inhibit lysosomal enzymes.
2023-07-13 | GSE237309 | GEO
Project description:Oxidative stress has been implicated in the pathogenesis of age-related macular degeneration(AMD), the leading cause of blindness in the elderly, with retinal pigment epithelial (RPE) cells playing a key role. To better understand the cytotoxic mechanisms underlying oxidative stress, we used cell culture and mouse models of iron overload, as iron can catalyze reactive oxygen species formation in the RPE. Iron-loading of cultured iPS-RPE cells increased lysosomal abundance, impaired proteolysis, and reduced the activity of a subset of lysosomal enzymes,including lysosomal acid lipase and acid sphingomyelinase. In a murine model of systemiciron overload, RPE cells accumulated lipid peroxidation adducts and lysosomes, developed progressive hypertrophy, and underwent cell death. Proteomic and lipidomic analyses revealed accumulation of lysosomal proteins, ceramide biosynthetic enzymes, and ceramides. The proteolytic enzyme cathepsin D had impaired maturation. A large proportion oflysosomes were galectin-3 positive, suggesting cytotoxic lysosomal membrane permeabilization (LMP). Collectively, these results demonstrate that iron overload induces lysosomal accumulation and impairs lysosomal function, likely due to iron-induced lipid peroxides that can inhibit lysosomal enzymes.
2023-07-19 | PXD043613 | Pride
Project description:Stargardt retinopathy is an inherited form of macular degeneration caused by mutations in gene ABCA4 and characterized by the accumulation of lipid-rich deposits in the retinal pigment epithelium (RPE), RPE atrophy, and photoreceptor cell death. Inadequate mechanistic insights into pathophysiological changes occurring in Stargardt RPE have contributed to its lacking treatments. Here we show that ABCA4 knockout or Stargardt patient’s induced pluripotent stem cells-derived RPE (STGD1-iRPE) differentiate normally but display intracellular lipid and ceramide deposits reminiscent of the disease phenotype. STGD1-iRPE also shows defective photoreceptor outer segment (POS) processing and reduced cathepsin B activity, indicating higher lysosomal pH. Lipid deposits in STGD1-iRPE are reduced by increasing the activity of ABCA1, a lipid transporter, and ABCA4 ortholog. Overall, our work suggests that ABCA4 is involved in POS and lipid handling in RPE cells and provides guidance for ongoing gene therapy approaches to target both RPE and photoreceptor cells for an effective treatment.
2022-11-16 | GSE198362 | GEO
Project description:We used human fibroblasts loaded with sucrose as a simple model of lysosomal accumulation, and we verified by high-throughput RNA sequencing which processes were altered by the treatment at two different time points. Moreover, we hypothesized that ceramide production due to the ectopic catabolism of plasma-membrane glycosphingolipids is involved in cell cycle arrest upon sucrose treatment. To test this hypothesis, we treated sucrose-loaded fibroblasts with two specific inhibitors for the β-glucosidases GBA1 and GBA2, and analyzed the cell responses using high-throughput RNA sequence.
2019-11-06 | GSE106928 | GEO
Project description:To evaluate the effect of oxidative stress on transcript localization in the retinal pigment epithelium (RPE), we performed poly-A RNA sequencing on nuclear and cytoplasmic fractions from induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE) cells exposed to hydrogen peroxide, as well as untreated controls.
2022-06-23 | GSE158909 | GEO
Project description:We have used human umbilical vein endothelial cell line (HUVEC) as a model to investigate the effect of ultrasound (US) activated micro-bubbles on sensitizing the tumour response to radiation and to check the implication of this approach on gene expression. The use of micro-bubbles in cancer therapy is a novel approach that is being recently investigated, and patterns of gene expressions were not yet characterized. Gene analysis has identified about 19,264 genes; where, 239 – 517 genes have shown more than two folds up-regulation in response to the different treatments. This included a number of genes that are known to be involved in apoptosis and ceramide-induced apoptosis pathways, such as Sphingomyelin phosphodiesterase 2, neutral sphingomyelinase (SMPD2), sphingomyelin phosphodiesterase 1, acid lysosomal (ASM; SMPD1), UDP-galactose ceramide galactosyltransferase (UGT8), cytochrome c oxidase (COX6B1), Caspase-9 and mitogen-activated protein kinase kinase 1 (MAP2K1). To verify these results, we have performed Real time RT-PCR, which indicated an up-regulation that ranged from 2 to 200 folds, specifically in the combined treatment of US, 3.3% micro-bubbles and 8 Gy. The observed higher levels of expression of UGT8, SMPD2 and Caspase 9-alpha when compared to the control or to radiation only indicated the involvement of ceramide, which possibly induces apoptosis . These results support the hypothesis that micro-bubbles play a role in increasing sensitization to radiation, and can prove to be an effective cancer therapeutic approach. Gene analysis has identified about 19,264 genes; where, 239 – 517 genes have shown more than two folds up-regulation in response to the different treatments. This included a number of genes that are known to be involved in apoptosis and ceramide-induced apoptosis pathways, such as Sphingomyelin phosphodiesterase 2, neutral sphingomyelinase (SMPD2), sphingomyelin phosphodiesterase 1, acid lysosomal (ASM; SMPD1), UDP-galactose ceramide galactosyltransferase (UGT8), cytochrome c oxidase (COX6B1), Caspase-9 and mitogen-activated protein kinase kinase 1 (MAP2K1). To verify these results, we have performed Real time RT-PCR, which indicated an up-regulation that ranged from 2 to 200 folds, specifically in the combined treatment of US, 3.3% micro-bubbles and 8 Gy. The observed higher levels of expression of UGT8, SMPD2 and Caspase 9-alpha when compared to the control or to radiation only indicated the involvement of ceramide, which possibly induces apoptosis . These results support the hypothesis that micro-bubbles play a role in increasing sensitization to radiation, and can prove to be an effective cancer therapeutic approach.
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Project description:Biallelic DRAM2 mutations cause an autosomal recessive cone-rod dystrophy named CORD21 typically manifesting by the third decade of life. DRAM2 localises to the lysosomes of photoreceptor and retinal pigment epithelium (RPE) cells, however, it remains unclear how DRAM2 contributes to retinal degeneration. Herein, we have derived and characterised retinal organoids (ROs) and RPE cells from two CORD21 induced pluripotent stem cells (iPSCs) lines. CORD21 ROs and RPE manifested abnormal lipid metabolism, autophagic flux defects, aberrant lysosomal content accumulations and reduced lysosomal enzyme activity. A combined proteomics and western blot approach revealed the involvement of DRAM2 in vesicular trafficking that was further corroborated by the immunofluorescent co-localisation of DRAM2 with clathrin adaptor-related proteins AP-1 and AP-3 in ROs. Collectively, our data suggest an indispensable role for DRAM2 in the maintenance of photoreceptors and RPE cells by overseeing the transport of lysosomal enzymes.
2024-02-27 | MSV000094178 | MassIVE
Project description:Alcohol’s impairment of both hepatic lipid metabolism and insulin resistance (IR) are key drivers of alcoholic steatosis, the initial stage of alcoholic liver disease (ALD). Pharmacologic reduction of lipotoxic ceramide prevents alcoholic steatosis and glucose intolerance in mice, but potential off-target effects limit its strategic utility. Here, we employed a hepatic-specific acid-ceramidase (ASAH) overexpression model to reduce hepatic ceramides in a Lieber-DeCarli model of experimental alcoholic steatosis. We examined effects of alcohol on hepatic lipid metabolism, body composition, energy homeostasis and insulin sensitivity as measured by hyperinsulinemic-euglycemic clamp. Our results demonstrate that hepatic ceramide reduction ameliorates the effects of alcohol on hepatic lipid droplet accumulation by promoting VLDL secretion and lipophagy, the latter of which involves ceramide cross-talk between the lysosomal and lipid droplet compartments. We additionally demonstrate that hepatic ceramide reduction prevents alcohol’s inhibition of hepatic insulin signaling. These effects on the liver are associated with a reduction in oxidative stress markers and are relevant to humans, as we observe peri-lipid droplet ASAH expression in human ALD. Together, our results suggest a potential role for hepatic ceramide inhibition in preventing ALD.
2020-07-08 | GSE139015 | GEO
Project description:The bioactive sphingolipid ceramide, generated by ceramide synthase, is an adaptive stress effector induced in response to various stress stimuli such as aging. In fact, ceramide synthase (CerS) was first discovered in yeast as a longevity assurance gene (LAG1), as the reduced ceramide generation consequent to LAG1 deletion increased longevity. There are six homologues of mammalian LAG1/ceramide synthases (CerS1-6) that generate ceramides with different fatty acid chain lengths with distinct functions and hydrophobicity. Ceramide can be metabolized by sphingosine kinase 1 or 2 for the generation of pro-survival sphingosine 1-phosphate (S1P), which enhances immune cell egress to the blood stream, activating immune function. Ceramide is known to induce mitophagy and cell death. Here we investigated the role of this sphingolipid pathway in immune function, specifically T cell functions, via high throughput expression profiling.
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Project description:This is the proteomic analysis data of bovine retinal pigment epithelium cell lysate subjected to cryo-EM study.
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