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Updated: Jun 7, 2025
Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells
Published on: May 16, 2019
Protective effect of zinc against A2E-induced toxicity in ARPE-19 cells: Possible involvement of lysosomal
Jeong A Choi1, Bo-Ra Seo1, Jae-Young Koh1,2
1Neural Injury Research Center, Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea.
Abstract:
A key pathogenic mechanism of dry age-related macular degeneration (AMD) is lysosomal dysfunction in retinal pigment epithelium (RPE) cells, which results in the accumulation of lipofuscins such as A2E (N-retinylidene-N-retinylethanolamine) that further compromises lysosomal function. This vicious cycle leads to cell death and poor visual acuity. Here, we established an in vitro model of AMD by treating a human RPE cell line (ARPE-19) with A2E and examined whether raising zinc levels confers protective effects against lysosomal dysfunction and cytotoxicity. MTT assay showed that A2E induced apoptosis in ARPE-19 cells. pHrodo™ Red fluorescence staining showed that lysosomal pH increased in A2E-treated ARPE-19 cells. Treatment with a zinc ionophore (clioquinol) reduced A2E accumulation, restored lysosomal pH to the acidic range, and reduced A2E-induced cell death, all of which were reversed by the addition of a zinc chelator (TPEN). Consistent with the in vitro results, subretinal injections of A2E in mouse eyes resulted in the death of RPE cells as well as lysosomal dysfunction, all of which were reversed by co-treatment with clioquinol. Our results suggest that restoring the levels of intracellular zinc, especially in lysosomes, would be helpful in mitigating A2E-induced cytotoxic changes including lysosomal dysfunction in RPE cells in the pathogenesis of AMD.
Insights
Restoring zinc levels protects against dry age-related macular degeneration (AMD) by preventing lysosomal dysfunction and cell death in retinal pigment epithelium (RPE) cells damaged by A2E accumulation.
Area of Science:
- Ophthalmology
- Cell Biology
- Molecular Medicine
Background:
- Dry age-related macular degeneration (AMD) pathogenesis involves lysosomal dysfunction in retinal pigment epithelium (RPE) cells.
- Accumulation of lipofuscins, like A2E (N-retinylidene-N-retinylethanolamine), exacerbates lysosomal dysfunction, creating a detrimental cycle leading to RPE cell death and vision loss.
Purpose of the Study:
- To investigate the protective effects of elevated zinc levels against A2E-induced lysosomal dysfunction and cytotoxicity in RPE cells.
- To evaluate the therapeutic potential of zinc restoration in an in vitro AMD model and an in vivo mouse model.
Main Methods:
- Established an in vitro AMD model using ARPE-19 cells treated with A2E.
- Assessed cell viability using MTT assay and lysosomal pH using pHrodo™ Red fluorescence staining.
- Administered zinc ionophore (clioquinol) and zinc chelator (TPEN) to modulate intracellular zinc levels.
- Utilized subretinal injections of A2E and clioquinol in a mouse model to validate in vitro findings.
Main Results:
- A2E treatment induced apoptosis and increased lysosomal pH in ARPE-19 cells.
- Zinc ionophore treatment reduced A2E accumulation, normalized lysosomal pH, and decreased A2E-induced cell death.
- The protective effects of zinc were reversible with a zinc chelator.
- In vivo studies confirmed that clioquinol reversed A2E-induced RPE cell death and lysosomal dysfunction in mouse eyes.
Conclusions:
- Intracellular zinc, particularly in lysosomes, plays a crucial role in mitigating A2E-induced cytotoxic effects.
- Restoring zinc levels presents a potential therapeutic strategy for dry AMD by addressing lysosomal dysfunction and RPE cell death.
