A cellular model of age–related macular degeneration: Apoptosis of RPE induced by cytokine under conditions of zinc deficiency

Abstract

Abstract: : Purpose: To investigate mechanisms underlying the apoptosis of retinal pigment epithelium (RPE) induced by TNF–α under conditions of Zn²⁺ deficiency. Methods:Apoptotic cells were identified using nuclear staining with Hoechst 33342. Cells containing activated caspase(s) were localized by immunofluorescence staining using antibodies against the cleaved form of caspase(s), or by in situ staining with FAM–VAD–fmk (ApoFluor). Cytochrome c (cyt c) distribution was revealed using immunofluorescecne with antibodies specific to cyt c. To monitor Δψ_m or the production of reactive oxygen species (rOx), cells were loaded with JC–1 or carboxylated dichlorodihydrofluorescein diacetate, respectively. Fluorescence intensity measurements were made using a plate reader. Results: The apoptotic response of RPE cells was markedly enhanced when TNF–α plus actinomycin D (act–D) was co–applied with N, N, N’, N’–tetrakis(2–pyridylmethyl)ethylenediamine (TPEN), a Zn²⁺ chelator. Apoptosis was caspase–dependant, and a blockade of the mitochondrial permeability transition (MPT) with cyclosporin A (CsA) abolished caspase activation, indicating that apoptosis required the MPT, and that caspase activation is downstream to the MPT. Mitochondrial pathology (e.g. Δψ_m loss and cyt c release) observed during the apoptotic process was prevented when cells were pre–treated with either CsA or the pan–caspase inhibitor, z–VAD–fmk. This suggests that the apoptotic signaling was initiated by the MPT, and further amplified by downstream caspases through a feedback loop. A slow rise of rOx was observed in cells exposed to TNF–α + act–D + TPEN, and rOx production did not require the MPT or caspase activation. Additionally, application of antioxidants (Trolox + vitamin C + 4–OH–TEMPO) inhibited the rOx production, the mitochondrial dysfunction, and apoptosis, suggesting a possible role for rOx as a pro–apoptotic signal. Conclusions: Our data suggest that Zn²⁺ play an important role in modulating the apoptotic response to cytokine(s) under conditions of Zn²⁺ deficiency, and that apoptotic signaling is initiated by the MPT and then amplified by caspase activation through a mitochondrial feedback loop. These results provide a new model for the roles of Zn²⁺ deficiency, over–production of rOx, apoptosis, and drusen–induced local inflammation in the etiology of AMD.

Keywords: age–related macular degeneration • apoptosis/cell death • mitochondria