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Qing Chang, Marcus Peter, Michael A. Grassi; Fas-mediated Death of Photoreceptors in A Phototoxicity Model. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2569.
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© ARVO (1962-2015); The Authors (2016-present)
Death of photoreceptors is the pathological endpoint that leads to vision loss in various retinal degenerative diseases. Previously, we demonstrated that the prototype death receptor Fas was expressed in photoreceptors of human retina. Activation of Fas by the agonist or Fas ligand (FasL) in a surrogate photoreceptor culture model induced apoptosis. In this study, we investigated the role of Fas signaling in white light (WL)-induced cell death in photoreceptors.
Mouse-derived photoreceptor cells (661W) pre-treated with 9-cis retinal were exposed to WL with/without pharmacological manipulation of Fas signaling. Cells under continuous darkness were treated as negative control. Apoptosis and necrosis were examined by flow cytometry using Alexa Fluor 488 annexin V and propidium iodide labeling. Fractionated cellular extracts were prepared to detect protein expression by western blotting. Phosphorylation of the receptor interacting protein 1 (RIP1) kinase, a key regulator in the necrotic death pathway, was detected by immunoprecipitation using anti-phosphoserine antibody. Cell-Tracker dye-labeled live cells under maintenance of continuous darkness were incubated with the conditioned medium or cell suspensions from WL-exposed cells to detect the likelihood of apoptosis induction.
WL induced both necrotic and apoptotic death in 661W cells, which exhibited death kinetics that correlated with the duration of light exposure. Administration of the anti-FasL neutralizing antibody or caspase 8 inhibitor z-IETD substantially inhibited apoptosis. However, it simultaneously induced necroptosis, which could be blocked by the RIP1 kinase inhibitor, Necrostatin-1. WL exposure plus co-treatment with z-IETD caused hyper-phosphorylation of RIP1 kinase. WL exposure did not up-regulate expression of FasL or Fas. Cell-Tracker dye-labeled live cells when incubated with the conditioned medium or cell suspensions from WL-exposed cells induced apoptosis, which could be partially prevented by the anti-FasL neutralizing antibody.
The canonical Fas-mediated apoptotic death pathway is activated in photoreceptors under light stress. Although blockade of this pathway will prevent apoptosis, it would not improve the overall photoreceptor survival due to a compensatory activation of necroptosis. Prevention of photoreceptor loss from retinal photo-oxidative stress may benefit from targeting both Fas and R1P1. In addition to FasL, light stress also induces secretion of cytotoxic soluble factors that can contribute to the death of photoreceptors by apoptosis through paracrine signaling.
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