Abstract
PURPOSE: To investigate the mechanism of chloroquine-induced cell death in vitro in adult human retinal pigmented epithelial (ARPE-19) cells and murine 661W photoreceptor-derived cells and to evaluate a mouse model of acute chloroquine-induced retinal toxicity. METHODS: Human RPE cells and photoreceptor cells were cultured in vitro and treated with chloroquine; cell death was measured by lactate dehydrogenase release. Lysosomal-associated membrane protein-1 (LAMP-1) staining assessed lysosomal destabilization upstream of cell death initiation. The role of cathepsins, lysosomal proteases was examined by blocking their activity. Cell permeable caspase, and RIP1 kinase inhibitors were used to elucidate the role of downstream mediators of cell death signaling pathways. Adult mice were administered intravitreal injections of 50 micrograms of chloroquine. Fundus photography, optical coherence tomography, and electroretinography were used to evaluate drug-induced retinal changes after one week. Quantitative RT-PCR was used to evaluate changes in gene expression in mice retinal-pigmented epithelium and neurosensory retina. RESULTS: ARPE-19 and 661W cells showed cell death within 24 hours of treatment. LAMP-1 staining and inhibition of cathepsins B, D, and L activity in ARPE-19 cells significantly decreased cell death, indicating that lysosomal destabilization and cathepsin release occur prior to regulated cell death. Treatment of RPE cells with caspase inhibitors and Nec-1 showed primarily caspase-dependent cell death in RPE cells whereas treatment of photoreceptor cells with caspase inhibitors and Nec-1 showed significant rescue of cell death. Chloroquine-injected animals demonstrated functional visual loss consistent with marked abnormality and disruption of the photoreceptor layer. Gene expression changes showed a significant up regulation of markers specific to caspase mediated cell death in the RPE and necrosis and oxidative stress markers in the neurosensory retina. CONCLUSIONS: ARPE-19 cells underwent both apoptotic and pyroptotic cell death while 661W cells showed activation of apoptosis and necroptosis. Chloroquine-injected mice demonstrated characteristic phenotypes also observed in human patients with chloroquine toxicity and showed activation of various cell death mediators. By elucidating the mechanism of chloroquine toxicity, we believe our findings can shed light on potential therapeutic agents.