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Jie Fan, Jiali Liu, Jian Liu, Craig E Crosson; Effects of C2-ceramide on Human iPSC-derived Retinal Ganglion Cells and Optic Nerve Astrocytes.. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3750.
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© ARVO (1962-2015); The Authors (2016-present)
Sphingomyelinases (SMase) catalyze the hydrolysis of sphingomyelin to ceramide and play a central role in sphingolipid metabolism. Previous studies have shown that changes in SMase activities contributes to the sequela of events leading to retinal ganglion cell (RGC) degeneration in ocular hypertensive eyes. To determine if RGCs and optic nerve astrocytes viability is modulated by the SMase product, ceramide, we developed an in vitro model for evaluating ceramide-induced cytotoxicity in human iPSC-derived RGCs and primary cultured human optic nerve astrocytes.
Differentiated RGCs were generated from human pluripotent cells. Cultures of RGCs were enriched to levels of over 80% purity using Thy 1.2 labeled magnetic microbeads columns. RGCs were identified and characterized by RT-PCR and immune-histochemical analysis. Primary cultures of optic nerve head astrocytes were cultured from human donor eyes. Cultures were treated with C2-ceramide (3 μM) for 6 hours. Media were then collected and cells prepared for immunohistochemistry. Cell death and TNF-α secretion into the media were determined by cell counts and ELISA, respectively. Localization of acid and neutral SMase and activated caspase 3 were determined by immunohistochemistry.
Immunofluorescence and RT PCR analysis revealed that the enriched RGCs cultures expressed retinal markers CHX10, PAX6 and RGC markers Brn3a, Math 5, and Thy 1.2. The addition of C2-ceraimde significantly reduced RGC cell number by 33.4 ± 7.7% when compared to vehicle-treated cells. This decrease in RGC viability was associated with the increased detection of activated caspase 3 in remnant cells. The addition of C2-ceraimde to cultured astrocytes did not significantly alter cell number. However, C2-ceramide treated astrocytes exhibited 2.6 fold increases in TNF-a secretion. Both of acid and neutral SMase were identified in RGCs and astrocytes.
These results provide evidence that ceramide acts directly to induce RGC apoptosis, and secretion of TNFa from optic nerve astrocytes. The identification of SMase enzyme expression in the RGCs and optic nerve astrocyte supports the idea that locally generated ceramide in the optic nerve and retinal ganglion cells can act to induce RGC apoptosis and cytokine secretion in glaucomatous optic neuropathy.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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