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Joseph L Mertz, Xitiz Chamling, David J Clark, Kyung Cho Cho, Cynthia Berlinicke, Hui Zhang, Donald J Zack; Proteomic Approaches to Study Cell Death Mechanisms in Human Stem Cell-Derived Retinal Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 2019;60(9):629.
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© ARVO (1962-2015); The Authors (2016-present)
Glaucoma is characterized by progressive loss of retinal ganglion cells (RGCs) and while reasonable therapies exist – primarily to lower intraocular pressure – they are not always effective. To date, there are no FDA-approved neuroprotective agents that directly halt vision loss. Our goal is to increase our understanding of RGC neuroprotective signaling networks during axonal injury using global and phosphoproteomics in human stem cell-derived RGCs (hscRGCs), a highly disease-relevant model. Our primary focus is on the dual leucine zipper kinase (DLK)/leucine zipper kinase (LZK)/c-Jun N-terminal kinase (JNK)/stress activated protein kinase (SAPK) pathway and the phosphorylation events that bring about cell death upon axonal injury.
Human ES cells carrying genes for tdTomato and murine cell-surface protein THY1.2 driven by the BRN3B promoter were differentiated into RGCs for 35 days, immunopurified through THY1.2, and cultured as a purified population for 7 days. Axonal injury was induced by treatment with 1µM of a microtubule destabilizer, colchicine, for time periods ranging from 5 minutes to 48 hours, with a subset simultaneously subjected to pharmacological DLK inhibition by the small molecule VX-680. Viability, morphology, and SAPK pathway activation under these conditions were assessed. Protein was harvested and prepared for LC-MS/MS analysis including phosphopeptide enrichment by Fe3+ immobilized metal affinity chromatography (IMAC). Phospho and global LC-MS/MS analyses were performed on Orbitrap LC-MS/MS instruments.
At 48 hours, 1µM colchicine significantly decreased cell viability [CB1] [JM2] (13.1±0.9%vs ctl) and neurite length (6.9±2.1% vs ctl), and engaged SAPK signaling (increased phospho-cJun) after only 30 minutes. DLK inhibition by VX-680 strongly suppressed all of these effects (87.1±2.3% survival, 47.5±6.4% neurite length, and no detectable phospho-cJun). Protein has been purified from these samples and proteomic and phosphoproteomic studies are underway. Based on pilot studies, we expect to quantify over 8,000 proteins and 10,000 phosphorylation sites.
Multi-tiered proteomic analysis of the signaling networks involved in axon injury-induced cell death in conjunction with pharmacological modulation of these networks will provide insights into key signaling events in RGC death, and hopefully lead to improved therapeutic approaches.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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