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Abstract #94820 Published in IGR 22-2
Human primary retinal cells as an in-vitro model for investigating defective signalling caused by OPTN mutants associated with glaucoma
Sayyad Z; Vishwakarma S; Dave TV; Naik MN; Radha V; Kaur I; Swarup GNeurochemistry International 2021; 148: 105075
Studies carried out on the pathogenesis of glaucoma using murine cell lines and animal models require to be validated in human cells. Therefore, we explored the possibility of using human primary retinal cells (hPRCs) in culture as a model for molecular studies and testing of potential therapeutic drugs. For this purpose, central retinal tissue, obtained from the enucleated eyes of patients with anterior staphyloma, was digested with trypsin and grown in a medium containing supplements (basic fibroblast growth factor and fetal bovine serum). hPRCs at passage 1 and 2, show expression of either GFAP, a glial cell marker, or β-III tubulin, a retinal ganglion cell (RGC)-specific marker. But at passages 3-5 nearly all of hPRCs express several RGC-specific markers (Brn3 proteins, Thy-1, β-III tubulin, RBPMS and NeuN) but not GFAP. Expression of these markers indicated that these cells may have functional properties of RGCs. As RGCs are sensitive to glaucoma-associated mutants of OPTN, we analysed the survival of hPRCs upon overexpression of OPTN mutants. Glaucoma-associated mutants, E50K-OPTN and M98K-OPTN, induced significantly higher cell death in hPRCs compared to WT-OPTN, whereas an amyotrophic lateral sclerosis-associated mutant, E478G-OPTN, did not. TBK1 inhibitor Amlexanox protected hPRCs from E50K-OPTN and M98K-OPTN induced cell death. M98K-OPTN induced cell death was suppressed by inhibitors of CaMKKβ and AMPK in hPRCs as well as in 661W, a mouse cell line that expresses several markers of RGCs and RGC precursor cells. Our results suggest that hPRCs under appropriate culture condition show RGC-like properties. These cells can be used to explore the molecular mechanisms of cell death relevant for glaucoma pathogenesis and for testing of cytoprotective compounds.
CSIR- Centre for Cellular and Molecular Biology, Hyderabad, 500007, India.
Full articleClassification:
3.6 Cellular biology (Part of: 3 Laboratory methods)
3.4.2 Gene studies (Part of: 3 Laboratory methods > 3.4 Molecular genetics)
11.8 Neuroprotection (Part of: 11 Medical treatment)
