Abstract
Purpose :
E50K and M98K mutants of OPTN associated with glaucoma induce cell death specifically in mouse retinal ganglion cells (RGCs) but not in other cell types. Studies carried out on the pathogenesis of glaucoma using murine cell lines and animal models require to be validated in human cells. Human primary retinal cells (hPRCs) in culture as a model for molecular studies and screening of potential therapeutic drugs have not been explored. Transition in conditions from in-vivo to ex-vivo can potentially alter the characteristics of retinal cells. Hence, we profiled adult hPRCs in culture for expression of various retinal cell specific markers and tested their sensitivity to disease associated mutants of OPTN. The signalling kinases associated with autophagy were examined for their involvement in cell death.
Methods :
Central retinal tissue was obtained from the enucleated eye of patients with anterior staphyloma. After chopping, retinal tissue was digested with trypsin (0.25%) and seeded in DMEM containing 10% FCS, antibiotics and bFGF (1ng/ml) for two weeks to obtain primary cells. Expression of markers was analysed by immunofluorescence and western blotting. Cell death assay was carried out by transient overexpression of OPTN and mutants followed by Annexin V/7AAD staining. hPRCs at passages 3 to 5 were used for all experiments.
Results :
hPRCs showed heterogeneity in size and shape. Almost all hPRCs expressed RGC specific markers Brn3a, Brn3b, Brn3c, Thy-1, β-III tubulin and NeuN. They also expressed photoreceptor specific marker OPN1MW, glial cell marker GFAP and neuronal precursor marker, Nestin. E50K-OPTN and M98K-OPTN overexpression resulted in significantly higher cell death in hPRCs compared to WT-OPTN, whereas an ALS associated mutant E478G-OPTN failed to do so. M98K-OPTN induced cell death was suppressed by pharmacological inhibitors of TBK1, CaMKKβ and AMPK, kinases known to regulate autophagy.
Conclusions :
Our results suggest that hPRCs under appropriate cell culture condition appear to dedifferentiate and show RGC precursor-like properties. Glaucoma associated mutants of OPTN selectively induce cell death in these cells. M98K-OPTN induced cell death signalling involves TBK1, CaMKKβ and AMPK. These cells can be used to explore the molecular mechanisms of glaucoma pathogenesis and for drug testing.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.