Development of human cellular model for ectopic calcification to study the physiopathological mechanism for Optic Disc Drusen (ODD)

Abstract

Purpose : Ectopic calcification is thought to be involved in the pathogenesis of optic disc drusen (ODD) and age-related macular degeneration. In ODD, calcified deposits in the anterior optic nerve result in optic nerve head elevation, hyperautofluorescence, and vision loss in the majority of patients. Although we do not know what causes ODD, a common mechanism in ectopic calcification in atherosclerosis is elevated levels of phosphate, leading to mitochondrial, cellular, and extracellular matrix calcification. In this study, we aim to establish the first cellular model of ODD using human cells.

Methods : We treated 3 human cell lines primary skin fibroblasts, SH-SY5Y neurons, and human embryonic stem cell-derived retinal ganglion cells (hES-RGC) with high potassium phosphate or osteogenic differentiation. We assessed calcification using Alizarine red S stain and cell survival using MTT assay and Calcein-AM staining. Mitochondrial dynamics were elucidated by Mito tracker, TMRM staining, and cryogenic electron microscopy (Cryo-EM). The molecular analysis was performed by qRT-PCR to study gene expression associated with calcification, cellular stress, and apoptosis. The effect of calcification was also studied based on cell morphology and axon structure of neurons.

Results : Exposure to potassium phosphate or osteogenic factors resulted in avid intracellular calcification within 7 days without impacting cell viability. SH-SY5Y and hES-RGCs showed abnormal neurite morphology showing initial signs of neurite degeneration. Treated cells resulted in the upregulation of osteogenic markers (BMP2, osteopontin, alkaline phosphatase, and Runx2) and apoptotic markers (Bax, Bcl2, Caspase3, P53), (P≤0.05). Qualitative analysis reveals calcification altered mitochondrial dynamics and membrane potential. Cryo-EM revealed abnormal mitochondrial structure with electron-dense granules at the site of mitochondrial ATP synthesis.

Conclusions : We show a novel cellular model of ectopic calcification using human RGCs and other cell lines. Ectopic calcification was associated with altered gene expression related to osteogenesis, cellular stress, mitochondrial dysfunction, and further alteration in axons – phenomena that likely underlie the pathogenesis of ODD.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.