Effect of CTG repeat expansion in TCF4 on the pathophysiology of Fuchs endothelial corneal dystrophy

Abstract

Purpose : CTG trinucleotide repeat (TNR) expansion is frequently found in TCF4 in Fuchs endothelial corneal dystrophy (FECD), and the potential involvement of TNR expansion in the pathophysiology has also been proposed. We previously reported that in FECD patients, TGF-β is highly expressed in corneal endothelial cells (CECs) and activation of TGF-β signaling induced cell death in an FECD cell model. Here we investigated the effect of CTG TNR expansion and expression of TCF4 on cell death and production of extracellular matrix production in FECD.

Methods : After obtaining patient consent, CECs were isolated from FECD patients, and then cultured and immortalized to produce an FECD cell model (iFECD). TP-PCR was used to determine the TNR length. CRISPR/Cas9 was used to delete CTG TNR (TCF4ΔCTG) or to knockout TCF4 (CTG TNR not deleted) (TCF4-/- iFECD). Cells were treated with TGF-β2 (10 ng/mL), and cell death was evaluated by phase-contrast microscopy and flow cytometry. Activation of apoptotic related proteins (caspase 3 and PARP) and expression of Snail1 and fibronectin was evaluated by western blotting. Aggresome was stained to evaluate the formation of unfolded protein.

Results : TP-PCR showed that iFECD harbored CTG repeat > 50. Phase-contrast microscopy showed that TGF-β induced cell death in iFECD and TCF4ΔCTG. However, the cell death was suppressed in TCF4-/- iFECD. Flow cytometry showed that Annexin V-positive apoptotic cells were significantly increased from 2.9% to 15.3% in iFECD and from 4.5% to 12.5% in TCF4ΔCTG by TGF-β (p<0.01). In contrast, Annexin V-positive cells were not increased by TGF-β in TCF4-/- iFECD. Western blotting showed that caspase 3 and PARP were cleaved (activated) by TGF-β in iFECD and TCF4ΔCTG, and that this activation did not occur in TCF4-/- iFECD. The expression level of Snail1 and fibronectin was increased in iFECD and TCF4ΔCTG by TGF-β. In contrast, that increased expression of Snail1 and fibronectin was suppressed in TCF4-/- iFECD. Aggresome staining showed that TGF-β mediated aggresome accumulation in iFECD and TCF4ΔCTG, but not in TCF4-/- iFECD.

Conclusions : Our current findings suggest that TCF4, but not CTG TNR expansion, is responsible for TGF-β mediated cell death in a cell model of FECD. Further study is needed to clarify the role of TCF4 and CTG TNR expansion in the pathophysiology of FECD.

This is a 2020 ARVO Annual Meeting abstract.