Investigation of the functional impact of CHED- and FECD4-associated SLC4A11 mutations in human corneal endothelial cells

Abstract

Purpose : To characterize the impact of SLC4A11 mutations associated with congenital hereditary endothelial dystrophy (CHED) and Fuchs endothelial corneal dystrophy type 4 (FECD4) on corneal endothelial cell (CEnC) function and SLC4A11 protein localization.

Methods : Human SLC4A11^-/- CEnC lines were generated by CRISPR-Cas9 mediated gene editing. SLC4A11 wildtype (SLC4A11^WT) and SLC4A11 mutant (SLC4A11^MU) human CEnC lines were generated by stable transduction with lentiviruses containing either SLC4A11^WT or SLC4A11^MU expression vectors harboring CHED-/FECD4-associated SLC4A11 mutations (CHED: c.374G>A, c.1813C>T, c.2263C>T; FECD4: c.2224G>A). Functional assays were performed to assess cell migration, proliferation, viability after induced oxidative stress and NH₄Cl-induced membrane conductance in the generated SLC4A11^WT, SLC4A11^MU and SLC4A11^-/- CEnC lines. Cell barrier function in SLC4A11^WT and SLC4A11^-/- CEnC were assessed by electric cell-substrate impedance sensing (ECIS). Immunostaining was performed to determine the subcellular localization of SLC4A11 protein in the generated SLC4A11^WT and SLC4A11^MU CEnC lines and human primary CEnC.

Results : SLC4A11^-/- CEnC and the majority of the SLC4A11^MU CEnC lines exhibited significantly increased migration rates, altered proliferation, and decreased cell viability under oxidative stress compared to SLC4A11^WT CEnC. Induction with 10mM NH₄Cl led SLC4A11^WT CEnC to depolarize; conversely, SLC4A11^-/- CEnC hyperpolarized and the majority of SLC4A11^MU CEnC either hyperpolarized or had minimal membrane potential changes following NH₄Cl induction. Based on ECIS modeling, SLC4A11^WT CEnC demonstrated increased cell-substrate adhesion and membrane capacitances compared to SLC4A11^-/- CEnC. Immunostaining of primary CEnC and SLC4A11^WT CEnC lines for SLC4A11 demonstrated predominately cell surface staining with partial colocalization with mitochondrial marker COX4 within punctate subcellular structures. SLC4A11^MU CEnC lines also displayed mainly cell surface staining of SLC4A11, except for SLC4A11^MU c.2263C>T CEnC, which exhibited mostly perinuclear staining.

Conclusions : CHED- and FECD4-associated SLC4A11 mutations likely lead to CEnC dysfunction, and ultimately CHED and FECD4, by interfering with cell migration, proliferation, viability, membrane conductance, barrier function, and/or cell surface localization of the SLC4A11 protein in CEnC.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.