Concepts:

• CHED
• endothelial corneal dystrophy
• CRISPR-Cas9
• SLC4A11
• electrophysiology

Source

Abstract:

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. Human SLC4A11^-/- CEnC lines were generated by CRISPR-Cas9 mediated gene editing. SLC4A11 wildtype (SLC4A11WT) and SLC4A11 mutant (SLC4A11MU) human CEnC lines were generated by stable transduction with lentiviruses containing either SLC4A11WT or SLC4A11MU 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 SLC4A11WT, SLC4A11MU and SLC4A11^-/- CEnC lines. Cell barrier function in SLC4A11WT 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 SLC4A11WT and SLC4A11MU CEnC lines and human primary CEnC. SLC4A11^-/- CEnC and the majority of the SLC4A11MU CEnC lines exhibited significantly increased migration rates, altered proliferation, and decreased cell viability under oxidative stress compared to SLC4A11WT CEnC. Induction with 10mM NH4Cl led SLC4A11WT CEnC to depolarize; conversely, SLC4A11^-/- CEnC hyperpolarized and the majority of SLC4A11MU CEnC either hyperpolarized or had minimal membrane potential changes following NH4Cl induction. Based on ECIS modeling, SLC4A11WT CEnC demonstrated increased cell-substrate adhesion and membrane capacitances compared to SLC4A11^-/- CEnC. Immunostaining of primary CEnC and SLC4A11WT CEnC lines for SLC4A11 demonstrated predominately cell surface staining with partial colocalization with mitochondrial marker COX4 within punctate subcellular structures. SLC4A11MU CEnC lines also displayed mainly cell surface staining of SLC4A11, except for SLC4A11MU c.2263C>T CEnC, which exhibited mostly perinuclear staining. 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.