An alpha 2 collagen VIII transgenic knock-in mouse model of Fuchs endothelial corneal dystrophy shows early endothelial cell unfolded protein response and apoptosis

Abstract

Fuchs endothelial corneal dystrophy (FECD) is a leading indication for corneal transplantation. FECD is characterized by progressive alterations in endothelial cell morphology, excrescences (guttae) and thickening of the endothelial basement membrane and cell death. Ultimately, these changes lead to corneal edema and vision loss. Due to the lack of vision loss in early disease stages and the decades long disease course, early pathophysiology in FECD is virtually unknown as studies of pathologic tissues have been limited to end-stage tissues obtained at transplant. The first genetic defect shown to cause FECD was a point mutation causing a glutamine to lysine substitution at amino acid position 455 (Q455K) in the alpha 2 collagen 8 gene (COL8A2) which results in an early onset form of the disease. Homozygous mutant knock-in mice with this mutation (Col8a2(Q455K/Q455K)) show features strikingly similar to human disease, including progressive alterations in endothelial cell morphology, cell loss and basement membrane guttae. Ultrastructural analysis shows the predominant defect as dilated endoplasmic reticulum (ER), suggesting ER stress and unfolded protein response (UPR) activation. Immunohistochemistry, western blotting, quantitative reverse transcriptase polymerase chain reaction and terminal deoxynucleotidyl transferase 2-deoxyuridine, 5-triphosphate nick end-labeling analyses support UPR activation and UPR-associated apoptosis in the Col8a2(Q455K/Q455K) mutant corneal endothelium. This study confirms the Q455K substitution in the COL8A2 gene as being sufficient to cause FECD in the first mouse model of this disease and supports the role of the UPR and UPR-associated apoptosis in the pathogenesis of FECD caused by COL8A2 mutations.

Endothelial Cell Whole Genome Expression Analysis in a Mouse Model of Early-Onset Fuchs' Endothelial Corneal Dystrophy

“…These include progressive loss of CEC density, alterations in endothelial cell morphology, and basement membrane guttae. 14 Our microarray analysis revealed 334 differentially regulated genes (162 up-regulated, 172 down-regulated) in the corneal endothelium of 12-month-old mutant mice. Real-time PCR experiments reinvestigating four respective up-or downregulated transcripts in Col8a2 mutant and wild-type animals validated our results and demonstrated good accordance with the microarray data.…”

“…14 The point mutation harbored by this animal model causes a glutamine to lysine substitution at amino acid position 455 (Q455K) of the Col8a2 gene, and the equivalent mutation in the human COL8A2 gene has been associated with early-onset FECD. 14,19 Homozygous Col8a2 Q455K/Q455K mutant mice exhibit an endothelial phenotype comprising important characteristics of the human disease. These include progressive loss of CEC density, alterations in endothelial cell morphology, and basement membrane guttae.…”

“…We consider this as potential further evidence for endothelial ER stress resulting in activation of the UPR, supporting results from our previous studies in the same mouse model and in a population of genetically heterogeneous late-onset FECD patients. 13,14 The hypothesis that the misfolding or aggregation of proteins in FECD contributes to cellular stress and apoptosis induction in CECs has also been proposed in studies investigating other genes affected in late-onset FECD like SLC4A11 and LOXHD1. 20,21 Increased transcriptional regulation of other important stress-associated genes such as Ptgs2 (Cox2), Hmox1, Serpine1, and Sod3 (complete list in Table 3) was found in the corneal endothelium of mutant animals.…”

“…Recent investigations proposed that CEC oxidative stress and stress of the endoplasmic reticulum (ER) play critical pathogenetic roles and may result in endothelial apoptosis induction. [11][12][13][14] Recently, we have reported the development of the first transgenic knock-in mouse model of FECD harboring a point mutation in the Col8a2 gene that has previously been associated with early-onset human disease. 14 Using this mouse model, we have demonstrated that the Col8a2 Q455K mutation is sufficient to elicit an FECD-like endothelial morphology; to activate the unfolded protein response (UPR), a cytoprotective signaling cascade; and to induce CEC apoptosis.…”

L450W and Q455KCol8a2Knock-In Mouse Models of Fuchs Endothelial Corneal Dystrophy Show Distinct Phenotypes and Evidence for Altered Autophagy

“…[10][11][12] Transmission electron microscopy (TEM) of corneal tissues obtained from FECD patients with unknown genotypes reveal enlarged rough endoplasmic reticulum (RER), suggestive of endoplasmic reticulum (ER) stress caused by the accumulation of misfolded proteins in the ER lumen. [13][14][15] Previous studies in human FECD corneal tissue 13 and a Col8a2 Q455K/Q455K mutant mouse model of FECD 16 show activation of ER stress and the unfolded protein response (UPR).…”