Torin 1 alleviates impairment of TFEB-mediated lysosomal biogenesis and autophagy in TGFBI (p.G623_H626del)-linked Thiel-Behnke corneal dystrophy.

ABSTRACT

Thiel-Behnke corneal dystrophy (TBCD) is an epithelial-stromal TGFBI dystrophy caused by mutations in the TGFBI (transforming growth factor beta induced) gene, though the underlying mechanisms and pathogenesis of TBCD are still obscure. The study identifies a novel mutation in the TGFBI gene (p.Gly623_His626del) in a TBCD pedigree. Characteristics of the typical vacuole formation, irregular corneal epithelial thickening and thinning, deposition of eosinophilic substances beneath the epithelium, and involvement of the anterior stroma were observed in this pedigree via transmission electron microscopy (TEM) and histological staining. Tgfbi-p.Gly623_Tyr626del mouse models of TBCD were subsequently generated via CRISPR/Cas9 technology, and the above characteristics were further verified via TEM and histological staining. Lysosomal dysfunction and downregulation of differential expression protein CTSD (cathepsin D) were observed using LysoTracker Green DND-26 and proteomic analysis, respectively. Hence, lysosomal dysfunction probably leads to autophagic flux obstruction in TBCD; this was supported by enhanced LC3-II and SQSTM1 levels and decreased CTSD. TFEB (transcription factor EB) was prominently decreased in TBCD corneal fibroblasts and administration of ATP-competitive MTOR inhibitor torin 1 reversed this decline, resulting in the degradation of accumulated mut-TGFBI (mutant TGFBI protein) via the ameliorative lysosomal function and autophagic flux owing to elevated TFEB activity as measured by western blot, confocal microscopy, and flow cytometry. Transfected HEK 293 cells overexpressing human full-length WT-TGFBI and mut-TGFBI were generated to further verify the results obtained in human corneal fibroblasts. Amelioration of lysosome dysfunction may therefore have therapeutic efficacy in the treatment of TBCD.Abbreviations AS-OCT anterior segment optical coherence tomography; ATP adenosine triphosphate; Cas9 CRISPR-associated protein 9; CLEAR coordinated lysosomal expression and regulation; CRISPR clustered regularly interspaced short palindromic repeats; CTSB cathepsin B; CTSD cathepsin D; CTSF cathepsin F; CTSL cathepsin L; DNA deoxyribonucleic acid; ECM extracellular matrix; Fas1 fasciclin 1; FC flow cytometry; GAPDH glyceraldeyde-3-phosphate dehydrogenase; GCD2 granular corneal dystrophy type 2; HE hematoxylin and eosin; LAMP2 lysosomal-associated membrane protein; MT mutation type; MTOR mechanistic target of rapamycin kinase; MTORC1 MTOR complex 1; mut-TGFBI mutant TGFBI protein; SD standard deviation; TBCD Thiel-Behnke corneal dystrophy; TEM transmission electron microscopy; TFEB transcription factor EB; TGFBI transforming growth factor beta induced; WT wild type.