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

 抽象的

Thiel-Behnke角膜营养不良（TBCD）是一种由TGFBI （转化生长因子β诱导）基因突变引起的上皮间质TGFBI营养不良，尽管TBCD的潜在机制和发病机制仍不清楚。该研究在 TBCD 谱系中发现了TGFBI基因 (p.Gly623_His626del) 的新突变。通过透射电子显微镜（TEM）和组织学染色观察该家系的典型空泡形成、不规则角膜上皮增厚和变薄、上皮下嗜酸性物质沉积以及前基质受累的特征。随后通过CRISPR/Cas9技术构建了TBCD的Tgfbi -p.Gly623_Tyr626del小鼠模型，并通过TEM和组织学染色进一步验证了上述特征。分别使用 LysoTracker Green DND-26 和蛋白质组分析观察溶酶体功能障碍和差异表达蛋白 CTSD（组织蛋白酶 D）的下调。因此，溶酶体功能障碍可能导致 TBCD 中的自噬通量受阻； LC3-II 和 SQSTM1 水平升高以及 CTSD 降低支持了这一点。 TBCD 角膜成纤维细胞中的 TFEB（转录因子 EB）显着减少，给予 ATP 竞争性 MTOR 抑制剂 torin 1 逆转了这种下降，通过改善溶酶体功能和自噬通量，导致积累的 mut-TGFBI（突变型 TGFBI 蛋白）降解。通过蛋白质印迹、共聚焦显微镜和流式细胞术测量，TFEB 活性升高。 产生过表达人全长WT- TGFBI和mut -TGFBI的转染HEK 293细胞以进一步验证在人角膜成纤维细胞中获得的结果。因此，改善溶酶体功能障碍可能对 TBCD 的治疗具有疗效。

缩写AS-OCT：眼前段光学相干断层扫描； ATP：三磷酸腺苷； Cas9：CRISPR相关蛋白9；明确：协调溶酶体表达和调节； CRISPR：成簇的规则间隔的短回文重复序列； CTSB：组织蛋白酶B； CTSD：组织蛋白酶D； CTSF：组织蛋白酶F； CTSL：组织蛋白酶L； DNA：脱氧核糖核酸； ECM：细胞外基质； Fas1: 成束蛋白 1; FC：流式细胞术； GAPDH：3-磷酸甘油醛脱氢酶； GCD2：颗粒性角膜营养不良2型； HE：苏木精和伊红； LAMP2：溶酶体相关膜蛋白； MT：突变型； MTOR：雷帕霉素激酶的机制靶点； MTORC1：MTOR 复合物 1； mut-TGFBI：突变型 TGFBI 蛋白； SD：标准差； TBCD：Thiel-Behnke 角膜营养不良； TEM：透射电子显微镜； TFEB：转录因子EB； TGFBI：转化生长因子β诱导； WT：野生型