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

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

泰尔-宾克角膜营养不良（Thiel-Behnke corneal dystrophy, TBCD）是一类上皮-基质型TGFBI营养不良，由TGFBI（transforming growth factor beta induced，转化生长因子β诱导蛋白）基因突变引发，但目前其潜在发病机制与病理过程仍不明晰。本研究在一个TBCD家系中鉴定出TGFBI基因的一种全新突变（p.Gly623_His626del）。通过透射电子显微镜（transmission electron microscopy, TEM）与组织染色技术，该家系患者呈现出典型的空泡形成、角膜上皮不规则增厚与变薄、上皮下嗜酸性物质沉积以及前基质受累等特征。随后本研究通过CRISPR/Cas9技术构建了TBCD的Tgfbi-p.Gly623_Tyr626del小鼠模型，并通过TEM与组织染色进一步验证了上述表型特征。分别通过LysoTracker Green DND-26染色与蛋白质组学分析，发现存在溶酶体功能异常以及差异表达蛋白组织蛋白酶D（cathepsin D, CTSD）的表达下调。因此，溶酶体功能异常可能导致TBCD中的自噬流受阻，这一结论得到了LC3-II与SQSTM1水平升高、CTSD水平降低的实验结果支持。在TBCD角膜成纤维细胞中，转录因子EB（transcription factor EB, TFEB）的表达显著降低；而ATP竞争性MTOR抑制剂torin 1可逆转这一下调趋势，通过提升TFEB活性改善溶酶体功能与自噬流，进而降解蓄积的突变型TGFBI（mutant TGFBI, mut-TGFBI）蛋白，该结果通过蛋白质印迹、共聚焦显微镜与流式细胞术（flow cytometry, FC）得到验证。为进一步验证在人角膜成纤维细胞中获得的实验结果，本研究构建了过表达人全长野生型（wild type, WT）TGFBI与mut-TGFBI的转染HEK 293细胞模型。综上，改善溶酶体功能障碍或可成为TBCD的潜在治疗策略。缩写说明如下：AS-OCT：前节光学相干断层扫描（anterior segment optical coherence tomography）；ATP：三磷酸腺苷（adenosine triphosphate）；Cas9：CRISPR相关蛋白9（CRISPR-associated protein 9）；CLEAR：溶酶体表达与调控协同通路（coordinated lysosomal expression and regulation）；CRISPR：成簇规律间隔短回文重复序列（clustered regularly interspaced short palindromic repeats）；CTSB：组织蛋白酶B（cathepsin B）；CTSD：组织蛋白酶D（cathepsin D）；CTSF：组织蛋白酶F（cathepsin F）；CTSL：组织蛋白酶L（cathepsin L）；DNA：脱氧核糖核酸（deoxyribonucleic acid）；ECM：细胞外基质（extracellular matrix）；Fas1：Fasciclin 1；FC：流式细胞术（flow cytometry）；GAPDH：甘油醛-3-磷酸脱氢酶（glyceraldeyde-3-phosphate dehydrogenase）；GCD2：颗粒状角膜营养不良2型（granular corneal dystrophy type 2）；HE：苏木精-伊红染色（hematoxylin and eosin）；LAMP2：溶酶体相关膜蛋白2（lysosomal-associated membrane protein）；MT：突变类型（mutation type）；MTOR：雷帕霉素机制性靶标激酶（mechanistic target of rapamycin kinase）；MTORC1：MTOR复合物1（MTOR complex 1）；mut-TGFBI：突变型TGFBI蛋白（mutant TGFBI protein）；SD：标准差（standard deviation）；TBCD：泰尔-宾克角膜营养不良（Thiel-Behnke corneal dystrophy）；TEM：透射电子显微镜（transmission electron microscopy）；TFEB：转录因子EB（transcription factor EB）；TGFBI：转化生长因子β诱导蛋白（transforming growth factor beta induced）；WT：野生型（wild type）