Institute of Biochemistry, Christian-Albrechts-University Kiel, Kiel, Germany.
Institute of Anatomy, Christian-Albrechts-University Kiel, Kiel, Germany.
Autophagy. 2020 May;16(5):811-825. doi: 10.1080/15548627.2019.1637200. Epub 2019 Jul 16.
CTSD (cathepsin D) is one of the major lysosomal proteases indispensable for the maintenance of cellular proteostasis by turning over substrates of endocytosis, phagocytosis and autophagy. Consequently, CTSD deficiency leads to a strong impairment of the lysosomal-autophagy machinery. In mice and humans CTSD dysfunction underlies the congenital variant (CLN10) of neuronal ceroid lipofuscinosis (NCL). NCLs are distinct lysosomal storage disorders (LSDs) sharing various hallmarks, namely accumulation of protein aggregates and ceroid lipofuscin leading to neurodegeneration and blindness. The most established and clinically approved approach to treat LSDs is enzyme replacement therapy (ERT) aiming to replace the defective hydrolase with an exogenously applied recombinant protein. Here we reveal that recombinant human pro-CTSD produced in a mammalian expression system can be efficiently taken up by a variety of cell models, is correctly targeted to lysosomes and processed to the active mature form of the protease. In proof-of-principle experiments we provide evidence that recombinant human CTSD (rhCTSD) can improve the biochemical phenotype of CTSD-deficient hippocampal slice cultures and retinal cells . Furthermore, we demonstrate that dosing of rhCTSD in the murine CLN10 model leads to a correction of lysosomal hypertrophy, storage accumulation and impaired autophagic flux in the viscera and central nervous system (CNS). We establish that direct delivery of the recombinant protease to the CNS is required for improvement of neuropathology and lifespan extension. Together these data support the continuation of the pre-clinical studies for the application of rhCTSD in the treatment of NCL. AIF1/IBA1: allograft inflammatory factor 1; BBB: blood brain barrier; CNS: central nervous system; CTSB: cathepsin B; CTSD: cathepsin D; CTSL: cathepsin L; ERT: enzyme replacement therapy; GFAP: glial fibrillary acidic protein; INL: inner nuclear layer; LAMP1: lysosomal-associated membrane protein 1; LAMP2: lysosomal-associated membrane protein 2; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; LDL: low-density lipoprotein; LRP1: low density lipoprotein receptor-related protein 1; LSD: lysosomal storage disorder; MEFs: mouse embryonic fibroblasts; M6P: mannose 6-phosphate; mCTSD: mature CTSD; NCL: neuronal ceroid lipofuscinosis; ONL: outer nuclear layer; PB: phosphate buffer; proCTSD: pro-cathepsin D; LRPAP1: low density lipoprotein receptor-related protein associated protein 1; rhCTSD: human recombinant CTSD; SAPC: saposin C; SAPD: saposin D; ATP5G1: ATP synthase, H+ transporting, mitochondrial F0 complex, subunit C1 (subunit 9); SQSTM1/p62: sequestosome 1; TPP1: tripeptidyl peptidase I.
组织蛋白酶 D (CTSD) 是一种主要的溶酶体蛋白酶，对于通过翻转内吞作用、吞噬作用和自噬作用的底物来维持细胞蛋白质稳态是必不可少的。因此，CTSD 缺乏会导致溶酶体-自噬机制严重受损。在小鼠和人类中，CTSD 功能障碍是神经元蜡样脂褐质沉积症 (NCL) 的先天性变异 (CLN10) 的基础。NCL 是不同的溶酶体贮积症 (LSD)，具有各种特征，即蛋白质聚集体和蜡样脂褐素的积累，导致神经退行性变和失明。治疗 LSD 的最成熟和临床批准的方法是酶替代疗法 (ERT)，旨在用外源性应用的重组蛋白替代有缺陷的水解酶。在这里，我们揭示了在哺乳动物表达系统中产生的重组人前 CTSD (pro-CTSD) 可以被多种细胞模型有效摄取，并正确靶向溶酶体并加工成蛋白酶的活性成熟形式。在原理验证实验中，我们提供了证据表明重组人 CTSD (rhCTSD) 可以改善 CTSD 缺乏的海马片培养物和视网膜细胞的生化表型。此外，我们证明 rhCTSD 在 CLN10 模型中的给药可纠正内脏和中枢神经系统 (CNS) 中的溶酶体肥大、储存积累和受损的自噬流。我们确定直接向 CNS 递送重组蛋白酶对于改善神经病理学和延长寿命是必需的。这些数据共同支持继续进行 rhCTSD 在 NCL 治疗中的应用的临床前研究。AIF1/IBA1：同种异体炎症因子 1；BBB：血脑屏障；CNS：中枢神经系统；CTSB：组织蛋白酶 B；CTSD：组织蛋白酶 D；CTSL：组织蛋白酶 L；ERT：酶替代疗法；GFAP：神经胶质纤维酸性蛋白；INL：内核层；LAMP1：溶酶体相关膜蛋白 1；LAMP2：溶酶体相关膜蛋白 2；MAP1LC3/LC3：微管相关蛋白 1 轻链 3；LDL：低密度脂蛋白；LRP1：低密度脂蛋白受体相关蛋白 1；LSD：溶酶体贮积症；MEFs：小鼠胚胎成纤维细胞；M6P：甘露糖 6-磷酸；mCTSD：成熟 CTSD；NCL：神经元蜡样脂褐质沉积症；ONL：外核层；PB：磷酸盐缓冲液；proCTSD：前组织蛋白酶 D；LRPAP1：低密度脂蛋白受体相关蛋白辅助蛋白 1；rhCTSD：人重组 CTSD；SAPC：脑苷脂 C；SAPD：脑苷脂 D；ATP5G1：ATP 合酶，H+转运，线粒体 F0 复合物，亚基 C1（亚基 9）；SQSTM1/p62：自噬体相关蛋白 1；TPP1：三肽基肽酶 I。
