Proteolysis mediated by lysosomal cathepsin proteases maintains a physiological flow in autophagy, phagocytosis and endocytosis. Neuronal Ceroid Lipofuscinosis (NCL) is a childhood neurodegenerative disorder characterized by disturbed autophagic flow and pathological accumulation of proteins. We demonstrated a therapeutic clearance of protein aggregates after dosing NCL10 mice with recombinant human pro-cathepsin-D. Prompted by these results and speculating that cathepsins may act in a redundant and in an hierarchical manner we envisaged that a treatment with human recombinant cysteine proteases pro-cathepsin-L (proCTSL) and pro-cathepsin-B (proCTSB) could similarly be used to induce protein degradation. Both enzymes were taken up by mannose 6-phosphate receptor- and LRP-receptor-mediated endocytosis and processed to the lysosomal mature cathepsins. In murine NCL10 astrocytes an abnormal increase in LAMP1 and saposin expression was revealed. Although proCTSB application did not improve this phenotype, proCTSL treatment led to reduced saposin-C levels in this model as well as in an acute brain slice model. Intracerebral dosing in a NCL10 mouse model revealed cellular and lysosomal uptake of both enzymes. Only proCTSL mildly reduced saposin-C levels and attenuated reactive astrogliosis. Application of both proteases did not improve weight loss and mortality of mutant mice. Our data reveal that although recombinant lysosomal proteases can be efficiently delivered to neuronal lysosomes cysteine proteases are less efficient in protein aggregates clearance as compared to the cathepsin-D treatment. Our data including in vitro degradation assays support the idea that bulk proteolysis requires a hierarchical process in which both aspartyl and cysteine hydrolases play a role.

由溶酶体组织蛋白酶介导的蛋白水解在自噬、吞噬作用和内吞作用中维持生理流动。神经元蜡样脂褐质沉积症 (NCL) 是一种儿童神经退行性疾病，其特征是自噬流紊乱和蛋白质病理性积累。我们证明了在用重组人组织蛋白酶原 D 给 NCL10 小鼠给药后蛋白质聚集体的治疗清除。受这些结果的启发，并推测组织蛋白酶可能以冗余和分层方式起作用，我们设想用人重组半胱氨酸蛋白酶 pro-cathepsin-L (proCTSL) 和 pro-cathepsin-B (proCTSB) 进行治疗可以类似地用于诱导蛋白质降解。两种酶都被甘露糖 6-磷酸受体和 LRP 受体介导的内吞作用吸收并加工成溶酶体成熟组织蛋白酶。在鼠 NCL10 星形胶质细胞中发现 LAMP1 和 saposin 表达异常增加。尽管 proCTSB 应用没有改善这种表型，proCTSL 治疗导致该模型以及急性脑切片模型中的 saposin-C 水平降低。NCL10 小鼠模型的脑内给药显示两种酶的细胞和溶酶体摄取。只有 proCTSL 轻度降低 saposin-C 水平并减轻反应性星形胶质细胞增生症。两种蛋白酶的应用都没有改善突变小鼠的体重减轻和死亡率。我们的数据显示，虽然重组溶酶体蛋白酶可以有效地传递到神经元溶酶体，但与组织蛋白酶-D 治疗相比，半胱氨酸蛋白酶在清除蛋白质聚集体方面的效率较低。我们的数据包括体外降解试验支持大量蛋白水解需要天冬氨酰和半胱氨酸水解酶都起作用的分级过程的观点。