Enzyme replacement therapy with recombinant pro-CTSD (cathepsin D) corrects defective proteolysis and autophagy in neuronal ceroid lipofuscinosis.

ABSTRACT

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 in vitro and retinal cells in vivo. 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.Abbreviations 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.