Recombinant pro-CTSD (cathepsin D) enhances SNCA/α-Synuclein degradation in α-Synucleinopathy models.

ABSTRACT

Parkinson disease (PD) is a neurodegenerative disorder characterized by the abnormal intracellular accumulation of SNCA/α-synuclein. While the exact mechanisms underlying SNCA pathology are not fully understood, increasing evidence suggests the involvement of autophagy as well as lysosomal deficiencies. Because CTSD (cathepsin D) has been proposed to be the major lysosomal protease involved in SNCA degradation, its deficiency has been linked to the presence of insoluble SNCA conformers in the brain of mice and humans as well as to the transcellular transmission of SNCA aggregates. We here postulate that SNCA degradation can be enhanced by the application of the recombinant human proform of CTSD (rHsCTSD). Our results reveal that rHsCTSD is efficiently endocytosed by neuronal cells, correctly targeted to lysosomes and matured to an enzymatically active protease. In dopaminergic neurons derived from induced pluripotent stem cells (iPSC) of PD patients harboring the A53T mutation within the SNCA gene, we confirm the reduction of insoluble SNCA after treatment with rHsCTSD. Moreover, we demonstrate a decrease of pathological SNCA conformers in the brain and within primary neurons of a ctsd-deficient mouse model after dosing with rHsCTSD. Boosting lysosomal CTSD activity not only enhanced SNCA clearance in human and murine neurons as well as tissue, but also restored endo-lysosome and autophagy function. Our findings indicate that CTSD is critical for SNCA clearance and function. Thus, enzyme replacement strategies utilizing CTSD may also be of therapeutic interest for the treatment of PD and other synucleinopathies aiming to decrease the SNCA burden.Abbreviations aa amino acid; SNCA/α-synuclein synuclein alpha; APP amyloid beta precursor protein; BBB blood brain barrier; BF basal forebrain; CBB Coomassie Brilliant Blue; CLN neuronal ceroid lipofuscinosis; CNL10 neuronal ceroid lipofuscinosis type 10; Corr. corrected; CTSD cathepsin D; CTSB cathepsin B; DA dopaminergic; DA-iPSn induced pluripotent stem cell-derived dopaminergic neurons; dox doxycycline; ERT enzyme replacement therapy; Fx fornix, GBA/ß-glucocerebrosidase glucosylceramidase beta; h hour; HC hippocampus; HT hypothalamus; i.c. intracranially; IF immunofluorescence; iPSC induced pluripotent stem cell; KO knockout; LAMP1 lysosomal associated membrane protein 1; LSDs lysosomal storage disorders; MAPT microtubule associated protein tau; M6P mannose-6-phosphate; M6PR mannose-6-phosphate receptor; MB midbrain; mCTSD mature form of CTSD; neurofil. neurofilament; PD Parkinson disease; proCTSD proform of CTSD; PRNP prion protein; RFU relative fluorescence units; rHsCTSD recombinant human proCTSD; SAPC Saposin C; SIM structured illumination microscopy; T-insol Triton-insoluble; T-sol Triton-soluble; TEM transmission electron microscopy, TH tyrosine hydroxylase; Thal thalamus.