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/β-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

Disrupting the α-synuclein-ESCRT interaction with a peptide inhibitor mitigates neurodegeneration in preclinical models of Parkinson’s disease

“…The optimized peptide derived from our proteomic screens, PDpep1.3, directly disrupts the a-syn-CHMP2B interaction, breaking the feedback loop and thereby restoring ESCRT function and lysosomal degradation. In neurons, PDpep1.3 normalizes derangements in the endolysosomal pathway caused by a-syn as demonstrated by a return to baseline levels of LAMP1, Rab7, cathepsin B, and cathepsin D. Although others have shown that overexpression of some of these proteins may rescue a-syn toxicity 56 , 57 in the context of a-syn accumulation, PDpep1.3 is reversing the deleterious effect of a-syn on this pathway. The net overall effect of PDpep1.3 is an increase in endolysosomal flux and consequently enhanced clearance of a-syn, including a-syn oligomers.…”

Accumulation of α-synuclein mediates podocyte injury in Fabry nephropathy

“…In a reverse approach primary urinary cells of Fabry patients did not depict increased SNCA levels upon Gb3 stimulation, moreover, indicating a substrate independent mechanism as a cause of SNCA accumulation. SNCA is produced in many cells and constantly degraded through chaperonemediated autophagy (48) and cathepsin degradation (49). This protein has been implicated in other lysosomal storage diseases (50) and is well-known in synucleinopathy related neurodegenerative diseases such as Parkinson disease (51,52), where SNCA is proposed to form a negative feedback loop that leads to decreased enzymatic degradation (53)(54)(55).…”

Cathepsin L regulates oocyte meiosis and preimplantation embryo development

“…Lactoferrin from bovine milk (LF; #L9507, Sigma) was dissolved in water and added to IVM/IVC medium at 10, 100 or 1000 μg/mL to inhibit CTSL activity in good quality oocytes/embryos while recombinant human active CTSL (rCTSL; #ab198444, Abcam, MA, USA) was supplemented to IVM/IVC medium at 100, 200 or 400 ng/mL to enhance CTSL activity in good quality oocytes/embryos. The cellular uptake of the recombinant cathepsins has been previously demonstrated 38,39 …”