Recombinant cathepsins B and L promote α-synuclein clearance and restore lysosomal function in human and murine models with α-synuclein pathology

The autophagy-lysosomal pathway is crucial for maintaining homeostasis and survival of neurons, hence defects in this system have been associated with neurodegeneration, including Parkinson's disease (PD). The cysteine proteases cathepsin B (CTSB) and cathepsin L (CTSL) are involved in the clearance of various neurodegenerative disease-related proteins such as amyloid-[Formula: see text], huntingtin and the prion protein. While there are studies implicating CTSB and CTSL as mediators of α-synuclein/SNCA clearance, their exact roles remain unclear. We previously demonstrated that recombinant procathepsin D can enhance the clearance of pathological-aggregates of SNCA both in vitro and in vivo, as well as restoring autophagy function. These results prompted us to investigate the role of the two cysteine proteases CTSB and CTSL regarding SNCA degradation by dosing recombinant human procathepsin B (rHsCTSB) and procathepsin L (rHsCTSL) alone or in combination. We here demonstrate that both proteases are efficiently endocytosed by neuronal cells and transported to lysosomes, where they undergo maturation into active enzymes. Treatment with either rHsCTSB or rHsCTSL resulted in a reduction of different SNCA species, present in Triton-insoluble protein fractions as well as sensitive for various pathology- and structure-specific antibodies analyzed via Western blot, immunofluorescence and ELISA. These effects were found to be similar in all models used here: dopaminergic neurons derived from induced pluripotent stem cells (iPSC) of PD patients harboring the SNCA A53T mutation, ex vivo organotypic brain slices and primary neuronal cultures of human SNCA overexpressing Thy1 mice. Interestingly, our data so far do not indicate a synergistic effect of both cysteine cathepsins when applied together. As proof-of-concept for future therapeutic studies, intracranial injections of both recombinant enzymes reduced SNCA in brains of a transgenic mouse model (Ctsd knockout) harboring SNCA pathology. Moreover, treatment with recombinant CTSB and CTSL improved lysosomal/autophagy functions indicated by recovery of β-glucocerebrosidase (GCase) activity and SQSTM1 (p62) level. Further, SNCA-dependent synaptic defects as well as toxicity was reduced after treatment of neuronal cells. These findings suggest that enhancing lysosomal CTSB or CTSL effectively degrades pathology-associated SNCA, suggesting a potential therapeutic protease-based strategy for PD and other synucleinopathies. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13024-025-00886-1.