Abstract
Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by α-synuclein aggregation and lysosomal dysfunction, with GBA1 mutations representing the most common genetic risk factor. Reduced glucocerebrosidase (GCase) activity is observed in both familial and sporadic PD, promoting α-synuclein accumulation and neuronal toxicity. Here, we developed a GBA1-HiBiT tagged knock-in HEK293T reporter system, providing a highly sensitive, real-time quantitative measurement of GCase dynamics. From a leech-derived peptide library, we identified a cell-penetrating peptide hirunipin 4 that significantly enhanced GCase protein levels and enzymatic activity. Moreover, hirunipin 4 alleviated α-synuclein preformed fibrils (PFF)-induced lysosomal dysfunction and glucosylceramide accumulation. Thus, hirunipin 4 was able to reduce PFF-induced pathological α-synuclein accumulation and neurotoxicity in both SH-SY5Y and primary cortical neurons. Mechanistically, hirunipin 4 promoted nuclear translocation of TFEB and enhanced GCase protein stability. These findings highlight the utility of the GBA1-HiBiT platform for peptide-based screening and identify hirunipin 4 as a promising candidate for restoring lysosomal function in PD.