Mutations in the GBA1 gene are the most common of the known risk factors for Parkinson disease (PD). While clinical studies argue a strong case towards a link between GBA1 mutations and the development of PD, mechanistic insights have been lacking. GBA1 encodes glucocerebrosidase (GCase), a lysosomal enzyme which hydrolyzes glucosylceramide (GluCer) into glucose and ceramide and is deficient in Gaucher disease (GD). Recent research suggests a relationship between GCase and the PD-related amyloid-forming protein, alpha-synuclein (alpha-syn); however, the specific molecular mechanisms responsible for association remain elusive. In our work, we focused on the structure-function relationship of alpha-syn and GCase interaction in the lysosome. We have evaluated enzymatic activity, characterized the membrane-bound protein complex by neutron reflectometry, and assessed the effect of Saposin C, an activator for GCase, on complex formation and GCase activity. In defining the molecular interactions that drive the reciprocal relationship between GCase and alpha-syn levels in vivo, we have turned to investigate how alpha-syn is degraded in the lysosome. As the lysosome removes aggregation-prone species or excess levels of alpha-syn, molecular interactions that occur within the lysosome such as with GCase would be pertinent. Such interactions could modulate proteolysis efficiency by altering availability of alpha-syn cleavage sites and dictate protease specificity and efficacy. We are testing hypotheses on how lysosomes contribute to alpha-syn proteostasis under healthy and disease-related conditions. Specifically, we are investigating the relationship between alpha-syn and lysosomal enzymes, cysteine cathepsins and GCase. Our work points to a direct role of cysteine cathepsins in the lysosomal clearance of alpha-syn. We are interested in how these enzymes could be targeted as an intervention strategy in PD progression. With cysteine cathepsins, our data are especially compelling for the potential for cathepsin L (CtsL) to degrade alpha-syn fibrils. For GCase, the enhancement of its levels or activity appears to ameliorate alpha-syn toxicity in cell-based and animal PD models; however, the molecular basis for why this occurs is not well understood. Our research efforts are addressing both these fronts: to evaluate whether CtsL could be a viable therapeutic agent towards cellular clearance of alpha-syn and to elucidate the mechanism(s) responsible for the observed correlation between risk for PD and GCase concentration. To understand how CtsL degrades alpha-syn fibrils, a detailed kinetics study has been carried out by tracking the degradation process of alpha-syn fibrils in the presence of purified CtsL using peptide mapping and electron microscopy (EM). Using mass spectrometry, early time points indicated alpha-syn fibrils are first truncated at the N- and C-termini, generating peptides such as 1-114, 6-114, 10-114, 1-126, 1-134, consistent with prior literature showing that the C- and N-termini are flexible and disordered in the fibrillar state. Prolonging the incubation reveals cleavage sites within the amyloidogenic region (between residues 30-100) of alpha-syn. Interestingly, the majority of these positions contains a glycine, which is known to be involved in beta-turns in a fibril and likely to be more accessible to the protease. By EM visualization, fibrils show imperfections along the fibril axis, which coincides with peptide mapping data showing CtsL cutting within the amyloidogenic core. Some of the images reveal missing density within the fibril, suggesting the protease is eating away at the fibril. Results from this study will be pivotal in developing this protease as a therapeutic agent for PD.
