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. A growing number of studies show a correlation between GCase deficiency and increased alpha-syn levels, leading some to speculate that GluCer accumulation affects normal alpha-syn turnover. However, since only a minority of GD patients and carriers develop PD, other factors are also expected to play a role in promoting pathogenesis. In our work, we had discovered a specific physical interaction exists between alpha-syn and GCase both in solution and on the lipid membrane, resulting in efficient enzyme inhibition. It is currently unresolved whether reduced GCase activity alone leads to increased alpha-syn levels. Based on our finding that N370S, the most common GD-related mutant, and CBE (conduritol-beta-epoxide)-inhibited enzyme had weaker association with alpha-syn, we hypothesized that this newly identified interaction influences cellular levels of alpha-syn and is potentially beneficial. Mutations that decrease the amount of enzyme reaching the lysosome or weaken the interaction, as seen with the N370S mutant, could reduce this potential benefit and thus explain the reciprocal relationship between GCase and alpha-syn levels in vivo. While compelling, further data are needed to support or refute this notion. Moreover, the possibility that reduction of enzymatic activity and hence accumulation of glycosphingolipid substrate (e.g. GluCer) is also affecting normal alpha-syn turnover warrants exploration. While the effect of GCase on alpha-syn homeostasis is the subject of considerable work, understanding of how lysosomes degrade alpha-syn has not been established. As the lysosome takes care of aggregation-prone species or excess levels of alpha-syn, conditions related to disease, molecular interactions that occur within the lysosome such as with intralumenal vesicles or GCase would be pertinent. Such interactions could modulate proteolysis efficiency by altering availability of alpha-syn cleavage sites and dictate protease specificity and efficacy. In this research period, we investigated the degradation process of recombinant alpha-syn by purified mouse brain and liver lysosomes as well as purified human cathepsins by peptide mapping using liquid chromatography mass spectrometry. The degradation process of alpha-syn in lysosomal extracts has been completely mapped by mass spectrometry. Selective protease inhibition experiments with purified lysosomes established that cysteine cathepsins are involved in lysosomal degradation of recombinant alpha-syn. Using purified human proteases as references, peptide mapping showed that the cysteine cathepsin activity involve cathepsin B and L. Both enzymes cleave within the amyloid core region of alpha-syn and inhibit aggregation. Prior to this work, cathepsin D (CtsD) is the only lysosomal protease implicated in alpha-syn proteolysis in vivo. However, CtsD does not completely proteolyze alpha-syn and generates amyloid-forming C-terminally truncated species in vitro. In order for CtsD to have improved efficacy, the presence of anionic lipids was required. This result is of biological relevance as intralysosomal vesicles may contain up to 30% of anionic BMP (bis(monooleoylglycero)phosphate) lipid; moreover, it could reconcile prior in vivo and in vitro results, as lipids were absent in the in vitro studies. Nevertheless, our data indicate that CtsL is the most efficient in degrading alpha-syn and remarkably, CtsL even fully degrades alpha-syn amyloid fibrils. In summary, our study has revealed the role of individual cathepsins: both soluble and membrane-bound alpha-syn is digested by both CtsB and CtsL whereas CtsD can only fully degrade the membrane-bound form, and aggregated alpha-syn can only be dealt with by CtsL. This work sets the framework for future studies in testing hypotheses connecting GCase, GluCer, and cathepsins in PD etiology.
