Cysteine cathepsins are essential in lysosomal degradation of α-synuclein

Identifying factors that regulate the degradation of α-synuclein, the protein at the center of Parkinson’s disease etiology, is vital in designing therapeutic strategies. This study provides new mechanistic insights into α-synuclein clearance in the lysosome, a cellular site for proteolysis by using purified mouse lysosomes and purified lysosomal proteases. Cathepsins B and L are identified to be vital through peptide mapping by mass spectrometry. Importantly, cathepsin L degrades α-synuclein amyloid fibrils, materials associated with neurodegeneration, and, thus, changes in its activity or levels could provide a promising avenue for intervention. Additional results on the stimulatory effect of anionic phospholipids on cathepsin activity suggest that changes in lipid metabolism may also contribute to lysosomal dysfunction leading to pathogenesis.

A cellular feature of Parkinson’s disease is cytosolic accumulation and amyloid formation of α-synuclein (α-syn), implicating a misregulation or impairment of protein degradation pathways involving the proteasome and lysosome. Within lysosomes, cathepsin D (CtsD), an aspartyl protease, is suggested to be the main protease for α-syn clearance; however, the protease alone only generates amyloidogenic C terminal-truncated species (e.g., 1–94, 5–94), implying that other proteases and/or environmental factors are needed to facilitate degradation and to avoid α-syn aggregation in vivo. Using liquid chromatography–mass spectrometry, to our knowledge, we report the first peptide cleavage map of the lysosomal degradation process of α-syn. Studies of purified mouse brain and liver lysosomal extracts and individual human cathepsins demonstrate a direct involvement of cysteine cathepsin B (CtsB) and L (CtsL). Both CtsB and CtsL cleave α-syn within its amyloid region and circumvent fibril formation. For CtsD, only in the presence of anionic phospholipids can this protease cleave throughout the α-syn sequence, suggesting that phospholipids are crucial for its activity. Taken together, an interplay exists between α-syn conformation and cathepsin activity with CtsL as the most efficient under the conditions examined. Notably, we discovered that CtsL efficiently degrades α-syn amyloid fibrils, which by definition are resistant to broad spectrum proteases. This work implicates CtsB and CtsL as essential in α-syn lysosomal degradation, establishing groundwork to explore mechanisms to enhance their cellular activity and levels as a potential strategy for clearance of α-syn.