title: Cathepsin D is the main lysosomal enzyme involved in the degradation of alpha-synuclein and generation of its carboxy-terminally truncated species.
abstract: Alpha-synuclein is likely to play a key role in the development of Parkinson's disease as well as other synucleinopathies. In animal models, overexpression of full-length or carboxy-terminally truncated alpha-synuclein has been shown to produce pathology. Although the proteosome and lysosome have been proposed to play a role in the degradation of alpha-synuclein, the enzyme(s) involved in alpha-synuclein clearance and generation of its carboxy-terminally truncated species have not been identified. In this study, the role of cathepsin D and calpain I in these processes was analyzed. In vitro experiments, using either recombinant or endogenous alpha-synuclein as substrates and purified cathepsin D or lysosomes, demonstrated that cathepsin D degraded alpha-synuclein very efficiently, and that limited proteolysis resulted in the generation of carboxy-terminally truncated species. Purified calpain I also cleaved alpha-synuclein, but carboxy-terminally truncated species were not the main cleavage products, and calpain I activity present in cellular lysates was not able to degrade the protein. Knockdown of cathepsin D in cells overexpressing wild-type alpha-synuclein increased total alpha-synuclein levels by 28% and lysosomal alpha-synuclein by 2-fold. In in vitro experiments, pepstatin A completely blocked the degradation of alpha-synuclein in purified lysosomes. Furthermore, lysosomes isolated from cathepsin D knockdown cells showed a marked reduction in alpha-synuclein degrading activity, indicating that cathepsin D is the main lysosomal enzyme involved in alpha-synuclein degradation. Our findings suggest that upregulation of cathepsin D could be an additional therapeutic strategy to lessen alpha-synuclein burden in synucleinopathies.
authors.affiliation: Department of Neuroscience, Mayo Clinic College of Medicine, 4500 San Pablo Road, Jacksonville, Florida 32224, USA. sevlever.daniel@mayo.edu
authors.forename: Daniel
authors.initials: D
authors.lastname: Sevlever
authors.forename: Peizhou
authors.initials: P
authors.lastname: Jiang
authors.forename: Shu-Hui C
authors.initials: SH
authors.lastname: Yen
mesh_terms: D000818:Animals; D002154:Calpain / Q000235:genetics / Q000378:metabolism; D002402:Cathepsin D / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D004195:Disease Models, Animal; D017353:Gene Deletion; D006801:Humans; D008247:Lysosomes / Q000201:enzymology*; D051379:Mice; D010300:Parkinson Disease / Q000201:enzymology* / Q000235:genetics; D051844:alpha-Synuclein / Q000235:genetics / Q000378:metabolism*
chemical_list: D051844:alpha-Synuclein; D002154:Calpain; D002402:Cathepsin D