Abstract
A pathological feature of Parkinson's disease (PD) is Lewy bodies (LBs) composed of α-synuclein (α-syn) amyloid fibrils. α-Syn is a 140 amino acids–long protein, but truncated α-syn is enriched in LBs. The proteolytic processes that generate these truncations are not well-understood. On the basis of our previous work, we propose that these truncations could originate from lysosomal activity attributable to cysteine cathepsins (Cts). Here, using a transgenic SNCAA53T mouse model, overexpressing the PD-associated α-syn variant A53T, we compared levels of α-syn species in purified brain lysosomes from nonsymptomatic mice with those in age-matched symptomatic mice. In the symptomatic mice, antibody epitope mapping revealed enrichment of C-terminal truncations, resulting from CtsB, CtsL, and asparagine endopeptidase. We did not observe changes in individual cathepsin activities, suggesting that the increased levels of C-terminal α-syn truncations are because of the burden of aggregated α-syn. Using LC-MS and purified α-syn, we identified C-terminal truncations corresponding to amino acids 1–122 and 1–90 from the SNCAA53T lysosomes. Feeding rat dopaminergic N27 cells with exogenous α-syn fibrils confirmed that these fragments originate from incomplete fibril degradation in lysosomes. We mimicked these events in situ by asparagine endopeptidase degradation of α-syn fibrils. Importantly, the resulting C-terminally truncated fibrils acted as superior seeds in stimulating α-syn aggregation compared with that of the full-length fibrils. These results unequivocally show that C-terminal α-syn truncations in LBs are linked to Cts activities, promote amyloid formation, and contribute to PD pathogenesis.
Highlights
A pathological feature of Parkinson’s disease (PD) is Lewy bodies (LBs) composed of ␣-synuclein (␣-syn) amyloid fibrils. ␣-Syn is a 140 amino acids–long protein, but truncated ␣-syn is enriched in LBs
Using LC-MS and purified ␣-syn, we identified C-terminal truncations corresponding to amino acids 1–122 and 1–90 from the SNCAA53T lysosomes
These results unequivocally show that C-terminal ␣-syn truncations in LBs are linked to Cts activities, promote amyloid formation, and contribute to PD pathogenesis
Summary
␣-Syn⌬C species are enriched in lysosomes isolated from symptomatic SNCAA53T mice. Mice overexpressing human SNCAA53T were euthanized when symptoms developed, typically at ϳ16 months of age [34]. Cut sites in the N terminus were seen at Phe-4/Met-5, Ser-9/Lys-10, Gly-14/Val-15, and Ala-17/Ala-18, corresponding to fragments 5–122, 10 –122, 15–122, and 18 –122 These peptides were observed using fibrils formed in vitro from the PD-associated A53T ␣-syn mutant (Fig. S8; Table S6), indicating that this point mutation does not change fibril degradation at the N and C terminus. These data complement results from lysosomes from SNCAA53T mice showing that the 12-kDa species is derived from C-terminal cleavage at Asn-122/Glu-123 and can be attributed to cysteine cathepsin digestion. TEM images did not discern obvious morphological differences after a second round of seeding, it is clear that the seeded samples are more homogenous than the nonseeded controls (Fig. S10)
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