Abstract
α-Synuclein (αsyn) aggregates into toxic fibrils in multiple neurodegenerative diseases where these fibrils form characteristic pathological inclusions such as Lewy bodies (LBs). The mechanisms initiating αsyn aggregation into fibrils are unclear, but ubiquitous post-translational modifications of αsyn present in LBs may play a role. Specific C-terminally (C)-truncated forms of αsyn are present within human pathological inclusions and form under physiological conditions likely in lysosome-associated pathways, but the roles for these C-truncated forms of αsyn in inclusion formation and disease are not well understood. Herein, we characterized the in vitro aggregation properties, amyloid fibril structures, and ability to induce full-length (FL) αsyn aggregation through prion-like mechanisms for eight of the most common physiological C-truncated forms of αsyn (1-115, 1-119, 1-122, 1-124, 1-125, 1-129, 1-133, and 1-135). In vitro, C-truncated αsyn aggregated more readily than FL αsyn and formed fibrils with unique morphologies. The presence of C-truncated αsyn potentiated aggregation of FL αsyn in vitro through co-polymerization. Specific C-truncated forms of αsyn in cells also exacerbated seeded aggregation of αsyn. Furthermore, in primary neuronal cultures, co-polymers of C-truncated and FL αsyn were potent prion-like seeds, but polymers composed solely of the C-truncated protein were not. These experiments indicated that specific physiological C-truncated forms of αsyn have distinct aggregation properties, including the ability to modulate the prion-like aggregation and seeding activity of FL αsyn. Proteolytic formation of these C-truncated species may have an important role in both the initiation of αsyn pathological inclusions and further progression of disease with strain-like properties.
Highlights
Lewy body dementia (LBD)2 and Parkinson’s disease (PD) are pathologically characterized by neuronal Lewy body (LB) inclu
2 The abbreviations used are: LBD, Lewy body dementia; LB, Lewy body; PD, Parkinson’s disease; ␣syn, ␣-synuclein; FBS, fetal bovine serum; FL, fullsions composed of amyloidogenic ␣-synuclein (␣syn), along with associated gliosis and neurodegeneration [1,2,3,4]
The propensity of ␣syn to aggregate into -sheet–rich fibrils has been posited to play a crucial role in the initiation and progression of disease, as a number of familial ␣syn missense mutants that cause hereditary PD/LBD accelerate this aggregation process [5,6,7,8,9]
Summary
To investigate differences in aggregate formation kinetics in vitro between human FL ␣syn and the eight major C-truncated ␣syn species that form under physiologic conditions (Fig. 1), monomers of each were diluted to 150 M in PBS and incubated at 37 °C with shaking over the course of 96 h. The rate of ␣syn amyloid formation was monitored using fluorometry, as ␣syn fibrils are amyloidogenic [59], and their formation can be quantified using the fluorescent amyloid binding dyes K114 and thioflavin T (ThT) [59, 60]. Serial fluorometric readings with both ThT and K114 (Fig. 2, B and C) revealed a sigmoidal transition from soluble monomers to insoluble fibrils for all C-truncated forms of ␣syn, which is characteristic of in vitro amyloid formation processes [17]. The 1–122, 1–124, and 1–129 ␣syn truncations fibrillized more readily than FL ␣syn, as evidenced by significantly increased normalized ThT fluorescence at 24 h (Table 1); the effect was less compared with the 1–115, 1–119, and 1–125 ␣syn truncations. In addition to the rate of formation, the total extent of aggregation for each C-truncated form of ␣syn was assessed using centrifugal sedimentation analysis at
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