Role of α-Synuclein Carboxy-Terminus on Fibril Formation in Vitro^†

Alpha-synuclein (α-syn) is the major component of intracellular inclusions in several neurodegenerative diseases, and the conversion of soluble α-syn into filamentous aggregates may contribute to disease pathogenesis. Since mechanisms leading to the formation of α-syn inclusions are unclear, in vitro models of α-syn aggregation may yield insights into this process. To that end, we examined the consequences on the progressive deletion of the carboxy-terminus of α-syn in regulating fibril formation, and we show here that carboxy-terminal truncated α-syn proteins aggregate faster than the full-length molecule. Protease digestion and immunoelectron microscopy indicate that the α-syn amino- and carboxy-termini are more solvent exposed than the central core and that filaments formed from carboxy-terminal truncated α-syn are narrower in diameter than the full-length molecule. Moreover, seeding experiments under conditions where full-length α-syn did not readily aggregate revealed that carboxy-truncated α-syn extending from amino acids 1−102 and 1−110 but not 1−120 were efficient in seeding full-length α-syn aggregation over a range of concentrations. Using site-directed mutagenesis, the negatively charged residues 104, 105 and 114, 115 in the carboxy-terminus were implicated in this reduced aggregation and the lack of seeding of full-length α-syn fibrillogenesis by 1−120. Our data support the view that the middle region of α-syn forms the core of α-syn filaments and that negative charges in the carboxy-terminus counteract α-syn aggregation. Thus, the carboxy-terminus of α-syn may regulate aggregation of full-length α-syn and determine the diameter of α-syn filaments.