title: Analysis of sheep α-synuclein provides a molecular strategy for the reduction of fibrillation.
abstract: Parkinson's disease (PD) presents with neuropathological inclusions called Lewy bodies, which are primarily composed of fibrillar α-synuclein. Recently, we characterized sheep with Gaucher disease and since GBA1 mutations represent the highest genetic risk factor for PD, we have investigated α-synuclein fibrillation in the sheep. Here we demonstrate that differences in six amino acid residues between sheep and human α-synuclein significantly alter in vitro fibril formation. Circular dichroism of recombinant human and sheep α-synuclein show that both proteins adopt the same secondary structure. Fibrils from human and sheep α-synuclein formed at pH7.0 or 4.5 were analyzed by Transmission Electron Microscopy (TEM). Unexpectedly, sheep α-synuclein form fibrils much less readily than human α-synuclein and this difference was more pronounced at the lysosomal pH of 4.5. Aggregation-propensity and intrinsic-solubility analysis revealed that sheep α-synuclein had lower aggregation-propensity and higher solubility. As a result of these observations, TEM was used to analyze fibrils formed at pH4.5 of various "sheep-like" human or "human-like" sheep mutant α-synucleins, together with their wild-type forms. Thioflavin T was used to monitor in situ α-synuclein fibril formation at pH7.0 and 4.5. Results show that "sheep-like" human α-synuclein has substantially lower fibril aggregation, and "human-like" sheep α-synuclein aggregates faster than wild-type forms, respectively. Seeding with WT human α-synuclein showed that "sheep-like" human α-synuclein could not be seeded, providing further evidence that sheep sequence is resistant to fibrillation. These findings provide new avenues to prevent/reduce fibrillation in PD, which may aid in the development of therapies.
authors.affiliation: Lysosomal Diseases Research Unit, South Australian Health and Medical Research Institute, North Terrace, Adelaide, South Australia 5000, Australia. Electronic address: lungisa.bickle@sahmri.com.
authors.forename: Lungisa
authors.initials: L
authors.lastname: Bickle
authors.affiliation: Lysosomal Diseases Research Unit, South Australian Health and Medical Research Institute, North Terrace, Adelaide, South Australia 5000, Australia.
authors.forename: John J
authors.initials: JJ
authors.lastname: Hopwood
authors.affiliation: Lysosomal Diseases Research Unit, South Australian Health and Medical Research Institute, North Terrace, Adelaide, South Australia 5000, Australia.
authors.forename: Litsa
authors.initials: L
authors.lastname: Karageorgos
mesh_terms: D000595:Amino Acid Sequence; D000682:Amyloid / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D052160:Benzothiazoles; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016631:Lewy Bodies / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010300:Parkinson Disease / Q000235:genetics / Q000378:metabolism; D066263:Protein Aggregation, Pathological / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012756:Sheep / Q000235:genetics / Q000378:metabolism*; D012995:Solubility; D013844:Thiazoles / Q000378:metabolism; D051844:alpha-Synuclein / Q000235:genetics / Q000378:metabolism*
chemical_list: D000682:Amyloid; D052160:Benzothiazoles; D013844:Thiazoles; D051844:alpha-Synuclein; C009462:thioflavin T
keywords: Aggregation; Amyloid; Fibril; Parkinson's disease; α-Synuclein