title: A sensitive assay reveals structural requirements for α-synuclein fibril growth.
abstract: The accumulation of α-synuclein (α-syn) fibrils in neuronal inclusions is the defining pathological process in Parkinson's disease (PD). A pathogenic role for α-syn fibril accumulation is supported by the identification of dominantly inherited α-syn (SNCA) gene mutations in rare cases of familial PD. Fibril formation involves a spontaneous nucleation event in which soluble α-syn monomers associate to form seeds, followed by fibril growth during which monomeric α-syn molecules sequentially associate with existing seeds. To better investigate this process, we developed sensitive assays that use the fluorescein arsenical dye FlAsH (fluorescein arsenical hairpin binder) to detect soluble oligomers and mature fibrils formed from recombinant α-syn protein containing an N-terminal bicysteine tag (C2-α-syn). Using seed growth by monomer association (SeGMA) assays to measure fibril growth over 3 h in the presence of C2-α-syn monomer, we observed that some familial PD-associated α-syn mutations (i.e.H50Q and A53T) greatly increased growth rates, whereas others (E46K, A30P, and G51D) decreased growth rates. Experiments with wild-type seeds extended by mutant monomer and vice versa revealed that single-amino acid differences between seed and monomer proteins consistently decreased growth rates. These results demonstrate that α-syn monomer association during fibril growth is a highly ordered process that can be disrupted by misalignment of individual amino acids and that only a subset of familial-PD mutations causes fibril accumulation through increased fibril growth rates. The SeGMA assays reported herein can be utilized to further elucidate structural requirements of α-syn fibril growth and to identify growth inhibitors as a potential therapeutic approach in PD.
authors.affiliation: From the Department of Neurology and Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, Missouri 63110.
authors.forename: Dhruva D
authors.identifier: 0000-0002-0096-1085
authors.initials: DD
authors.lastname: Dhavale
authors.affiliation: From the Department of Neurology and Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, Missouri 63110.
authors.forename: Christina
authors.initials: C
authors.lastname: Tsai
authors.affiliation: From the Department of Neurology and Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, Missouri 63110.
authors.forename: Devika P
authors.initials: DP
authors.lastname: Bagchi
authors.affiliation: From the Department of Neurology and Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, Missouri 63110.
authors.forename: Laura A
authors.initials: LA
authors.lastname: Engel
authors.affiliation: From the Department of Neurology and Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, Missouri 63110.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Sarezky
authors.affiliation: From the Department of Neurology and Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, Missouri 63110 kotzbauerp@wustl.edu.
authors.forename: Paul T
authors.initials: PT
authors.lastname: Kotzbauer
mesh_terms: D019943:Amino Acid Substitution; D000682:Amyloid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020125:Mutation, Missense; D010300:Parkinson Disease; D066263:Protein Aggregation, Pathological; D051844:alpha-Synuclein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000682:Amyloid; C497604:SNCA protein, human; D051844:alpha-Synuclein
keywords: FlAsH; Parkinson's disease; alpha-synuclein; biarsenical tetracysteine; fibril; mutations; neurodegeneration; oligomers; protein aggregation; protein labeling