Abstract
Assembled α-synuclein in nerve cells and glial cells is the defining pathological feature of neurodegenerative diseases called synucleinopathies. Seeds of α-synuclein can induce the assembly of monomeric protein. Here, we used sucrose gradient centrifugation and transiently transfected HEK 293T cells to identify the species of α-synuclein from the brains of homozygous, symptomatic mice transgenic for human mutant A53T α-synuclein (line M83) that seed aggregation. The most potent fractions contained Sarkosyl-insoluble assemblies enriched in filaments. We also analyzed six cases of idiopathic Parkinson's disease (PD), one case of familial PD, and six cases of multiple system atrophy (MSA) for their ability to induce α-synuclein aggregation. The MSA samples were more potent than those of idiopathic PD in seeding aggregation. We found that following sucrose gradient centrifugation, the most seed-competent fractions from PD and MSA brains are those that contain Sarkosyl-insoluble α-synuclein. The fractions differed between PD and MSA, consistent with the presence of distinct conformers of assembled α-synuclein in these different samples. We conclude that α-synuclein filaments are the main driving force for amplification and propagation of pathology in synucleinopathies.
Highlights
Assembled ␣-synuclein in nerve cells and glial cells is the defining pathological feature of neurodegenerative diseases called synucleinopathies
We found that following sucrose gradient centrifugation, the most seed-competent fractions from Parkinson’s disease (PD) and multiple system atrophy (MSA) brains are those that contain Sarkosylinsoluble ␣-synuclein
Addition of seeds induced the formation of Triton X-100 –insoluble ␣-synuclein that was positive with an antibody specific for phosphorylation at serine 129 (Fig. 1C)
Summary
Recombinant full-length [1–140] and N terminally truncated (6 –140) human A53T ␣-synucleins were expressed, purified, and assembled into filaments (Fig. 1, A and B). Sucrose gradient fractionated brain extracts from heterozygous M83 mice were unable to seed aggregation of human A53T ␣-synuclein in transfected HEK 293T cells (Fig. S1). Immunoelectron microscopy using an antibody specific for ␣-synuclein phosphorylated at Ser-129 was used to study the presence of ␣-synuclein assemblies in sucrose gradient fractions from the brains of mice transgenic for human A53T ␣-synuclein (Fig. 3). Western blot analysis with Syn-1 showed monomeric ␣-synuclein in all fractions and higher molecular weight ␣-synuclein in the 50% and pellet fractions (Fig. 7, A–D). Nondenaturing gel electrophoresis showed the presence of high-molecular weight assemblies of ␣-synuclein that did not migrate into the gel in the 50% and pellet fractions, and confirmed the presence of monomeric ␣-synuclein in the less dense fractions (Fig. 7E).
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
