Structural heterogeneity of α-synuclein fibrils amplified from patient brain extracts (original) (raw)
Related papers
Structured Regions of α-Synuclein Fibrils Include the Early-Onset Parkinson's Disease Mutation Sites
Journal of Molecular Biology, 2011
α-Synuclein (AS) fibrils are the major component of Lewy bodies, the pathological hallmark of Parkinson's disease (PD). Here, we use results from an extensive investigation employing solidstate NMR to present a detailed structural characterization and conformational dynamics quantification of full-length AS fibrils. Our results show that the core extends with a repeated structural motif. This result disagrees with the previously proposed fold of AS fibrils obtained with limited solid-state NMR data. Additionally, our results demonstrate that the three single point mutations associated with early-onset PD-A30P, E46K and A53T-are located in structured regions. We find that E46K and A53T mutations, located in rigid β-strands of the wild-type fibrils, are associated with major and minor structural perturbations, respectively.
PLoS ONE, 2013
Parkinson's disease (PD) is pathologically characterized by the presence of Lewy bodies (LBs) in dopaminergic neurons of the substantia nigra. These intracellular inclusions are largely composed of misfolded a-synuclein (AS), a neuronal protein that is abundant in the vertebrate brain. Point mutations in AS are associated with rare, early-onset forms of PD, although aggregation of the wild-type (WT) protein is observed in the more common sporadic forms of the disease. Here, we employed multidimensional solid-state NMR experiments to assess A53T and E46K mutant fibrils, in comparison to our recent description of WT AS fibrils. We made de novo chemical shift assignments for the mutants, and used these chemical shifts to empirically determine secondary structures. We observe significant perturbations in secondary structure throughout the fibril core for the E46K fibril, while the A53T fibril exhibits more localized perturbations near the mutation site. Overall, these results demonstrate that the secondary structure of A53T has some small differences from the WT and the secondary structure of E46K has significant differences, which may alter the overall structural arrangement of the fibrils.
Structurally Distinct α‐Synuclein Fibrils Induce Robust Parkinsonian Pathology
Movement Disorders, 2019
Objective: Alpha-synuclein (α-syn) is a major component of Lewy bodies, which are the pathological hallmark in Parkinson's disease, and its genetic mutations cause familial forms of Parkinson's disease. Patients with α-syn G51D mutation exhibit severe clinical symptoms. However, in vitro studies showed low propensity for α-syn with the G51D mutation. We studied the mechanisms associated with severe neurotoxicity of α-syn G51D mutation using a murine model generated by G51D α-syn fibril injection into the brain. Methods: Structural analysis of wild-type and G51D α-syn-fibrils were performed using Fourier transform infrared spectroscopy. The ability of α-syn fibrils forming aggregates was first assessed in in vitro mammalian cells. An in vivo mouse model with an intranigral injection of α-syn fibrils was then used to evaluate the propagation pattern of α-syn and related cellular changes.
Journal of fluorescence, 2016
The etiology of Parkinson's disease involves the interplay between the environmental and genetic factors. Here in this study human α-synuclein upon exposure to 100 μM pendimethalin for 12 h in vitro passes through a partially folded state which proceeds to the aggregated state and terminally ends in the fibrillar phase. Variations in the ANS fluorescence intensities led to the detection of intermediate and aggregated states at 6 and 10 h respectively. Far-UV CD analysis depicted significant α-helical content for intermediate state at 6 h in presence of 100 μM pendimethalin. Further increasing the incubation time to 12 h resulted in a predominant β-sheet content which was confirmed to be fibrillar by TEM. Turbidity, Rayleigh scattering analysis, Congo red assay and ThT measurements supported the TEM data i.e. the formation of fibrillar structure of human α-synuclein upon 12 h incubation. Thus, our observation could suggest a possible underlying molecular basis for Parkinson's...
PLoS ONE, 2013
Amyloid fibrils of a-synuclein are the main constituent of Lewy bodies deposited in substantial nigra of Parkinson's disease brains. a-Synuclein is an intrinsically disordered protein lacking compact secondary and tertiary structures. To enhance the understanding of its structure and function relationship, we utilized temperature treatment to study a-synuclein conformational changes and the subsequent effects. We found that after 1 hr of high temperature pretreatment, .80uC, asynuclein fibrillization was significantly inhibited. However, the temperature melting coupled with circular dichroism spectra showed that a-synuclein was fully reversible and the NMR studies showed no observable structural changes of a-synuclein after 95uC treatment. By using cross-linking and analytical ultracentrifugation, rare amount of pre-existing a-synuclein oligomers were found to decrease after the high temperature treatment. In addition, a small portion of C-terminal truncation of a-synuclein also occurred. The reduction of pre-existing oligomers of a-synuclein may contribute to less seeding effect that retards the kinetics of amyloid fibrillization. Overall, our results showed that the pre-existing oligomeric species is a key factor contributing to a-synuclein fibrillization. Our results facilitate the understanding of a-synuclein fibrillization.
Communications Biology
Parkinson’s disease (PD) and Multiple System Atrophy (MSA) are progressive and unremitting neurological diseases that are neuropathologically characterized by α-synuclein inclusions. Increasing evidence supports the aggregation of α-synuclein in specific brain areas early in the disease course, followed by the spreading of α-synuclein pathology to multiple brain regions. However, little is known about how the structure of α-synuclein fibrils influence its ability to seed endogenous α-synuclein in recipient cells. Here, we aggregated α-synuclein by seeding with homogenates of PD- and MSA-confirmed brain tissue, determined the resulting α-synuclein fibril structures by cryo-electron microscopy, and characterized their seeding potential in mouse primary oligodendroglial cultures. The combined analysis shows that the two patient material-amplified α-synuclein fibrils share a similar protofilament fold but differ in their inter-protofilament interface and their ability to recruit endogen...
Journal of Biological Chemistry, 1999
Parkinson's disease (PD) is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies, the major components of which are filaments consisting of ␣-synuclein. Two recently identified point mutations in ␣-synuclein are the only known genetic causes of PD. ␣-Synuclein fibrils similar to the Lewy body filaments can be formed in vitro, and we have shown recently that both PDlinked mutations accelerate their formation. This study addresses the mechanism of ␣-synuclein aggregation: we show that (i) it is a nucleation-dependent process that can be seeded by aggregated ␣-synuclein functioning as nuclei, (ii) this fibril growth follows first-order kinetics with respect to ␣-synuclein concentration, and (iii) mutant ␣-synuclein can seed the aggregation of wild type ␣-synuclein, which leads us to predict that the Lewy bodies of familial PD patients with ␣-synuclein mutations will contain both, the mutant and the wild type protein. Finally (iv), we show that wild type and mutant forms of ␣-synuclein do not differ in their critical concentrations. These results suggest that differences in aggregation kinetics of ␣-synucleins cannot be explained by differences in solubility but are due to different nucleation rates. Consequently, ␣-synuclein nucleation may be the rate-limiting step for the formation of Lewy body ␣-synuclein fibrils in Parkinson's disease.
Defining α-synuclein species responsible for Parkinson disease phenotypes in mice
Journal of Biological Chemistry
Edited by Paul E. Fraser Parkinson's disease (PD) is a neurodegenerative disorder characterized by fibrillar neuronal inclusions composed of aggregated ␣-synuclein (␣-syn). These inclusions are associated with behavioral and pathological PD phenotypes. One strategy for therapeutic interventions is to prevent the formation of these inclusions to halt disease progression. ␣-Synuclein exists in multiple structural forms, including disordered, nonamyloid oligomers, ordered amyloid oligomers, and fibrils. It is critical to understand which conformers contribute to specific PD phenotypes. Here, we utilized a mouse model to explore the pathological effects of stable -amyloid-sheet oligomers compared with those of fibrillar ␣-synuclein. We biophysically characterized these species with transmission EM, atomic-force microscopy, CD spectroscopy, FTIR spectroscopy, analytical ultracentrifugation, and thioflavin T assays. We then injected these different ␣-synuclein forms into the mouse striatum to determine their ability to induce PD-related phenotypes. We found that -sheet oligomers produce a small but significant loss of dopamine neurons in the substantia nigra pars compacta (SNc). Injection of small -sheet fibril fragments, however, produced the most robust phenotypes, including reduction of striatal dopamine terminals, SNc loss of dopamine neurons, and motor-behavior defects. We conclude that although the -sheet oligomers cause some toxicity, the potent effects of the short fibrillar fragments can be attributed to their ability to recruit monomeric ␣-synuclein and spread in vivo and hence contribute to the development of PD-like phenotypes. These results suggest that strategies to reduce the formation and propagation of -sheet fibrillar species could be an important route for therapeutic intervention in PD and related disorders. This work was supported in part by the Michael J. Fox Foundation (to L. V.-D. and N. C.) and Grant P50NS108675 (Alabama Udall Center). The authors declare that they have no conflicts of interest with the contents of this article. This article was selected as one of our Editors' Picks. This article contains Figs. S1-S5.
The EMBO …, 2009
The relation of a-synuclein (aS) aggregation to Parkinson's disease (PD) has long been recognized, but the mechanism of toxicity, the pathogenic species and its molecular properties are yet to be identified. To obtain insight into the function different aggregated aS species have in neurotoxicity in vivo, we generated aS variants by a structurebased rational design. Biophysical analysis revealed that the aS mutants have a reduced fibrillization propensity, but form increased amounts of soluble oligomers. To assess their biological response in vivo, we studied the effects of the biophysically defined pre-fibrillar aS mutants after expression in tissue culture cells, in mammalian neurons and in PD model organisms, such as Caenorhabditis elegans and Drosophila melanogaster. The results show a striking correlation between aS aggregates with impaired b-structure, neuronal toxicity and behavioural defects, and they establish a tight link between the biophysical properties of multimeric aS species and their in vivo function.
Proceedings of the National Academy of Sciences, 2000
The Parkinson's disease (PD) substantia nigra is characterized by the presence of Lewy bodies containing fibrillar α-synuclein. Early-onset PD has been linked to two point mutations in the gene that encodes α-synuclein, suggesting that disease may arise from accelerated fibrillization. However, the identity of the pathogenic species and its relationship to the α-synuclein fibril has not been elucidated. In this in vitro study, the rates of disappearance of monomeric α-synuclein and appearance of fibrillar α-synuclein were compared for the wild-type (WT) and two mutant proteins, as well as equimolar mixtures that may model the heterozygous PD patients. Whereas one of the mutant proteins (A53T) and an equimolar mixture of A53T and WT fibrillized more rapidly than WT α-synuclein, the other (A30P) and the corresponding equimolar mixture with WT fibrillized more slowly. However, under conditions that ultimately produced fibrils, the A30P monomer was consumed at a comparable rate or s...