Misfolded α-synuclein amyloid fibrils are the principal components of Lewy bodies and neurites, hallmarks of Parkinson’s disease (PD). Here we present a high-resolution structure of an α-synuclein fibril, in a form that induces robust pathology in primary neuronal culture, determined by solid-state NMR spectroscopy and validated by electron microscopy and X-ray fiber diffraction. Over 200 unique long-range distance restraints define a consensus structure with common amyloid features including parallel in-register β-sheets and hydrophobic core residues, but also substantial complexity, arising from diverse structural features: an intermolecular salt bridge, a glutamine ladder, close backbone interactions involving small residues, and several steric zippers stabilizing a novel, orthogonal Greek-key topology. These characteristics contribute to the robust propagation of this fibril form, as evidenced by structural similarity of early-onset PD mutants. The structure provides a framework for understanding the interactions of α-synuclein with other proteins and small molecules to diagnose and treat PD.

SUMMARY Many neurodegenerative diseases are characterized by the accumulation of insoluble protein aggregates, including neurofibrillary tangles comprised of tau in Alzheimer’s disease and Lewy bodies composed of α-synuclein in Parkinson’s disease. Moreover, different pathological proteins frequently codeposit in disease brains. To test whether aggregated α-synuclein can directly cross-seed tau fibrillization, we administered preformed α-synuclein fibrils assembled from recombinant protein to primary neurons and transgenic mice. Remarkably, we discovered two distinct strains of synthetic α-synuclein fibrils that demonstrated striking differences in the efficiency of cross-seeding tau aggregation, both in neuron cultures and in vivo. Proteinase K digestion revealed conformational differences between the two synthetic α-synuclein strains and also between sarkosyl-insoluble α-synuclein extracted from two subgroups of Parkinson’s disease brains. We speculate that distinct strains of pathological α-synuclein likely exist in neurodegenerative disease brains and may underlie the tremendous heterogeneity of synucleinopathies.

In Lewy body (LB) diseases, including Parkinson’s disease (PD), without and with dementia (PDD), dementia with Lewy bodies (DLB) and Alzheimer’s disease (AD) patients with LB co-pathology1, α-synuclein (α-Syn) aggregates in neurons as LBs and Lewy neurites (LNs)2, while in multiple system atrophy (MSA), α-Syn mainly accumulates in oligodendrocytes as glial cytoplasmic inclusions (GCIs)3. Here, we report that pathological α-Syn in GCIs and LBs (GCI-α-Syn and LB-α-Syn) are conformationally and biologically distinct. GCI-α-Syn forms more compact structures and is ~1,000-fold more potent than LB-α-Syn in seeding α-Syn aggregation, consistent with the highly aggressive nature of MSA. Surprisingly, GCI-α-Syn and LB-α-Syn show no cell type preference in seeding α-Syn pathology, raising the question of why they demonstrate different cell type distributions in LB disease versus MSA. Strikingly, we found that oligodendrocytes but not neurons transform misfolded α-Syn into a GCI-like strain, highlighting that distinct α-Syn strains are generated by different intracellular milieus. Moreover, GCI-α-Syn maintains its high seeding activity when propagated in neurons. Thus, α-Syn strains are determined by both misfolded seeds and intracellular environments.

Summary The accumulation and propagation of misfolded α-Synuclein (α-Syn) is a central feature of Parkinson’s disease (PD) and other synucleinopathies. Molecular compatibility between a fibrillar seed and its native protein state is a major determinant of amyloid self-replication. We show that cross-seeded aggregation of human (Hu) and mouse (Ms) α-Syn is bidirectionally restricted. Although fibrils formed by Hu-Ms-α-Syn chimeric mutants can overcome this inhibition in cell-free systems, sequence homology poorly predicts their efficiency in inducing α-Syn pathology in primary neurons or after intracerebral injection into wildtype mice. Chimeric α-Syn fibrils demonstrate enhanced or reduced pathogenicities compared to wildtype Hu- or Ms-α-Syn fibrils. Furthermore, α-Syn mutants induced to polymerize by fibrillar seeds inherit the functional properties of their template, suggesting transferable pathogenic and non-pathogenic states likely influence the initial engagement between exogenous α-Syn seeds with endogenous neuronal α-Syn. Thus, transmission of synucleinopathies is regulated by biological processes in addition to molecular compatibility.

Keywords allylic oxidation; C-H activation; enantioselectivity C-H oxidation reactions have the potential to significantly streamline synthetic processes. However, to be useful for the synthesis of complex molecules, these reactions must proceed with high levels of chemo-, regio-, and stereoselectivity, Chiral bisoxazoline/copper-catalyzed systems have shown promising levels of asymmetric induction in the enantioselective allylic C-H esterification of symmetrical, cyclic olefins. Application of these systems to complex substrates is limited by a lack of chemo-and regioselectivity as well as the need to use a large excess of reactant (4 to 10 equiv).[1] A direct allylic C-H oxidation route would significantly increase the efficiency of producing chiral allylic esters; their syntheses generally require lengthy sequences of functional-group manipulations from preoxidized materials. [2,3] We have recently reported a collection of mild, highly regio-and chemoselective, allylic C-H esterification[4] and amination[5] reactions of α-olefins, and have demonstrated their utility in streamlining the synthesis of complex molecules.[6] These allylic oxidation reactions are catalyzed by Pd II systems with weakly coordinating sulfoxide and quinone ligands that are poorly suited for effecting asymmetric induction.[7] Herein we disclose a novel chiral Lewis acid strategy for generating an asymmetric environment about a metal center in electrophilic, oxidative reactions that do not tolerate strongly coordinating ligands. In our approach a chiral Lewis acid is used which selectively interacts with an organopalladium intermediate to accelerate and induce asymmetry in the C-O bond-forming step. Significantly, by using this strategy we have achieved the highest enantioselection observed to date for the allylic C-H oxidation of terminal olefins (Scheme 1). [8] This system represents the first example of asymmetric induction from an organometallic intermediate that is effected by a chiral Lewis acid, as well as a rare example of catalytic enantioselective C-H activation by palladium. [9] Conventional approaches toward asymmetric organometallic reactions make use of strongly coordinating chiral ligands. The oxidation of terminal olefins using 1 to give branched allylic compounds has been demonstrated to proceed by a serial ligand catalysis mechanism in which weakly coordinating bis(sulfoxide) and 1,4-benzoquinone (BQ) ligands sequentially interact with the Pd center to promote the C-H bond-cleavage and C-O bond-forming steps, ** Financial support was provided by NIH/NIGMS (grant no. GM076153) and kind gifts were received from Eli Lilly, Bristol-Myers Squibb, Pfizer, Amgen, and Merck Research Laboratories. D.J.C. is a recipient of a Roger Adams and a C.S. Marvel graduate fellowship. We thank the Aldrich Chemical Company for a generous gift of commercial bis(sulfoxide)/Pd(OAc) 2 catalyst 1. We thank A. Young for checking the experimental procedure outlined in Table 2, entry 4. We thank Dr. N. Prabagaran for preliminary experiments.Supp...

Aims The aim of this study was to test the hypothesis that different conformations of misfolded α-synuclein (α-syn) are present in Parkinson’s disease (PD) brain. Methods Using two previously characterized conformations of α-syn fibrils, we generated new conformation-selective, α-syn monoclonal antibodies (mAbs). We then interrogated multiple brain regions in a well-characterized autopsy cohort of PD patients (n = 49) with these mAbs, Syn7015 and Syn9029. Results Syn7015 detects Lewy bodies (LBs) and Lewy neurites (LNs) formed by pathological α-syn in all brain regions tested, and is particularly sensitive to LNs and small Lewy dots, inclusions believed to form early in the disease. Further, we observed co-localization between Syn7015 and an early marker of α-syn pathology formation, phospho-Ser129-α-syn, and a lack of extensive co-localization with markers of more mature pathology. In comparison, Syn9029 detects Lewy pathology in all regions examined, but indicates significantly fewer LNs than Syn7015. In addition, co-localization of Syn9029 with later markers of α-syn pathology maturation (ubiquitin and P62) suggests that the pathology detected by Syn9029 is older. Semi-quantitative scoring of both LN and LB pathology in nine brain regions further established this trend, with Syn7015 LN scores consistently higher than Syn9029 LN scores. Conclusions Our data indicate that different conformations of α-syn pathology are present in PD brain and correspond to different stages of maturity for Lewy pathology. Regional analysis of Syn7015 and Syn9029 immunostaining also provides support for the Braak hypothesis that α-syn pathology advances through the brain.

Characterization of the amyloidogenic Parkinson’s Disease protein α-synuclein (αS) has proven difficult due to its structural plasticity. Here, we present a number of complementary methods to site-specifically introduce fluorescent probes to examine αS fibril formation and cellular uptake. By using various combinations of conventional Cys modification, amber codon suppression, transferase mediated N-terminal modification, and native chemical ligation, several variants of singly- and doubly-labeled αS were produced. We validated the nonperturbative nature of the label by a combination of in vitro aggregation kinetics measurements and imaging of the resulting fibrils. The labeled αS can then be used to monitor conformational changes during fibril formation or cellular uptake of αS fibrils in models of disease propagation.

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