Cellular Milieu Imparts Distinct Pathological α-Synuclein Strains in α-Synucleinopathies

Introduction: In Lewy body diseases-including Parkinson\u27s disease, without or with dementia, dementia with Lewy bodies, and Alzheimer\u27s disease with Lewy body co-pathology -α-synuclein (α-Syn) aggregates in neurons as Lewy bodies and Lewy neurites. By contrast, in multiple system atrophy α-Syn accumulates mainly in oligodendrocytes as glial cytoplasmic inclusions (GCIs) Objective: Our objective was to determine the conformational and biological profiles of a-Syn strains. Methods: The following methods were used to collect and analyze data: Recombinant α-Syn purification and in vitro fibrillization. Preparation of sarkosyl-insoluble fractions from disease and control brains. Sandwich ELISA. Cell cultures. Stereotaxic injection of sarkosyl-insoluble fraction of pathological α-Syn and α-Syn PFFs. Immunohistochemistry. Purification and depletion of α-Syn from the sarkosyl-insoluble fraction by immunoprecipitation. Results: GCI-α-Syn forms structures that are more compact and it is about 1,000-fold more potent than LB-α-Syn in seeding α-Syn aggregation, consistent with the highly aggressive nature of multiple system atrophy. We found that oligodendrocytes but not neurons transform misfolded α-Syn into a GCI-like strain. 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. Discussion: Here we report that pathological α-Syn in GCIs and Lewy bodies (GCI-α-Syn and LB- α-Syn, respectively) is conformationally and biologically distinct. Furthermore, we showed that distinct α-Syn strains had no cell type preference in seeding a-Syn pathology and are generated by different intracellular milieu

Improving synthetic efficiency through C???H activation

Selective C???H activation methods provide a complementary approach for synthesizing complex small molecules, which traditionally are constructed by chemists using C???C bond forming reactions to join preoxidized fragments. Furthermore, the strategic application of C???H activation reactions has considerable potential for improving the overall efficiency of synthetic endeavors by introducing functionality directly into preassembled hydrocarbon frameworks, mitigating the effect of having to carry reactive functionality throughout a reaction sequence. With this goal in mind, this work describes a series of projects that develop and implement novel C???H oxidation reactions and strategies. Firstly, a mild and efficient oxidation strategy for the preparation of chiral polyols is presented and validated through an enantioselective synthesis of differentially protected L-galactose. This synthesis is enabled by the development of a highly regio- and stereoselective linear allylic C???H oxidation reaction that generates 4-methoxybenzoate derivatives of chiral (E)-2-butene-1,4-diols directly from readily available chiral homoallylic alcohols and carboxylic acids. Secondly, this work details the discovery of a heterobimetallic Pd(II)bis-sulfoxide/(Salen)Cr(III)F catalyst system for asymmetric allylic C???H oxidation of terminal olefins. Evidence is provided that supports a model in which a chiral Lewis acid co-catalyst interacts with an organometallic intermediate and influences the stereochemical course of the catalytic process. Additionally, this work establishes that the asymmetric branched allylic oxidation reaction can be combined with other enantioselective transformations to afford enantiopure, polyoxygenated allylic alcohols rapidly and in good yields. Thirdly, this work outlines the development of a novel catalytic palladium(II)-based method for the conversion of ketones, ketoesters, and aldehydes directly to their unsaturated homologs, without the need for prior activation of the carbonyl. Importantly, this reaction shows good to excellent reactivity and unprecedented selectivities for a number of substrates with a diverse array of functional groups. Preliminary mechanistic studies suggest the reaction proceeds through a Pd-enolate intermediate that undergoes successive beta-hydride elimination to give the desired unsaturated carbonyl compounds, and that the acid additive is a key promoter of the reaction

Sequential Allylic C–H Amination/Vinylic C–H Arylation: A Strategy for Unnatural Amino Acid Synthesis from α-Olefins

Tandem reaction sequences that selectively convert multiple C–H bonds of abundant hydrocarbon feedstocks to functionalized materials enable rapid buildup of molecular complexity in an economical way. A tandem C–H amination/vinylic C–H arylation reaction sequence is described under Pd(II)/sulfoxide-catalysis that furnishes a wide range of α- and β-homophenylalanine precursors from commodity α-olefins and readily available aryl boronic acids. General routes to enantiopure amino acid esters and densely functionalized homophenylalanine derivatives are demonstrated

Molecular and Biological Compatibility with Host Alpha-Synuclein Influences Fibril Pathogenicity

The accumulation and propagation of misfolded α-synuclein (α-Syn) is a central feature of Parkinson’s disease 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 wild-type mice. Chimeric α-Syn fibrils demonstrate enhanced or reduced pathogenicities compared with wild-type Hu- or Ms-α-Syn fibrils. Furthermore, α-Syn mutants induced to polymerize by fibrillar seeds inherit the functional properties of their template, suggesting that transferable pathogenic and non-pathogenic states likely influence the initial engagement between exogenous α-Syn seeds and endogenous neuronal α-Syn. Thus, transmission of synucleinopathies is regulated by biological processes in addition to molecular compatibility

Solid-state NMR structure of a pathogenic fibril of full-length human alpha-synuclein

Misfolded a-synuclein amyloid fibrils are the principal components of Lewy bodies and neurites, hallmarks of Parkinson’s disease (PD). We present a high-resolution structure of an a-synuclein fibril, in a form that induces robust pathology in primary neuronal culture, determined by solid-state NMR spectroscopy and validated by EM and X-ray fiber diffraction. Over 200 unique longrange distance restraints define a consensus structure with common amyloid features including parallel, in-register b-sheets and hydrophobic-core residues, and with substantial complexity arising from diverse structural features including an intermolecular salt bridge, a glutamine ladder, close backbone interactions involving small residues, and several steric zippers stabilizing a new orthogonal Greek-key topology. These characteristics contribute to the robust propagation of this fibril form, as supported by the structural similarity of early-onset-PD mutants. The structure provides a framework for understanding the interactions of asynuclein with other proteins and small molecules, to aid in PD diagnosis and treatment.This study was supported by the US National Institutes of Health (NIH) (grants R01-GM073770 to C.M.R., P50-NS053488 to V.M.Y.L. and P01-AG002132 to G.S.) and used SSNMR instrumentation procured with the support of grant S10-RR025037 (to C.M.R.) from the NIH National Center for Research Resources (NCRR). M.D.T., A.J.N. and A.M.B. were supported as members of the NIH Molecular Biophysics Training Grant at the University of Illinois at UrbanaChampaign (T32-GM008276), and D.J.C. is supported by grant T32-AG000255