Bioinorganic chemistry of Parkinson's disease: structural determinants for the copper-mediated amyloid formation of alpha-synuclein

The aggregation of alpha-synuclein (AS) is a critical step in the etiology of Parkinson's disease (PD). A central, unresolved question in the pathophysiology of PD relates to the role of AS-metal interactions in amyloid fibril formation and neurodegeneration. Our previous works established a hierarchy in alpha-synuclein-metal ion interactions, where Cu(II) binds specifically to the protein and triggers its aggregation under conditions that might be relevant for the development of PD. Two independent, non-interacting copper-binding sites were identified at the N-terminal region of AS, with significant difference in their affinities for the metal ion. In this work we have solved unknown details related to the structural binding specificity and aggregation enhancement mediated by Cu(II). The high-resolution structural characterization of the highest affinity N-terminus AS-Cu(II) complex is reported here. Through the measurement of AS aggregation kinetics we proved conclusively that the copper-enhanced AS amyloid formation is a direct consequence of the formation of the AS-Cu(II) complex at the highest affinity binding site. The kinetic behavior was not influenced by the His residue at position 50, arguing against an active role for this residue in the structural and biological events involved in the mechanism of copper-mediated AS aggregation. These new findings are central to elucidate the mechanism through which the metal ion participates in the fibrillization of AS and represent relevant progress in the understanding of the bioinorganic chemistry of PD.     

Copper(II) binding to alpha-synuclein, the Parkinson's protein

Variations in tryptophan fluorescence intensities confirm that copper(II) interacts with alpha-synuclein, a protein implicated in Parkinson's disease. Trp4 fluorescence decay kinetics measured for the F4W protein show that Cu(II) binds tightly (Kd 100 nM) near the N-terminus at pH 7. Work on a F4W/H50S mutant indicates that a histidine imidazole is not a ligand in this high-affinity site.     

Spermine binding to Parkinson's protein alpha-synuclein and its disease-related A30P and A53T mutants

Aggregation of alpha-synuclein (alpha-syn), a protein implicated in Parkinson's disease (PD), is believed to progress through formation of a partially folded intermediate. Using nanoelectrospray ionization (nano-ESI) mass spectrometry combined with ion mobility measurements we found evidence for a highly compact partially folded family of structures for alpha-syn and its disease-related A53T mutant with net charges of -6, -7, and -8. For the other early onset PD mutant, A30P, this highly compact population was only evident when the protein had a net charge of -6. When bound to spermine near physiologic pH, all three proteins underwent a charge reduction from the favored solution charge state of -10 to a net charge of -6. This charge reduction is accompanied by a dramatic size reduction of about a factor of 2 (cross section of 2600 A2 (-10 charge state) down to 1430 A2 (-6 charge state)). We conclude that spermine increases the aggregation rate of alpha-syn by inducing a collapsed conformation, which then proceeds to form aggregates.     

Chemically induced Parkinson's disease. III: A study of a possible role of singlet molecular oxygen in Parkinson's disease.

The near IR emission at 1270 nm following pulsed laser excitation of methylene blue in deuterium oxide, was used to study the interaction of a singlet molecular oxygen (1O2) with (i) 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its oxidation products, and (ii) biosubstrates of relevance in Parkinson's disease. Steady state irradiation of methylene blue and MPTP led to a product with an absorption profile consistent with that of 1-methyl-4-phenyl-2,3-dihydropyridinium ion. This may suggest that even if monoamine oxidase enzyme activity is inhibited by the use of drugs such as Deprenyl and Paragyline the underlying conversion of MPTP to its neurotoxic oxidation product via 1O2 may still take place.     

Role of the ubiquitin proteasome system in Parkinson's disease

Parkinson's disease (PD) is the most common neurodegenerative movement disorder. Although a subject of intense research, the etiology of PD remains poorly understood. Recently, several lines of evidence have implicated an intimate link between aberrations in the ubiquitin proteasome system (UPS) and PD pathogenesis. Derangements of the UPS, which normally functions as a type of protein degradation machinery, lead to alterations in protein homeostasis that could conceivably promote the toxic accumulation of proteins detrimental to neuronal survival. Not surprisingly, various cellular and animal models of PD that are based on direct disruption of UPS function reproduce the most prominent features of PD. Although persuasive, new developments in the past few years have in fact raised serious questions about the link between the UPS and PD. Here I review current thoughts and controversies about their relationship and discuss whether strategies aimed at mitigating UPS dysfunction could represent rational ways to intervene in the disease. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com).     

Proteomics of Caenorhabditis elegans over-expressing human alpha-synuclein analyzed by fluorogenic derivatization-liquid chromatography/tandem mass spectrometry: identification of actin and several ribosomal proteins as negative markers at early Parkinson's disease stages

It has been known that the over-expression of alpha-synuclein, the main protein of Lewy bodies in Parkinson's disease (PD), leads to neurodegeneration in PD models. In this study, the changes in protein expression between the transgenic over-expressing human alpha-synuclein wild type (alpha-synWT) and the control Caenorhabditis elegans were elucidated by fluorogenic derivatization-liquid chromatography/tandem mass spectrometry (FD-LC-MS/MS) proteome analysis, which is a highly selective, sensitive, repeatable and quantitative method for protein identification. Because the alpha-synuclein wild-type worms showed moderate levels of dopamine loss without overt behavioral abnormalities, it was suggested that the changes in proteins in the alpha-synWT are related in the sequence of the formation of Lewy bodies. Among more than 400 protein peaks detected, actin and several ribosomal proteins were identified for the first time as negative markers at early PD stages. Actin was suggested to be one of the important targets in the elucidation of the etiology of neuronal diseases such as PD or other synucleinopathies.     

Changes in interfacial properties of alpha-synuclein preceding its aggregation

Parkinson's disease (PD) is associated with the formation and deposition of amyloid fibrils of the protein alpha-synuclein (AS). It has been proposed that oligomeric intermediates on the pathway to fibrilization rather than the fibrils themselves are the pathogenic agents of PD, but efficient methods for their detection are lacking. We have studied the interfacial properties of wild-type AS and the course of its aggregation in vitro using electrochemical analysis and dynamic light scattering. The oxidation signals of tyrosine residues of AS at carbon electrodes and the ability of fibrils to adsorb and catalyze hydrogen evolution at hanging mercury drop electrodes (HMDEs) decreased during incubation. HMDEs were particularly sensitive to pre-aggregation changes in AS. Already after 1 h of a standard aggregation assay in vitro (stirring at 37 degrees C), the electrocatalytic peak H increased greatly and shifted to less negative potentials. Between 3 and 9 h of incubation, an interval during which dynamic light scattering indicated AS oligomerization, peak H diminished and shifted to more negative potentials, and AS adsorbability decreased. We tentatively attribute the very early changes in the interfacial behavior of the protein after the first few hours of incubation to protein destabilization with disruption of long-range interactions. The subsequent changes can be related to the onset of oligomerization. Our results demonstrate the utility of electrochemical methods as new and simple tools for the investigation of amyloid formation.     

Tertiary contact formation in alpha-synuclein probed by electron transfer

To explore tertiary contact formation in alpha-synuclein, a natively unfolded protein implicated in Parkinson's disease, we have measured the rates of reaction between a powerful electron donor, the tryptophan (W) triplet excited state, and an acceptor, 3-nitro-tyrosine (Y(NO2)) in six different variants, probing loop sizes between 15 and 132 residues. Electron transfer rates decrease with loop size with the fastest contact time of 140 ns for the N-terminal pair and the slowest of 1.2 mus for the N- to C-terminal pair. Diffusion coefficients ranging from approximately 2 x 10-6 to approximately 10-5 cm2 s-1 were extracted from simultaneous fits of the W to Y(NO2) electron (triplet excited state) and energy transfer (singlet excited state) kinetics.     

Nontargeted metabolomics reveals sequential changes in amino acid and ferroptosis-related metabolism in Parkinson's disease

Parkinson's disease (PD) is inseparable from metabolic disorders but lacks assessment of specific metabolite alteration. To explore the sequential metabolic changes in PD progression, we evenly divided 78 C57BL/6 mice (10 weeks) into six groups (one control group and five experimental groups) and collected the hippocampus tissue of mice after treating with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, and probenecid (twice a week) at five periods (1, 2, 3, 4, and 5 weeks) for metabolome analysis. Our study identified 567 differentially abundant metabolites (DAMs) (total 4348 metabolites). Compared with controls, 145, 146, 171, 208, and 213 DAMs were obtained from the five experimental groups, respectively. Notably, 40 shared DAMs were present in five experimental groups, of which 22 shared DAMs formed a new metabolic network based on amino acid metabolism. Compared with group W3, 84 DAMs were identified in group W5, including 12 unique DAMs. DAMs in different stages of PD were significantly enriched in amino acid metabolism pathway, lipid metabolism pathway, and ferroptosis pathway. l -Glutamine, spermidine, and l -tryptophan were the key hubs in the whole metabolic process of PD. N-Formyl-l -methionine gradually increased in abundance with PD progression, whereas 5-methylcytosine gradually decreased. The study emphasized the sequential changes in DAMs in PD progression, stimulating subsequent studies.     

Rifampicin inhibits alpha-synuclein fibrillation and disaggregates fibrils

The aggregation of alpha-synuclein in dopaminergic neurons of the substantia nigra is a critical step in the pathogenesis of Parkinson's disease. We show that the antibiotic rifampicin inhibited alpha-synuclein fibrillation and disaggregated existing fibrils in a concentration-dependent manner. Size-exclusion chromatography data indicated that rifampicin stabilized alpha-synuclein as both a monomer and soluble oligomers comprised of partially folded alpha-synuclein. Experiments using aged samples of rifampicin indicated that the most active species in inhibiting fibrillation and disaggregating fibrils is an oxidation product of rifampicin, which was confirmed in experiments under anaerobic conditions. These results indicate that rifampicin-mediated inhibition of alpha-synuclein fibrillation and disaggregation of fibrils involves preferential stabilization of monomeric and soluble oligomeric forms, and that rifampicin potentially may have therapeutic application for Parkinson's disease.     

Antiparallel arrangement of the helices of vesicle-bound alpha-synuclein

alpha-Synuclein (alphaS) is the main component of Lewy bodies from Parkinson's disease. That alphaS binds to membranes is known, but the conformation it adopts is still unclear. Pulsed EPR on doubly spin-labeled variants of alphaS sheds light on the most likely structure. For alphaS bound to vesicles large enough to accommodate also the extended conformation, an antiparallel helix conformation is found. This suggests that the bent structure shown is the preferred conformation of alphaS on membranes.     

Inter-helix distances in lysophospholipid micelle-bound alpha-synuclein from pulsed ESR measurements

We demonstrate the use of pulsed ESR spectroscopy to measure intramolecular distances in the Parkinson's disease-associated protein alpha-synuclein bound to detergent and lysophospholipid micelles. We show that the inter-helical separation between the two helices formed upon binding to micelles is dependent on micelle composition, with micelles formed from longer acyl chains leading to an increased splaying of the two helices. Our data suggest that the topology of alpha-synuclein is not strongly constrained by the linker region between the two helices and instead depends on the geometry of the surface to which the protein is bound.     

Urine biomarkers discovery by metabolomics and machine learning for Parkinson's disease diagnoses

Parkinson’s disease(PD) is a complex neurological disorder that typically worsens with age. A wide range of pathologies makes PD a very heterogeneous condition, and there are currently no reliable diagnostic tests for this disease. The application of metabolomics to the study of PD has the potential to identify disease biomarkers through the systematic evaluation of metabolites. In this study, urine metabolic profiles of 215 urine samples from 104 PD patients and 111 healthy individuals were ass...     

Microglia, major player in the brain inflammation: their roles in the pathogenesis of Parkinson's disease

Inflammation, a self-defensive reaction against various pathogenic stimuli, may become harmful self-damaging process. Increasing evidence has linked chronic inflammation to a number of neurodegenerative disorders including Alzheimer's disease (AD), Parkinson's disease (PD), and multiple sclerosis. In the central nervous system, microglia, the resident innate immune cells play major role in the inflammatory process. Although they form the first line of defense for the neural parenchyma, uncontrolled activation of microglia may directly toxic to neurons by releasing various substances such as inflammatory cytokines (IL-1beta, TNF-alpha, IL-6), NO, PGE(2), and superoxide. Moreover, our recent study demonstrated that activated microglia phagocytose not only damaged cell debris but also neighboring intact cells. It further supports their active participation in self-perpetuating neuronal damaging cycles. In the following review, we discuss microglial responses to damaging neurons, known activators released from injured neurons and how microglia cause neuronal degeneration. In the last part, microglial activation and their role in PD are discussed in depth.     

Dopamine promotes formation and secretion of non-fibrillar alpha-synuclein oligomers

Parkinson's disease (PD) is characterized by selective and progressive degeneration of dopamine (DA)-producing neurons in the substantia nigra pars compacta (SNpc) and by abnormal aggregation of α-synuclein. Previous studies have suggested that DA can interact with α-synuclein, thus modulating the aggregation process of this protein; this interaction may account for the selective vulnerability of DA neurons in patients with PD. However, the relationship between DA and α-synuclein, and the role in progressive degeneration of DA neurons remains elusive. We have shown that in the presence of DA, recombinant human α-synuclein produces non-fibrillar, SDS-resistant oligomers, while β-sheet-rich fibril formation is inhibited. Pharmacologic elevation of the cytoplasmic DA level increased the formation of SDS-resistant oligomers in DA-producing neuronal cells. DA promoted α-synuclein oligomerization in intracellular vesicles, but not in the cytosol. Furthermore, elevation of DA levels increased secretion of α-synuclein oligomers to the extracellular space, but the secretion of monomers was not changed. DA-induced secretion of α-synuclein oligomers may contribute to the progressive loss of the dopaminergic neuronal population and the pronounced neuroinflammation observed in the SNpc in patients with PD.     

Characterization of surface-confined alpha-synuclein by surface plasmon resonance measurements

Urea-driven denaturation and renaturation of surface-bound alpha-synuclein are monitored by surface plasmon resonance (SPR) spectroscopy. The differential SPR angle shift (Delta Theta(SPR))(Net) enables us to estimate the Gibbs free energy change (DeltaG(o)) for the denaturation of the supported alpha-synuclein. DeltaG(o) for the denaturation of the supported alpha-synuclein, which is indirectly related to its biological activity can be increased significantly by the mixed self-assembled monolayers of 11-mercaptoundecanoic acid and 1,6-hexanedithiol. These SPR measurements of surface-bound biomolecules suggested herein can be further utilized to design effective biological scaffold for biosensor, biocatalyst, and possible diagnosis.     

Phosphorylation of an alpha-synuclein peptide fragment enhances metal binding

By using Tb3+ as a luminescent probe, we demonstrate that the phosphorylation state of a 14-residue peptide fragment of alpha-synuclein, a protein implicated in Parkinson's Disease, dramatically affects the metal ion affinity of the peptide. Whereas the unphosphorylated peptide and its phosphoserine analogue show weak Tb3+ binding, its phosphotyrosine analogue shows tight 1:1 binding as well as 2:1 and 3:1 Tb:peptide adducts. Our data suggest that the phosphorylated amino acid must be appropriately positioned among additional ligating residues to establish this phosphorylation-dependent metal binding.     

Raman spectroscopic characterization of secondary structure in natively unfolded proteins: alpha-synuclein

The application of Raman spectroscopy to characterize natively unfolded proteins has been underdeveloped, even though it has significant technical advantages. We propose that a simple three-component band fitting of the amide I region can assist in the conformational characterization of the ensemble of structures present in natively unfolded proteins. The Raman spectra of alpha-synuclein, a prototypical natively unfolded protein, were obtained in the presence and absence of methanol, sodium dodecyl sulfate (SDS), and hexafluoro-2-propanol (HFIP). Consistent with previous CD studies, the secondary structure becomes largely alpha-helical in HFIP and SDS and predominantly beta-sheet in 25% methanol in water. In SDS, an increase in alpha-helical conformation is indicated by the predominant Raman amide I marker band at 1654 cm(-1) and the typical double minimum in the CD spectrum. In 25% HFIP the amide I Raman marker band appears at 1653 cm(-1) with a peak width at half-height of approximately 33 cm(-1), and in 25% methanol the amide I Raman band shifts to 1667 cm(-1) with a peak width at half-height of approximately 26 cm(-1). These well-characterized structural states provide the unequivocal assignment of amide I marker bands in the Raman spectrum of alpha-synuclein and by extrapolation to other natively unfolded proteins. The Raman spectrum of monomeric alpha-synuclein in aqueous solution suggests that the peptide bonds are distributed in both the alpha-helical and extended beta-regions of Ramachandran space. A higher frequency feature of the alpha-synuclein Raman amide I band resembles the Raman amide I band of ionized polyglutamate and polylysine, peptides which adopt a polyproline II helical conformation. Thus, a three-component band fitting is used to characterize the Raman amide I band of alpha-synuclein, phosvitin, alpha-casein, beta-casein, and the non-A beta component (NAC) of Alzheimer's plaque. These analyses demonstrate the ability of Raman spectroscopy to characterize the ensemble of secondary structures present in natively unfolded proteins.