Quantitative proteomics of tau and Aß in detergent fractions from Alzheimer's disease brains.

The two hallmarks of Alzheimer's disease (AD) are amyloid-ß (Aß) plaques and neurofibrillary tangles marked by phosphorylated tau. Increasing evidence suggests that aggregating Aß drives tau accumulation, a process that involves synaptic degeneration leading to cognitive impairment. Conversely, there is a realization that non-fibrillar (oligomeric) forms of Aß mediate toxicity in AD. Fibrillar (filamentous) aggregates of proteins across the spectrum of the primary and secondary tauopathies were the focus of recent structural studies with a filament structure-based nosologic classification, but less emphasis was given to non-filamentous co-aggregates of insoluble proteins in the fractions derived from post-mortem human brains. Here, we revisited sarkosyl-soluble and -insoluble extracts to characterize tau and Aß species by quantitative targeted mass spectrometric proteomics, biochemical assays, and electron microscopy. AD brain sarkosyl-insoluble pellets were greatly enriched with Aß42 at almost equimolar levels to N-terminal truncated microtubule-binding region (MTBR) isoforms of tau with multiple site-specific post-translational modifications (PTMs). MTBR R3 and R4 tau peptides were most abundant in the sarkosyl-insoluble materials with a 10-fold higher concentration than N-terminal tau peptides. This indicates that the major proportion of the enriched tau was the aggregation-prone N-terminal and proline-rich region (PRR) of truncated mixed 4R and 3R tau with more 4R than 3R isoforms. High concentration and occupancies of site-specific phosphorylation pT181 (~22%) and pT217 (~16%) (key biomarkers of AD) along with other PTMs in the PRR and MTBR indicated a regional susceptibility of PTMs in aggregated tau. Immunogold labelling revealed that tau may exist in globular non-filamentous form (N-terminal intact tau) co-localized with Aß in the sarkosyl-insoluble pellets along with tau filaments (N-truncated MTBR tau). Our results suggest a model that Aß and tau interact forming globular aggregates, from which filamentous tau and Aß emerge. These characterizations contribute towards unravelling the sequence of events which lead to end-stage AD changes.

Citrullination of Amyloid-ß Peptides in Alzheimer's Disease.

Protein citrullination (deimination of arginine residue) is a well-known biomarker of inflammation. Elevated protein citrullination has been shown to colocalize with extracellular amyloid plaques in postmortem AD patient brains. Amyloid-ß (Aß) peptides which aggregate and accumulate in the plaques of Alzheimer's disease (AD) have sequential N-terminal truncations and multiple post-translational modifications (PTM) such as isomerization, pyroglutamate formation, phosphorylation, nitration, and dityrosine cross-linking. However, no conclusive biochemical evidence exists whether citrullinated Aß is present in AD brains. In this study, using high-resolution mass spectrometry, we have identified citrullination of Aß in sporadic and familial AD brains by characterizing the tandem mass spectra of endogenous N-truncated citrullinated Aß peptides. Our quantitative estimations demonstrate that ∼ 35% of pyroglutamate3-Aß pool was citrullinated in plaques in the sporadic AD temporal cortex and ∼ 22% in the detergent-insoluble frontal cortex fractions. Similarly, hypercitrullinated pyroglutamate3-Aß (∼ 30%) was observed in both the detergent-soluble as well as insoluble Aß pool in familial AD cases. Our results indicate that a common mechanism for citrullination of Aß exists in both the sporadic and familial AD. We establish that citrullination of Aß is a remarkably common PTM, closely associated with pyroglutamate3-Aß formation and its accumulation in AD. This may have implications for Aß toxicity, autoantigenicity of Aß, and may be relevant for the design of diagnostic assays and therapeutic targeting.

Quantification of N-terminal amyloid-ß isoforms reveals isomers are the most abundant form of the amyloid-ß peptide in sporadic Alzheimer's disease.

Plaques that characterize Alzheimer's disease accumulate over 20 years as a result of decreased clearance of amyloid-ß peptides. Such long-lived peptides are subjected to multiple post-translational modifications, in particular isomerization. Using liquid chromatography ion mobility separations mass spectrometry, we characterized the most common isomerized amyloid-ß peptides present in the temporal cortex of sporadic Alzheimer's disease brains. Quantitative assessment of amyloid-ß N-terminus revealed that > 80% of aspartates (Asp-1 and Asp-7) in the N-terminus was isomerized, making isomerization the most dominant post-translational modification of amyloid-ß in Alzheimer's disease brain. Total amyloid-ß1-15 was ∼85% isomerized at Asp-1 and/or Asp-7 residues, with only 15% unmodified amyloid-ß1-15 left in Alzheimer's disease. While amyloid-ß4-15 the next most abundant N-terminus found in Alzheimer's disease brain, was only ∼50% isomerized at Asp-7 in Alzheimer's disease. Further investigations into different biochemically defined amyloid-ß-pools indicated a distinct pattern of accumulation of extensively isomerized amyloid-ß in the insoluble fibrillar plaque and membrane-associated pools, while the extent of isomerization was lower in peripheral membrane/vesicular and soluble pools. This pattern correlated with the accumulation of aggregation-prone amyloid-ß42 in Alzheimer's disease brains. Isomerization significantly alters the structure of the amyloid-ß peptide, which not only has implications for its degradation, but also for oligomer assembly, and the binding of therapeutic antibodies that directly target the N-terminus, where these modifications are located.

Modulating Protein Phosphatase 2A Rescues Disease Phenotype in Neurodegenerative Tauopathies.

Alzheimer's disease (AD) is the leading cause of dementia worldwide accounting for around 70% of all cases. There is currently no treatment for AD beyond symptom management and attempts at developing disease-modifying therapies have yielded very little. These strategies have traditionally targeted the peptide Aß, which is thought to drive pathology. However, the lack of clinical translation of these Aß-centric strategies underscores the need for diverse treatment strategies targeting other aspects of the disease. Metal dyshomeostasis is a common feature of several neurodegenerative diseases such as AD, Parkinson's disease, and frontotemporal dementia, and manipulation of metal homeostasis has been explored as a potential therapeutic avenue for these diseases. The copper ionophore glyoxalbis-[N4-methylthiosemicarbazonato]Cu(II) (CuII(gtsm)) has previously been shown to improve the cognitive deficits seen in an AD animal model; however, the molecular mechanism remained unclear. Here we report that the treatment of two animal tauopathy models (APP/PS1 and rTg4510) with CuII(gtsm) recovers the cognitive deficits seen in both neurodegenerative models. In both models, markers of tau pathology were significantly reduced with CuII(gtsm) treatment, and in the APP/PS1 model, the levels of Aß remained unchanged. Analysis of tau kinases (GSK3ß and CDK5) revealed no drug induced changes; however, both models exhibited a significant increase in the levels of the structural subunit of the tau phosphatase, PP2A. These findings suggest that targeting the tau phosphatase PP2A has therapeutic potential for preventing memory impairments and reducing the tau pathology seen in AD and other tauopathies.

A randomized, exploratory molecular imaging study targeting amyloid ß with a novel 8-OH quinoline in Alzheimer's disease: The PBT2-204 IMAGINE study.

INTRODUCTION: We are developing a second generation 8-OH quinoline (2-(dimethylamino) methyl-5, 7-dichloro-8-hydroxyquinoline [PBT2, Prana Biotechnology]) for targeting amyloid ß (Aß) in Alzheimer's disease (AD). In an earlier phase IIa, 3 month trial, PBT2 lowered cerebrospinal fluid Aß by 13% and improved cognition (executive function) in a dose-related fashion in early AD. We, therefore, sought to learn whether PBT2 could alter the Aß-PET signal in subjects with prodromal or mild AD, in an exploratory randomized study over a 12-month phase in a double-blind and a 12-month open label extension phase trial design. METHODS: For inclusion, the usual clinical criteria for prodromal or probable AD, Mini-Mental State Examination ≥20, and global Pittsburgh compound B (PiB)-PET standardized uptake volume ratio (SUVR) >1.7 were used. As this was an exploratory study, we included contemporaneous matched control data from the Australian Imaging Biomarker and Lifestyle Study (AIBL). Other measures included fluorodeoxyglucose-positron emission tomography, magnetic resonance imaging volumetrics, blood Aß biomarkers, and cognition and function. RESULTS: Forty subjects completed the first 12-month double-blind phase (placebo = 15, PBT2 = 25), and 27 subjects completed the 12-month open label extension phase (placebo = 11, PBT2 = 16). Overall, PTB2 250 mg/day was safe and well tolerated. The mean PiB-PET SUVR at baseline was 2.51 ± 0.59. After adjusting for baseline SUVR, in the double-blind phase, the placebo group showed a nonsignificant decline in PiB-PET SUVR, whereas the PBT2 group declined significantly (P = .048). Subjects who did not enter or complete the extension study had a significantly higher 12-month Aß-PET SUVR (2.68 ± 0.55) compared with those who completed (2.29 ± 0.48). Both groups differed significantly from the rate of change over 12 months in the AIBL control group. In the open label 12-month extension study, the PiB-SUVR stabilized. There were no significant differences between PBT2 and controls in fluorodeoxyglucose-positron emission tomography, magnetic resonance imaging volumetrics, blood Aß biomarkers, or cognition/function over the course of the double-blind phase. DISCUSSION: There was no significant difference between PBT2 and controls at 12 months, likely due to the large individual variances over a relatively small number of subjects. PBT2 was associated with a significant 3% PiB-PET SUVR decline in the double-blind phase and a stabilization of SUVR in the open-label phase. From this exploratory study, we have learned that the entry criterion of SUVR should have been set at ≥ 1.5 and <2.0, where we know from the AIBL study that subjects in this band are accumulating Aß in a linear fashion and that subjects who withdrew from this type of study have much higher SUVRs, which if not taken into account, could distort the final results. Because of large individual variations in SUVR, future studies of PBT2 will require larger numbers of subjects (n > 90 per arm) over a longer period (18 months or more). Further evaluation of higher doses of PBT2 in earlier stages of AD is warranted. TRIAL REGISTRATION: ACTRN 12611001008910 and ACTRN 12613000777796.

Peripheral α-defensins 1 and 2 are elevated in Alzheimer's disease.

Biomarkers enabling the preclinical identification of Alzheimer's disease (AD) remain one of the major unmet challenges in the field. The blood cellular fractions offer a viable alternative to current cerebrospinal fluid and neuroimaging modalities. The current study aimed to replicate our earlier reports of altered binding within the AD-affected blood cellular fraction to copper-loaded immobilized metal affinity capture (IMAC) arrays. IMAC and anti-amyloid-ß (Aß) antibody arrays coupled with mass spectrometry were used to analyze blood samples collected from 218 participants from within the AIBL Study of Aging. Peripheral Aß was fragile and prone to degradation in the AIBL samples, even when stored at -80°C. IMAC analysis of the AIBL samples lead to the isolation and identification of alpha-defensins 1 and 2 at elevated levels in the AD periphery, validating earlier findings. Alpha-defensins 1 and 2 were elevated in AD patients indicating that an inflammatory phenotype is present in the AD periphery; however, peripheral Aß levels are required to supplement their prognostic power.

High order W02-reactive stable oligomers of amyloid-ß are produced in vivo and in vitro via dialysis and filtration of synthetic amyloid-ß monomer.

Oligomeric forms of amyloid-ß (Aß) are thought to be responsible for the pathogenesis of Alzheimer's disease. While many oligomers of Aß are thought to be naturally occurring in the brain of humans and/or transgenic animals, it is well known that Aß oligomers are also readily produced in vitro in the laboratory. In recent studies, we discovered that synthetic monomeric Aß (4.7 kDa) could be transformed by microdialysis to higher molecular weight species (approximately 56 kDa, by western blot). Surface-enhanced laser desorption/ionization mass spectrometry and electron microscopy further identified these species' as potential Aß oligomers. The production of similar species could also be produced by centrifugal filtration and this formation was concentration and pore-size dependent. These higher order species of Aß were resistant to dissolution in NaOH, HFIP, formic acid, urea, and guanidine. We postulate that we have identified a novel way of producing a high order species of oligomeric Aß and we provide evidence to suggest that Aß oligomers can quite easily be a product of normal laboratory practices. These data suggest that the experimental detection of higher order oligomers in tissues derived from Alzheimer's disease brains or from animal models of disease could, in some cases, be a product the method of analysis.

A commonly used Drosophila model of Alzheimer's disease generates an aberrant species of amyloid-ß with an additional N-terminal glutamine residue.

Expression of human amyloid-ß (Aß) in Drosophila is frequently used to investigate its toxicity in vivo. We expressed Aß1-42 in the fly using a secretion signal derived from the Drosophila necrotic gene, as described in several previous publications. Surface-enhanced laser desorption/ionization TOF MS analysis revealed that the Aß produced contained an additional glutamine residue at the N-terminus. AßQ+1-42 was found to have increased protein abundance and to cause more severe neurodegenerative effects than wild type Aß1-42 as assessed by locomotor activity and lifespan assays. These data reveal that a commonly used model of Alzheimer's disease generates incorrect Aß peptide.

Do current therapeutic anti-Aß antibodies for Alzheimer's disease engage the target?

Reducing amyloid-ß peptide (Aß) burden at the pre-symptomatic stages of Alzheimer's disease (AD) is currently the advocated clinical strategy for treating this disease. The most developed method for targeting Aß is the use of monoclonal antibodies including bapineuzumab, solanezumab and crenezumab. We have synthesized these antibodies and used surface plasmon resonance (SPR) and mass spectrometry to characterize and compare the ability of these antibodies to target Aß in transgenic mouse tissue as well as human AD tissue. SPR analysis showed that the antibodies were able to bind Aß with high affinity. All of the antibodies were able to bind Aß in mouse tissue. However, significant differences were observed in human brain tissue. While bapineuzumab was able to capture a variety of N-terminally truncated Aß species, the Aß detected using solanezumab was barely above detection limits while crenezumab did not detect any Aß. None of the antibodies were able to detect any Aß species in human blood. Immunoprecipitation experiments using plasma from AD subjects showed that both solanezumab and crenezumab have extensive cross-reactivity with non-Aß related proteins. Bapineuzumab demonstrated target engagement with brain Aß, consistent with published clinical data. Solanezumab and crenezumab did not, most likely as a result of a lack of specificity due to cross-reactivity with other proteins containing epitope overlap. This lack of target engagement raises questions as to whether solanezumab and crenezumab are suitable drug candidates for the preventative clinical trials for AD.

Changes in plasma amyloid beta in a longitudinal study of aging and Alzheimer's disease.

BACKGROUND: A practical biomarker is required to facilitate the preclinical diagnosis of Alzheimer's disease (AD). METHODS: Plasma amyloid beta (Aß)1-40, Aß1-42, Aßn-40, and Aßn-42 peptides were measured at baseline and after 18 months in 771 participants from the Australian Imaging Biomarkers and Lifestyle (AIBL) study of aging. Aß peptide levels were compared with clinical pathology, neuroimaging and neuropsychological measurements. RESULTS: Although inflammatory and renal function covariates influenced plasma Aß levels significantly, a decrease in Aß1-42/Aß1-40 was observed in patients with AD, and was also inversely correlated with neocortical amyloid burden. During the 18 months, plasma Aß1-42 decreased in subjects with mild cognitive impairment (MCI) and in those transitioning from healthy to MCI. CONCLUSION: Our findings are consistent with a number of published plasma Aß studies and, although the prognostic value of individual measures in any given subject is limited, the diagnostic contribution of plasma Aß may demonstrate utility when combined with a panel of peripheral biomarkers.

Oligomers, fact or artefact? SDS-PAGE induces dimerization of ß-amyloid in human brain samples.

The formation of low-order oligomers of ß-amyloid (Aß) within the brain is widely believed to be a central component of Alzheimer's disease (AD) pathogenesis. However, despite advances in high-throughput and high-resolution techniques such as xMAP and mass spectrometry (MS), investigations into these oligomeric species have remained reliant on low-resolution Western blots and enzyme-linked immunosorbent assays. The current investigation compared Aß profiles within human cortical tissue using sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE), xMAP and surface enhanced laser desorption/ionization time-of-flight MS and found that whilst there was significant correlation across the techniques regarding levels of monomeric Aß, only SDS-PAGE was capable of detecting dimeric isoforms of Aß. The addition of synthetic di-tyrosine cross-linked Aß(1-40)Met(35)(O) to the AD tissue demonstrated that the MS methodology was capable of observing dimeric Aß at femto-molar concentrations, with no noticeable effect on monomeric Aß levels. Focus turned to the association between SDS-PAGE and levels of observable dimeric Aß within the AD brain tissue. These investigations revealed that increased levels of dimeric Aß were observed with increasing concentrations of SDS in the sample buffer. This finding was subsequently confirmed using synthetic Aß(1-42) and suggests that SDS was inducing the formation of dimeric Aß. The findings that SDS promotes Aß dimerization have significant implications for the putative role of low-order oligomers in AD pathogenesis and draw into question the utility of oligomeric Aß as a therapeutic target.

Variability in blood-based amyloid-beta assays: the need for consensus on pre-analytical processing.

Effective therapeutic interventions for Alzheimer's disease (AD) will require treatment regimes to move toward the earliest stages of the disease. For this to occur the field has to identify biomarkers that are able to accurately identify individuals at risk for progression toward AD in the presymptomatic stage. One very significant implication is that some form of population-based screening will need to be undertaken in order to identify those at risk. To date, efforts in neuroimaging brain amyloid-ß (Aß) and changes in cerebrospinal fluid Aß and tau levels shows promise, however, it is questionable as to whether these methods are applicable for screening the general population. The Aß peptide is also found in blood which is the most economical and efficient biological fluid to analyze. Unfortunately, investigations into blood-based diagnostic markers have produced mixed results. This variability is likely to be the result of differences in the preanalytical processing of samples and as such is delaying progress in the field. Reported preanalytical processing techniques from 87 recent articles focusing on the measurement of Aß in blood were compared, to investigate whether basic sample-handling techniques were comparable between studies. This comparison revealed that not only is it likely that some of the variability in blood-based results is attributable to discrepancies in preanalytical methodologies but also that the field is failing to adequately report sample processing techniques. This review highlights the current shortcomings in methodological reporting and recommends a standardized blood collection methodology based on the limited consensus of the reviewed articles.

Elucidating the role of metals in Alzheimer's disease through the use of Surface-Enhanced Laser Desorption/Ionisation time-of-flight mass spectrometry.

Alzheimer's disease (AD) is a highly heterogeneous and progressive dementia which is characterised by a progressive decline in cognitive functioning, selective neuronal atrophy, and loss of cortical volume in areas involved in learning and memory. However, recent research has indicated that the AD-affected brain is also besieged by increases in oxidative stress as well as perturbations to the homeostasis of biometals, such as copper and iron. These metals are known to interact with the neuropathological hallmark of AD, the ß-amyloid peptide (Aß), in a manner which increases Aß's neurotoxic effects. This knowledge has led to the development of therapeutic measures which act to restore biometal homeostasis within the AD brain. This chapter outlines how Surface-Enhanced Laser Desorption/Ionisation Time-of-Flight Mass Spectrometry can be used to monitor Aß levels within biological systems as well as describing the use of immobilised metal affinity capture in the observation of synthetic Aß peptides.

Stereospecific interactions are necessary for Alzheimer disease amyloid-ß toxicity.

Previous studies suggest membrane binding is a key determinant of amyloid ß (Aß) neurotoxicity. However, it is unclear whether this interaction is receptor driven. To address this issue, a D-handed enantiomer of Aß42 (D-Aß42) was synthesized and its biophysical and neurotoxic properties were compared to the wild-type Aß42 (L-Aß42). The results showed D- and L-Aß42 are chemically equivalent with respect to copper binding, generation of reactive oxygen species and aggregation profiles. Cell binding studies show both peptides bound to cultured cortical neurons. However, only L-Aß42 was neurotoxic and inhibited long term potentiation indicating L-Aß42 requires a stereospecific target to mediate toxicity. We identified the lipid phosphatidylserine, as a potential target. Annexin V, which has very high affinity for externalized phosphatidylserine, significantly inhibited L-Aß42 but not D-Aß42 binding to the cultured cortical neurons and significantly rescued L-Aß42 neurotoxicity. This suggests that Aß mediated toxicity in Alzheimer disease is dependent upon Aß binding to phosphatidylserine on neuronal cells.

Increasing the predictive accuracy of amyloid-ß blood-borne biomarkers in Alzheimer's disease.

Diagnostic measures for Alzheimer's disease (AD) commonly rely on evaluating the levels of amyloid-ß (Aß) peptides within the cerebrospinal fluid (CSF) of affected individuals. These levels are often combined with levels of an additional non-Aß marker to increase predictive accuracy. Recent efforts to overcome the invasive nature of CSF collection led to the observation of Aß species within the blood cellular fraction, however, little is known of what additional biomarkers may be found in this membranous fraction. The current study aimed to undertake a discovery-based proteomic investigation of the blood cellular fraction from AD patients (n = 18) and healthy controls (HC; n = 15) using copper immobilized metal affinity capture and Surface Enhanced Laser Desorption/Ionisation Time-Of-Flight Mass Spectrometry. Three candidate biomarkers were observed which could differentiate AD patients from HC (ROC AUC > 0.8). Bivariate pairwise comparisons revealed significant correlations between these markers and measures of AD severity including; MMSE, composite memory, brain amyloid burden, and hippocampal volume. A partial least squares regression model was generated using the three candidate markers along with blood levels of Aß. This model was able to distinguish AD from HC with high specificity (90%) and sensitivity (77%) and was able to separate individuals with mild cognitive impairment (MCI) who converted to AD from MCI non-converters. While requiring further characterization, these candidate biomarkers reaffirm the potential efficacy of blood-based investigations into neurodegenerative conditions. Furthermore, the findings indicate that the incorporation of non-amyloid markers into predictive models, function to increase the accuracy of the diagnostic potential of Aß.

Iron-export ferroxidase activity of ß-amyloid precursor protein is inhibited by zinc in Alzheimer's disease.

Alzheimer's Disease (AD) is complicated by pro-oxidant intraneuronal Fe(2+) elevation as well as extracellular Zn(2+) accumulation within amyloid plaque. We found that the AD ß-amyloid protein precursor (APP) possesses ferroxidase activity mediated by a conserved H-ferritin-like active site, which is inhibited specifically by Zn(2+). Like ceruloplasmin, APP catalytically oxidizes Fe(2+), loads Fe(3+) into transferrin, and has a major interaction with ferroportin in HEK293T cells (that lack ceruloplasmin) and in human cortical tissue. Ablation of APP in HEK293T cells and primary neurons induces marked iron retention, whereas increasing APP695 promotes iron export. Unlike normal mice, APP(-/-) mice are vulnerable to dietary iron exposure, which causes Fe(2+) accumulation and oxidative stress in cortical neurons. Paralleling iron accumulation, APP ferroxidase activity in AD postmortem neocortex is inhibited by endogenous Zn(2+), which we demonstrate can originate from Zn(2+)-laden amyloid aggregates and correlates with Aß burden. Abnormal exchange of cortical zinc may link amyloid pathology with neuronal iron accumulation in AD.

Blood-borne amyloid-beta dimer correlates with clinical markers of Alzheimer's disease.

Alzheimer's disease (AD) is the most common age-related dementia. Unfortunately due to a lack of validated biomarkers definitive diagnosis relies on the histological demonstration of amyloid-beta (Abeta) plaques and tau neurofibrillary tangles. Abeta processing is implicated in AD progression and many therapeutic strategies target various aspects of this biology. While Abeta deposition is the most prominent feature of AD, oligomeric forms of Abeta have been implicated as the toxic species inducing the neuronal dysfunction. Currently there are no methods allowing routine monitoring of levels of such species in living populations. We have used surface enhanced laser desorption ionization time of flight (SELDI-TOF) mass spectrometry incorporating antibody capture to investigate whether the cellular membrane-containing fraction of blood provides a new source of biomarkers. There are significant differences in the mass spectra profiles of AD compared with HC subjects, with significantly higher levels of Abeta monomer and dimer in the blood of AD subjects. Furthermore, levels of these species correlated with clinical markers of AD including brain Abeta burden, cognitive impairment and brain atrophy. These results indicate that fundamental biochemical events relevant to AD can be monitored in blood, and that the species detected may be useful clinical biomarkers for AD.

The structure of dopamine induced alpha-synuclein oligomers.

Inclusions of aggregated alpha-synuclein (alpha-syn) in dopaminergic neurons are a characteristic histological marker of Parkinson's disease (PD). In vitro, alpha-syn in the presence of dopamine (DA) at physiological pH forms SDS-resistant non-amyloidogenic oligomers. We used a combination of biophysical techniques, including sedimentation velocity analysis, small angle X-ray scattering (SAXS) and circular dichroism spectroscopy to study the characteristics of alpha-syn oligomers formed in the presence of DA. Our SAXS data show that the trimers formed by the action of DA on alpha-syn consist of overlapping worm-like monomers, with no end-to-end associations. This lack of structure contrasts with the well-established, extensive beta-sheet structure of the amyloid fibril form of the protein and its pre-fibrillar oligomers. We propose on the basis of these and earlier data that oxidation of the four methionine residues at the C- and N-terminal ends of alpha-syn molecules prevents their end-to-end association and stabilises oligomers formed by cross linking with DA-quinone/DA-melanin, which are formed as a result of the redox process, thus inhibiting formation of the beta-sheet structure found in other pre-fibrillar forms of alpha-syn.