The involvement of dityrosine crosslinking in α-synuclein assembly and deposition in lewy bodies in parkinson’s disease

ABSTRACT Parkinson’s disease (PD) is characterized by intracellular, insoluble Lewy bodies composed of highly stable α-synuclein (α-syn) amyloid fibrils. α-synuclein is an intrinsically

disordered protein that has the capacity to assemble to form β-sheet rich fibrils. Oxidiative stress and metal rich environments have been implicated in triggering assembly. Here, we have

explored the composition of Lewy bodies in post-mortem tissue using electron microscopy and immunogold labeling and revealed dityrosine crosslinks in Lewy bodies in brain tissue from PD

patients. _In vitro_, we show that dityrosine cross-links in α-syn are formed by covalent ortho-ortho coupling of two tyrosine residues under conditions of oxidative stress by fluorescence

and confirmed using mass-spectrometry. A covalently cross-linked dimer isolated by SDS-PAGE and mass analysis showed that dityrosine dimer was formed via the coupling of Y39-Y39 to give a

homo dimer peptide that may play a key role in formation of oligomeric and seeds for fibril formation. Atomic force microscopy analysis reveals that the covalent dityrosine contributes to

the stabilization of α-syn assemblies. Thus, the presence of oxidative stress induced dityrosine could play an important role in assembly and toxicity of α-syn in PD. SIMILAR CONTENT BEING

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DERIVED FROM HUMAN LEWY BODY DEMENTIA TISSUE Article Open access 29 March 2024 INTRODUCTION Parkinson’s disease (PD) is the second most common neurodegenerative disease after Alzheimer’s

disease (AD) in humans and both diseases are associated with the aggregation of proteins into amyloid deposits. PD is characterized by the formation of intracelluar amyloid fibril deposition

of α-synuclein (α-syn) in Lewy bodies that accumulate in the substantia nigra of sufferers1. α-syn is an 140 amino-acid intrinsically disordered protein that can self-assemble to form

β-sheet rich oligomers and amyloid fibrils1. It has been proposed that small early oligomeric forms of α-syn may lead to local degeneration of the dopaminergic cells of the substantia

nigra2,3 and permeation of lipid bilayers4 whilst the fibrils themselves may cause structural damage to cellular membranes5. Oxidative stress has been implicated in the pathogenesis of a

number of neurodegenerative diseases, including AD and PD6,7. Oxidative stress can lead to a multitude of protein, lipid and nucleic acid damage and is implicated in the formation of

dityrosine cross-links in the Amyloid-β (Aβ) peptide in amyloid plaques in AD8,9. Dityrosine cross-linking is formed by the ortho-ortho coupling of tyrosine residues and can take place

intra- or inter-molecularly. Similar to Aβ, dityrosine cross-links can also be formed _in vitro_ via metal catalyzed oxidation (MCO) of α-syn under physiological conditions10,11, and have

been identified as biomarkers of oxidative stress in an 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD12,13. Tyrosines are found at four positions in the α-syn

sequence at residue 39, 125, 133 and 136 and previous work has highlighted the importance of Y39 in self-assembly14. Elevated levels of many metal ions such as iron and zinc have been

reported in substantia nigra15,16. Studying the influence of dityrosine cross-links on α-syn conformation and the effects during amyloid formation will, therefore, provide insights into the

importance of oxidative modification of tyrosine residues in the pathogenesis of PD. Here, immunogold electron microscopy (EM) was used to examine the prevalence of dityrosines in Lewy

bodies in PD brain tissue, and revealed the presence of dityrosine in α-syn in Lewy bodies. The ability of Cu2+ to promote the formation of _in vitro_ dityrosine cross-linked α-syn was

explored and the effect of the Cu2+ ion on α-syn amyloid self-assembly and conformation was investigated. The presence of dityrosine cross-links was examined using fluorescence spectroscopy,

liquid chromatography electrospray ionization mass-spectrometry (LC-ESIMS/MS), SDS-PAGE, transmission electron microscopy (TEM), atomic force microscopy (AFM) and X-ray fibre diffraction

(XRFD). Our results show that oxidative stress induces dityrosine cross-links, which could play an important role in assembly, stability and toxicity of α-syn in PD. RESULTS DETECTION OF

DITYROSINE IN LEWY BODIES IN BRAIN SECTIONS FROM PD AFFECTED PATIENTS Dityrosine cross-linking has been identified previously in α-syn and has been suggested to play important role in the

pathogenesis of PD17,18. However, previous studies have not demonstrated direct physiological links between PD and dityrosine crosslinking. Here, post-mortem substantia nigra brain sections

from PD patients were immunogold labeled using anti-dityrosine mouse monoclonal antibody and anti-α-syn within Lewy bodies in the brain sections and then visualized under TEM to explore the

colocalization of gold-conjugated antibodies. Three cases were examined (see Table 1) and of these, two cases (PD028 and PD041) showed Lewy bodies which were examined further. The TEM images

show the presence of dityrosine antibody labeling alongside extensive α-syn labeling in Lewy bodies (Fig. 1). In contrast, no labeling was observed for either dityrosine or α-syn outside of

the Lewy bodies (circled in Fig. 1c(iii)). A very high density of α-syn labeling was observed compared to dityrosine labeling (Fig. 1c(iii)), and this difference may suggest that some, but

not all α-syn molecules contain dityrosine. Elevated levels of several metals have been reported in the substantia nigra of PD brains15,16,19, suggesting the mechanism by which dityrosine

can be formed may be metal-induced. Therefore, Cu2+ was tested for the ability to induce the dityrosine cross-linked α-syn and the structural consequences of these dityrosine cross-links

were investigated _in vitro_. OXIDATION OF Α-SYN ENHANCES DITYROSINE CROSS-LINK FORMATION The formation of dityrosine cross-linked α-syn was enhanced by incubating 50 μM recombinant α-syn

with Cu2+ and hydrogen peroxide at pH 7.4 and for 24 hours at 37 °C with agitation (Fig. 2a). Controls were prepared by incubating 50 μM recombinant α-syn in buffer alone or with buffer

supplemented with Cu2+ or H2O2 only (Fig. 2c). The incubation of α-syn with Cu2+/H2O2 leads to rapid loss of the intrinsic tyrosine fluorescence signal (305 nm) concomitant with an

enhancement in fluorescence emission intensity at wavelength of 405–410 nm, indicating dityrosine cross-link formation (Fig. 2a) whilst α-syn with buffer only (Fig. 2c(i)), Cu2+ only (Fig.

2c(ii)) or H2O2 only (Fig. 2c(iii)) showed no change in tyrosine or dityrosine fluorescence intensity over 24 hour incubation period. The assignment of the fluorescence to dityrosine was

further confirmed using an excitation wavelength of 320 nm and emission at 405 nm (inset Fig. 2a) supported by published data obtained at a similar pH17 and showed increased dityrosine

signal over 24 hours incubation (Fig. 2b). The formation of dityrosine is rapid, with a signal appearing within 10 mins and increasing over 1 hour (Fig. 2b inset). The Cu2+/H2O2 oxidized

α-syn was quenched using EDTA after 24 hours, acid hydrolysed and LC-ESIMS/MS was acquired using MRM mode to further confirm the identity of the resulting dityrosine cross-link (Fig. 2d).

The mass chromatogram of α-syn hydrolysate revealed a peak with retention time of 5.8 min, which corresponded to the retention time for authentic dityrosine (Fig. 2d). CHARACTERIZING THE

NATURE OF THE DITYROSINE CROSS-LINKED Α-SYN Fluorescence spectra (Fig. 2a) showed that incubating α-syn under oxidative conditions rapidly induces dityrosine cross-links in α-syn and 1 h was

enough to observe a dityrosine signal with high intensity. Because α-syn has four tyrosine residues located at 39, 125, 133, 136, this raises the possibility that α-syn oxidation can lead

to the formation of both intra- and inter-molecular dityrosine cross-links. Cu2H2O2 oxidized α-syn incubated for 5 h was analyzed by SDS gel electrophoresis and visualized by silver staining

(Fig. 3a). Both oxidized and control samples displayed the presence of high levels of monomeric α-syn running at the expected molecular weight of 14.5 kDa. After five hours incubation under

oxidation conditions α-syn also ran at a higher molecular weight band at around 35 kDa, which may represent covalently cross-linked dimer through tyrosine-tyrosine coupling. The molecular

weight of this band is slightly higher than the expected 29–30 kDa and may be due to an altered conformation of the protein in SDS conditions20. This “dimer” band was not present in the

non-oxidized control (Fig. 3a). To investigate whether the monomeric or dimeric α-syn contains dityrosine cross-links, Western blotting was conducted using the anti-dityrosine antibody. The

α-syn samples were centrifuged to separate the fibril containing pellet from the soluble species in the supernatant. Figure 3b shows two bands at molecular weights 37 kDa and 70 kDa which

are approximately consistent with dimer and tetramer in the supernatant and a dimer band in the pellet. Also both lanes show some high molecular weight species which are presumably

protofibrillar and fibrillar structures. This result confirms that the dimeric species is dityrosine cross-linked and showing that this dimer can assemble further into tetramer and

dityrosine containing fibrils, consistent with the observation that these dimers go on to form fibrillar structures. IDENTIFICATION OF DITYROSINE CROSS-LINKED Α-SYN DIMER USING NANOLC-MS/MS

To investigate which tyrosine residue(s) are involved in the formation of dityrosine cross-links, both intact oxidized and non-oxidized α-syn were analyzed using nanoLC-MS/MS from trypsin

digested peptides extracted from the SDS PAGE gel (Fig. 3a). To identify the α-syn peptides, the Mascot search engine was used and the search results revealed 100% sequence recovery of

monomeric α-syn and 89% of dimeric α-syn (Fig. 3c). Interestingly, in the dimer band sample two regions were not mapped, corresponding to 7GLSKAK12 and 35EGVLYVGSK43 (Fig. 3c). The reason

for the missing 7–12 region is unclear, however, the second region contained Y39, and one explanation for the absence of this sequence is that Y39 is oxidized and as a result a dityrosine

cross-link was formed. As dityrosine exhibits high protease resistance, this peptide fragment was not released and instead a dimeric form of the 35EGVLYVGSK43 peptide is released, indicating

the involvement of Y39 to form a homodimer α-syn. This was confirmed by the appearance of a peak at _m/z_ 1179.6127 and identified to be a dimer peptide of (33TKEGVLYVGSK43) as shown in

(Fig. 3d). The potential involvement of Y39 in the formation of a heterodimer, i.e. Y39-Y125, Y39-Y133 or Y136, was also investigated but the data showed no evidence of formation of side

chain links. Furthermore, the Mascot data showed the presence of the regions that contained Y125, Y133, and Y136 in both monomeric and dimeric α-syn (Fig. 3c) indicating that the α-syn dimer

results from cross-linking between Y39 from two molecules to form a dimer. Α-SYN CONFORMATION AND AMYLOID FORMATION IN THE PRESENCE OF CU2+ IONS At low concentration, α-syn does not

self-assemble within a short time frame. Therefore, we examined dityrosine formation and assembly of α-syn by increasing the concentration to 100 μM. 100 μM α-syn incubated buffer alone

(Fig. 4a) or with Cu2+ only (Fig. 4b), revealed the appearance of a fluorescence peak around 405 nm after 7 days of incubation which increased to 14 days. A higher intensity fluorescence

signal was observed for those incubated with Cu2+ (Fig. 4b). The samples were also monitored over two weeks using ThT fluorescence, negative-stain TEM and immunogold labeling for dityrosine.

ThT fluorescence spectra of Cu2+-induced α-syn fibrils revealed a significant increase in intensity over the two week incubation period (Fig. 4c), revealing α-syn fibril formation after

seven days of incubation, and this was further confirmed by observation of fibrils in TEM images (Fig. 4d). After one week of incubation, α-syn fibrils formed when incubated in the presence

of Cu2+, whilst very few α-syn fibrils were detected by TEM or ThT fluorescence in the sample incubated under control conditions. The distribution of dityrosine was further examined with

immunogold labeling using an anti-dityrosine antibody which revealed sparse distribution of dityrosine labeling on the buffer incubated α-syn fibrils (Fig. 4d) compared to a regular

distribution of gold labels along the fibrils incubated in Cu2+ only (Fig. 4d). Also, the morphology of the fibrils differed whereby those incubated with Cu2+ were more plentiful and showed

some lateral association or crossing over of straight, well ordered filaments compared to the more twisted fibrils formed in buffer alone. In order to further explore the structure of

dityrosine cross-linked α-syn, a high concentration of fibrillar α-syn was obtained by incubation of 400 μM monomeric recombinant human α-syn in the absence or presence of 400 μM Cu2+. ThT

fluorescence assay confirmed the assembly of α-syn in the presence and absence of Cu2+ over 120 hours, and showed that the presence of Cu2+ appeared to enhance the rate of assembly (Fig.

5a). Fluoresence showed increased intensity at 405 nm in the Cu2+ samples confirming the presence of dityrosine (Fig. 5b). Electron micrographs of α-syn fibrils formed under control and

Cu2+-containing buffer conditions after 120 h incubation (Fig. 5c) again revealed that incubation with Cu2+ markedly accelerates fibril formation (Fig. 5a). TEM micrographs revealed a higher

density of Cu2+-enhanced α-syn fibrils that also appeared to have a more ordered, rigid appearance (Fig. 5c). This provided further evidence that the Cu2+ ion plays a role in accelerating

α-syn fibril formation and may affect the final structure. To verify that the samples contained the expected cross-β amyloid fibrils, XRFD patterns were collected for both α-syn fibrils

incubated with Cu2+ ions and those assembled without Cu2+ ions. The XRFD patterns showed the expected amyloid cross-β pattern21,22,23, for both Cu2+-enhanced α-syn fibrils and control α-syn

fibrils (Fig. 5d). The expected cross-β meridional reflections at 4.76 and 2.4 Å were observed in patterns from fibrils assembled under both control (i.e. without Cu2+) and Cu2+-containing

conditions. Moreover, a set of reflections at ~8.1, 9.5 and 19.4 Å were observed on the equator, similar to those reported in a previous study24, confirming the formation of α-syn fibrils

with cross-β structure. The equatorial diffraction signals arise from structural distances perpendicular to the fibre axis, such as sheet spacing, protofilament packing and protofilament

size. However, the XRFD for Cu2+-enhanced α-syn fibrils (Fig. 5d) revealed a new reflection at ~35 Å that may arise from regular lateral packing of protofilaments and also showed more

intense reflections attributable to the formation of more aligned fibrils compared to those assemblies incubated under control conditions. MORPHOLOGY AND STABILITY OF DITYROSINE CROSS-LINKED

Α-SYN To compare α-syn fibrils containing cross-links with those without cross-links, α-syn was allowed to self-assemble for two weeks in the presence of Cu2+ or EDTA was used to deplete

the Cu2+. Cu2+ depleted α-syn was confirmed to be lacking dityrosine crosslinks from fluorescence assays (data not shown). Following mechanical stirring to disrupt the preformed fibrils, the

structure and morphologies of the α-syn fibrils formed in Cu2+ oxidizing and Cu2+ depleted environments were next analyzed using AFM. Height images of both Cu2+ oxidized and Cu2+ depleted

fibrils deposited on mica surfaces were acquired over 30 × 30 μm surface areas imaged at resolution of 2048 × 2048 pixels for qualitative analysis (Fig. 6a). The height distribution of the

pixels of the fibril particles extracted from the AFM images are shown in Fig. 6b. α-syn fibrils grown in the presence of Cu2+ often appeared to be linked, crossed-over or attached to form

star shaped structures, whilst those grown without Cu2+ appeared shorter and more dispersed. The average height 6.6 ± 2.0 nm SD and 7.1 ± 2.3 nm SD for fibrils grown in Cu2+ oxidizing and

Cu2+ depleted environment, respectively, suggests that aggregation in both a Cu2+ oxidizing and Cu2+ depleted environment results in fibrils of similar morphologies and width. However, the

extracted contour length distributions for the fibril samples (Fig. 6c), displayed an increased average length for fibrils formed in a Cu2+ oxidizing environment compared with fibrils formed

in Cu2+ depleted conditions. This is consistent with the idea that Cu2+ mediates enhancement of aggregation through stabilization of the aggregates. Fibrils formed in a Cu2+ depleted

environment display a weighted average contour length of 220 ± 10 nm SE. This is considerably shorter than fibrils formed in Cu2+ oxidized conditions, which present a weighted average

contour length of 880 ± 30 nm SE. This increased fibril length of fibrils formed in a Cu2+ oxidized enviroment supports the formation of dityrosine cross-linked dimers as a critical step in

the early stages of α-syn aggregation. The ability of α-syn to form fibrils of increased average length in the presence of Cu2+, therefore, further supports an important role of dityrosine

cross-linked dimers in the formation of amyloid assemblies, as the presence of additional core interactions provided by the dityrosine cross-links may responsible for an increase in the

stabilization of the amyloid core and accelerated amyloid assembly. _In vitro_ growth of α-syn in the presence of Cu2+ was monitored after prolonged incubation using electron microscopy

(Fig. 7a) and atomic force microscopy (Fig. 7c) and reveals the formation of networks and star-like structures. We propose that these structures may form the basis for the distinctive Lewy

body shape (Fig. 7). DISCUSSION PD is the second most common neurodegenerative disease after AD25. Cu2+ has been found to decrease in the substantia nigra and increase in the CSF of PD

patients26,27. Oxidative stress is implicated in the pathogenesis of PD and modification of tyrosine residues may play a role in the α-syn aggregation via protein cross-linking. We have

revealed that Lewy bodies in PD patient brain tissues contain α-syn and dityrosine cross-links. These structures may serve to allow the fibrils within Lewy body deposits to persist.

Furthermore, the formation of dityrosine cross-linked dimers may play an important role in oligomer toxicity in animal models4. Dityrosine cross-linked α-syn oligomers generated using PICUP

using tris(bipyridine) ruthenium(II) chloride complex as a photosensitizer have been shown to exhibit high toxicity on differentiated neuronal-like SH-SY5Y cells, indicating the importance

of the tyrosine residue oxidative modification in the etiology of PD17. Previous work has revealed that disordered, soluble α-syn can persist in mammalian cells and Y39 was identified as

playing a role in maintaining a compact structure28. To further characterize the role of the dityrosine cross-links in self-assembly and structure, α-syn was examined _in vitro_ using

fluorescence spectroscopy, LC-ESIMS/MS, TEM, XRFD and AFM following incubation in oxidative conditions and with Cu2+. We showed that the rate of assembly was enhanced for α-syn incubated

with Cu2+. This was further confirmed by electron micrographs showing increased formation of α-syn fibrils in the presence of Cu2+ compared with control, which shows the accumulation of

amorphous or spherical aggregates of α-syn after one week and fibrils forming after 2 weeks. Formation of dityrosine, detected using fluorescence, showed an increased intensity after 7 days

incubation for α-syn with Cu2+. α-syn without Cu2+ was also able to form dityrosine, but at a much slower rate. This indicates a role for metal catalyzed oxidation (MCO) in initiating the

α-syn amyloid formation process _in vitro_, and may implicate a metal catalyzed mechanism _in vivo_. AFM results reveal that the dityrosine cross-links may also stabilize the amyloid core

resulting in accelerated assembly and thereby fibrils of longer contour lengths. It has been shown previously that dityrosine forms in α-syn fibrils that have been aged in the absence and

presence of Cu2+ in an aerobic environment, whilst α-syn aged anaerobically did not form dityrosine cross-links29. Furthermore, incubation of α-syn in solutions that contain methionine

and/or EDTA, which help to prevent or reduce the level of protein oxidation, led to lengthened lag times for fibril formation18 and here reduced the length of α-syn fibrils revealed by AFM.

The results show that α-asyn is able to assemble in the absence of dityrosine cross-link formation but that the assembly is slowed. It has been shown previously that stable α-syn polymers

can be generated using nitrating agents and these polymers are stabilized due to the formation of dityrosine cross-link30. Collectively our results indicate that Cu2+ ions strongly promote

the oxidation of α-syn, resulting in dityrosine cross-linked α-syn. It has been found that seeding α-syn with dityrosine cross-linked dimers accelerates α-syn fibril growth, suggesting that

the critical rate-limiting step in the nucleation of α-syn fibrils is the formation of dityrosine cross-linked dimeric species18. Here, a covalently cross-linked dimer was isolated by

SDS-PAGE and confirmed to be dityrosine by western blotting and using nanoLC-MS/MS following tryptic digestion. The mass analysis showed that dityrosine dimer was formed via the coupling of

Y39-Y39 to give a homodimer. This observation is supported by previous work showing that a α-syn mutant Y125W/Y133F/Y136F where a single Y remained at position 39 showed the same ability to

form fibrils as wild type α-syn31, reflecting the important role played by Y39 in the α-syn fibrillation. Quenched hydrogen/deuterium exchange NMR spectroscopy was used previously to study

the structure of α-syn in its amyloid state and identified five β-strands within the fibril core, comprising residues 35–96 and termed aSβ1, aSβ2, aSβ3, aSβ4 and aSβ532. Tyrosine at position

39 is found within aSβ1, while tyrosine residues at 125, 133, and 136 did not contribute towards the formation of any β-strand. Recently, the first region consisting of 37VLYVGSKT44 was

studied using XRFD and it was found that at high concentrations the peptide forms nanotubular cross-β assemblies33. Interestingly, the proposed structural model of these nanotubes revealed

that peptide packing and inter-sheet Tyr interactions were involved to stabilize the tape width33. Also, the model shows that the orientation of the phenol groups of Tyr residues is in

agreement with the formation of inter-sheet dityrosine cross-links, however, it is important to keep in mind this may be not be true for fibrils formed by full length α-syn. Indeed, a very

recent structure of the α-syn fibril elucidated using solid state NMR combined with X-ray fibre diffraction revealed that residues 44–96 form the core of the fibril. Residue 39 in this

structural context is found at the edge of the structured core and within a disordered region and therefore is available for dityrosine cross-linking between and within fibrils34. These

results are in good agreement with other published data18,29,30, and support the notion that dityrosine cross-linked α-syn oligomers can serve as seeds in the α-syn amyloid formation process

and are able to stabilize the amyloid oligomers and fibrils. This may be important in the formation of Lewy bodies which persist in PD brain. CONCLUDING REMARKS Many studies have

demonstrated that dityrosine cross-linked α-syn species reduce the yield of amyloid fibrils14,17, while dityrosine formation has been described to be the critical rate-limiting step in α-syn

assembly18. These results appear to be in conflict and this could be interpreted by the variation of the _in vitro_ oxidative stress conditions that have been used to form the dityrosine

cross-links. Also, there are many factors that can affect the lag duration such as α-syn concentration, buffer composition and pH, and temperature35,36,37. In order to gain better

understanding of the relationship between dityrosine oligomer formation and α-syn assembly, we have used _in vitro_ oxidative stress conditions that mimic the _in vivo_ conditions and

revealed dityrosine cross-linked α-syn _in vivo_ in Lewy bodies in PD brain. MATERIALS AND METHODS IMMUNOGOLD LABELING TEM OF TISSUE SECTIONS PD brain tissue from substantia nigra was

obtained from Parkinson’s disease UK Brain Bank. Tissue was removed according to Local Ethics Committee guidelines, and informed consent for brain donation was obtained from the next of kin

and stored at -80 °C until required (Table 1). Brain tissue blocks were prepared for immunogold labeling TEM by minimal cold fixation and embedding protocols, as previously described38.

Immunogold labeling was performed using an established methodology39, with PBS+ buffer being used for all dilutions of immunoreagents and for rinsing. A modified phosphate-buffered saline,

pH 8.2, containing 1% BSA, 500 μl/l Tween-20, 10 mM Na EDTA, and 0.2 g/l NaN3 (henceforward termed PBS+), was used throughout all the following procedures for all dilutions of antibodies and

secondary gold probes. Thin sections were collected upon TEM support grids, then incubated with normal goat serum (1.10 dilution) for 30 min at room temperature to block non-specific

secondary antibody binding. Grids were then labeled with (10 μg/ml IgG) anti-dityrosine mouse monoclonal antibody (Japan Institute for the Control of Aging JaICA, Shizuoka, Japan) or

double-labeled using a mixture of (10 μg/ml IgG) anti-α-syn (C-20)-R rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc.) and (10 μg/ml IgG) anti-dityrosine mouse monoclonal antibody

and incubated overnight at 4 °C. After 3 × 2 min PBS+ rinses, sections were then immunolabeled with GaM10 (10 nm diameter) or a mixture of GaR5 (5 nm diameter) and GaM15 (15 nm diameter)

secondary probes (all 1.10 dilution), for 1 h at room temperature. After 3 × 10 min PBS+ and 4 × 5 min distilled water rinses, the grids were post-stained in 0.22 μM-filtered 0.5% (w/v)

aqueous uranyl acetate for 1 h. The grids were examined on a Hitachi 7100 TEM (Hitachi, Germany) fitted with a Gatan Ultrascan 1000 CCD camera (Gatan, Abingdon, UK), and operating with a

voltage of 100 kV. SYNTHESIS OF A DITYROSINE STANDARD A dityrosine standard was synthesized using horseradish peroxidase and _N_-acetyl-3,5-diiodo-L-tyrosine as a starting material as

described previously9. PREPARATION OF Α-SYN The lyophilized full-length human recombinant α-syn (1 mg) in final buffer of 10 mM Tris-HCl (pH 7.4) was purchased from rPeptide (Bogart, GA,

USA) or prepared as previously described40. α-syn was of high purity and does not show any cysteine misincorporation to tyrosine at position 136, which has been observed for about 20% of

human α-syn expressed in _Escherichia coli_41. The peptide was resuspended in Milli-Q filtered water at concentration of 2 mg/ml, and then the Tris-HCl buffer was removed using Vivaspin-500

concentrator tube, 3000 MWCO PES (Sartorius stedim biotech, Germany). Briefly, the resuspended α-syn was centrifuged at 14,000 g for 30 min, subsequently the obtained concentrated α-syn was

again diluted with Milli-Q filtered water at a concentration of 2 mg/ml and the concentrating process was repeated. The resulting α-syn solution in water was collected and filtered using

0.22 μm syringe filter to remove any preformed aggregates. Finally, the concentration was determined using a molar extinction coefficient of 5120 M−1cm−1 and the absorbance was measured at a

wavelength of 280 nm using an Eppendorf Biophotometer (Eppendorf UKLtd., Cambridge, UK)29. CU2+- CATALYZED OXIDATION OF Α-SYN Prior to oxidation, α-syn stock solution was prepared as

described above, to remove any preformed aggregates and fibrils. Soluble α-syn monomer was incubated with Cu2+ using molar ratio of 1.1 and concentration of 50 μM. To initiate the oxidation

reaction, H2O2 (1.25 mM) was added. The oxidation reaction was performed in 20 mM HEPES (pH 7.4) at 37 °C with agitation of 400 rpm. After 24 h, the oxidation reaction was stopped using EDTA

at a final concentration of 1.25 mM. The dityrosine formation was monitored using a fluorescence spectrophotometer (Varian Ltd., Oxford, UK). Controls were obtained by incubation of 50 μM

α-syn alone, with H2O2 (1.25 mM) only and with Cu2+ only in 20 mM HEPES buffer pH 7.4 at 37 °C with agitation of 400 rpm. EXPLORING THE EFFECT OF CU2+ ON THE Α-SYN FIBRILLOGENESIS AND

STRUCTURE To examine the slow oxidizing effect of Cu2+ only on the α-syn fibrillogenesis, two concentrations of α-syn (100 and 400 μM) were examined, whilst maintaining a molar ratio of 1.1

with Cu2+. The samples were then further analyzed as described below. FLUORESCENCE SPECTROSCOPY The fibrils were resuspended by agitation and fluorescence spectra were collected at time

intervals. Fluorescence measurements were carried out on a Varian Cary Eclipse fluorimeter (Varian Ltd., Oxford, UK) using a 1 cm path length quartz cuvette (Starna, Essex, UK), and

dityrosine fluorescence was monitored using an excitation wavelength of 320 nm. Dityrosine emission was monitored between 340 and 500 nm, with maximum fluorescence intensity at around

405–410 nm at a controlled temperature of 21 °C. To detect dityrosine fluorescence at early time points, 130 μl of the reaction mixture was removed and EDTA was added to final concentration

of 1.25 mM. Tyrosine fluorescence signal was monitored using an excitation wavelength of 280 nm and emission wavelength of 305 nm. Excitation and emission slits were both set to 10 nm, and

the scan rate was set to 300 nm/min with 2.5 nm data intervals and an averaging time of 0.5 s. The photomultiplier tube detector voltage was set at 500 V. SAMPLE PREPARATION FOR LC-ESIMS/MS

ANALYSIS For LC-ESIMS/MS analysis, 24 h incubated, oxidized α-syn (50 μM) was first desalted to remove the HEPES buffer that could affect the mass spectrum. The desalting was performed using

Amicon ultra-4 centrifugal filter units, 10 k NMWL (M Millipore, USA) and centrifuged at 3900 g and a temperature of 4 °C. The resulting α-syn was then hydrolyzed using evacuated sealed

tubes under acidic conditions of (6 M) HCl, 10% TFA, and 1% phenol at 110 °C for 24 h. The resulting hydrolysate was then dried under nitrogen gas, dissolved in 100 μl of 0.1% formic acid in

water and then filtered using a Millipore 0.22 μm filter into a 0.2 ml tube. DETECTION OF DITYROSINE BY LC-ESIMS/MS 20 μl of oxidized α-syn hydrolysate was injected on to a Phenomenex

Gemini 3 u C6-phenyl 110 (150 mm × 4.6 mm, 3 micron) column using a high performance liquid chromatography (HPLC) system (Waters Alliance 2695, Ireland) coupled to the mass spectrometer

(MicroMass Quattro Premier, Waters, Ireland) operated in the multiple reaction-monitoring (MRM) mode with positive electrospray ionization (ESI). The solvents for the mobile phase were A.

0.1% formic acid in water; and solvent B. 0.1% formic acid in acetonitrile. The gradients were as follows. t = 0 min, 0% B; t = 1 min, 0% B; t = 15 min, 100% B; t = 20 min, 100% B; t = 25 

min, 0% B; t = 30 min, 0% B, and the flow rate was 200 μl/min. Mass spectrometric detection was performed by positive electrospray ionization (ESI) tandem mass spectrometry on a triple

quadrupole mass spectrometer (MicroMass Quattro Premier, Waters, Ireland). The conditions for the mass spectrometer were as follows; electrospray ionization spray voltage 3.5 kV, the cone

voltage 35 V, the source temperature at 100 °C, whereas the desolvation temperature was 400 °C. Argon was used as the collision gas at 5.95 e−003 mbar at 26 ev collision energy. THIOFLAVIN T

FLUORESCENCE ASSAY The fibril formation of α-syn was monitored using ThT fluorescence. A 0.2 μm filtered (3.14 mM) aqueous ThT stock solution was prepared and stored frozen at −20 °C in

1–10 μl aliquots until required. ThT was added to a 10 μM α-syn sample (20 mM HEPES buffer pH 7.4) to a final concentration of 20 μM, and then EDTA was added to a final concentration of 250 

μM. The resulting mixture was then gently vortexed, and allowed to bind for 3 minutes before a reading was taken. Using a microvolume cuvette of 1 cm path length, ThT fluorescence was

measured using a Varian Cary Eclipse fluorimeter (Varian, Oxford, UK) with excitation wavelength of 450 nm. The emission spectrum was recorded between 460–600 nm at 21 °C. HEPES buffer

baselines were subtracted from the data. Excitation and emission slits were set to 5 nm and 10 nm respectively. The scan rate was 600 nm/min with 1 nm data intervals and an averaging time of

0.1 s. The voltage on the photomultiplier tube was set to high (800 v) and experiments were carried out in triplicate to confirm trends. IMMUNOGOLD LABELING, NEGATIVE STAIN TEM FOR FIBRILS

The α-syn fibrils were immunogold-labeled ‘on grid’ for dityrosine9 using a monoclonal anti-dityrosine antibody (JaICA, cat. no. MDT-020P). The antibody has been fully characterized and

shown to be highly specific and does not show any cross-reactivity with other tyrosine derivatives such as nitrotyrosine, chlorotyrosine42. The specificity and the antibody were also checked

in our previous work using the identical procedure and IgG concentrations, with an irrelevant antibody to hair cell antigen (MAb10)9. The antibody will detect any protein containing

dityrosine. Briefly, 4 μl aliquots of the α-syn fibrils were pipetted onto Formvar/carbon coated 400 mesh copper TEM support grids (Agar Scientific, Essex, UK), left for 1 min, the excess

was removed by filter paper, and then blocked in normal goat serum (1.10 in PBS+) for 15 min. Grids were then incubated with (10 μg/ml IgG) mouse dityrosine monoclonal antibody (JaICA,

Shizuoka, Japan) for 2 h at room temperature, rinsed in 3×2 min PBS+, and then immunolabeled in a 10 nm gold particle-conjugated goat anti-mouse IgG secondary probe (GaM10 British BioCell

International, Cardiff, UK; 1.10 dilution) for 1 h at room temperature. After 5 × 2 min PBS+ and 5 × 2 min distilled water rinses, the grids were negatively stained as described in negative

stain TEM methods below. NEGATIVE STAIN TEM Four μl aliquots of α-syn samples were placed onto Formvar/carbon coated 400-mesh copper grids (Agar Scientific, Essex, UK) for 1 min, and the

excess was removed using filter paper. Subsequently the grid was washed using 4 μl of Milli-Q water filtered with 0.22 μM filter and blotted dry, then negatively stained twice with 4 μl of

filtered 2% (w/v) uranyl acetate for 1 min and blotted dry. The grid was allowed to air-dry before examination on a Hitachi 7100 transmission electron microscope (Hitachi, Germany) fitted

with a Gatan Ultrascan 1000 CCD camera (Gatan, Abingdon, UK) at an operating voltage of 100 kV. SDS GEL ELECTROPHORESIS The separation gel consisted of 12% (w/v) acrylamide, 0.32% (w/v)

bis-acrylamide, 0.1% SDS, and 0.38 M Tris-HCl (pH 8.8). The polymerization was initiated by addition of 5 μl TEMED and 50 μl freshly prepared ammonium persulfate (APS) solution per 10 ml of

the gel solution. The stacking gel consisted of 5% (w/v) acrylamide, 0.13% (w/v) bis-acrylamide, 0.1% (w/v) SDS, and 0.38 M Tris-HCl (pH 6.8) and polymerization was initiated by addition of

5 μl TEMED and 20 μl freshly prepared ammonium persulfate (10% w/v) per 4 ml of the gel solution. Electrophoresis buffer consisted of 25 mM Tris-HCl (pH 8.3), 19 mM glycine, and 0.1% (w/v)

SDS. Laemmli sample buffer (Sigma-Aldrich, UK), which consisted of 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue and 0.125 M Tris- HCl (pH 6.8), was used in

preparation of the protein samples using 1.1 volume ratio, then the resulting mixture was boiled for 5 min at 100 °C and centrifuged for 1 min. The SDS electrophoresis analysis was performed

using Bio-Rad chamber (Bio-Rad, Hercules) under constant voltage (200 V). A protein marker standard (Sigma-Aldrich, UK) with range of 6,500–200,000 Da was run alongside the samples. The

separated protein bands were visualized using Silver staining. The silver staining was performed according to the manufacture’s instructions (Bio-Rad, U.S.). WESTERN BLOTTING α-synuclein was

oxidized using 100 μM alpha-syn with 100 μM Cu2+ in 20 mM HEPES buffer at pH 7.4 and incubated at 37 °C with 400 rpm agitation for 30 days. Samples were centrifuged at 14,000 g at 4 °C for

20 minutes to separate the supernatant and pellet. The concentration of the pellet was estimated by subtracting the concentration of protein in the supernatant from the total concentration

using a molar extinction coefficient of 5120 M−1cm−1 and the absorbance was measured at a wavelength of 280 nm using an Eppendorf Biophotometer (Eppendorf UK Ltd., Cambridge, UK)29. An

aliquot of 5 μg of protein of the sample was mixed with 4x Laemmli sample buffer (containing 2-mercaptoethanol) and heated to 95 °C for 5 mins. Samples were loaded and run on SDS-PAGE

(4-20%Tris-Glycine gel (Bio-Rad)) and transferred to PVDF membrane, blocked with 5% milk (w/v) in Tris-buffered saline, 0.1% Tween 20 (TBST) and then probed with anti-dityrosine (DT)

monoclonal antibody (IC3; JaICA diluted 1/1000) overnight at 4 °C. The blot was incubated with an anti-mouse HRP conjugate (7076 S from Cell Signaling diluted 1/1000) for 1 hour at room

temperature and developed with ECL reagent (Bio-Rad) following the manufacturer’s instructions. IN GEL DIGESTION PROTOCOL The α-syn oxidation mixture (after 5 h of oxidation) were separated

by SDS-PAGE and stained using Coomassie Blue, then the separated bands (monomer and dimer) were excised and divided into 3–4 pieces and de-stained with 30% acetonitrile for 15 min with

agitation followed by 50% acetonitrile/25 mM ammonium bicarbonate for 15 min with agitation. This step was repeated until the gel pieces were completely de-stained. The de-stained gel pieces

were then dehydrated using vacuum centrifugation for 5 min without heating. 12.5 ng/ml of trypsin solution in 25 mM ammonium bicarbonate was added and the gel pieces were allowed to

rehydrate for 5 min, then the excess trypsin solution was removed and the gel pieces covered by 25 mM ammonium bicarbonate and incubated at 37 °C for 6 h. Formic acid was added to ~5% (v/v)

and the resulting mixture was vortexed and centrifuged at 1000 g for 1 min and the supernatant was removed, the remaining peptides were extracted from the gel pieces using 50% acetonitrile

with agitation and brief sonication. This step was repeated and all supernatants were pooled and the volume was reduced by vacuum centrifugation. The resultant sample solution was stored at

−80 °C until analyzed by nanoLC-LTQ-OrbitrapXL mass spectrometry. Α-SYN FIBRIL PREPARATION FOR X-RAY FIBRE DIFFRACTION α-syn fibrils were formed in 20 mM HEPES buffer (pH 7.4) by incubation

with and without Cu2+ at molar ratio of 1.1 and concentration of 400 μM for 5 days at 37 °C and with agitation of 450 rpm. To remove HEPES buffer, which can affect the quality of X-ray fibre

diffraction data, the resulting fibrils were centrifuged for 30 min at 20,000 g and 4 °C, then the pellet was re-suspended in 10 μl Milli-Q water and aligned as described below. X-RAY FIBRE

DIFFRACTION (XRFD) Fibre diffraction specimens were prepared by suspending a 10 μl droplet of α-syn fibril solution, which is prepared as described above, between two wax-tipped 1.2 mm O.D,

0.94 mm I.D borosilicate capillaries (Harvard apparatus), then left at room temperature in a parafilm sealed petri dish until dry. X-ray diffraction patterns were collected using a Rigaku

007HF CuKα (λ 1.5419 Å) rotating anode generator with a Saturn 944+ CCD detector with exposure times of 10–120 seconds and specimen to detector distances of 50 or 100 mm. The images were

displayed and examined using Mosflm43. ATOMIC FORCE MICROSCOPY AFM α-syn stock solution was prepared as described above, to remove any preformed aggregates and fibrils. Soluble α-syn monomer

was incubated with Cu2+ using a molar ratio of 1.1 and concentration of 100 μM in 20 mM HEPES (pH 7.4) at 37 °C with agitation of 400 rpm for two weeks. To compare between dityrosine

crosslinked and non-crosslinked α-syn, dityrosine formation was prevented by depleting Cu2+ by adding EDTA (final concentration 2.5 mM) to 100 μM α-syn in 20 mM HEPES buffer pH 7.4 and

incubating at 37 °C with agitation of 400 rpm for two weeks. To prepare the samples for AFM, the fibril stock was diluted to 2 μM with milliQ water. Samples were then stirred at 1000 rpm for

1 hour in 1.5-ml glass vials containing 38 mm polytetrafluoroethylene-coated magnetic stirring bars to cause mechanical distruption. 20 μl of the samples were subsequently deposited on

freshly cleaved mica and allowed to incubate for 8 minutes. The mica surfaces were then washed with a 1 ml filter sterilized milli-Q water, and dried using filter paper to absorb residual

water followed by gentle stream of nitrogen gas. The samples were imaged with a Bruker multimode 8 scanning probe microscope operating under ScanAsyst peak force tapping mode equipped with

SCANASYST-AIR, silicon nitride cantilever probes with a nominal tip radius of 2 nm and nominal spring constant of 0.4 N/m (Bruker Corporation, Massachusetts). Images were collected at a

resolution of 2048 × 2048 pixels at a scan size of 30 × 30 μm and a scan rate of 0.4 Hz. The Images were processed using Nanoscope analysis software (version 1.4, Bruker Corporation,

Massachusetts) to flatten and thereby removing surface tilt and bow on the images. Height and contour length was quantified in the AFM images using an in-house Matlab script that traces

individual fibril particles44. DECLARATIONS ETHICS Ethics approval was obtained locally for this work (University of Sussex) and the tissue was obtained from Parkinson’s Disease UK (PDUK)

brain bank under a Material transfer agreement. Informed consent is sought by PDUK. ADDITIONAL INFORMATION HOW TO CITE THIS ARTICLE: Al-Hilaly, Y. K. _et al_. The involvement of dityrosine

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Scholar  Download references ACKNOWLEDGEMENTS The authors would like to acknowledge help with synthesis and characterization of dityrosine from Dr Matthew Stanley, Dr Iain Day, Prof. Mark

Bagley and Dr Steve Sweet. Brain tissue was provided by the Parkinson’s UK brain bank and, respectfully, we thank the anonymous tissue donors and their next of kin. The authors thank Prof.

Guy Richardson and Dr Richard Goodyear for MAb10 antibody. This work was supported by funding from Alzheimer’s research UK and Biotechnology and Biological Sciences Research Council, UK

(WFX. BB/J008001/1), Medical research council (LCS. MR/K004999/1) and Alzheimer’s society (AS-DTC-2014-003 for LB and LCS). YA is supported by funding from Ministry of Higher Education and

Scientific Research in Iraq. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * School of Life Sciences, University of Sussex, Falmer, BN1 9QG, UK Youssra K. Al-Hilaly, Luca Biasetti, Saskia J.

Pollack, Alaa Abdul-Sada, Julian R. Thorpe & Louise C. Serpell * Chemistry Department, College of Sciences, Al-Mustansiriyah University, Baghdad, Iraq Youssra K. Al-Hilaly * School of

Biosciences, University of Kent, CT2 7NJ, UK Ben J. F. Blakeman & Wei-Feng Xue * Laboratory of Molecular Biology, MRC Centre, Hills Rd, Cambridge, CB2 OQH, UK Shahin Zibaee Authors *

Youssra K. Al-Hilaly View author publications You can also search for this author inPubMed Google Scholar * Luca Biasetti View author publications You can also search for this author

inPubMed Google Scholar * Ben J. F. Blakeman View author publications You can also search for this author inPubMed Google Scholar * Saskia J. Pollack View author publications You can also

search for this author inPubMed Google Scholar * Shahin Zibaee View author publications You can also search for this author inPubMed Google Scholar * Alaa Abdul-Sada View author publications

You can also search for this author inPubMed Google Scholar * Julian R. Thorpe View author publications You can also search for this author inPubMed Google Scholar * Wei-Feng Xue View

author publications You can also search for this author inPubMed Google Scholar * Louise C. Serpell View author publications You can also search for this author inPubMed Google Scholar

CONTRIBUTIONS Y.A. conducted and analyzed the experiments and wrote the paper. W.F.X. and B.B. collected data, analyzed results and edited the paper S.Z., L.B. and S.P. conducted

experiments. S.A. supervised the LCMS/MS. J.T. conducted experiments and analysis, L.C.S. managed the research and wrote the paper. ETHICS DECLARATIONS COMPETING INTERESTS The authors

declare no competing financial interests. RIGHTS AND PERMISSIONS This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party

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and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Al-Hilaly, Y., Biasetti, L., Blakeman, B. _et al._ The involvement of dityrosine crosslinking in α-synuclein assembly and deposition in

Lewy Bodies in Parkinson’s disease. _Sci Rep_ 6, 39171 (2016). https://doi.org/10.1038/srep39171 Download citation * Received: 13 July 2016 * Accepted: 18 November 2016 * Published: 16

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