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.

Figure 1. Immunogold labeling TEM within Lewy bodies from PD brains.

(a) Shows a Lewy body taken from PD041 case (Table 1) and (b,c) show representative micrographs showing double labeling using the dityrosine antibody (15 nm) and anti-α-syn (5 nm) antibody on a PD substantia nigra brain section taken from PD028 case (Table 1) and independent areas at increasing magnifications. The micrographs reveal very dense anti-α-syn labeling of the Lewy bodies and confirm colocalization of dityrosine with α-syn within Lewy bodies. (b) (ii) reveals fibrillar structures labeled with both 5 and 15 nm gold particles supporting the view that dityrosine crosslinks are present within the fibrils in the Lewy bodies (white arrows). (c) (i, ii and iii) highlight these structures by increasing magnification to show the presence of both 5 and 15 nm gold labels and the 15 nm (red) and 5 nm (blue) have been highlighted in (c) (iii). An area outside of the Lewy body is highlighted (dotted circle) to show the absence of gold labeling.

Figure 2. α-syn forms dityrosine cross-links in the presence of Cu²⁺/H₂O₂.

(a) 50 μM of monomeric α-syn was incubated for 24 h with Cu²⁺/H₂O₂ at 37 °C and agitation of 400 rpm. A fluorescence spectra was collected with excitation wavelength of 280 nm to explore tyrosine and dityrosine signals. Fluorescence spectra was also collected using excitation wavelength of 320 nm to focus on dityrosine signals and to record the early time points (inset). After one hour of incubation, the tyrosine fluorescence signal appeared to decline with simultaneous appearance of a new increasing signal at 405–410 nm, typical of the dityrosine fluorophore. (b) shows the development of dityrosine signal (at 405 nm) (ex. 320 nm) over time for 24 h and over 1 hour (inset). (c) Using an excitation wavelength of 280 nm, no change in the tyrosine signal and an absence of dityrosine fluorescence signal was observed over 24 h of incubation of 50 μM α-syn alone (i), with 50 μM Cu²⁺ only (ii) or with 1.25 mM H₂O₂ only (iii). (d) LC-ESIMS/MS (MRM) was used to detect dityrosine in the oxidized α-syn hydrolysate. The oxidized α-syn was obtained by oxidation of (50 μM) monomeric α-syn for 24 h with Cu²⁺/H₂O₂. Data is shown in a chromatogram as relative abundance against retention time showing the dityrosine standard for comparison.

Figure 3

(a) SDS-PAGE analysis of α-syn samples. 100 μM of purified recombinant human α-syn was oxidized using Cu²⁺/H₂O₂ in 20 mM HEPES buffer, pH 7.4, at 37 °C with agitation of 400 rpm for 5 hours and EDTA was added to quench the oxidation process. SDS-sample buffer was added to the samples and then boiled for 5 min at 100 °C, and 10 μl of the boiled sample loaded on 12% Tris-glycine gel and then silver stained. The figure shows the appearance of a band with molecular weight about 35 kDa after 5 h oxidation indicating the presence of a dimer, which is not present in the non-oxidized control. (b) Shows western blot showing the separated pellet and supernatant labeled using the anti-dityrosine primary antibody. The antibody positive bands are highlighted by arrows which are likely to be dimer and tetramer in supernatant and only dimer and fibrillar species in the pellet. NanoLC-MS/MS analysis of tryptic digested α-syn monomer and dimer that were extracted from electrophoresis gel boxed in panel (a), (c) shows analysis from Mascot Engine. Sequence from the monomer band was complete, but two sequences were missing from dimer band in the oxidized sample, TKEGVLYVGSK and GLSKAK (highlighted in red). (d) Mass spectrum of tryptic digested α-syn dimer showing the appearance of a peak at m/z 1179.6127 that corresponds to the dimer of the peptide with sequence of (₃₃TKEGVLYVGSK₄₃) containing Y39.

Figure 4

Slow oxidation of α-syn incubation in HEPES buffer only (a) and with Cu²⁺ (b). Incubation of 100 μM α-syn with or without Cu²⁺ only, showed the appearance of a dityrosine signal at 405 nm after 7 days of incubation which increases over 14 days. The presence of Cu²⁺ significantly increases dityrosine formation compared to buffer only. (c) ThT fluorescence assay to monitor fibril formation showed that the intensity increased significantly more for the α-syn incubated with 100 μM Cu²⁺ compared to α-syn without Cu²⁺ over 14 days. (d) TEM micrographs show comparison of α-syn incubated with and without Cu²⁺. The Cu²⁺ incubated α-syn assemblies display characteristic fibril morphology by negative stain TEM whilst sparse assemblies are observed for α-syn incubated without Cu²⁺. Immunogold labeling TEM for dityrosine cross-links reveals increased gold labels in α-syn incubated with Cu²⁺ and very few when Cu²⁺ is absent. Gold labels are evenly distributed along the fibrils (inserts).

Figure 5. Cu²⁺ ions affect α-syn fibril growth and structure.

400 μM α-syn fibrils grown in 20 mM HEPES buffer, pH 7.4 with and without Cu²⁺ with agitation of 450 rpm. (a) ThT fluorescence assay over 120 hours revealed increased fluorescence for α-syn incubated with Cu²⁺ compared to α-syn without Cu²⁺. (b) Shows fluorescence at 405 nm following incubation of α-syn for 120 hours confirming that dityrosine forms in 400 μM α-syn and that Cu²⁺ enhances its formation. (c) TEM micrographs reveal no significant morphological changes between α-syn with or without Cu²⁺, although there appear to be increased number of fibrils in Cu²⁺ induced samples, consistent with the ThT results. (d) X-ray fibre diffraction patterns collected from partially aligned α-syn fibrils incubated at 400 μM α-syn without and with Cu²⁺ reveals the characteristic cross-β pattern. Inset shows a zoom of ~35 Å reflection close to the back stop.

Figure 6. Quantitative AFM imaging analysis of α-syn fibrils grown for 2 weeks in Cu²⁺ oxidized or Cu²⁺ depleted conditions (using EDTA).

(a) AFM height images of α-syn fibril samples formed in Cu²⁺ oxidized and Cu²⁺ depleted conditions, respectively. The scale bars indicate the length of 10 μm. Magnified (4X) images are shown as insets. (b) Height distributions of the pixels of the fibril particles are shown as histograms, indicating the width of the fibrils formed in Cu²⁺ oxidized and Cu²⁺ depleted conditions, respectively. (c) Length distributions of fibril particles formed in Cu²⁺ oxidized and Cu²⁺ depleted conditions, respectively. The length distributions are show as 1-cumulative distribution functions on a semi-log plot to enable visual comparison for the wide length distributions of the samples. A total of 781 fibrils formed in Cu²⁺ oxidized conditions and 409 fibrils formed in Cu²⁺ depleted conditions were analyzed. 100 μM α-syn shaken for 2 weeks at 37 °C with 100 μM Cu²⁺ or with EDTA (2.5 mM) was diluted to 2 μM for AFM imaging.

Figure 7. Dityrosine cross-links form between fibrils to generate intertwined and interconnected structures.

(a) Schematic showing fibrils connected via dityrosine cross-links, (b) electron micrograph showing dityrosine immunogold labeling of fibrils and (c) AFM image showing interconnected fibrils. α-syn fibrils were formed using 100 uM α-syn incubated with 100 uM Cu²⁺ in 20 mM HEPES buffer at pH 7.4 with 400 rpm agitation for 30 days incubation.