Lipid peroxidation product 4-hydroxy-2-nonenal promotes seeding-capable oligomer formation and cell-to-cell transfer of α-synuclein

Abstract

Aims: Abnormal accumulation of α-synuclein aggregates is one of the key pathological features of many neurodegenerative movement disorders and dementias. These pathological aggregates propagate into larger brain regions as the disease progresses, with the associated clinical symptoms becoming increasingly severe and complex. However, the factors that induce α-synuclein aggregation and spreading of the aggregates remain elusive. Herein, we have evaluated the effects of the major lipid peroxidation byproduct 4-hydroxy-2-nonenal (HNE) on α-synuclein oligomerization and cell-to-cell transmission of this protein.

Results: Incubation with HNE promoted the oligomerization of recombinant human α-synuclein via adduct formation at the lysine and histidine residues. HNE-induced α-synuclein oligomers evidence a little β-sheet structure and are distinct from amyloid fibrils at both conformation and ultrastructure levels. Nevertheless, the HNE-induced oligomers are capable of seeding the amyloidogenesis of monomeric α-synuclein under in vitro conditions. When neuronal cells were treated with HNE, both the translocation of α-synuclein into vesicles and the release of this protein from cells were increased. Neuronal cells can internalize HNE-modified α-synuclein oligomers, and HNE treatment increased the cell-to-cell transfer of α-synuclein proteins.

Innovation and conclusion: These results indicate that HNE induces the oligomerization of α-synuclein through covalent modification and promotes the cell-to-cell transfer of seeding-capable oligomers, thereby contributing to both the initiation and spread of α-synuclein aggregates.

FIG. 1.

Increased oligomerization of α-synuclein by 4-hydroxy-2-nonenal (HNE). (A) Size exclusion column chromatography (SEC) of α-synuclein proteins incubated with HNE (red trace) and an equal volume of ethanol (blue trace) or phosphate buffered saline (green trace) for 7 days. The black square and circle mark the void volume and monomer positions, respectively. (B) Western blot analysis of the SEC fractions. Note that α-synuclein proteins in the void volume fractions are mostly sodium dodecyl sulfate (SDS)–stable oligomers. (C) Time course of oligomerization. HNE/α-synuclein mixture (70 μM) was incubated for up to 7 days, and the samples were analyzed on SEC at the indicated times. (To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars.)

FIG. 2.

HNE-α-synuclein adduct formation and oligomerization. (A, B) Western analysis of α-synuclein (70 μM) after incubation with different concentrations of HNE, diluted by a factor of 2 from 5 mM to 0.078125 mM and 0 mM, using either an antibody specific for HNE-protein adducts (A) or an antibody for α-synuclein (B). (C) Quantification of HNE-α-synuclein adducts (closed circles) and α-synuclein oligomers (open squares) from the data in (A) and (B), respectively. For oligomer quantification, monomer bands were excluded from measurement.

FIG. 3.

Mass spectrometry of HNE-modified α-synuclein. (A) Amino acid sequence of human α-synuclein. Enlarged bold letters indicate positions modified by HNE. (B–D) MS/MS spectra of peptides contained HNE-modified His50 (B), Lys60 (C), and Lys 96 (D).

FIG. 3.

Mass spectrometry of HNE-modified α-synuclein. (A) Amino acid sequence of human α-synuclein. Enlarged bold letters indicate positions modified by HNE. (B–D) MS/MS spectra of peptides contained HNE-modified His50 (B), Lys60 (C), and Lys 96 (D).

FIG. 4.

Analysis of conformation of HNE-induced α-synuclein oligomers. (A) Circular dichroism spectroscopy. α-Synuclein monomers (solid line), HNE-induced oligomers (interrupted line), and fibrils (interrupted line with spots). On the right, the graph represents the molecular ellipticities at 218 nm of α-synuclein monomers, HNE-induced oligomers, and fibrils. (B) Thioflavin T binding assay with α-synuclein monomers (M), fibrils (F), and HNE-induced oligomers (HNE-O). (C) Dot blot analysis using a conformation-specific antibody, Fila-4, which recognizes β-sheet-rich α-synuclein.

FIG. 5.

Ultrastructural analysis of HNE-induced α-synuclein oligomers. (A) Atomic force microscopy. (B) Electron microscopy. Scale bar: 200 nm. Oligomer images in white rectangles on the left images are enlarged in the tables on the right. (To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars.)

FIG. 6.

Seeding of α-synuclein fibrillation by HNE-induced oligomers. (A) Thioflavin T binding assay. Different amounts of HNE-induced oligomers (0%, circle; 5%, square; 10%, triangle) were added to 70 μM of α-synuclein monomers, and fibrillation was measured by thioflavin T over time. (B) Electron microscopy of samples in (A) at indicated times. Scale bars: 200 nm.

FIG. 7.

HNE promotes the translocation of α-synuclein into vesicles in neuronal cells. (A) Immunofluorescence microscopy. Differentiated SH-SY5Y human neuroblastoma cells overexpressing α-synuclein were treated with either ethanol (top images) or 10 μM HNE (bottom images) for 2 days. Green: α-synuclein; blue: nuclei. Scale bars: 20 μm. (B) Vesicle fractionation. Differentiated SH-SY5Y cells overexpressing α-synuclein were treated with either ethanol (−) or 10 μM HNE (+), and vesicles were separated from the cytosol via flotation centrifugation (β-act: β-actin; ST: synaptotagmin). Right panel: vesicle preparations and recombinant α-synuclein monomers (α-syn) were subjected to proteinase K digestion to confirm the intravesicular localization of α-synuclein. (C) Levels of α-synuclein in the cytosol and vesicles. The Western data as shown in (B) were quantified. The data were normalized by the levels of β-actin (cytosol) and synaptotagmin (vesicle). Relative values were obtained by setting one of the control values as being 100 (n=3, *p<0.05). (To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars.)

FIG. 8.

Increased secretion of α-synuclein from HNE-treated cells. (A) Enzyme-linked immunosorbent assay (ELISA) of secreted α-synuclein in culture media of differentiated SH-SY5Y cells overexpressing α-synuclein (n=3, *p<0.05). (B) Western blotting of cellular α-synuclein in cell lysates. (C) ELISA analysis of HNE-modified α-synuclein in the culture media (n=3, *p<0.05). (D) Western blotting of the endogenous α-synuclein in the cytosol and culture media of primary mouse cortical neurons after incubation with different concentrations of HNE, diluted by a factor of 5 from 500 μM to 20 μM and 0 μM. (E) ELISA analysis of the total α-synuclein and HNE-modified α-synuclein in the cytosol of primary cortical neurons treated with HNE. (F) ELISA analysis of the total α-synuclein and HNE-modified α-synuclein in culture media of primary cortical neurons treated with HNE. (G) Cells overexpressing his-tagged α-synuclein were incubated with 100 μM hydrogen peroxide and 1 mM iron sulfate for 2 days, and α-synuclein proteins were pulled down from the cell lysates and culture media and analyzed via Western blotting with α-synuclein antibody and HNE-adduct antibody. The y-axis of the graph indicates the fold increase of HNE-modified α-synuclein relative to the level of HNE-modified α-synuclein in control cell extract (n=3, *p<0.05; ext: cell extract).

FIG. 9.

Internalization of HNE-induced α-synuclein oligomers into differentiated SH-SY5Y cells. (A) Cells were incubated with 1 μM α-synuclein preincubated with HNE for indicated times, and cell lysates were analyzed with ELISA for the total α-synuclein and HNE-modified α-synuclein (n=3). (B) Removal of surface-bound proteins. Cells were treated as in (A) for 5 min and washed with 2 N hydrochloric acid to remove surface-bound proteins. Internalized α-synuclein was analyzed via ELISA for the total α-synuclein and HNE-modified α-synuclein (n=3).

FIG. 10.

HNE promotes cell-to-cell transfer of α-synuclein. (A) Differentiated SH-SY5Y cells overexpressing α-synuclein (donor cells with α-synuclein staining in the entire cytoplasm; green) were cocultured with recipient cells without ectopic expression of α-synuclein (labeled with Qtracker; red). Green dots in the recipient cells represent transferred α-synuclein (arrows). Blue: nuclei. Scale bar: 20 μm. (B) Quantification of cell-to-cell transfer. Graph on the left: percent recipient cells with transferred α-synuclein (n=4, *p<0.05). Three hundred cells were analyzed in each experiment. Graph on the right: green fluorescence intensity per μm² in the recipient cells (n=4, *p<0.05). Three hundred cells were analyzed in each experiment.