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. 2020 Feb 4;59(4):425-435.
doi: 10.1021/acs.biochem.9b00655. Epub 2019 Dec 30.

Interactions between Soluble Species of β-Amyloid and α-Synuclein Promote Oligomerization while Inhibiting Fibrillization

Jason Candreva  1 , Edward Chau  1 , Margaret E Rice  2 , Jin Ryoun Kim  1
Affiliations

Interactions between Soluble Species of β-Amyloid and α-Synuclein Promote Oligomerization while Inhibiting Fibrillization

Jason Candreva et al. Biochemistry. .

Abstract

Aggregations of β-amyloid (Aβ) and α-synuclein (αS) into oligomeric and fibrillar assemblies are the pathological hallmarks of Alzheimer's and Parkinson's diseases, respectively. Although Aβ and αS affect different regions of the brain and are separated at the cellular level, there is evidence of their eventual interaction in the pathology of both disorders. Characterization of interactions of Aβ and αS at various stages of their aggregation pathways could reveal mechanisms and therapeutic targets for the prevention and cure of these neurodegenerative diseases. In this study, we comprehensively examined the interactions and their molecular manifestations using an array of characterization tools. We show for the first time that αS monomers and oligomers, but not αS fibrils, inhibit Aβ fibrillization while promoting oligomerization of Aβ monomers and stabilizing preformed Aβ oligomers via coassembly, as judged by Thioflavin T fluorescence, transmission electron microscopy, and SDS- and native-PAGE with fluorescently labeled peptides/proteins. In contrast, soluble Aβ species, such as monomers and oligomers, aggregate into fibrils, when incubated alone under the otherwise same condition. Our study provides evidence that the interactions with αS soluble species, responsible for the effects, are mediated primarily by the C-terminus of Aβ, when judged by competitive immunoassays using antibodies recognizing various fragments of Aβ. We also show that the C-terminus of Aβ is a primary site for its interaction with αS fibrils. Collectively, these data demonstrate aggregation state-specific interactions between αS and Aβ and offer insight into a molecular basis of synergistic biological effects between the two polypeptides.

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Figures

Figure 1.
Figure 1.
Characterizations of Aβ monomers (M) mixed with αS monomers (M), αS oligomers (O) or αS fibrils (F) incubated for 7 days at 37 °C, as examined by (A) ThT fluorescence, (B-D) TEM, and (E-F) in-gel fluorescence imaging of (E) SDS-PAGE and (F) native-PAGE. In (A-F), the concentration of Aβ monomers was 70 μM. The concentrations of αS monomers and αS fibrils were 350 μM and that of αS oligomers was 17 μM. Concentrations of oligomers and fibrils were monomer-equivalent concentrations. In (A), the data on Aβ monomers mixed with αS monomers (filled red diamonds), αS oligomers (filled blue triangles) and αS fibrils (filled gray circles) are shown along with those on αS monomers alone (empty red diamonds), αS oligomers alone (empty blue triangles) and αS fibrils alone (empty gray circles) taken from Figure S7A. The data on Aβ monomers alone (filled black squares) taken from Figure S3A are also shown for comparison. Error bars: 1 standard deviation of triplicates. In (B-D), representative TEM images of mixtures containing (B) Aβ monomers and αS monomers, (C) Aβ monomers and αS oligomers and (D) Aβ monomers and αS fibrils are shown with scale bars of 200 nm. In (E-F), samples contained HiLyte Fluor 488-labeled Aβ (green) and Alexa Fluor 647-labeled αS (red). Each panel was taken from a bigger gel image (see Figure S5) and reassembled for better presentation. In (E), T: total fraction and S: soluble fraction. In (F), the images in the right provide brighter upper portions of the native-PAGE gels – enclosed by a blue box in the left – obtained by renormalizing the brightness from the monomer to the oligomer bands, which often contained lower percentages of fluorophores. G: green channel and R: red channel.
Figure 2.
Figure 2.
Characterizations of Aβ oligomers (O) mixed with αS monomers (M), αS oligomers (O) or αS fibrils (F) incubated for 7 days at 37 °C, as examined by (A) ThT fluorescence, (B-D) TEM, and (E-F) in-gel fluorescence imaging of (E) SDS-PAGE and (F) native-PAGE. In (A-F), the concentration of Aβ oligomers was 70 μM. The concentrations of αS monomers and αS fibrils were 350 μM and that of αS oligomers was 17 μM. Concentrations of oligomers and fibrils were monomer-equivalent concentrations. In (A), the data on Aβ oligomers mixed with αS monomers (filled red diamonds), αS oligomers (filled blue triangles) and αS fibrils (filled gray circles) are shown along with those on αS monomers alone (empty red diamonds), αS oligomers alone (empty blue triangles) and αS fibrils alone (empty gray circles) taken from Figure S7A. The data on Aβ oligomers alone (filled black squares) taken from Figure S3A are also shown for comparison. Error bars: 1 standard deviation of triplicates. In (B-D), representative TEM images of mixtures containing (B) Aβ oligomers and αS monomers, (C) Aβ oligomers and αS oligomers and (D) Aβ oligomers and αS fibrils are shown with scale bars of 200 nm. In (E-F), samples contained HiLyte Fluor 488-labeled Aβ (green) and Alexa Fluor 647-labeled αS (red). Each panel was taken from a bigger gel image (see Figure S5) and reassembled for better presentation. In (E), T: total fraction and S: soluble fraction. In (F), the images in the right provide brighter upper portions of the native-PAGE gels – enclosed by a blue box in the left – obtained by renormalizing the brightness from the monomer to the oligomer bands, which often contained lower percentages of fluorophores. G: green channel and R: red channel.
Figure 3.
Figure 3.
Characterizations of Aβ fibrils (F) mixed with αS monomers (M), αS oligomers (O) or αS fibrils (F) incubated for 7 days at 37 °C, as examined by (A) ThT fluorescence, (B-D) TEM, and (E-F) in-gel fluorescence imaging of (E) SDS-PAGE and (F) native-PAGE. In (A-F), the concentration of Aβ fibrils was 70 μM. The concentrations of αS monomers and αS fibrils were 350 μM and that of αS oligomers was 17 μM. Concentrations of oligomers and fibrils were monomer-equivalent concentrations. In (A), the data on Aβ fibrils mixed with αS monomers (filled red diamonds), αS oligomers (filled blue triangles) and αS fibrils (filled gray circles) are shown along with those on αS monomers alone (empty red diamonds), αS oligomers alone (empty blue triangles) and αS fibrils alone (empty gray circles) taken from Figure S7A. The data on Aβ fibrils alone (filled black squares) taken from Figure S3A are also shown for comparison. Error bars: 1 standard deviation of triplicates. In (B-D), representative TEM images of mixtures containing (B) Aβ fibrils and αS monomers, (C) Aβ fibrils and αS oligomers and (D) Aβ fibrils and αS fibrils are shown with scale bars of 200 nm. In (E-F), samples contained HiLyte Fluor 488-labeled Aβ (green) and Alexa Fluor 647-labeled αS (red). Each panel was taken from a bigger gel image (see Figure S5) and reassembled for better presentation. In (E), T: total fraction and S: soluble fraction. In (F), the images in the right provide brighter upper portions of the native-PAGE gels – enclosed by a blue box in the left – obtained by renormalizing the brightness from the monomer to the oligomer bands, which often contained lower percentages of fluorophores. G: green channel and R: red channel.
Figure 4.
Figure 4.
Competitive binding dot blot assay using Aβ sequence-specific antibodies of (A) 6E10 recognizing Aβ1-16, (B) 4G8 recognizing Aβ17-22, (C) Anti-Aβ (22-35) recognizing Aβ22-35, and (D) 5C3 recognizing Aβ32-40 of Aβ monomers (top row), Aβ oligomers (middle row), and Aβ fibrils (bottom row) mixed with αS monomers (left column), αS oligomers (middle column), and αS fibrils (right column). M, O and F represent monomers, oligomers and fibrils, respectively.
Figure 5.
Figure 5.
Summary of experimental findings described in this study. Aβ monomers self-assemble into Aβ oligomers, which then further aggregate to form Aβ fibrils. Addition of αS soluble species (i.e., monomers and oligomers) promotes oligomerization of Aβ monomers and stabilizes pre-formed Aβ oligomers via co-assembly. Monomeric, oligomeric and fibrillar forms of Aβ can be incorporated to αS fibrils. M, O and F represent monomers, oligomers and fibrils, respectively.
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