Amyloids of α-Synuclein Promote Chemical Transformations of Neuronal Cell Metabolites

Abstract

The assembly of α-synuclein into cross-β structured amyloid fibers results in Lewy body deposits and neuronal degeneration in Parkinson's disease patients. As the cell environment is highly crowded, interactions between the formed amyloid fibers and a range of biomolecules can occur in cells. Although amyloid fibers are considered chemically inert species, recent in vitro work using model substrates has shown α-synuclein amyloids, but not monomers, to catalyze the hydrolysis of ester and phosphoester bonds. To search for putative catalytic activity of α-synuclein amyloids on biologically relevant metabolites, we here incubated α-synuclein amyloids with neuronal SH-SY5Y cell lysates devoid of proteins. LC-MS-based metabolomic (principal component and univariate) analysis unraveled distinct changes in several metabolite levels upon amyloid (but not monomer) incubation. Of 63 metabolites identified, the amounts of four increased (3-hydroxycapric acid, 2-pyrocatechuic acid, adenosine, and NAD), and the amounts of seventeen decreased (including aromatic and apolar amino acids, metabolites in the TCA cycle, keto acids) in the presence of α-synuclein amyloids. Many of these metabolite changes match what has been reported previously in Parkinson's disease patients and animal-model metabolomics studies. Chemical reactivity of α-synuclein amyloids may be a new gain-of-function that alters the metabolite composition in cells and, thereby, modulates disease progression.

Keywords: LC-MS; Parkinson’s disease; amyloid; metabolomics; α-synuclein.

Figure 1

Sample preparation scheme for LC-MS metabolomic analysis. Neuronal cell lysate samples (with proteins and larger molecules removed) were treated with 20 µM freshly gel-filtered monomeric α-synuclein or pre-formed amyloids for 0 (no incubation, control), 1, and 6 h. Methanol (MetOH) was used to precipitate α-synuclein and, thus, quench any ongoing reactions. This also facilitated the removal of the protein by centrifugation prior to LC-MS analysis of the resulting metabolites.

Figure 2

Multivariate PCA of the 63 identified metabolites with coloring according to (A) Treatment: black, lysate + buffer; red, lysate + monomer; blue, lysate + fiber, (B) Incubation time: black, 0 h; red, 1 h; blue, 6 h, and (C) Biological replicate (lysate batch): black, batch 1; red, batch 2; blue, batch 3.

Figure 3

Time-dependent changes (1 and 6 h as compared to 0 h) of metabolites which showed increased levels with amyloid fiber treatment compared to monomer treatment. α-synuclein fibers (red) or monomers (black) treatments are shown as box plots. (A) 3-hydroxycapric acid; (B) 2-pyrocatechuic acid/2,4 dihydroxybenzoic acid; (C) adenosine; (D) NAD. The boxes cover the full range of the data; the line denotes the median. Chemical structures of metabolites are shown as insets.

Figure 4

Time-dependent changes in metabolites which showed decreased levels with amyloid fiber treatment compared to monomer treatment. α-synuclein fiber (red) or monomer (black) treatments are shown as box plots. (A) Hypoxanthine; (B) Merged data for TCA cycle metabolites (oxoglutaric acid, succinic acid, malic acid, citric acid, and L-2-hydroxyglutaric acid/3-hydroxiglutaric acid); (C) Merged data for amino acids (Phe, Tyr, Ile, Leu, Ac-Ala, and Ac-Ser); (D) Merged data for α-ketoisovaleraic acid and ketoleucine; (E) Trigonelline; (F) Pantothenic acid (vitamin b5). For (A,E,F), boxes cover the full range of measured data, while for (B–D), the boxes cover the 25–75% range of the data. The line denotes the median. Chemical structures of some metabolites are shown as insets.