3,4-Dihydroxyphenylacetaldehyde Is More Efficient than Dopamine in Oligomerizing and Quinonizing α-Synuclein - PubMed (original) (raw)

3,4-Dihydroxyphenylacetaldehyde Is More Efficient than Dopamine in Oligomerizing and Quinonizing _α_-Synuclein

Yunden Jinsmaa et al. J Pharmacol Exp Ther. 2020 Feb.

Abstract

Lewy body diseases such as Parkinson's disease involve intraneuronal deposition of the protein _α_-synuclein (AS) and depletion of nigrostriatal dopamine (DA). Interactions of AS with DA oxidation products may link these neurohistopathologic and neurochemical abnormalities via two potential pathways: spontaneous oxidation of DA to dopamine-quinone and enzymatic oxidation of DA catalyzed by monoamine oxidase to form 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is then oxidized to DOPAL-Q. We compared these two pathways in terms of the ability of DA and DOPAL to modify AS. DOPAL was far more potent than DA both in oligomerizing and forming quinone-protein adducts with (quinonizing) AS. The DOPAL-induced protein modifications were enhanced similarly by pro-oxidation with Cu(II) or tyrosinase and inhibited similarly by antioxidation with _N_-acetylcysteine. Dopamine oxidation evoked by Cu(II) or tyrosinase did not quinonize AS. In cultured MO3.13 human oligodendrocytes DOPAL resulted in the formation of numerous intracellular quinoproteins that were visualized by near-infrared spectroscopy. We conclude that of the two routes by which oxidation of DA modifies AS and other proteins the route via DOPAL is more prominent. The results support developing experimental therapeutic strategies that might mitigate deleterious modifications of proteins such as AS in Lewy body diseases by targeting DOPAL formation and oxidation. SIGNIFICANCE STATEMENT: Interactions of the protein _α_-synuclein with products of dopamine oxidation in the neuronal cytoplasm may link two hallmark abnormalities of Parkinson disease: Lewy bodies (which contain abundant AS) and nigrostriatal DA depletion (which produces the characteristic movement disorder). Of the two potential routes by which DA oxidation may alter AS and other proteins, the route via the autotoxic catecholaldehyde 3,4-dihydroxyphenylacetaldehyde is more prominent; the results support experimental therapeutic strategies targeting DOPAL formation and DOPAL-induced protein modifications.

U.S. Government work not protected by U.S. copyright.

PubMed Disclaimer

Conflict of interest statement

The authors have no conflicts of interest to disclose.

Figures

None

Graphical abstract

Fig. 1.

Fig. 1.

Time courses of DOPAL and DA effects on AS quinonization and oligomerization. (A) AS (3 _μ_M) was incubated with DOPAL or DA (30 _μ_M each) at 37°C and samples were taken after 120 minutes (A) and 300 minutes (B) of incubation. (A and B) Quinonized AS was detected by nIRF spectroscopy (red). (A and C) Oligomerized AS was detected by western blotting (green). (D) Protein staining was used to demonstrate decreased AS monomer during incubation of AS with DOPAL. Lanes = order of the gel lanes, Groups: 1 = AS alone as a control (CON), 2 = DOPAL, 3 = DA. DOPAL time dependently increased AS quinonization and oligomerization, whereas DA did not elicit AS quinonization and produced a slight smear of high molecular weight AS.

Fig. 2.

Fig. 2.

Effects of enzymatic oxidation of DA and DOPAL with tyrosinase on AS quinonization and oligomerization. DA or DOPAL (30 _μ_M each) was incubated with tyrosinase (+Tyr) or without tyrosinase (no Tyr) for 20 minutes at room temperature and then incubated with AS (3 _μ_M) for 1 hour at 37°C. (A and B) Concentration course of DA or DOPAL (10, 30, and 100 _μ_M each) oxidation with Tyr. (A and C) Quinonized AS was detected by nIRF spectroscopy (red). (B and D) Oligomerized AS was detected by western blotting (green). N = number of replicates; 1 = DA; 2 = DOPAL. Enzymatic oxidization augmented DOPAL-induced oligomerization and quinonization of AS. Incubation of AS with DA and Tyr resulted in a smear of high molecular weight AS. DA did not quinonize AS even in the setting of enzymatic oxidation by Tyr.

Fig. 3.

Fig. 3.

Effects of Cu(II) on DOPAL-induced AS quinonization and NAC effect. (A and B) AS (3 _μ_M) was incubated with 30 _μ_M DOPAL and 1–100 _μ_M Cu(II) for 1 hour at 37°C. (C and D) AS was incubated with DOPAL and 30 _μ_M Cu(II) and 0–1000 μ_M NAC for 1 hour at 37°C. Quinonized AS was detected by nIRF spectroscopy (red). N, number of replicates; Fx, fractions of integrated intensities of AS monomers compared with DOPAL alone. Statistical analyses were done by one-way ANOVA with Dunnett’s post-hoc test. Mean values are expressed as ± S.E.M. ****P < 0.0001, ***P < 0.001 compared with DOPAL alone; ☨☨☨_P < 0.001 vs. DOPAL + Cu(II) compared with no NAC. Cu(II) concentration dependently augmented DOPAL-induced AS quinonization and oligomerization. NAC attenuated this effect.

Fig. 4.

Fig. 4.

Comparisons of DA- vs. DOPAL-induced AS modifications in the presence of Cu(II). (A) Incubation of AS (3 _µ_M) with 30 _µ_M each of DA and DOPAL and 1 _µ_M Cu(II). (B) Incubation of AS with DA or DOPAL and 30 _µ_M Cu(II). (A–C) Quinonized AS was detected by nIRF spectroscopy (red). (A and B) Oligomerized AS was detected by western blotting (green). ΔIntegrated intensity, the difference in integrated intensity of signal at each time point minus the integrated intensity at 0 minutes; Lanes, order of the gel lanes. Cu(II) at 30 _µ_M accelerated and enhanced DOPAL-induced oligomerization and quinonization of AS. Incubation of Cu(II) (30 _µ_M) with 30 _µ_M DA and AS resulted in a smear of high molecular weight AS.

Fig. 5.

Fig. 5.

(A and B) DOPAL-induced quinonization of mutant A53T vs. WT AS. WT or A53T mutant AS (3 _μ_M) was incubated with 30 μ_M each of DOPAL and Cu(II) (with or without) or 300 μ_M NAC (with or without) for 1 hour at 37°C. Quinonized AS was detected by nIRF spectroscopy. Fx, fractions of integrated intensities of AS monomers compared with DOPAL alone; N, number of replicates. Statistical analyses were done by one-way ANOVA with Dunnett’s post-hoc test. Mean values are expressed as ± S.E.M. ***P < 0.001; **P < 0.01 compared with DOPAL alone; ☨☨☨_P < 0.001; ☨☨_P < 0.05 compared with no NAC; ++P < 0.01 for A53T compared with WT. DOPAL quinonized both A53T and WT AS, with about twice as large an effect on A53T AS. The enhancing effects were attenuated by NAC.

Fig. 6.

Fig. 6.

DOPAL-induced quinonization of intracellular proteins in MO3.13 cells and NAC effect. (A and B) MO3.13 cells (1.5 × 105 cells/well) were exposed to DOPAL (100 _μ_M) or DOPAL + Cu(II) (10 and 30 _μ_M) for 24 hours and then lysed in radioimmunoprecipitation assay buffer with protease inhibitors. (C and D) MO3.13 cells were exposed to DOPAL and 30 µ_M Cu(II), with NAC (0–300 μ_M) added at the start of incubation. DOPAL-quinonized proteins were detected and quantified by nIRF spectroscopy (red). Fx, fractions of integrated intensities of each column compared with CON (B) or DOPAL alone (D) groups normalized to the protein of each lanes. N, number of replicates. Statistical analyses were done by one-way ANOVA with Dunnett’s post-hoc test. Mean values are expressed as ± S.E.M. **P < 0.001 compared with DOPAL alone; ☨☨☨_P < 0.001; ☨☨_P < 0.01 compared with no NAC. Cu(II) augmented DOPAL-induced quinonization of intracellular proteins and NAC attenuated these effects.

Fig. 7.

Fig. 7.

Visualization of intracellular DOPAL-induced quinoproteins. MO3.13 cells were cultured in slide chambers (8 × 104 cells/slides) for 24 hours and treated with Cu(II) (30 _μ_M) and 0–100 _μ_M DOPAL for 5 hours. Cells were then stained with 4,6-diamidino-2-phenylindole (1:2000) (blue) and human tubulin antibody (1:1500) (green). Immunofluorescence and nIRF were visualized microscopically. Scale bar in images is 20 _μ_m. Treatment with DOPAL produced nIRF signals, suggesting the presence of quinoproteins.

Similar articles

Cited by

References

    1. Anderson DG, Florang VR, Schamp JH, Buettner GR, Doorn JA. (2016) Antioxidant-mediated modulation of protein reactivity for 3,4-dihydroxyphenylacetaldehyde, a toxic dopamine metabolite. Chem Res Toxicol 29:1098–1107. - PMC - PubMed
    1. Anderson DG, Mariappan SV, Buettner GR, Doorn JA. (2011) Oxidation of 3,4-dihydroxyphenylacetaldehyde, a toxic dopaminergic metabolite, to a semiquinone radical and an ortho-quinone. J Biol Chem 286:26978–26986. - PMC - PubMed
    1. Asanuma M, Miyazaki I, Ogawa N. (2003) Dopamine- or L-DOPA-induced neurotoxicity: the role of dopamine quinone formation and tyrosinase in a model of Parkinson’s disease. Neurotox Res 5:165–176. - PubMed
    1. Badillo-Ramírez I, Saniger JM, Rivas-Arancibia S. (2019) 5-S-cysteinyl-dopamine, a neurotoxic endogenous metabolite of dopamine: implications for Parkinson’s disease. Neurochem Int 129:104514. - PubMed
    1. Banerjee K, Munshi S, Sen O, Pramanik V, Roy Mukherjee T, Chakrabarti S. (2014) Dopamine cytotoxicity involves both oxidative and nonoxidative pathways in SH-SY5Y cells: potential role of alpha-synuclein overexpression and proteasomal inhibition in the etiopathogenesis of Parkinson’s disease. Parkinsons Dis 2014:878935. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources