DT-Diaphorase Prevents Aminochrome-Induced Alpha-Synuclein Oligomer Formation and Neurotoxicity

Abstract

It was reported that aminochrome induces the formation of alpha synuclein (SNCA) oligomers during dopamine oxidation. We found that DT-diaphorase (NQO1) prevents the formation of SNCA oligomers in the presence of aminochrome determined by Western blot, transmission electron microscopy, circular dichroism, and thioflavin T fluorescence, suggesting a protective role of NQO1 by preventing the formation of SNCA oligomers in dopaminergic neurons. In order to test NQO1 protective role in SNCA neurotoxicity in cellular model, we overexpressed SNCA in both RCSN-3 cells (wild-type) and RCSN-3Nq7 cells, which have constitutive expression of a siRNA against NQO1. The expression of SNCA in RCSN-3SNCA and RCSN-3Nq7SNCA cells increased 4.2- and 4.4-fold, respectively. The overexpression of SNCA in RCSN-3Nq7SNCA cells induces a significant increase in cell death of 2.8- and 3.2-fold when they were incubated with 50 and 70 µM aminochrome, respectively. The cell death was found to be of apoptotic character determined by annexin/propidium iodide technique with flow cytometry and DNA laddering. A Western blot demonstrated that SNCA in RCSN-3SNCA is only found in monomer form both in the presence of 20 µM aminochrome or cell culture medium contrasting with RCSN-3Nq7SNCA cells where the majority SNCA is found as oligomer. The antioligomer compound scyllo-inositol induced a significant decrease in aminochrome-induced cell death in RCSN-3Nq7SNCA cells in comparison to cells incubated in the absence of scyllo-inositol. Our results suggest that NQO1 seems to play an important role in the prevention of aminochrome-induced SNCA oligomer formation and SNCA oligomers neurotoxicity in dopaminergic neurons.

Keywords: NQO1; Parkinsons’s disease; alpha synuclein; aminochrome; dopamine; dopaminergic neurons; neuroprotection; oligomers.

FIG. 1.

Conformational changes of SNCA in the presence of aminochrome and NQO1. A, The effect of NQO1 on aminochrome-induced conformational changes. Circular dichroism was used to determine the possible conformational changes in SNCA to a beta-sheet structure after addition of aminochrome. The circular dichroism spectra of 100 µM SNCA (1); 100 µM SNCA with 100 µM aminochrome (2); 100 µM SNCA, 100 µM aminochrome, 1.2 µg NQO1, and 500µM NADH (3) were obtained by incubated for 24 (A), 48 (B), and 72 h (C) at 37°C with stirring as described in Materials and Methods section. B, Fibrillization and beta-sheet formation kinetics of SNCA incubated with aminochrome in the presence of NQO1. Fibrillization and beta-sheet formation were determined with the ThT fluorescence assay. The incubation mixtures contained 100µM SNCA (•) show SNCA fibrils formation; 100µM SNCA together with 100µM aminochrome (○) show the formation of oligomers; 100µM SNCA, 100µM aminochrome, 1.2 µg NQO1, and 500µM NADH (▾) show that SNCA is in the monomer state. Measurements were performed at 37°C with agitation as described in the Experimental Procedures.

FIG. 2.

Transmission electron microscopy of aminochrome-induced SNCA aggregation. A, Human recombinant SNCA (100µM) incubated 48 h at 37°C with stirring is shown. B, SNCA (100µM) incubated with 100µM aminochrome show the formation of small globular structures corresponding SNCA globular aggregation. C, SNCA (100µM) incubated with 100µM aminochrome in the presence of 1.2 µg NQO1 and 500µM NADH show the absence of small globular structures. The magnification was ×30 000 and the scale bar represents 0.25 µm. The white arrowheads show SNCA oligomers and the white arrows show SNCA globular aggregations.

FIG. 3.

Overexpression of SNCA in RCSN-3 cells. A, We have constructed the lentiviral plasmid pLvGFP-SNCA that was transduced into RCSN-3 and RCSN-3Nq7 cells to overexpress SNCA. We generated 2 new cell lines RCSN-3SNCA (WT) and RCSN-3Nq7SNCA (Nq7SNCA) which have permanent expression of a siRNA against NQO1. The overexpression of SNCA was determined by using dot blot technique and immunostaining that show the total amount of alpha synuclein but you cannot see the different forms of SNCA such as monomer and oligomers (B). The result of dot blot was plotted by using the SNCA/GAPDH values (C). A significant decrease in enzymatic activity of NQO1 was observed in RCSN-3Nq7SNCA (D). Quantitative real-time PCR confirmed the over-expression of SNCA (SNCA) in both cell lines and the decrease of mRNA expression of NQO1 (NQO1) in RCSN-3Nq7SNCA cells (E). The statistical significance was assessed using analysis of variance (ANOVA) for multiple comparisons (*P < 0.05; **P < 0.01; ***P < 0.001).

FIG. 4.

The effect of aminochrome on RCSN-3SNCA and RCSN-3Nq7SNCA cells. A, The effect of aminochrome at different concentrations (0, 10, 30, 50, and 70 µM) was determined on RCSN-3 (WT), RCSN-3SNCA (WT-SNCA), RCSN-3Nq7 (Nq7), and RCSN-3Nq7SNCA (Nq7-SNCA) cells at 24 h. B, The effect of 20 µM aminochrome was determined on RCSN-3SNCA (WT) and RCSN-3Nq7SNCA (Nq7SNCA) cells at 24 h. The values are the mean ± SD with n = 3. The statistical significance was assessed using ANOVA for multiple comparisons (*P < 0.05; **P < 0.01; ***P < 0.001).

FIG. 5.

Determination of the role of apoptosis in aminochrome-induced cell death in RCSN-3SNCA and RCSN-3Nq7SNCA cells. The possible role of apoptosis on aminochrome-induced cell death in RCSN-3SNCA and RCSN-3Nq7SNCA cells was determined by using DNA laddering (A and B) and flow cytometry with Alexa Fluor 488-annexin and propidium iodide technique (C–G). DNA fragmentation was only observed in RCSN-3Nq7SNCA cells (B) when the cells were treated with 20 µM aminochrome (AM) for 24 h contrasting with the lack of DNA fragmentation in RCSN-3SNCA cells treated under the same conditions (A). As positive control we used 3.4 mM staurosporine (C+) and as negative control the cells were incubated in the absence of aminochrome (C). The flow cytometry with Alexa Fluor 488 annexin V and propidium iodide revealed that only RCSN-3Nq7SNCA cells treated with 20 µM aminochrome (F) presented similar localization as cells treated with 3.4 mM staurosporine that is a positive control for apoptosis (G). RCSN-3SNCA (C) and RCSN-3Nq7SNCA (E) cells incubated in the absence of aminochrome or RCSN-3SNCA cells incubated with 20 µM aminochrome (D) for 24 h don’t have the same localization as cells treated with 3.4 mM staurosporine. The results were plotted in H. The values are the mean ± SD with n = 3. The statistical significance was assessed using ANOVA for multiple comparisons (*P < 0.05).

FIG. 6.

Aminochrome induces the formation of SNCA oligomers in RCSN-3Nq7SNCA cells and the effect of scyllo-inositol on aminochrome-induced cell death. The formation of oligomers were determined by using Western blot technique (A). In RCSN-3SNCA cells (WT-SNCA) SNCA is found in monomer state both in the presence or absence of aminochrome. However, SNCA monomer significantly decreases in RCSN-3Nq7SNCA cells both in untreated and treated with 20 µM aminochrome (B). SNCA oligomers were also detected by using inmunofluorescence (C) and the results were plotted in (D) where we can observe a significant increase in RCSN-3Nq7SNCA cells. (E) The incubation of RCSN-3Nq7SNCA (Nq7-SNCA) cells with 1.5 µM scyllo-inositol decreases aminochrome-induced cell death. No effect of scyllo-inositol was observed in RCSN-3SNCA (WT-SNCA) cells revealing the protective role of NQO1 against SNCA-aminochrome oligomers. The cells were incubated with 20 µM aminochrome during 24 h. The values are the mean ± SD with n = 3. The statistical significance was assessed using ANOVA for multiple comparisons (*P < 0.05; **P < 0.01; ***P < 0.001).

FIG. 7.

A possible mechanism for aminochrome-induced cell death. NQO1 prevents the formation of aminochrome-dependent SNCA oligomers by 2-electron reduction of aminochrome that it is a neuroprotective reaction. When NQO1 is silenced by siRNA aminochrome forms adducts with SNCA inducing the formation of SNCA oligomers that induce neurotoxicity, apoptosis, and finally cell death.