Protective effects of amphetamine and methylphenidate against dopaminergic neurotoxicants in SH-SY5Y cells

Abstract

Full treatment of the second most common neurodegenerative disorder, Parkinson's disease (PD), is still considered an unmet need. As the psychostimulants, amphetamine (AMPH) and methylphenidate (MPH), were shown to be neuroprotective against stroke and other neuronal injury diseases, this study aimed to evaluate their neuroprotective potential against two dopaminergic neurotoxicants, 6-hydroxydopamine (6-OHDA) and paraquat (PQ), in differentiated human dopaminergic SH-SY5Y cells. Neither cytotoxicity nor mitochondrial membrane potential changes were seen following a 24-hour exposure to either therapeutic concentration of AMPH or MPH (0.001-10 μM). On the other hand, a 24-hour exposure to 6-OHDA (31.25-500 μM) or PQ (100-5000 μM) induced concentration-dependent mitochondrial dysfunction, assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and lysosomal damage, evaluated by the neutral red uptake assay. The lethal concentrations 25 and 50 retrieved from the concentration-toxicity curves in the MTT assay were 99.9 µM and 133.6 µM for 6-OHDA, or 422 µM and 585.8 µM for PQ. Both toxicants caused mitochondrial membrane potential depolarization, but only 6-OHDA increased reactive oxygen species (ROS). Most importantly, PQ-induced toxicity was partially prevented by 1 μM of AMPH or MPH. Nonetheless, neither AMPH nor MPH could prevent 6-OHDA toxicity in this experimental model. According to these findings, AMPH and MPH may provide some neuroprotection against PQ-induced neurotoxicity, but further investigation is required to determine the exact mechanism underlying this protection.

Keywords: 6-Hydroxydopamine; Amphetamine; Methylphenidate; Neuroprotection; Paraquat; SH-SY5Y cells.

Graphical abstract

Fig. 1

Concentration-response curves for 6-OHDA. Differentiated SH-SY5Y cells were exposed to 31.25, 62.5, 125, 250, and 500 µM of 6-OHDA for 24 hours and the NR uptake (A) and the MTT reduction (B) assays were performed. Results are presented as mean ± SD (N = 24 to 28 different wells from 7 independent experiments). Mitochondrial membrane potential evaluated by JC-1 probe in differentiated SH-SY5Y cells incubated with 62.5 or 125 µM of 6-OHDA (C) after 24-hour exposure. Results are presented as mean ± SD (N = 6 different wells from 2 independent experiments). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test (*p < 0.05, ***p < 0.001, ****p < 0.0001 vs. Control; ^#p < 0.05, ^####p < 0.0001 vs. previous lower concentration tested).

Fig. 2

Concentration-response curves for PQ. Differentiated SH-SY5Y cells were exposed to 100, 200, 300, 400, 500, 1000, 2500, and 5000 µM of PQ for 24 hours and the NR uptake (A) and MTT reduction (B) assays were performed. Results are presented as mean ± SD (N = 16 to 43 different wells from 4 to 7 independent experiments). Mitochondrial membrane potential evaluated by JC-1 probe in differentiated SH-SY5Y cells incubated with 300 or 500 µM of PQ (C) after 24-hour exposure. Results are presented as mean ± SD (N = 6 different wells from 2 independent experiments). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test (*p < 0.005, **p < 0.01, **** p < 0.0001 vs. Control; ^#p < 0.05, ^##p < 0.001, ^####p < 0.0001 vs. previous lower concentration tested).

Fig. 3

Morphological analysis by phase contrast microscopy (left side) of differentiated SH-SY5Y cells exposed for 24 hours to 6-OHDA 125 µM (C) or PQ 500 µM (E), and the control group (A). Microphotographs from Hoechst nuclear staining (right side) were taken after a 24-hour exposure period to 6-OHDA 125 µM (D) or PQ 500 µM (F), and control group (B). Inserted red arrows point to the pyknotic nuclei, while green arrows indicate chromatin condensation. Representative microphotographs were taken of randomly chosen fields in 48-well plates (scale bar 100 μm) from 3 independent experiments.

Fig. 4

Cellular cytotoxicity evaluated by the NR uptake (A) and MTT reduction (B) assays in differentiated SH-SY5Y cells pre-incubated with 1 µM of AMPH or MPH and then exposed to 125 µM of 6-OHDA for 24 hours. Results are presented as mean ± SD (N = 13 to 16 different wells from 4 independent experiments). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test (****p < 0.0001 vs. Control).

Fig. 5

Assessment of reactive oxygen species (ROS) production, using the 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) probe, on differentiated SH-SY5Y cells exposed to 6-OHDA 125 µM, AMPH 1 µM, MPH 1 µM and 6-OHDA 125 µM pre-incubated with AMPH 1 µM or MPH 1 µM until 24-hour exposure. Results are presented as mean ± SD (N = 15 different wells from 5 independent experiments). Statistical analysis was performed using two-way ANOVA repeated measurements followed by Tukey’s multiple comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001 ****p < 0.0001 vs. Control).

Fig. 6

Cellular cytotoxicity evaluated by the NR uptake and MTT reduction assays in differentiated SH-SY5Y cells pre-incubated with 1 µM of AMPH or MPH and then exposed to 300 µM (A, C) or 500 µM (B, D) of PQ for 24 hours. Results are presented as mean ± SD (N = 24 to 32 different wells from 6 (B, D) to 7 (A, B) independent experiments). Mitochondrial membrane potential evaluated by JC-1 probe in differentiated SH-SY5Y cells exposed to 500 µM of PQ (E) during 24 hours. Results are presented as mean ± SD (N = 18 to 24 different wells from 6 independent experiments). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison tests (*p < 0.05, **p < 0.01, ****p < 0.0001 vs. Control; ^$$p < 0.01 vs PQ 500 µM; ^$$$p < 0.001, ^$$$$p < 0.0001 vs PQ 300 µM; ^#p < 0.05 vs. PQ 500 μM).