Do Autophagy Enhancers/ROS Scavengers Alleviate Consequences of Mild Mitochondrial Dysfunction Induced in Neuronal-Derived Cells?

Abstract

Mitochondrial function is at the nexus of pathways regulating synaptic-plasticity and cellular resilience. The involvement of brain mitochondrial dysfunction along with increased reactive oxygen species (ROS) levels, accumulating mtDNA mutations, and attenuated autophagy is implicated in psychiatric and neurodegenerative diseases. We have previously modeled mild mitochondrial dysfunction assumed to occur in bipolar disorder (BPD) using exposure of human neuronal cells (SH-SY5Y) to rotenone (an inhibitor of mitochondrial-respiration complex-I) for 72 and 96 h, which exhibited up- and down-regulation of mitochondrial respiration, respectively. In this study, we aimed to find out whether autophagy enhancers (lithium, trehalose, rapamycin, and resveratrol) and/or ROS scavengers [resveratrol, N-acetylcysteine (NAC), and Mn-Tbap) can ameliorate neuronal mild mitochondrial dysfunction. Only lithium (added for the last 24/48 h of the exposure to rotenone for 72/96 h, respectively) counteracted the effect of rotenone on most of the mitochondrial respiration parameters (measured as oxygen consumption rate (OCR)). Rapamycin, resveratrol, NAC, and Mn-Tbap counteracted most of rotenone's effects on OCR parameters after 72 h, possibly via different mechanisms, which are not necessarily related to their ROS scavenging and/or autophagy enhancement effects. The effect of lithium reversing rotenone's effect on OCR parameters is compatible with lithium's known positive effects on mitochondrial function and is possibly mediated via its effect on autophagy. By-and-large it may be summarized that some autophagy enhancers/ROS scavengers alleviate some rotenone-induced mild mitochondrial changes in SH-SY5Y cells.

Keywords: ROS scavengers; autophagy enhancers; bipolar disorder; mitochondrial dysfunction; rotenone.

Figure 1

Effect of ROS scavengers/autophagy enhancers on cell viability pre and post exposure to 10 pM rotenone.

Figure 2

Effect of ROS scavengers/autophagy enhancers on mitochondrial respiration parameters pre and post exposure to 10 pM rotenone for 72 (A,C,E,G,I,K) or for 96 (B,D,F,H,J,L) h.

Figure 2

Effect of ROS scavengers/autophagy enhancers on mitochondrial respiration parameters pre and post exposure to 10 pM rotenone for 72 (A,C,E,G,I,K) or for 96 (B,D,F,H,J,L) h.

Figure 3

Effect of ROS scavengers/autophagy enhancers on ATP Levels pre and post exposure to 10 pM rotenone.

Figure 4

Effect of autophagy enhancers/ROS scavengers on LC3-II protein levels pre and post exposure to 10 pM rotenone Results represent means ± S.E.M. of four independent experiments, each in duplicate. Rot. = rotenone; Li = lithium; Tre. = trehalose; Rap. = rapamycin; Res. = resveratrol; NAC = N-acetylcysteine; Mn. = Mn-Tbap. ^зRot. for 72/96 h vs. control, p = 0.05—as previously described [23] and confirmed now. One sample of each of rotenone’s effect, NAC’s effect and Mn-Tbap’s effect for 72 h exceeding mean ± 2SD was omitted. A. Lithium effects, Two-way ANOVA: Treatment—F_3,25 = 3.02, p = 0.04; duration—F_1,25 = 42.7, p = 0.00001; TreatmentXDuration interaction—F_3,25 = 7.6, p = 0.0008; *Fisher’s LSD post-hoc test: 24 h of Li vs. control, p < 0.00002; Rot. for 72 h + Li. last 24 h vs. control, p = 0.008; Rot. for 96 h + Li. last 48 h vs. Rot. for 96 h, p = 0.05. B. Trehalose effects, Two-way ANOVA: TreatmentXDuration interaction—F_3,26 = 2.5, p = 0.07. C. Rapamycin effects, Two-way ANOVA: TreatmentXDuration interaction—F_3,26 = 4.2, p = 0.01. D. Resveratrol effects, Two-way ANOVA: TreatmentXDuration interaction—F_3,24 = 3.2, p = 0.03. E. NAC effects, Two-way ANOVA: TreatmentXDuration interaction—F_3,26 = 3.8, p = 0.02; *Fisher’s LSD post-hoc test: 72 h of Rot. + NAC last 24 h vs. 72 h of Rot, p < 0.005. F. Mn-Tbap effects, Two-way ANOVA: Duration—F_1,25 = 4.7, p = 0.03; TreatmentXDuration interaction—F_3,25 = 3.9, p = 0.01; *Fisher’s LSD post-hoc test: 72 h of Rot.+Mn. last 24 h vs. control, p = 0.02.

Figure 4

Effect of autophagy enhancers/ROS scavengers on LC3-II protein levels pre and post exposure to 10 pM rotenone Results represent means ± S.E.M. of four independent experiments, each in duplicate. Rot. = rotenone; Li = lithium; Tre. = trehalose; Rap. = rapamycin; Res. = resveratrol; NAC = N-acetylcysteine; Mn. = Mn-Tbap. ^зRot. for 72/96 h vs. control, p = 0.05—as previously described [23] and confirmed now. One sample of each of rotenone’s effect, NAC’s effect and Mn-Tbap’s effect for 72 h exceeding mean ± 2SD was omitted. A. Lithium effects, Two-way ANOVA: Treatment—F_3,25 = 3.02, p = 0.04; duration—F_1,25 = 42.7, p = 0.00001; TreatmentXDuration interaction—F_3,25 = 7.6, p = 0.0008; *Fisher’s LSD post-hoc test: 24 h of Li vs. control, p < 0.00002; Rot. for 72 h + Li. last 24 h vs. control, p = 0.008; Rot. for 96 h + Li. last 48 h vs. Rot. for 96 h, p = 0.05. B. Trehalose effects, Two-way ANOVA: TreatmentXDuration interaction—F_3,26 = 2.5, p = 0.07. C. Rapamycin effects, Two-way ANOVA: TreatmentXDuration interaction—F_3,26 = 4.2, p = 0.01. D. Resveratrol effects, Two-way ANOVA: TreatmentXDuration interaction—F_3,24 = 3.2, p = 0.03. E. NAC effects, Two-way ANOVA: TreatmentXDuration interaction—F_3,26 = 3.8, p = 0.02; *Fisher’s LSD post-hoc test: 72 h of Rot. + NAC last 24 h vs. 72 h of Rot, p < 0.005. F. Mn-Tbap effects, Two-way ANOVA: Duration—F_1,25 = 4.7, p = 0.03; TreatmentXDuration interaction—F_3,25 = 3.9, p = 0.01; *Fisher’s LSD post-hoc test: 72 h of Rot.+Mn. last 24 h vs. control, p = 0.02.