Distinct effects of rotenone, 1-methyl-4-phenylpyridinium and 6-hydroxydopamine on cellular bioenergetics and cell death

Abstract

Parkinson's disease is characterized by dopaminergic neurodegeneration and is associated with mitochondrial dysfunction. The bioenergetic susceptibility of dopaminergic neurons to toxins which induce Parkinson's like syndromes in animal models is then of particular interest. For example, rotenone, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite 1-methyl-4-phenylpyridinium (MPP(+)), and 6-hydroxydopamine (6-OHDA), have been shown to induce dopaminergic cell death in vivo and in vitro. Exposure of animals to these compounds induce a range of responses characteristics of Parkinson's disease, including dopaminergic cell death, and Reactive Oxygen Species (ROS) production. Here we test the hypothesis that cellular bioenergetic dysfunction caused by these compounds correlates with induction of cell death in differentiated dopaminergic neuroblastoma SH-SY5Y cells. At increasing doses, rotenone induced significant cell death accompanied with caspase 3 activation. At these concentrations, rotenone had an immediate inhibition of mitochondrial basal oxygen consumption rate (OCR) concomitant with a decrease of ATP-linked OCR and reserve capacity, as well as a stimulation of glycolysis. MPP(+) exhibited a different behavior with less pronounced cell death at doses that nearly eliminated basal and ATP-linked OCR. Interestingly, MPP(+), unlike rotenone, stimulated bioenergetic reserve capacity. The effects of 6-OHDA on bioenergetic function was markedly less than the effects of rotenone or MPP(+) at cytotoxic doses, suggesting a mechanism largely independent of bioenergetic dysfunction. These studies suggest that these dopaminergic neurotoxins induce cell death through distinct mechanisms and differential effects on cellular bioenergetics.

Figure 1. Caspase 3 activation and cell viability in response to rotenone, MPP⁺ and 6-OHDA.

Whole cell lysates were collected after 24 hr exposure with increasing concentrations of rotenone, MPP⁺ and 6-OHDA. Western blot analysis for activated caspase 3 was performed using actin as a loading control, for increasing concentrations of rotenone (A), MPP⁺ (C), and 6-OHDA (E). Cell viability was assessed by trypan blue exclusion for rotenone (B), MPP⁺ (D), and 6-OHDA (F) after 24 hr exposure. Data are expressed as percent normalized to 0 µM treatment. Data  =  mean ± SEM, n = 3. *p<0.05, Student t-test compared to 0 µM treatment.

Figure 2. Concentration-dependent effects of rotenone, MPP⁺, and 6-OHDA on basal OCR.

Using the XF24 analyzer, the mitochondrial oxygen consumption rate (OCR) was determined for 4 basal readings with 80,000 cells plated per well. OCRs were between 8–12 pmol O₂/min/µg protein. Then rotenone (A), MPP⁺ (B), and 6-OHDA (C) were injected. (A) ▪ control, □ 1 nM rotenone, • 10 nM rotenone, and ○ 100 nM rotenone. (B) ▪ control, □ 500 nM MPP⁺, •5 µM MPP⁺, and ○ 500 µM MPP⁺. (C) ▪ control, □ 50 µM 6-OHDA, and ○ 200 µM 6-OHDA. Data are expressed as percent of the basal OCR prior to injection of neurotoxins. Data  =  mean ± SEM, n = 3. In some cases, the error bars are smaller than the symbols.

Figure 3. Concentration-dependent effects of rotenone, MPP⁺ and 6-OHDA on basal OCR after 2 hr exposure.

Changes in basal OCR after rotenone (A), MPP⁺ (B), and 6-OHDA (C) are shown and are expressed as percent normalized to OCR before injection. Data  =  mean ± SEM, n = 3. *p<0.05, Student t-test compared to 0 µM treatment. The relationship between 24 hr % viability and 2 hr % of OCR was plotted for (D) rotenone, (E) MPP⁺ and (F) 6-OHDA. (D) ▪ control, ◊ 0.1 nM rotenone, □ 1 nM rotenone, • 10 nM rotenone, and ○ 100 nM rotenone. (E) ▪ control, ◊ 5 nM MPP⁺, □ 500 nM MPP⁺, • 5 µM MPP⁺, and ○ 500 µM MPP⁺. (F) ▪ control, □ 50 µM 6-OHDA, • 100 µM 6-OHDA, and ○ 200 µM 6-OHDA.

Figure 4. Concentration-dependent effects of rotenone, MPP⁺ and 6-OHDA on OCR and ECAR.

The OCR and ECAR data are taken from the 2 hr time point shown in Figure 2 and are expressed as percent normalized to basal OCR or basal ECAR before injection of increasing doses of rotenone (A), MPP⁺ (B), and 6-OHDA (C). ECAR values ranged between 35–110 mpH/min before normalization and 0.5–3 mpH/min/µg protein. (A) ▪ control, □ 1 nM rotenone, • 10 nM rotenone, and ○ 100 nM rotenone. (B) ▪ control, □ 500 nM MPP⁺, • 5 µM MPP⁺, and ○ 500 µM MPP⁺. (C) ▪ control, □ 50 µM 6-OHDA, • 100 µM 6-OHDA, and ○ 200 µM 6-OHDA. Data are expressed as percent normalized to OCR and ECAR before injection. Data  =  mean ± SEM, n = 3. *p<0.05, Student t-test compared to 0 µM treatment OCR; #p<0.05, Student t-test compared to 0 µM treatment ECAR.

Figure 5. Changes in ATP-linked and proton leak OCR in response to 2 hr exposure to rotenone, MPP⁺ and 6-OHDA.

After exposure to rotenone (A), MPP⁺ (B), and 6-OHDA (C) for 2 hr, OCRs were measured after injection of oligomycin (O), FCCP (F) and antimycin A (A). ATP-linked OCR was plotted for rotenone at 0, 0.1, 1, 10 and 100 nM (D), MPP⁺ at 0, 0.005, 0.5, 5 and 500 µM (E), and 6-OHDA at 0, 50, 100 and 200 µM (F); and proton leak OCR for rotenone (G), MPP⁺ (H), and 6-OHDA (I) over the same ranges of increasing doses of neurotoxins as in panels D–F. Data are expressed as percent normalized to OCR before injection of rotenone, MPP⁺, and 6-OHDA. Data  =  mean ± SEM, n = 3. *p<0.05, Student t-test compared to 0 µM treatment.

Figure 6. Changes in maximal, reserve capacity and non-mitochondrial OCR in response to 2 hr exposure to rotenone, MPP⁺ and 6-OHDA.

Using the OCR traces shown in Figure 5 A–C, maximal OCR, reserve capacity, and non-mitochondrial OCR were determined. Maximal OCR for rotenone (A), MPP⁺ (B), and 6-OHDA (C), reserve capacity for rotenone (D), MPP⁺ (E), and 6-OHDA (F), and non-mitochondrial OCR for rotenone (G), MPP⁺ (H), and 6-OHDA (I) are shown. Data are expressed as percent normalized to OCR before injection of rotenone, MPP⁺, and 6-OHDA. Data  =  mean ± SEM, n = 3. *p<0.05, Student t-test compared to 0 µM treatment.