The unintended mitochondrial uncoupling effects of the FDA-approved anti-helminth drug nitazoxanide mitigates experimental parkinsonism in mice

Abstract

Mitochondria play a primary role in the pathophysiology of Parkinson's disease (PD), and small molecules that counteract the initial stages of disease may offer therapeutic benefit. In this regard, we have examined whether the off-target effects of the Food and Drug Administration (FDA)-approved anti-helminth drug nitazoxanide (NTZ) on mitochondrial respiration could possess any therapeutic potential for PD. Results indicate that MPP⁺-induced loss in oxygen consumption rate (OCR) and ATP production by mitochondria were ameliorated by NTZ in real time by virtue of its mild uncoupling effect. Pretreatment of cells with NTZ mitigated MPP⁺-induced loss in mitochondrial OCR and reactive oxygen species (ROS). Similarly, addition of NTZ to cells pretreated with MPP⁺ could reverse block in mitochondrial OCR and reactive oxygen species induced by MPP⁺ in real time. The observed effects of NTZ were found to be transient and reversible as removal of NTZ from incubation medium restored the mitochondrial respiration to that of controls. Apoptosis induced by MPP⁺ was ameliorated by NTZ in a dose-dependent manner. In vivo results demonstrated that oral administration of NTZ (50 mg/kg) in an acute MPTP mouse model of PD conferred significant protection against the loss of tyrosine hydroxylase (TH)-positive neurons of substantia nigra. Based on the above observations we believe that repurposing of NTZ for PD may offer therapeutic benefit.

Keywords: MPTP; Parkinson's disease; bioenergetics; mitochondria; neurodegeneration; nitazoxanide; oxidative stress; uncoupler.

Figure 1.

Effect of NTZ and its chemical moieties on mitochondrial OCR. A, chemical structures of nitazoxanide, O-acetyl salicylamide, and 2-amino-5-nitrothiazole. B, dose-dependent effect of NTZ on mitochondrial OCR: SK-N-SH cells in 24-well plates were treated with different concentrations of NTZ (0.25–10 μm) and cellular OCR was measured using Seahorse extracellular flux analyzer. C, effect of NTZ (1 and 5 μm) and its chemical moieties, salicylamide (1, 5, and 10 μm) and 2-amino-5-nitrothiazole (ANT) (1, 5, and 10 μm), on cellular OCR was measured using Seahorse extracellular flux analyzer. Control cells were treated with equal volumes of buffer in place of compounds. Data are representative of the mean ± S.D. of samples analyzed in triplicates (n = 3) from two independent experiments.

Figure 2.

Effect of NTZ on overall mitochondrial OCR and at individual respiratory complexes. A and B, human SK-N-SH neuroblastoma cells were incubated with different concentrations of NTZ (1, 5, and 10 μm) on (A) pyruvate and malate-driven respiration and (B) OCR at individual respiratory complexes and was monitored by electron flow assay in permeabilized cells as mentioned in “Experimental Procedures.” Data are representative of the mean ± S.D. of samples analyzed in triplicates (n = 3) from three independent experiments.

Figure 3.

NTZ mitigates MPP⁺-induced mitochondrial dysfunction. A, SK-N-SH cells were treated with MPP⁺ in the presence and absence of NTZ (1 μm) and Mito Stress assay was performed as described in “Experimental Procedures.” B and C, bar diagrams representing (B) ATP production and (C) protein leak from the data obtained from (A). D, SK-N-SH cells were incubated with NTZ (1 μm and 5 μm) for 60 min before exposure to MPP⁺ for 4, 8, and 24 h and cellular ATP content was analyzed by luciferase-based kit as mentioned in the “Experimental Procedures” section. Data shown are the representative of mean ± S.E. from triplicate measurements per each condition (n = 3) for (A) from three independent experiments. B–D, values indicated are the mean ± S.E. of triplicate measurements per each condition from two different experiments (n = 6). ***, p < 0.001; **, p < 0.01; *, p < 0.05 as compared with controls. ###, p < 0.001; ##, p < 0.01; #, p < 0.05 as compared with MPP⁺ group as analyzed by two-way ANOVA with Tukey's multiple comparisons test.

Figure 4.

NTZ-induced uncoupling effects are reversible and do not involve UCPs or adenine nucleotide translocase. A, SK-N-SH cells were treated with NTZ (1 μm) for 1 h in a 24-well Seahorse assay plate. In few wells the NTZ containing medium was replaced with fresh assay medium after 1 h incubation and Mito Stress assay was performed as described in “Experimental Procedures.” B, bar diagrams representing basal respiration, proton leak, and ATP production from the data obtained from (A). C, cells treated with the UCP inhibitor GTP (4 mm) for 20 min, and NTZ (5 μm) or FCCP (3 μm) was added and OCR was recorded further for 25 min. Control cells were treated with equal volumes of buffer under similar conditions. D, cells were treated with the adenine nucleotide translocase inhibitor CAT (3 μg/ml) for 20 min, and later NTZ (1 and 5 μm) or FCCP (3 μm) was added and OCR was recorded further for 25 min. Control cells were treated with equal volumes of buffer under similar conditions. A, C, and D, data are the representative of mean ± S.E. of triplicate measurements per each condition (n = 3) from two independent experiments. B, values indicated are the mean ± S.E. of triplicate measurements per each condition from two independent experiments (n = 6). ***, p < 0.001; **, p < 0.01 as compared with controls. ###, p < 0.001; ##, p < 0.01 as compared with NTZ (medium) group as analyzed by two-way ANOVA with Tukey's multiple comparisons test.

Figure 5.

Reversal of MPP⁺-induced block in mitochondrial respiration by NTZ. A, following 20 min of recording basal OCR in SK-N-SH cells, MPP⁺ was added through port A and OCR was recorded further for 2 h. Later, NTZ (1 and 5 μm) was added through port B and OCR was measured. Buffer was added in place of MPP⁺ in controls, whereas, buffer was added in place of NTZ for MPP⁺-treated samples. Values indicated are the mean ± S.E. of triplicate measurements (n = 3) and the experiment was repeated two times. B, SK-N-SH cells were either pretreated with NTZ (1 μm) for 1 h before incubation with MPP⁺ (1 mm) for 12 h or posttreated for 1 h following 12 h incubation with MPP⁺ (1 mm). At the end of incubation MitoSOX staining was performed as described in “Experimental Procedures” and fluorescence images were captured from five different fields of view. C, the average intensity units from five different fields of view from two independent experiments (n = 8–10) were represented as mean ± S.D. *, p < 0.05 as compared with controls. ##, p < 0.01; #, p < 0.05 as compared with MPP⁺ group by two-way ANOVA with Sidak's multiple comparisons test.

Figure 6.

NTZ suppressed MPP⁺-induced loss of mitochondrial membrane potential, ROS generation, and cellular apoptosis. Rat mesencephalic N27 cells were treated with NTZ (1 μm) for 1 h before exposure to MPP⁺ (500 μm) for 24 h. A, mitochondrial membrane potential was analyzed by JC-1 staining. B, ROS production was determined by H₂DCF-DA as described in “Experimental Procedures.” C, caspase-3 activity in cells treated with MPP⁺ in the presence and absence of NTZ (1, 5, and 10 μm). D, DPPH radical scavenging activity of NTZ and vitamin C was employed as a positive control. A and B, data are the representative of three independent experiments. C and D, values are the mean ± S.E. of triplicate measurements from two independent experiments (n = 6). ***, p < 0.001; **, p < 0.01 as compared with controls. ###, p < 0.001 as compared with MPP⁺ group. For (C) two-way ANOVA with Sidak's test and for (D) one-way ANOVA with Dunnett's test was performed.

Figure 7.

NTZ mitigates MPTP-induced loss of TH neurons and locomotor activity in vivo. Effects of NTZ on MPTP-induced loss of dopamine neurons as measured by TH-positive staining in the SNPc and striatum of mouse brain in an acute mouse model of PD. A, schematic representation of in vivo experiment depicting the therapeutic and preventive models employed. B and C, TH-positive staining and TH-positive fiber density in the SNPc and striatum of mice. D and E, densitometric analysis of TH-positive staining from SNPc and striatum from three different mice (n = 3). F, locomotor activity of animals as measured by open field test at the end of experiment (n = 4). D and E, data are the mean ± S.E. (n = 3). F, values indicated are the mean ± S.E. from four animals (n = 4). ***, p < 0.001; **, p < 0.01 as compared with controls. ###, p < 0.001 as compared with MPTP 7D group. $$, p < 0.01; $, p < 0.05 as compared with MPTP 14D group as analyzed by two-way ANOVA with Tukey's multiple comparisons test.