Nicotinamide nucleotide transhydrogenase (Nnt) links the substrate requirement in brain mitochondria for hydrogen peroxide removal to the thioredoxin/peroxiredoxin (Trx/Prx) system

Abstract

Mitochondrial reactive oxygen species are implicated in the etiology of multiple neurodegenerative diseases, including Parkinson disease. Mitochondria are known to be net producers of ROS, but recently we have shown that brain mitochondria can consume mitochondrial hydrogen peroxide (H2O2) in a respiration-dependent manner predominantly by the thioredoxin/peroxiredoxin system. Here, we sought to determine the mechanism linking mitochondrial respiration with H2O2 catabolism in brain mitochondria and dopaminergic cells. We hypothesized that nicotinamide nucleotide transhydrogenase (Nnt), which utilizes the proton gradient to generate NADPH from NADH and NADP(+), provides the link between mitochondrial respiration and H2O2 detoxification through the thioredoxin/peroxiredoxin system. Pharmacological inhibition of Nnt in isolated brain mitochondria significantly decreased their ability to consume H2O2 in the presence, but not absence, of respiration substrates. Nnt inhibition in liver mitochondria, which do not require substrates to detoxify H2O2, had no effect. Pharmacological inhibition or lentiviral knockdown of Nnt in N27 dopaminergic cells (a) decreased H2O2 catabolism, (b) decreased NADPH and increased NADP(+) levels, and (c) decreased basal, spare, and maximal mitochondrial oxygen consumption rates. Nnt-deficient cells possessed higher levels of oxidized mitochondrial Prx, which rendered them more susceptible to steady-state increases in H2O2 and cell death following exposure to subtoxic levels of paraquat. These data implicate Nnt as the critical link between the metabolic and H2O2 antioxidant function in brain mitochondria and suggests Nnt as a potential therapeutic target to improve the redox balance in conditions of oxidative stress associated with neurodegenerative diseases.

Keywords: Mitochondria; Nicotinamide Nucleotide Transhydrogenase; Oxidative Stress; Parkinson Disease; Reactive Oxygen Species (ROS); Thioredoxin Reductase.

FIGURE 1.

Pharmacological inhibition of Nnt results in substrate-dependent decreases in TrxR and Trx activity in isolated brain mitochondria. A, a representative Western blot to indicate the purity of isolated mitochondria conducted in these studies. Trx (B) or TrxR (C) activity assay was conducted as described under “Experimental Procedures.” There was a significant decrease in TrxR and Trx activity when respiration substrates malate and glutamate (M&G) were present, and Nnt was pharmacologically inhibited with palmitoyl-CoA (Palm). Data are represented as mean ± S.E. (n = 4–11). *, p < 0.05; **, p < 0.005; ***, p < 0.001 as determined by one-way ANOVA.

FIGURE 2.

N27 cells with Nnt pharmacologically inhibited results in a decrease in H₂O₂ catabolism. N27 cells were exposed to 100 μm palmitoyl-CoA, and H₂O₂ removal rate was measured. A, there was a significant decrease in Nnt activity (n = 7–9). B, N27 cells exposed to 100 μm palmitoyl-CoA had a significant decrease in the ability to consume 3 μm exogenous H₂O₂ (n = 5–6). Data are represented as mean ± S.E. *, p < 0.05; ***, p < 0.001 as determined by Student's t test.

FIGURE 3.

Generation of Nnt-deficient cell line. N27 cells were transfected with Nnt shRNA (Nnt-deficient) and compared with mock-transfected cells (mock). A, Nnt mRNA expression in Nnt-deficient cells had an ∼95% decrease in Nnt mRNA compared with mock-transfected cells (n = 3). B, Nnt activity was measured in mock control and Nnt-deficient cells, and there was an ∼75% decrease in activity (n = 9). All bars represent mean ± S.E.; ***, p < 0.0001 (Student's t test).

FIGURE 4.

Nnt-deficient N27 cells have increased NADP⁺ and decreased NADPH levels, H₂O₂ removal rates, and GSH levels. NADP⁺ levels (A) and NADPH levels (B) were determined in Nnt-deficient cells and compared with mock control via HPLC. There was a significant increase in NADP⁺ levels coupled with a decrease in NADPH levels. Each individual NADP⁺ value was divided by the average NADPH value to determine the ratio (C), which was significantly oxidized in the Nnt-deficient cells (n = 4–9). D, Nnt-deficient cells showed an ∼40% decrease in the ability to remove exogenous H₂O₂ (n = 10). Additionally, Nnt-deficient cells showed a decrease in GSH levels (E) and an increase in GSSG level (F; n = 3). G, there was no change in isocitrate dehydrogenase 2 (IDH) activity (n = 5–8). *, p < 0.05; **, p < 0.005; ***, p < 0.0005 (Student's t test). Data are represented as mean ± S.E.

FIGURE 5.

Nnt-deficient N27 cells had a significant increase in oxidation of Prx3 compared with mock controls. A, a representative oxidized versus reduced Western blot. B, quantification of the redox blot for mitochondrial Prx3 from five separate samples. C, a representative SDS-PAGE blot for total Prx3 levels in mock and Nnt-deficient cells. D, quantification from four to five separate samples normalized to β-actin levels for total levels of Prx3. *, p < 0.05 as determined by Student's t test. Bars represent mean ± S.E.

FIGURE 6.

Summarized trace of decreased OCR in Nnt deficient cells compared with mock cells. Using a Seahorse 24XF analyzer, mitochondrial OCR was measured in Nnt-deficient and mock control N27 cells under different respiratory parameters. Data are represented as mean ± S.E. (n = 18–19). Oligo, oligonucleotide; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; Anti-A, antimyicin A.

FIGURE 7.

Decreased mitochondrial membrane potential leading to increased susceptibility to paraquat toxicity in Nnt-deficient compared with mock control N27 cells. A, mitochondrial membrane potential was measured via tetramethylrhodamine ethyl ester assay, and there was a significant decrease in the membrane potential in the Nnt-deficient cells compared with mock control N27 cells (n = 35–36). B, Nnt-deficient and mock control cells were exposed to various concentrations of paraquat for 24 h, and the amount of H₂O₂ produced was measured via Amplex Red assay. Nnt cells produced significantly more H₂O₂ alone and when exposed to various concentrations of paraquat (n = 10). As indicated in C, there was a significant shift in lactate dehydrogenase (LDH) released in Nnt-deficient cells exposed to various concentrations of paraquat (PQ) compared with mock after 48 h of exposure (n = 5–10). *, p < 0.05; **, p < 0.01; ***, p < 0.001 as determined by two-way ANOVA. Data are represented as mean ± S.E.

FIGURE 8.

A proposed model of Nnt activity in linking respiration-dependent H₂O₂ removal by the Trx/Prx antioxidant system in isolated brain mitochondria. When respiration substrates are present the citric acid cycle (TCA) and electron transport chain (ETC) can actively generate NADH and maintain the proton (H⁺) gradient. Nnt will then utilize the H⁺ gradient and NADH to create NADPH, which will then be utilized by TrxR to keep the Trx/Prx antioxidant system in a reduced state to detoxify H₂O₂. Thus, Nnt links the energy side of the schematic to the redox side.