Thioredoxin reductase deficiency potentiates oxidative stress, mitochondrial dysfunction and cell death in dopaminergic cells

Abstract

Mitochondria are considered major generators of cellular reactive oxygen species (ROS) which are implicated in the pathogenesis of neurodegenerative diseases such as Parkinson's disease (PD). We have recently shown that isolated mitochondria consume hydrogen peroxide (H₂O₂) in a substrate- and respiration-dependent manner predominantly via the thioredoxin/peroxiredoxin (Trx/Prx) system. The goal of this study was to determine the role of Trx/Prx system in dopaminergic cell death. We asked if pharmacological and lentiviral inhibition of the Trx/Prx system sensitized dopaminergic cells to mitochondrial dysfunction, increased steady-state H₂O₂ levels and death in response to toxicants implicated in PD. Incubation of N27 dopaminergic cells or primary rat mesencephalic cultures with the Trx reductase (TrxR) inhibitor auranofin in the presence of sub-toxic concentrations of parkinsonian toxicants paraquat; PQ or 6-hydroxydopamine; 6OHDA (for N27 cells) resulted in a synergistic increase in H₂O₂ levels and subsequent cell death. shRNA targeting the mitochondrial thioredoxin reductase (TrxR2) in N27 cells confirmed the effects of pharmacological inhibition. A synergistic decrease in maximal and reserve respiratory capacity was observed in auranofin treated cells and TrxR2 deficient cells following incubation with PQ or 6OHDA. Additionally, TrxR2 deficient cells showed decreased basal mitochondrial oxygen consumption rates. These data demonstrate that inhibition of the mitochondrial Trx/Prx system sensitizes dopaminergic cells to mitochondrial dysfunction, increased steady-state H₂O₂, and cell death. Therefore, in addition to their role in the production of cellular H₂O₂ the mitochondrial Trx/Prx system serve as a major sink for cellular H₂O₂ and its disruption may contribute to dopaminergic pathology associated with PD.

Figure 1. Pharmacological inhibition of TrxR in primary mesencephalic cultures results in decreased TrxR activity, increased H₂O₂ production and cell death.

(a) Aur (100 and 300 nM) decreases the activity of TrxR after 6 hr of incubation *** =  p<0.0001 as determined by 1-way ANOVA (n = 10−18). (b) Subtoxic concentrations of Aur or PQ alone caused minimal increases in H₂O₂ production after 24 hrs. Aur and PQ caused a synergistic increase in H₂O₂ production (n  = 4−6). (c) After 48 hrs of treatment there was minimal cell death into the media in either treatment alone. Aur and PQ caused an additive effect on cell death (n  = 12−16). Bars represent mean ± SEM. α =  p<0.05 compared to 0 nM Aur in same PQ treatment, β = p<0.05 compared to 100 nM Aur in same PQ treatment, χ = p<0.05 compared to 0 µM PQ in same Aur treatment, φ = p<0.01 compared to 100 µM PQ in same Aur treatment as determined by 2-way ANOVA.

Figure 2. Pharmacological inhibition of TrxR in N27 cells results in decreased TrxR activity and increased H₂O₂ production and cell death.

(a) Aur (100 and 300 nM) decreases the activity of TrxR after 6 hr of incubation in a concentration-dependent manner. * = p<0.005 *** =  p<0.0001 by 1-way ANOVA (n = 8−12). Subtoxic concentrations of Aur or PQ alone caused minimal increases in H₂O₂ production after 24 hrs (b) and cell death after 48 hrs (c). Aur and PQ caused a synergistic increase in H₂O₂ production and an additive effect on cell death. Bars represent mean ± SEM. α = p<0.05 compared to 0 nM Aur in same PQ treatment, β = p<0.05 compared to 100 nM Aur in same PQ treatment, χ = p<0.05 compared to 0 µM PQ in same Aur treatment,φ = p<0.01 compared to 100 µM PQ in same Aur treatment by 2-way ANOVA (n = 10−16).

Figure 3. Generation of TrxR2 deficiency in N27 Cells.

N27 cells were transfected with TrxR2 shRNA (TrxR2 deficient) and compared to mock-transfected cells (mock). (a) TrxR2 mRNA expression was measure by real-time PCR. Cells transfected with TrxR2 shRNA had a ∼60% decrease in TrxR2 mRNA compared to mock transfected cells (n = 3−6). (b) TrxR activity was measured in isolated mitochondria from mock and TrxR2 shRNA cells and there was a ∼95% loss in TrxR2 activity in the deficient vs. mock transfected cells (n = 3−6). To determine if the shRNA effect was mitochondrial specific TrxR1 mRNA (c) and TrxR activity (d) was measured in cytosolic fractions. There was no change in TrxR1 mRNA levels or activity (n = 2−6). (e) Mock and TrxR2 deficient cells were exposed to 3 µM exogenous H₂O₂ and the removal rates were determined with a Clark-type electrode. There was a significant (p<0.001) decrease in the TrxR2 deficient cells ability to remove exogenous H₂O₂ compared to mock controls (n = 9) Bars represent mean ± SEM. * =  <0.05, ** = p<0.005 as determined by two-tailed t-test.

Figure 4. Increased susceptibility to H₂O₂ production and cell death in TrxR2 deficient cells.

(a) TrxR2 deficient and mock control cells were exposed to varying concentration of PQ for 12 hr and H₂O₂ production was measured via Amplex Red. At 100 µM, 300 µM and 1 mM PQ concentrations there was a significant increase in H₂O₂ released in the deficient cells compared to mock controls (n = 6) * = p<0.05 as determined 2-way ANOVA. (b) Cell death was determined in mock and TrxR2 deficient cells after 24 hrs exposure to varying concentrations of PQ (n = 12−16). * = p<0.01, ** =  p<0.001 as determined by 2-way ANOVA. (c) TrxR2 deficient cells were exposed to varying concentrations of PQ alone and in combination with the 100 U catalase and 10 µM AEOL10150 for 24 hrs. Catalase was unable to rescue the PQ induced cell death but co-incubation with AEOL10150 was able to significantly decrease %LDH released in TrxR2 deficient cells * = p<0.05, *** = p<0.001 as determined by 2-way ANOVA compared to TrxR2 deficient cells with PQ alone. Data points represent mean ± SEM (n = 6−30).

Figure 5. Oxygen Consumption Rates (OCR) and respiration parameters in Aur treated N27 cells.

N27 cells were treated with 100 nM or 300 nM Aur alone or in combination with 100 µM PQ for 18 hrs. (a) Oxygen Consumption Rate (OCR) trace was determined using a Seahorse XF24 Analyzer. (b) Maximum Respiratory Capacity (c) Reserve Respiratory Capacity (d) Baseline Respiratory Capacity and (e) ATP Turnover where all decreased in cells treated with Aur and PQ and further decreased with the combined treatments. (e) Proton Leak was increased in cells treated with PQ alone or combined with Aur. α = p<0.05 compared to control, β = p<0.05 compared to 100 nM Aur, χ = p<0.05 compared to 300 nM Aur, φ = p<0.05 compared to 100 µM PQ (n = 5−15) as determined by 1-way ANOVA. Bars represent mean ± SEM.

Figure 6. Oxygen Consumption Rates (OCR) and respiration parameters in mock control and TrxR2 deficient cells.

Stably transfected cells were treated with 100 µM or 300 µM PQ for 6 hrs. (a) Oxygen Consumption Rate (OCR) trace was determined using a Seahorse XF24 Analyzer. (b) Maximal Respiratory Capacity (c) Reserve Respiratory Capacity (d) Baseline Respiratory Capacity and (f) Proton leak where all decreased in cells treated with 300 µM PQ. (e) ATP turnover was decreased in TrxR2 deficient cells with no PQ treatment compared to mock control. * = p<0.05 compared to mock control with same PQ concentration treatment. ** = p<0.01 mock control with same PQ concentration treatment (n = 7−9) as determined by two-tailed students t-test. Bars represent mean ± SEM.

Figure 7. Effects of Aur treated or TrxR2 deficient N27 cells with 6OHDA.

(a) N27 cells were exposed to varying concentration of 6OHDA and Aur for 24 hr and cell death was measured via percent LDH released (n = 8−16). * = p<0.001 compared to 0 nM Aur with same 6OHDA concentration; # = p<0.001 compared to 100 nM Aur with same 6OHDA concentration determined by 2-way ANOVA. (b) Maximal respiratory capacity and (c) Reserve respiratory capacity were measured with an XF analyzer after 18 hr incubation (n = 4−5) * = p<0.05, ** = p<0.01 as determined by 1-way ANOVA. (d) TrxR2 mock and deficient cells were treated with varying concentrations of 6OHDA and cell death was measured (n = 6−12). *** = p<0.001 as determined by 1-way ANOVA. (e) After 6 hr incubation with 6OHDA there was a significant decrease in both maximal respiratory capacity (e) and reserve capacity (f) in TrxR2 deficient cells alone and treated with 30 µM 6OHDA (n = 7−11) determined by two-tailed students t-test ** = p<0.005, *** = p<0.001. Bars represent mean ± SEM.