Differential effects of the mitochondrial uncoupling agent, 2,4-dinitrophenol, or the nitroxide antioxidant, Tempol, on synaptic or nonsynaptic mitochondria after spinal cord injury

Abstract

We recently documented the progressive nature of mitochondrial dysfunction over 24 hr after contusion spinal cord injury (SCI), but the underlying mechanism has not been elucidated. We investigated the effects of targeting two distinct possible mechanisms of mitochondrial dysfunction by using the mitochondrial uncoupler 2,4-dinitrophenol (2,4-DNP) or the nitroxide antioxidant Tempol after contusion SCI in rats. A novel aspect of this study was that all assessments were made in both synaptosomal (neuronal)- and nonsynaptosomal (glial and neuronal soma)-derived mitochondria 24 hr after injury. Mitochondrial uncouplers target Ca(2+) cycling and subsequent reactive oxygen species production in mitochondria after injury. When 2,4-DNP was injected 15 and 30 min after injury, mitochondrial function was preserved in both populations compared with vehicle-treated rats, whereas 1 hr postinjury treatment was ineffective. Conversely, targeting peroxynitrite with Tempol failed to maintain normal bioenergetics in synaptic mitochondria, but was effective in nonsynaptic mitochondria when administered 15 min after injury. When administered at 15 and 30 min after injury, increased hydroxynonenal, 3-NT, and protein carbonyl levels were significantly reduced by 2,4-DNP, whereas Tempol only reduced 3-NT and protein carbonyls after SCI. Despite such antioxidant effects, only 2,4-DNP was effective in preventing mitochondrial dysfunction, indicating that mitochondrial Ca(2+) overload may be the key mechanism involved in acute mitochondrial damage after SCI. Collectively, our observations demonstrate the significant role that mitochondrial dysfunction plays in SCI neuropathology. Moreover, they indicate that combinatorial therapeutic approaches targeting different populations of mitochondria holds great potential in fostering neuroprotection after acute SCI.

Fig. 1

Contusion SCI leads to mitochondrial dysfunction as demonstrated by oxygram traces for synaptic mitochondria isolated from either sham spinal cords, 24 hr after injury alone or treatment with 2,4-DNP (DNP) or Tempol at 15 min after injury. Mild mitochondrial uncoupling after injury with DNP significantly maintained mitochondrial bioenergetics after injury, whereas Tempol showed little effect.

Fig. 2

The RCR (calculated as the ratio of state III vs. state IV slopes) is a measure of mitochondrial integrity and coupling of the ETS to oxidative phosphorylation. The RCR for synaptic mitochondria was significantly decreased in vehicle-treated injured animals at 24 hr after injury, whereas 15- and 30-min postinjury treatment with 2,4-DNP (DNP) significantly maintained the RCR. Notably, the RCR for all the 2,4-DNP-treated groups remained significantly (P < 0.05) lower compared with sham-treated animals. Bars represent group mean ± SEM, n = 5–6 per group. ★P < 0.05 vs. vehicle-treated injured group.

Fig. 3

Respiration rates from synaptic mitochondria were calculated as nmol oxygen/min/mg protein in at 24 hr after SCI after treatment with either vehicle, 2,4-DNP (DNP) or Tempol at 15 min (A), 30 min (B), and 1 hr (C) after injury. Significantly impaired mitochondrial function was observed with vehicle treatment at all times points of administration. Notably, only mild mitochondrial uncoupling with DNP injections at 15 and 30 min after injury maintained normal mitochondrial function vs. vehicle-treated injured groups; notably, state III and state V–complex I respiration rates were comparable to shams (P > 0.05). Conversely, Tempol treatments were ineffective at all time points delivered. Bars represent group mean ± SEM, n = 5–6 per group. ★P < 0.05 vs. vehicle-treated injured group.

Fig. 4

Activities of complexes I, II and IV were assessed by using their specific substrates and calculated as nmol/min/mg protein. Activities are shown for synaptic mitochondria isolated from 15 min (A), 30 min (B), and 1 hr (C) postinjury treatment groups, respectively. With vehicle treatment, the activities of complexes I and IV were significantly decreased 24 hr after contusion SCI. Treatment with 2,4-DNP (DNP) at 15 and 30 min, but not 1 hr after injury, maintained normal levels of activities that were not significantly (P > 0.05) different than shams. At no time point of administration did Tempol demonstrate protective effects. Bars represent group mean ± SEM, n = 3 per group. ★P < 0.05 vs. vehicle-treated injured group.

Fig. 5

Synaptic mitochondrial oxidative stress was assessed by quantifying the oxidative markers 3-NT, 4-HNE and protein carbonyls by slot blot analysis. Data for synaptic mitochondria are expressed as intensity (AU). Data are shown at 24 hr after contusion SCI from the 15 min (A), 30 min (B), and 1 hr (C) postinjury treatment groups, respectively. Treatment with 2,4-DNP (DNP) at 15 and 30 min, but not 1 hr after injury, significantly decreased the increased levels of all the markers from vehicle-treated injured rats to levels comparable to shams (P > 0.05). Tempol treatment at 15 and 30 min after injury also significantly reduced 3-NT and protein carbonyls levels compared with vehicle-treated injured rats to levels comparable to shams (P > 0.05), but not when administered 1 hr after injury. Unlike with DNP treatment, HNE levels were not reduced by Tempol administration at any time point. Bars represent group mean ± SEM, n = 6 per group. ★P < 0.05 vs. vehicle-treated injured group.

Fig. 6

The RCR (calculated as the ratio of state III vs. state IV slopes) is a measure of mitochondrial integrity and coupling of the ETS to oxidative phosphorylation. The RCR for nonsynaptic mitochondria was significantly decreased in vehicle-treated injured animals at 24 hr after injury. Treatment with 2,4-DNP (DNP) at all the time points significantly maintained the RCR compared with vehicle-treated injured animals. Although the values with 15- and 30-min treatments were comparable to shams (P > 0.05), the RCR remained significantly lower than shams after 1 hr DNP treatment. Conversely, Tempol treatment only at 15 min after injury was significantly effective in maintaining RCR, comparable to shams (P > 0.05). Bars represent group mean ± SEM, n = 5–6 per group. ★P < 0.05 vs. vehicle-treated injured group.

Fig. 7

Respiration rates from nonsynaptic mitochondria were calculated as nmol oxygen/min/mg protein in at 24 hr after SCI after treatment with either vehicle (A), 2,4-DNP (DNP) (B), or Tempol (C) at 15 min, 30 min, and 1 hr after injury. Significantly impaired mitochondrial function was observed with vehicle treatment compared with sham-treated animals. Notably, mild mitochondrial uncoupling with DNP at all time points, as well as treatment with Tempol only at 15 min after injury, significantly maintained normal mitochondrial function compared with vehicle-treated injured animals. However, with DNP treatments at 30 min and 1 hr after injury, state III and state V–complex I respiration rates remained significantly lower compared with sham-treated animals (P < 0.05). Also, state III respiration rates with Tempol treatment at 15 min in the postinjury groups remained significantly lower than shams (P < 0.05). Bars represent group mean ± SEM, n = 5–6 per group. ★P < 0.05 vs. vehicle-treated injured group.

Fig. 8

Activities of complexes I, II, and IV were assessed by using their specific substrate and calculated as nmol/min/mg protein. Shown are activities for nonsynaptic mitochondria isolated from 15 min (A), 30 min (B), and 1 hr (C) postinjury treatment groups, respectively. Only complex I activity was significantly decreased after acute contusion SCI compared with sham-treated animals (P < 0.05). Treatment with 2,4-DNP (DNP) at all time points after injury significantly maintained complex I activity compared with vehicle-treated injured animals. With both 15 and 30 min treatments, complex I activity remained comparable to shams (P > 0.05), but not with treatment at 1 hr after injury. At no time point of administration did Tempol demonstrate protective effects. Bars represent group mean ± SEM, n = 3 per group. ★P < 0.05 vs. vehicle-treated injured group.

Fig. 9

Nonsynaptic mitochondrial oxidative stress was assessed by quantifying the oxidative markers 3-NT, 4-HNE and protein carbonyls by using slot blot analysis. Data for nonsynaptic mitochondria are presented as intensity (AU). Data are shown at 24 hr after contusion SCI from the 15 min (A), 30 min (B), and 1 hr (C) postinjury treatment groups, respectively. Compared with vehicle-treated injured animals, treatment with 2,4-DNP (DNP) at 15 and 30 min after injury significantly reduced the increased levels of HNE and 3-NT, comparable to shams (P > 0.05). Notably, Tempol administration at 15 and 30 min after injury was effective only in reducing increased 3-NT levels to sham values, but did not reduce HNE levels at all time points of administration. Both DNP and Tempol administration up to 1 hr after injury significantly decreased the increased levels of protein carbonyls vs. vehicle-treated injured animals, comparable to sham values (P > 0.05). Bars represent group mean ± SEM, n = 6 per group. ★P < 0.05 vs. vehicle-treated injured group.