Investigation of Mitochondrial Dysfunction by Sequential Microplate-Based Respiration Measurements from Intact and Permeabilized Neurons

Mitochondrial dysfunction is a component of many neurodegenerative conditions. Measurement of oxygen consumption from intact neurons enables evaluation of mitochondrial bioenergetics under conditions that are more physiologically realistic compared to isolated mitochondria. However, mechanistic analysis of mitochondrial function in cells is complicated by changing energy demands and lack of substrate control. Here we describe a technique for sequentially measuring respiration from intact and saponin-permeabilized cortical neurons on single microplates. This technique allows control of substrates to individual electron transport chain complexes following permeabilization, as well as side-by-side comparisons to intact cells. To illustrate the utility of the technique, we demonstrate that inhibition of respiration by the drug KB-R7943 in intact neurons is relieved by delivery of the complex II substrate succinate, but not by complex I substrates, via acute saponin permeabilization. In contrast, methyl succinate, a putative cell permeable complex II substrate, failed to rescue respiration in intact neurons and was a poor complex II substrate in permeabilized cells. Sequential measurements of intact and permeabilized cell respiration should be particularly useful for evaluating indirect mitochondrial toxicity due to drugs or cellular signaling events which cannot be readily studied using isolated mitochondria

Etude des effets mitochondriaux du monoxyde d'azote :<br />Régulation de l'oxydation phosphorylante et de la transition de perméabilité

Nitric oxide (NO) is produced from L-Arginine by the NO synthase family. Dysregulation of the synthesis of this signal molecule is involved in the development of numerous pathologies. The cellular effects of NO are partly due to the inhibition of the complexes of the mitochondrial respiratory chain. We studied the NO regulation of the oxidative phosphorylation and the permeability transition, two mitochondrial processes controlling cellular survival. We have demonstrated that the inhibition of cytochrome c oxidase (COX) by NO was associated with an inhibition of the respiration and the ATP synthesis. However, NO increased the yield of oxidative phosphorylation (ATP/O) due to a reduction in the « slipping ». Our work highlights an effect of NO which depends on the substrate used. Indeed, in the presence of NADH-linked substrates, NO does not have the beneficial effect on the yield of the oxidative phosphorylation, that obtained in the presence of FADH2-linked substrates with or without NADH-linked subtrates. The study of the redox status of the cytochromes of the COX revealed that in mitochondria energized with FADH2-linked substrates with or without NADH-linked subtrates, the cytochromes aa3 are more reduced than in the presence of NADHlinked subtrates alone.We can suggest that the « slipping » depends on the redox status of the cytochrome aa3. NO could thus physiologically increase oxidative phosphorylation efficiency. We also sought to support the hypothesis that the inhibition of complex I would have an antiapoptotic effect by inhibiting the PTP opening and the cytochrome c release. Because NO acts on the whole respiratory chain, we could not certify that this activating effect of the pore opening is due to an inhibition of a complex in particular. It seems that the permeability transition is sensitive to the complex II inhibition. Inhibition of the complex II by inhibitors like TTFA or malonate would have a pro-apoptotic effect despite the presence of powerful inhibitor of PTP such as cyclosporine. This results suggests that complex II belongs to the regulating proteic complexes of the PTP and that its inhibition prevails over the effect induced by the inhibition of complex I.Le monoxyde d'azote (NO) est formé à partir de L-arginine par la famille des NO synthases. La dysrégulation de la synthèse de cette molécule de signalisation cellulaire est impliquée dans le développement d'un grand nombre de pathologies. Les effets cellulaires du NO sont en partie dus à une inhibition des complexes de la chaîne respiratoire mitochondriale. Nous nous sommes intéressés aux effets du NO sur la régulation de l'oxydation phosphorylante et de le transition de perméabilité, deux processus mitochondriaux gouvernant la survie cellulaire. Nous avons ainsi démontré que l'inhibition de la cytochrome c oxydase (COX) par le NO était associée à une inhibition de la respiration et de la synthèse d'ATP. Cependant le NO augmente l'efficacité de l'oxydation phosphorylante (ATP/O) en diminuant le processus de « slipping ». Toutefois, notre travail a mis en exergue un effet du NO dépendant du substrat utilisé. En présence de NADH seul, le NO n'a pas l'effet bénéfique sur le rendement de l'oxydation phosphorylante, obtenu avec le FADH2 seul ou associé au NADH. L'étude de l'état d'oxydoréduction des cytochromes de la COX a révélé que dans les mitochondries énergisées avec du FADH2 seul ou en association avec le NADH, les cytochromes aa3 sont plus réduits qu'en présence de NADH seul. Nous pouvons suggérer que le « slipping » au niveau de la COX soit dépendant de l'état d'oxydoréduction de ses cytochromes. Le NO pourrait donc être un régulateur physiologique du processus d'oxydation phosphorylante. Nous avons également cherché à étayer l'hypothèse selon laquelle l'inhibition du complexe I aurait un effet anti-apoptotique en inhibant l'ouverture du PTP et la libération de cytochrome c. Le NO ayant un effet sur la chaîne respiratoire dans sa globalité, nous n'avons pu certifier que son effet activateur sur l'ouverture du pore soit dû à l'inhibition d'un complexe en particulier. Il semble que le phénomène de transition de perméabilité soit sensible à l'inhibition du complexe II. L'inhibition du complexe II par un inhibiteur comme le TTFA ou le malonate aurait un effet pro-apoptotique et ce malgré la présence d'inhibiteurs puissants du PTP comme la ciclosporine. Ces résultats suggèrent que le complexe II ferait partie des complexes protéiques régulateurs du PTP et que son inhibition soit dominante sur l'effet induit par l'inhibition du complexe I

Effect of the permeabilizing agent and time on FCCP-stimulated respiration.

<p>(<b>A</b>) Primary rat cortical neurons were permeabilized by saponin (sap, 25 µg/ml, filled squares) or digitonin (25 µg/ml, open squares, 50 µg/ml, open triangles, 100 µg/ml, open circles) plus EGTA (5 mM) in aCSF medium after three baseline O₂ consumption rate (OCR) measurements (first arrow). Pyruvate and malate (P/M, 5 mM each), ADP (1 mM), and excess K₂PHO₄ (3.6 mM for 4 mM final) were co-injected with saponin to measure complex I-dependent ADP-stimulated respiration. Oligomycin (oligo, 0.3 µg/ml), FCCP, (F, 2 µM) and antimycin A (AA, 1 µM) were added as indicated (arrows). (<b>B</b>) OCRs were measured and at the injection marked <i>a</i>, neurons were control-treated in the absence (filled squares) or presence of FCCP plus pyruvate (2 µM and 10 mM, respectively, open squares) or permeabilized using saponin (triangles). Complex I-linked respiration (P/M) in permeabilized neurons was stimulated by ADP (1 mM, filled triangles) or FCCP (F, 2 µM, open triangles). Intact (open squares) and permeabilized (open triangles) cells treated with FCCP received a second FCCP injection (1 µM) at <i>b</i> to insure respiration was maximally uncoupled, followed by a control injection at <i>c</i> and finally antimycin A (AA, 1 µM) at <i>d</i>. Control-treated intact cells (filled squares) and ADP-treated permeabilized cells (filled triangles) received injections of oligo, FCCP, (F, 2 µM) and AA in ports <i>b</i>, <i>c</i>, and <i>d</i>, respectively, with pyruvate (10 mM) included with FCCP for intact cells. OCRs in (<b>A</b>) and (<b>B</b>) are mean ± SD in quadruplicate, normalized to the third measurement point and expressed as % baseline OCR.</p

Respiration rates and respiratory control ratios for permeabilized neurons oxidizing various substrates.

<p>Initial, state 3, state 4, and uncoupled O₂ consumption rates (OCRs) are expressed in pmol O₂/min/80,000 plated cells. The initial OCRs are the rates measured just prior to permeabilization or control injection (intact). The state 4 rates were measured in the presence of oligomycin. The respiratory control ratio (RCR) is the ratio of the state 3 rate to the state 4 rate, calculated on an individual well basis prior to averaging. Data are mean ± SE, n = 4 experiments of 3–5 wells per treatment. One well (P/M) was excluded from analysis because the antimycin OCR was higher than the oligomycin OCR.</p>*<p>indicates a significant difference compared to intact cells (p<0.05). # indicates a significant difference compared to pyruvate/malate (p<0.05).</p

Nitric oxide increases oxidative phosphorylation efficiency.

International audienceWe have studied the effect of nitric oxide (NO) and potassium cyanide (KCN) on oxidative phosphorylation efficiency. Concentrations of NO or KCN that decrease resting oxygen consumption by 10-20% increased oxidative phosphorylation efficiency in mitochondria oxidizing succinate or palmitoyl-L-carnitine, but not in mitochondria oxidizing malate plus glutamate. When compared to malate plus glutamate, succinate or palmitoyl-L-carnitine reduced the redox state of cytochrome oxidase. The relationship between membrane potential and oxygen consumption rates was measured at different degrees of ATP synthesis. The use of malate plus glutamate instead of succinate (that changes the H(+)/2e(-) stoichiometry of the respiratory chain) affected the relationship, whereas a change in membrane permeability did not affect it. NO or KCN also affected the relationship, suggesting that they change the H(+)/2e(-) stoichiometry of the respiratory chain. We propose that NO may be a natural short-term regulator of mitochondrial physiology that increases oxidative phosphorylation efficiency in a redox-sensitive manner by decreasing the slipping in the proton pumps

Methyl succinate fails to rescue KB-R7943-inhibited respiration in intact neurons.

<p>(<b>A</b>) Primary rat cortical neurons were control-treated (con, filled squares) or treated with KB-7943 (K-BR, 10 µM, open circles, 20 µM, filled triangles, or 30 µM, open triangles) after three baseline O₂ consumption rate (OCR) measurements (first arrow). Subsequently, oligomycin (oligo, 0.3 µg/ml), FCCP plus pyruvate (F, 2 µM and 10 mM, respectively), and antimycin A (AA, 1 µM) were added as indicated (arrows). (<b>B</b>) Neurons were control-treated (con, filled squares) or treated with KB-7943 (30 µM, all other groups) as in (<b>A</b>). Other additions were as (A) except the FCCP+pyruvate injection after KB-R7943 addition also contained methyl succinate (MeS, 10 mM, open triangles), methyl succinate plus rotenone (10 mM/0.5 µM, MeS/R, open circles), or no additional substrate (open squares). OCRs in (<b>A</b>) and (<b>B</b>) are mean ± SD in triplicate, normalized to the third measurement point and expressed as % baseline OCR.</p

Methyl succinate is a poor substrate for complex II.

<p>(<b>A</b>) Primary rat cortical neurons were incubated in aCSF in the presence of glucose (15 mM, filled squares), no substrate (open circles), pyruvate (10 mM, filled triangles), or methyl succinate (10 mM, open squares). Cells were control-treated (con, filled squares) or treated with 2-deoxyglucose (2-DG, 2 mM, all other groups) after three baseline O₂ consumption rate (OCR) measurements (first arrow). Rotenone (0.5 µM) was subsequently added (second arrow) to inhibit complex I. (<b>B</b>) Neurons were control-treated (con, filled squares) or permeabilized by saponin (sap, 25 µg/ml) plus EGTA (5 mM) in aCSF medium after three baseline O₂ consumption rate (OCR) measurements (first arrow). Succinate/rotenone (S/R, 5 mM and 0.5 mM respectively) or methyl succinate/rotenone (MeS/R, 5 mM and 0.5 mM respectively) as substrate (sub), ADP (1 mM), and excess K₂PHO₄ (3.6 mM for 4 mM final) were co-injected with saponin to measure complex II-dependent ADP-stimulated respiration. Subsequently, oligomycin (oligo, 0.3 µg/ml), FCCP, (F, 2 µM) and antimycin A (AA, 1 µM) were added as indicated (arrows). Pyruvate (10 mM) was included with FCCP for intact cells (filled squares) to insure that substrate supply was not rate-limiting for uncoupled respiration. OCRs in (<b>A</b>) and (<b>B</b>) are mean ± SD in triplicate, normalized to the third measurement point and expressed as % baseline OCR.</p

Inhibition of respiration by KB-R7943 is reversed by the complex II substrate succinate.

<p>Primary rat cortical neurons were control-treated (filled symbols) or treated with KB-7943 (K-BR, 30 µM, open symbols) after three baseline O₂ consumption rate (OCR) measurements (first arrow). Neurons were then permeabilized by saponin (sap, 25 µg/ml) plus EGTA (5 mM) in aCSF medium containing glutamate/malate (G/M, 3 mM and 1 mM, respectively, circles) or succinate/glutamate (S/G, 3 mM each, triangles) as substrates (sub), ADP (1 mM), and excess K₂PHO₄ (3.6 mM for 4 mM final) to measure ADP-stimulated respiration (second arrow). Neurons permeabilized in the presence of G/M received a subsequent succinate (5 mM) addition (third arrow, circles) while neurons already exposed to succinate received a control (con) injection (triangles). OCRs are mean ± SD in triplicate, normalized to the third measurement point and expressed as % baseline OCR.</p

Comparison of saponin-permeabilized neuronal respiration in aCSF vs. KCl-based medium.

<p>(<b>A</b>) Primary rat cortical neurons were control-treated in aCSF (con, filled squares) or permeabilized by saponin (sap, 25 µg/ml) in aCSF (open squares) or KCl-based assay medium (open triangles) after three baseline O₂ consumption rate (OCR) measurements (first arrow). Respiration was stimulated by co-injection of ADP (1 mM) in the presence of the complex I substrates pyruvate and malate (P/M, 5 mM each). K₂HPO₄ was also co-injected with saponin to obtain a final concentration of 4 mM in each assay medium (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034465#s2" target="_blank">Methods</a>) and EGTA (5 mM) was included for cells assayed in aCSF. Oligomycin (oligo, 0.3 µg/ml), FCCP, (F, 2 µM) and antimycin A (AA, 1 µM) were subsequently added as indicated (arrows). Pyruvate (10 mM) was included with FCCP for intact cells (filled squares) here and in subsequent experiments to insure that substrate supply was not rate-limiting for uncoupled respiration. OCRs were measured in triplicate (mean ± SD). (<b>B</b>) OCRs in (<b>A</b>) baseline-normalized to the point prior to sap or con addition.</p

Magnesium sulfate protects against the bioenergetic consequences of chronic glutamate receptor stimulation.

Extracellular glutamate is elevated following brain ischemia or trauma and contributes to neuronal injury. We tested the hypothesis that magnesium sulfate (MgSO4, 3 mM) protects against metabolic failure caused by excitotoxic glutamate exposure. Rat cortical neuron preparations treated in medium already containing a physiological concentration of Mg(2+) (1 mM) could be segregated based on their response to glutamate (100 µM). Type I preparations responded with a decrease or small transient increase in oxygen consumption rate (OCR). Type II neurons responded with >50% stimulation in OCR, indicating a robust response to increased energy demand without immediate toxicity. Pre-treatment with MgSO4 improved the initial bioenergetic response to glutamate and ameliorated subsequent loss of spare respiratory capacity, measured following addition of the uncoupler FCCP, in Type I but not Type II neurons. Spare respiratory capacity in Type I neurons was also improved by incubation with MgSO4 or NMDA receptor antagonist MK801 in the absence of glutamate treatment. This finding indicates that the major difference between Type I and Type II preparations is the amount of endogenous glutamate receptor activity. Incubation of Type II neurons with 5 µM glutamate prior to excitotoxic (100 µM) glutamate exposure recapitulated a Type I phenotype. MgSO4 protected against an excitotoxic glutamate-induced drop in neuronal ATP both with and without prior 5 µM glutamate exposure. Results indicate that MgSO4 protects against chronic moderate glutamate receptor stimulation and preserves cellular ATP following treatment with excitotoxic glutamate