RESUMO
Idebenone is a synthetic quinone that on reduction in cells can bypass mitochondrial Complex I defects by donating electrons to Complex III. The drug is used clinically to treat the Complex I disease Leber's hereditary optic neuropathy (LHON), but has been less successful in clinical trials for other neurodegenerative diseases. NAD(P)H:quinone oxidoreductase 1 (NQO1) appears to be the main intracellular enzyme catalyzing idebenone reduction. However, NQO1 is not universally expressed by cells of the brain. Using primary rat cortical cells pooled from both sexes, we tested the hypotheses that the level of endogenous NQO1 activity limits the ability of neurons, but not astrocytes, to use idebenone as an electron donor to support mitochondrial respiration. We then tested the prediction that NQO1 induction by pharmacological activation of the transcription factor nuclear erythroid 2-related factor 2 (Nrf2) enables idebenone to bypass Complex I in cells with poor NQO1 expression. We found that idebenone stimulated respiration by astrocytes but reduced the respiratory capacity of neurons. Importantly, idebenone supported mitochondrial oxygen consumption in the presence of a Complex I inhibitor in astrocytes but not neurons, and this ability was reversed by inhibiting NQO1. Conversely, recombinant NQO1 delivery to neurons prevented respiratory impairment and conferred Complex I bypass activity. Nrf2 activators failed to increase NQO1 in neurons, but carnosic acid induced NQO1 in COS-7 cells that expressed little endogenous enzyme. Carnosic acid-idebenone combination treatment promoted NQO1-dependent Complex I bypass activity in these cells. Thus, combination drug strategies targeting NQO1 may promote the repurposing of idebenone for additional disorders.SIGNIFICANCE STATEMENT Idebenone is used clinically to treat loss of visual acuity in Leber's hereditary optic neuropathy. Clinical trials for several additional diseases have failed. This study demonstrates a fundamental difference in the way idebenone affects mitochondrial respiration in cortical neurons compared with cortical astrocytes. Cortical neurons are unable to use idebenone as a direct mitochondrial electron donor due to NQO1 deficiency. Our results suggest that idebenone behaves as an NQO1-dependent prodrug, raising the possibility that lack of neuronal NQO1 activity has contributed to the limited efficacy of idebenone in neurodegenerative disease treatment. Combination therapy with drugs able to safely induce NQO1 in neurons, as well as other brain cell types, may be able to unlock the neuroprotective therapeutic potential of idebenone or related quinones.
Assuntos
Antioxidantes/farmacologia , Astrócitos/enzimologia , Respiração Celular/fisiologia , Mitocôndrias/enzimologia , NAD(P)H Desidrogenase (Quinona)/metabolismo , Ubiquinona/análogos & derivados , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Células COS , Respiração Celular/efeitos dos fármacos , Células Cultivadas , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Feminino , Masculino , Mitocôndrias/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Ubiquinona/farmacologiaRESUMO
Cell-based respirometers, such as the Seahorse Extracellular Flux Analyzer, are valuable tools to assess the functionality of mitochondria within adherent neurons, as well as other cell types. The Mito Stress Test is the most frequently employed protocol of drug additions to evaluate mitochondrial bioenergetic function. Sequential exposure of cells to an ATP synthase inhibitor such as oligomycin and an uncoupler such as FCCP cause changes in oxygen consumption rate that allow estimation of the cellular efficiency and capacity for mitochondrial ATP synthesis. While a useful first step in assessing whether an experimental treatment or genetic manipulation affects mitochondrial energetics, the Mito Stress Test does not identify specific sites of altered respiratory chain function. This article discusses limitations of the Mito Stress Test, proposes a refined protocol for comparing cell populations that requires independent drug titrations at multiple cell densities, and describes a stepwise series of respirometry-based assays that "map" locations of electron transport deficiency. These include strategies to test for cytochrome c release, to probe the functionality of specific electron transport chain complexes within intact or permeabilized cells, and to measure NADH oxidation by the linked activity of Complexes I, III, and IV. To illustrate utility, we show that although UK5099 and ABT-737 each decrease the spare respiratory capacity of cortical neurons, the stepwise assays reveal different underlying mechanisms consistent with their established drug targets: deficient Complex I substrate supply induced by the mitochondrial pyruvate carrier inhibitor UK5099 and cytochrome c release induced by the anti-apoptotic BCL-2 family protein inhibitor ABT-737.
Assuntos
Bioensaio , Metabolismo Energético , Técnicas In Vitro , Doenças Mitocondriais , Animais , Linhagem Celular , Respiração Celular , Humanos , Neurônios , RatosRESUMO
Biological sex is thought to influence mitochondrial bioenergetic function. Previous respiration measurements examining brain mitochondrial sex differences were made at atmospheric oxygen using isolated brain mitochondria. Oxygen is 160 mm Hg (21%) in the atmosphere, while the oxygen tension in the brain generally ranges from â¼5 to 45 mm Hg (â¼1-6% O2). This study tested the hypothesis that sex and/or brain physiological oxygen tension influence the mitochondrial bioenergetic properties of primary rat cortical astrocytes and microglia. Oxygen consumption was measured with a Seahorse XF24 cell respirometer in an oxygen-controlled environmental chamber. Strikingly, male astrocytes had a higher maximal respiration than female astrocytes when cultured and assayed at 3% O2. Three percent O2 yielded a low physiological dissolved O2 level of â¼1.2% (9.1 mm Hg) at the cell monolayer during culture and 1.2-3.0% O2 during assays. No differences in bioenergetic parameters were observed between male and female astrocytes at 21% O2 (dissolved O2 of â¼19.7%, 150 mm Hg during culture) or between either of these cell populations and female astrocytes at 3% O2. In contrast to astrocytes, microglia showed no sex differences in mitochondrial bioenergetic parameters at either oxygen level, regardless of whether they were non-stimulated or activated to a proinflammatory state. There were also no O2- or sex-dependent differences in proinflammatory TNF-α or IL-1ß cytokine secretion measured at 18 h activation. Overall, results reveal an intriguing sex variance in astrocytic maximal respiration that requires additional investigation. Findings also demonstrate that sex differences can be masked by conducting experiments at non-physiological O2.
Assuntos
Astrócitos/metabolismo , Encéfalo/metabolismo , Metabolismo Energético/fisiologia , Microglia/metabolismo , Mitocôndrias/metabolismo , Consumo de Oxigênio/fisiologia , Caracteres Sexuais , Animais , Animais Recém-Nascidos , Células Cultivadas , Feminino , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Read the highlighted article 'Defective synthesis and release of astrocytic thrombospondin-1 mediates the neuronal TDP-43 proteinopathy, resulting in defects in neuronal integrity associated with chronic traumatic encephalopathy: in vitro studies' on page 645.