Idebenone Has Distinct Effects on Mitochondrial Respiration in Cortical Astrocytes Compared to Cortical Neurons Due to Differential NQO1 Activity.

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.

An in vitro model yields 'importin' new insights into chronic traumatic encephalopathy: damaged astrocytes stop 'thrombospondin' to the injury: An Editorial Highlight for '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'.

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Monoallelic loss of tumor suppressor GRIM-19 promotes tumorigenesis in mice.

Gene-associated with retinoid-interferon induced mortality-19 (GRIM-19), a STAT3-inhibitory protein, was isolated as a growth-suppressive gene product using a genome-wide expression knockdown screen. We and others have shown a loss of expression and occurrence of mutations in the GRIM-19 gene in a variety of primary human cancers, indicating its potential role as tumor suppressor. To help investigate its role in tumor development in vivo, we generated a genetically modified mouse in which Grim-19 can be conditionally inactivated. Deletion of Grim-19 in the skin significantly increased the susceptibility of mice to chemical carcinogenesis, resulting in development of squamous cell carcinomas. These tumors had high Stat3 activity and an increased expression of Stat3-responsive genes. Loss of Grim-19 also caused mitochondrial electron transport dysfunction resulting from failure to assemble electron transport chain complexes and altered the expression of several cellular genes involved in glycolysis. Surprisingly, the deletion of a single copy of the Grim-19 gene was sufficient to promote carcinogenesis and formation of invasive squamous cell carcinomas. These observations highlight the critical role of GRIM-19 as a tumor suppressor.

Idebenone and neuroprotection: antioxidant, pro-oxidant, or electron carrier?

Ubiquinone, commonly called coenzyme Q10 (CoQ), is a lipophilic electron carrier and endogenous antioxidant found in all cellular membranes. In the mitochondrial inner membrane it transfers electrons to complex III of the electron transport chain. The short chain CoQ analogue idebenone is in clinical trials for a number of diseases that exhibit a mitochondrial etiology. Nevertheless, evidence that idebenone ameliorates neurological symptoms in human disease is inconsistent. Although championed as an antioxidant, idebenone can also act as a pro-oxidant by forming an unstable semiquinone at complex I. The antioxidant function of idebenone is critically dependent on two-electron reduction to idebenol without the creation of unstable intermediates. Recently, cytoplasmic NAD(P)H: quinone oxidoreductase 1 (NQO1) was identified as a major enzyme catalyzing idebenone reduction. While reduction allows idebenone to act as an antioxidant, evidence also suggests that NQO1 enables idebenone to shuttle reducing equivalents from cytoplasmic NAD(P)H to mitochondrial complex III, bypassing any upstream damage to the electron transport chain. In this mini-review we discuss how idebenone can influence mitochondrial function within the context of cytoprotection. Importantly, in the brain NQO1 is expressed primarily by glia rather than neurons. As NQO1 is an inducible enzyme regulated by oxidative stress and the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway, optimizing NQO1 expression in appropriate cell types within a specific disease context may be key to delivering on idebenone's therapeutic potential.

Mapping mitochondrial respiratory chain deficiencies by respirometry: Beyond the Mito Stress Test.

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.

SIRT3, a metabolic target linked to ataxia-telangiectasia mutated (ATM) gene deficiency in diffuse large B-cell lymphoma.

Inactivation of Ataxia-telangiectasia mutated (ATM) gene results in an increased risk to develop cancer. We show that ATM deficiency in diffuse large B-cell lymphoma (DLBCL) significantly induce mitochondrial deacetylase sirtuin-3 (SIRT3) activity, disrupted mitochondrial structure, decreased mitochondrial respiration, and compromised TCA flux compared with DLBCL cells expressing wild type (WT)-ATM. This corresponded to enrichment of glutamate receptor and glutamine pathways in ATM deficient background compared to WT-ATM DLBCL cells. ATM-/- DLBCL cells have decreased apoptosis in contrast to radiosensitive non-cancerous A-T cells. In vivo studies using gain and loss of SIRT3 expression showed that SIRT3 promotes growth of ATM CRISPR knockout DLBCL xenografts compared to wild-type ATM control xenografts. Importantly, screening of DLBCL patient samples identified SIRT3 as a putative therapeutic target, and validated an inverse relationship between ATM and SIRT3 expression. Our data predicts SIRT3 as an important therapeutic target for DLBCL patients with ATM null phenotype.

Sex differences in the mitochondrial bioenergetics of astrocytes but not microglia at a physiologically relevant brain oxygen tension.

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 ∼5 to 45 mm Hg (∼1-6% 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 ∼1.2% (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 ∼19.7%, 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.

Role of hypoxia in Diffuse Large B-cell Lymphoma: Metabolic repression and selective translation of HK2 facilitates development of DLBCL.

Published molecular profiling studies in patients with lymphoma suggested the influence of hypoxia inducible factor-1 alpha (HIF1α) targets in prognosis of DLBCL. Yet, the role of hypoxia in hematological malignancies remains unclear. We observed that activation of HIF1α resulted in global translation repression during hypoxic stress in DLBCL. Protein translation efficiency as measured using 35S-labeled methionine incorporation revealed a ≥50% reduction in translation upon activation of HIF1α. Importantly, translation was not completely inhibited and expression of clinically correlated hypoxia targets such as GLUT1, HK2, and CYT-C was found to be refractory to translational repression under hypoxia in DLBCL cells. Notably, hypoxic induction of these genes was not observed in normal primary B-cells. Translational repression was coupled with a decrease in mitochondrial function. Screening of primary DLBCL patient samples revealed that expression of HK2, which encodes for the enzyme hexokinase 2, was significantly correlated with DLBCL phenotype. Genetic knockdown studies demonstrated that HK2 is required for promoting growth of DLBCL under hypoxic stress. Altogether, our findings provide strong support for the direct contribution of HK2 in B-cell lymphoma development and suggest that HK2 is a key metabolic driver of the DLBCL phenotype.

Author Correction: Role of hypoxia in Diffuse Large B-cell Lymphoma: Metabolic repression and selective translation of HK2 facilitates development of DLBCL.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.