SUMMARY Mitochondrial fission mediated by the GTPase dynamin-related protein-1 (Drp1) is an attractive drug target in numerous maladies that range from heart disease to neurodegenerative disorders. The compound mdivi-1 is widely reported to inhibit Drp1-dependent fission, elongate mitochondria, and mitigate brain injury. Here, we show that mdivi-1 reversibly inhibits mitochondrial Complex I-dependent O2 consumption and reverse electron transfer-mediated reactive oxygen species (ROS) production at concentrations (e.g. 50 μM) used to target mitochondrial fission. Respiratory inhibition is rescued by bypassing Complex I using yeast NADH dehydrogenase Ndi1. Unexpectedly, respiratory impairment by mdivi-1 occurs without mitochondrial elongation, is not mimicked by Drp1 deletion, and is observed in Drp1-deficient fibroblasts. In addition, mdivi-1 poorly inhibits recombinant Drp1 GTPase activity (Ki>1.2 mM). Overall, results suggest that mdivi-1 is not a specific Drp1 inhibitor. The ability of mdivi-1 to reversibly inhibit Complex I and modify mitochondrial ROS production may contribute to effects observed in disease models.

MBoC | ARTICLE Mitochondrial E3 ubiquitin ligase MARCH5 controls mitochondrial fission and cell sensitivity to stress-induced apoptosis through regulation of MiD49 protein ABSTRACT Ubiquitin-and proteasome-dependent outer mitochondrial membrane (OMM)-associated degradation (OMMAD) is critical for mitochondrial and cellular homeostasis. However, the scope and molecular mechanisms of the OMMAD pathways are still not well understood. We report that the OMM-associated E3 ubiquitin ligase MARCH5 controls dynamin-related protein 1 (Drp1)-dependent mitochondrial fission and cell sensitivity to stress-induced apoptosis. MARCH5 knockout selectively inhibited ubiquitination and proteasomal degradation of MiD49, a mitochondrial receptor of Drp1, and consequently led to mitochondrial fragmentation. Mitochondrial fragmentation in MARCH5 −/− cells was not associated with inhibition of mitochondrial fusion or bioenergetic defects, supporting the possibility that MARCH5 is a negative regulator of mitochondrial fission. Both MARCH5 re-expression and MiD49 knockout in MARCH5 −/− cells reversed mitochondrial fragmentation and reduced sensitivity to stress-induced apoptosis. These findings and data showing MARCH5-dependent degradation of MiD49 upon stress support the possibility that MARCH5 regulation of MiD49 is a novel mechanism controlling mitochondrial fission and, consequently, the cellular response to stress.

Mitochondrial reactive oxygen species (ROS) are implicated in signal transduction, inflammation, neurodegenerative disorders, and normal aging. Net ROS release by isolated brain mitochondria derived from a mixture of neurons and glia is readily quantified using fluorescent dyes. Measuring intracellular ROS in intact neurons or glia and assigning the origin to mitochondria are far more difficult. In recent years, the protonmotive force crucial to mitochondrial function has been exploited to target a variety of compounds to the highly negative mitochondrial matrix using the lipophilic triphenylphosphonium cation (TPP+) as a “delivery” conjugate. Among these, MitoSOX Red, also called mito-hydroethidine or mitodihydroethidium, is prevalently used for mitochondrial ROS estimation. Although the TPP+ moiety of MitoSOX enables the many-fold accumulation of ROS-sensitive hydroethidine in the mitochondrial matrix, the membrane potential sensitivity conferred by TPP+ creates a daunting set of challenges not often considered in the application of this dye. This chapter provides recommendations and cautionary notes on the use of potentiometric fluorescent indicators for the approximation of mitochondrial ROS in live neurons, with principles that can be extrapolated to non-neuronal cell types. It is concluded that mitochondrial membrane potential changes render accurate estimation of mitochondrial ROS using MitoSOX difficult to impossible. Consequently, knowledge of mitochondrial membrane potential is essential to the application of potentiometric fluorophores for the measurement of intramitochondrial ROS.

Background/Objectives To examine the relationships of plasma and tissue markers of systemic and vascular inflammation to obesity and insulin resistance and determine the effects of aerobic exercise training+weight loss (AEX+WL) and weight loss (WL) on these biomarkers. Design Prospective controlled study. Participants Seventy-seven overweight and obese sedentary postmenopausal women. Interventions Six months, 3d/wk AEX+WL (n=37) or WL (n=40). Measurements Total body dual-energy x-ray absorptiometry, abdominal computed tomography scans, hyperinsulinemic-euglycemic clamps, adipose tissue biopsies (n=28), and blood for Homeostasis model assessment-insulin resistance, and soluble forms of intracellular adhesion molecule (sICAM-1) and vascular CAM-1 (sVCAM-1), C-reactive protein (CRP), and serum amyloid A (SAA). Results Body weight, %fat, visceral fat, triglyceride levels and systolic blood pressure decreased comparably after WL and AEX+WL (P<0.05). VO2max increased 16% after AEX+WL (P<0.001). Insulin resistance decreased in both groups (P<0.01). Glucose utilization increased 10% (P< 0.05) after AEX+WL and 8% with WL (P=0.07). AEX+WL and WL decreased CRP by 29% and 21%, (P<0.05). SAA levels decreased two-fold more after AEX+WL (−19%, P<0.05) than with WL (−9%, P=0.08). Plasma sICAM-1 and sVCAM-1 levels did not change; however, women with the greatest reduction in plasma sICAM-1 levels had the greatest reductions in fasting glucose, insulin and insulin resistance (P<0.05). Gluteal ICAM mRNA levels decreased 27% after AEX+WL (P<0.05) and did not change after WL. Conclusion Obesity and insulin resistance worsen markers of systemic and vascular inflammation. A reduction in plasma sICAM-1 is important to improve insulin sensitivity. CRP and SAA and tissue ICAM decrease with exercise and weight loss, suggesting that exercise training is a necessary component of lifestyle modification in obese postmenopausal women.

MitoSOX Red is a fluorescent probe used for the detection of mitochondrial reactive oxygen species by live cell imaging. The lipophilic, positively charged triphenylphosphonium moiety within MitoSOX concentrates the superoxide-sensitive dihydroethidium conjugate within the mitochondrial matrix. Here we investigated whether common MitoSOX imaging protocols influence mitochondrial bioenergetic function in primary rat cortical neurons and microglial cell lines. MitoSOX dose-dependently uncoupled neuronal respiration, whether present continuously in the assay medium or washed following a ten minute loading protocol. Concentrations of 5-10 μM MitoSOX caused severe loss of ATP synthesis-linked respiration. Redistribution of MitoSOX to the cytoplasm and nucleus occurred concomitant to mitochondrial uncoupling. MitoSOX also dose-dependently decreased the maximal respiration rate and this impairment could not be rescued by delivery of a complex IV specific substrate, revealing complex IV inhibition. As in neurons, loading microglial cells with MitoSOX at low micromolar concentrations resulted in uncoupled mitochondria with reduced respiratory capacity whereas submicromolar MitoSOX had no adverse effects. The MitoSOX parent compound dihydroethidium also caused mitochondrial uncoupling and respiratory inhibition at low micromolar concentrations. However, these effects were abrogated by pre-incubating dihydroethidium with cation exchange beads to remove positively charged oxidation products, which would otherwise by sequestered by polarized mitochondria. Collectively, our results suggest that the matrix accumulation of MitoSOX or dihydroethidium oxidation products causes mitochondrial uncoupling and inhibition of complex IV. Because MitoSOX is inherently capable of causing severe mitochondrial dysfunction with the potential to alter superoxide production, its use therefore requires careful optimization in imaging protocols.

Introduction Despite increasing representation in surgery, women continue to lag behind men in important metrics. Little is known on how industry funding may also contribute to this ongoing disparity. This article seeks to quantify industry payments to academic plastic surgeons (APSs) by sex and examine the relationship between funding and academic achievement. Methods We conducted a cross-sectional analysis of industry payments disbursed to APSs in 2017. Faculty were identified using departmental listings of Accreditation Council for Graduate Medical Education plastic surgery residency programs. Payments were identified via the Center for Medicare and Medicaid Services open payment database. Academic achievement was assessed using rank (eg, assistant professor), leadership designation (eg, division head), and Scopus H-index and then controlled for time in practice. Results Of the 805 APSs, the majority were male (82% male vs 18% female, P < 0.0001). Significant sex differences emerged in average yearly industry contributions (men, US $3202, vs women, US $707; P < 0.0001). Across all academic ranks, men received significantly higher payments than women (P < 0.0500). Men constituted 93% of full professors and were almost twice as likely to hold additional leadership positions compared with women (odds ratio, 1.82; P = 0.0143). After adjustment for time in practice, there was no difference in H-indices between male and female APSs, although payment disparity persisted (P < 0.0001). Conclusions Substantial sex-based disparities exist among APSs' academic rank and leadership attainment, which is not attributed to differences in academic qualifications or experience. To better elucidate the sources of this disparity, future studies should assess sexed differences in payment types. Furthermore, we urge for increased transparency in the selection process for industry payments.

Background and Purpose Dynamin‐related protein 1 (Drp1) mediates mitochondrial fission and is thought to promote Bax/Bak‐induced cytochrome c release during apoptosis. Conformationally active Bax, Bak and Bax/Bak‐activating BH3‐only proteins, such as Bim, are restrained by anti‐apoptotic Bcl‐2 proteins in cells that are ‘primed for death’. Inhibition of Bcl‐2/Bcl‐xL/Bcl‐w by the antagonist ABT‐737 causes rapid apoptosis of primed cells. Hence, we determined whether Drp1 is required for cytochrome c release, respiratory alterations and apoptosis of cells that are already primed for death. Experimental Approach We tested the Drp1 inhibitor mdivi‐1 for inhibition of cytochrome c release in MCF10A cells primed by Bcl‐2 overexpression. We measured ATP synthesis‐dependent, ‐independent and cytochrome c‐limited maximal oxygen consumption rates (OCRs) and cell death of immortalized wild‐type (WT) and Drp1 knockout (KO) mouse embryonic fibroblasts (MEFs) treated with ABT‐737. Key Results Mdivi‐1 failed to attenuate ABT‐737‐induced cytochrome c release. ABT‐737 decreased maximal OCR measured in the presence of uncoupler in both WT and Drp1 KO MEF, consistent with respiratory impairment due to release of cytochrome c. However, Drp1 KO MEF were slightly less sensitive to this ABT‐737‐induced respiratory inhibition compared with WT, and were resistant to an initial ABT‐737‐induced increase in ATP synthesis‐independent O2 consumption. Nevertheless, caspase‐dependent cell death was not reduced. Pro‐apoptotic Bax was unaltered, whereas Bak was up‐regulated in Drp1 KO MEF. Conclusions and Implications The findings indicate that once fibroblast cells are primed for death, Drp1 is not required for apoptosis. However, Drp1 may contribute to ABT‐737‐induced respiratory changes and the kinetics of cytochrome c release. Linked Articles This article is part of a themed issue on Mitochondrial Pharmacology: Energy, Injury & Beyond. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2014.171.issue-8

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.