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Background: Mutations in several genes of Caenorhabditis elegans confer altered sensitivities to volatile anesthetics. A mutation in one gene, gas-1(fc21), causes animals to be immobilized at lower concentrations of all volatile anesthetics than in the wild type, and it does not depend on mutations in other genes to control anesthetic sensitivity. gas-1 confers different sensitivities to stereoisomers of isoflurane, and thus may be a direct target for volatile anesthetics. The authors have cloned and characterized the gas gene and the mutant allele fc21., Methods: Genetic techniques for nematodes were as previously described. Polymerase chain reaction, sequencing, and other molecular biology techniques were performed by standard methods. Mutant rescue was done by injecting DNA fragments into the gonad of mutant animals and scoring the offspring for loss of the mutant phenotype., Results: The gas-1 gene was cloned and identified. The protein GAS-1 is a homologue of the 49-kd (IP) subunit of the mitochondrial NADH-ubiquinone-oxidoreductase (complex I of the respiratory chain). gas-1(fc21) is a missense mutation replacing a strictly conserved arginine with lysine., Conclusions: The function of the 49-kd (IP) subunit of complex I is unknown. The finding that mutations in complex I increase sensitivity of C. elegans to volatile anesthetics may implicate this physiologic process in the determination of anesthetic sensitivity. The hypersensitivity of animals with a mutation in the gas-1 gene may be caused by a direct anesthetic effect on a mitochondrial protein or secondary effects at other sites caused by mitochondrial dysfunction., (Copyright © 2024 American Society of Anesthesiologists. All Rights Reserved.)

Aim: Leigh syndrome (LS), the most common paediatric presentation of genetic mitochondrial dysfunction, is a multi-system disorder characterised by severe neurologic and metabolic abnormalities. Symmetric, bilateral, progressive necrotizing lesions in the brainstem are defining features of the disease. Patients are often symptom free in early life but typically develop symptoms by about 2 years of age. The mechanisms underlying disease onset and progression in LS remain obscure. Recent studies have shown that the immune system causally drives disease in the Ndufs4(-/-) mouse model of LS: treatment of Ndufs4(-/-) mice with the macrophage-depleting Csf1r inhibitor pexidartinib prevents disease. While the precise mechanisms leading to immune activation and immune factors involved in disease progression have not yet been determined, interferon-gamma (IFNγ) and interferon gamma-induced protein 10 (IP10) were found to be significantly elevated in Ndufs4(-/-) brainstem, implicating these factors in disease. Here, we aimed to explore the role of IFNγ and IP10 in LS., Methods: To establish the role of IFNγ and IP10 in LS, we generated IFNγ and IP10 deficient Ndufs4(-/-)/Ifng(-/-) and Ndufs4(-/-)/IP10(-/-) double knockout animals, as well as IFNγ and IP10 heterozygous, Ndufs4(-/-)/Ifng(+/-) and Ndufs4(-/-)/IP10(+/-), animals. We monitored disease onset and progression to define the impact of heterozygous or homozygous loss of IFNγ and IP10 in LS., Results: Loss of IP10 does not significantly impact the onset or progression of disease in the Ndufs4(-/-) model. IFNγ loss significantly extends survival and delays disease progression in a gene dosage-dependent manner, though the benefits are modest compared to Csf1r inhibition., Conclusions: IFNγ contributes to disease onset and progression in LS. Our findings suggest that IFNγ targeting therapies may provide some benefits in genetic mitochondrial disease, but targeting IFNγ alone would likely yield only modest benefits in LS., (© 2024 The Authors. Neuropathology and Applied Neurobiology published by John Wiley & Sons Ltd on behalf of British Neuropathological Society.)

Background: Genetic mitochondrial diseases impact over 1 in 4000 individuals, most often presenting in infancy or early childhood. Seizures are major clinical sequelae in some mitochondrial diseases including Leigh syndrome, the most common pediatric presentation of mitochondrial disease. Dietary ketosis has been used to manage seizures in mitochondrial disease patients. Mitochondrial disease patients often require surgical interventions, leading to anesthetic exposures. Anesthetics have been shown to be toxic in the setting of mitochondrial disease, but the impact of a ketogenic diet on anesthetic toxicities in this setting has not been studied., Aims: Our aim in this study was to determine whether dietary ketosis impacts volatile anesthetic toxicities in the setting of genetic mitochondrial disease., Methods: The impact of dietary ketosis on toxicities of volatile anesthetic exposure in mitochondrial disease was studied by exposing young Ndufs4(-/-) mice fed ketogenic or control diet to isoflurane anesthesia. Blood metabolites were measured before and at the end of exposures, and survival and weight were monitored., Results: Compared to a regular diet, the ketogenic diet exacerbated hyperlactatemia resulting from isoflurane exposure (control vs. ketogenic diet in anesthesia mean difference 1.96 mM, Tukey's multiple comparison adjusted p = .0271) and was associated with a significant increase in mortality during and immediately after exposures (27% vs. 87.5% mortality in the control and ketogenic diet groups, respectively, during the exposure period, Fisher's exact test p = .0121). Our data indicate that dietary ketosis and volatile anesthesia interact negatively in the setting of mitochondrial disease., Conclusions: Our findings suggest that extra caution should be taken in the anesthetic management of mitochondrial disease patients in dietary ketosis., (© 2024 The Authors. Pediatric Anesthesia published by John Wiley & Sons Ltd.)

Background: Volatile anesthetics induce hyperpolarizing potassium currents in spinal cord neurons that may contribute to their mechanism of action. They are induced at lower concentrations of isoflurane in noncholinergic neurons from mice carrying a loss-of-function mutation of the Ndufs4 gene, required for mitochondrial complex I function. The yeast NADH dehydrogenase enzyme, NDi1, can restore mitochondrial function in the absence of normal complex I activity, and gain-of-function Ndi1 transgenic mice are resistant to volatile anesthetics. The authors tested whether NDi1 would reduce the hyperpolarization caused by isoflurane in neurons from Ndufs4 and wild-type mice. Since volatile anesthetic behavioral hypersensitivity in Ndufs4 is transduced uniquely by glutamatergic neurons, it was also tested whether these currents were also unique to glutamatergic neurons in the Ndufs4 spinal cord., Methods: Spinal cord neurons from wild-type, NDi1, and Ndufs4 mice were patch clamped to characterize isoflurane sensitive currents. Neuron types were marked using fluorescent markers for cholinergic, glutamatergic, and γ-aminobutyric acid-mediated (GABAergic) neurons. Norfluoxetine was used to identify potassium channel type. Neuron type-specific Ndufs4 knockout animals were generated using type-specific Cre-recombinase with floxed Ndufs4., Results: Resting membrane potentials (RMPs) of neurons from NDi1;Ndufs4, unlike those from Ndufs4, were not hyperpolarized by 0.6% isoflurane (Ndufs4, ΔRMP -8.2 mV [-10 to -6.6]; P = 1.3e-07; Ndi1;Ndufs4, ΔRMP -2.1 mV [-7.6 to +1.4]; P = 1). Neurons from NDi1 animals in a wild-type background were not hyperpolarized by 1.8% isoflurane (wild-type, ΔRMP, -5.2 mV [-7.3 to -3.2]; P = 0.00057; Ndi1, ΔRMP, 0.6 mV [-1.7 to 3.2]; P = 0.68). In spinal cord slices from global Ndufs4 animals, holding currents (HC) were induced by 0.6% isoflurane in both GABAergic (ΔHC, 81.3 pA [61.7 to 101.4]; P = 2.6e-05) and glutamatergic (ΔHC, 101.2 pA [63.0 to 146.2]; P = 0.0076) neurons. In neuron type-specific Ndufs4 knockouts, HCs were increased in cholinergic (ΔHC, 119.5 pA [82.3 to 156.7]; P = 0.00019) and trended toward increase in glutamatergic (ΔHC, 85.5 pA [49 to 126.9]; P = 0.064) neurons but not in GABAergic neurons., Conclusions: Bypassing complex I by overexpression of NDi1 eliminates increases in potassium currents induced by isoflurane in the spinal cord. The isoflurane-induced potassium currents in glutamatergic neurons represent a potential downstream mechanism of complex I inhibition in determining minimum alveolar concentration., (Copyright © 2023 American Society of Anesthesiologists. All Rights Reserved.)

Background: Volatile anaesthetics are widely used in human medicine. Although generally safe, hypersensitivity and toxicity can occur in rare cases, such as in certain genetic disorders. Anaesthesia hypersensitivity is well-documented in a subset of mitochondrial diseases, but whether volatile anaesthetics are toxic in this setting has not been explored., Methods: We exposed Ndufs4(-/-) mice, a model of Leigh syndrome, to isoflurane (0.2-0.6%), oxygen 100%, or air. Cardiorespiratory function, weight, blood metabolites, and survival were assessed. We exposed post-symptom onset and pre-symptom onset animals and animals treated with the macrophage depleting drug PLX3397/pexidartinib to define the role of overt neuroinflammation in volatile anaesthetic toxicities., Results: Isoflurane induced hyperlactataemia, weight loss, and mortality in a concentration- and duration-dependent manner from 0.2% to 0.6% compared with carrier gas (O 2 100%) or mock (air) exposures (lifespan after 30-min exposures ∗P<0.05 for isoflurane 0.4% vs air or vs O 2 , ∗∗P<0.005 for isoflurane 0.6% vs air or O 2 ; 60-min exposures ∗∗P<0.005 for isoflurane 0.2% vs air, ∗P<0.05 for isoflurane 0.2% vs O 2 ). Isoflurane toxicity was significantly reduced in Ndufs4(-/-) exposed before CNS disease onset, and the macrophage depleting drug pexidartinib attenuated sequelae of isoflurane toxicity (survival ∗∗∗P=0.0008 isoflurane 0.4% vs pexidartinib plus isoflurane 0.4%). Finally, the laboratory animal standard of care of 100% O 2 as a carrier gas contributed significantly to weight loss and reduced survival, but not to metabolic changes, and increased acute mortality., Conclusions: Isoflurane is toxic in the Ndufs4(-/-) model of Leigh syndrome. Toxic effects are dependent on the status of underlying neurologic disease, largely prevented by the CSF1R inhibitor pexidartinib, and influenced by oxygen concentration in the carrier gas., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: A variety of molecular targets for volatile anesthetics have been suggested, including the anesthetic-sensitive potassium leak channel, TREK-1. Knockout of TREK-1 is reported to render mice resistant to volatile anesthetics, making TREK-1 channels compelling targets for anesthetic action. Spinal cord slices from mice, either wild type or an anesthetic- hypersensitive mutant, Ndufs4, display an isoflurane-induced outward potassium leak that correlates with their minimum alveolar concentrations and is blocked by norfluoxetine. The hypothesis was that TREK-1 channels conveyed this current and contribute to the anesthetic hypersensitivity of Ndufs4. The results led to evaluation of a second TREK channel, TREK-2, in control of anesthetic sensitivity., Methods: The anesthetic sensitivities of mice carrying knockout alleles of Trek-1 and Trek-2, the double knockout Trek-1;Trek-2, and Ndufs4;Trek-1 were measured. Neurons from spinal cord slices from each mutant were patch clamped to characterize isoflurane-sensitive currents. Norfluoxetine was used to identify TREK-dependent currents., Results: The mean values for minimum alveolar concentrations (± SD) between wild type and two Trek-1 knockout alleles in mice (P values, Trek-1 compared to wild type) were compared. For wild type, minimum alveolar concentration of halothane was 1.30% (0.10), and minimum alveolar concentration of isoflurane was 1.40% (0.11); for Trek-1tm1Lex, minimum alveolar concentration of halothane was 1.27% (0.11; P = 0.387), and minimum alveolar concentration of isoflurane was 1.38% (0.09; P = 0.268); and for Trek-1tm1Lzd, minimum alveolar concentration of halothane was 1.27% (0.11; P = 0.482), and minimum alveolar concentration of isoflurane was 1.41% (0.12; P = 0.188). Neither allele was resistant for loss of righting reflex. The EC50 values of Ndufs4;Trek-1tm1Lex did not differ from Ndufs4 (for Ndufs4, EC50 of halothane, 0.65% [0.05]; EC50 of isoflurane, 0.63% [0.05]; and for Ndufs4;Trek-1tm1Lex, EC50 of halothane, 0.58% [0.07; P = 0.004]; and EC50 of isoflurane, 0.61% [0.06; P = 0.442]). Loss of TREK-2 did not alter anesthetic sensitivity in a wild-type or Trek-1 genetic background. Loss of TREK-1, TREK-2, or both did not alter the isoflurane-induced currents in wild-type cells but did cause them to be norfluoxetine insensitive., Conclusions: Loss of TREK channels did not alter anesthetic sensitivity in mice, nor did it eliminate isoflurane-induced transmembrane currents. However, the isoflurane-induced currents are norfluoxetine-resistant in Trek mutants, indicating that other channels may function in this role when TREK channels are deleted., (Copyright © 2023 American Society of Anesthesiologists. All Rights Reserved.)

One of the unsolved mysteries of medicine is how do volatile anesthetics (VAs) cause a patient to reversibly lose consciousness. In addition, identifying mechanisms for the collateral effects of VAs, including anesthetic-induced neurotoxicity (AiN) and anesthetic preconditioning (AP), has proven challenging. Multiple classes of molecules (lipids, proteins, and water) have been considered as potential VA targets, but recently proteins have received the most attention. Studies targeting neuronal receptors or ion channels had limited success in identifying the critical targets of VAs mediating either the phenotype of "anesthesia" or their collateral effects. Recent studies in both nematodes and fruit flies may provide a paradigm shift by suggesting that mitochondria may harbor the upstream molecular switch activating both primary and collateral effects. The disruption of a specific step of electron transfer within the mitochondrion causes hypersensitivity to VAs, from nematodes to Drosophila and to humans, while also modulating the sensitivity to collateral effects. The downstream effects from mitochondrial inhibition are potentially legion, but inhibition of presynaptic neurotransmitter cycling appears to be specifically sensitive to the mitochondrial effects. These findings are perhaps of even broader interest since two recent reports indicate that mitochondrial damage may well underlie neurotoxic and neuroprotective effects of VAs in the central nervous system (CNS). It is, therefore, important to understand how anesthetics interact with mitochondria to affect CNS function, not just for the desired facets of general anesthesia but also for significant collateral effects, both harmful and beneficial. A tantalizing possibility exists that both the primary (anesthesia) and secondary (AiN, AP) mechanisms may at least partially overlap in the mitochondrial electron transport chain (ETC).

The mechanisms of volatile anesthetic action remain among the most perplexing mysteries of medicine. Across phylogeny, volatile anesthetics selectively inhibit mitochondrial complex I, and they also depress presynaptic excitatory signaling. To explore how these effects are linked, we studied isoflurane effects on presynaptic vesicle cycling and ATP levels in hippocampal cultured neurons from wild-type and complex I mutant (Ndufs4(KO)) mice. To bypass complex I, we measured isoflurane effects on anesthetic sensitivity in mice expressing NADH dehydrogenase (NDi1). Endocytosis in physiologic concentrations of glucose was delayed by effective behavioral concentrations of isoflurane in both wild-type (τ [unexposed] 44.8 ± 24.2 s; τ [exposed] 116.1 ± 28.1 s; p < 0.01) and Ndufs4(KO) cultures (τ [unexposed] 67.6 ± 16.0 s; τ [exposed] 128.4 ± 42.9 s; p = 0.028). Increasing glucose, to enhance glycolysis and increase ATP production, led to maintenance of both ATP levels and endocytosis (τ [unexposed] 28.0 ± 14.4; τ [exposed] 38.2 ± 5.7; reducing glucose worsened ATP levels and depressed endocytosis (τ [unexposed] 85.4 ± 69.3; τ [exposed] > 1,000; p < 0.001). The block in recycling occurred at the level of reuptake of synaptic vesicles into the presynaptic cell. Expression of NDi1 in wild-type mice caused behavioral resistance to isoflurane for tail clamp response (EC 50 Ndi1(-) 1.27% ± 0.14%; Ndi1(+) 1.55% ± 0.13%) and halothane (EC 50 Ndi1(-) 1.20% ± 0.11%; Ndi1(+) 1.46% ± 0.10%); expression of NDi1 in neurons improved hippocampal function, alleviated inhibition of presynaptic recycling, and increased ATP levels during isoflurane exposure. The clear alignment of cell culture data to in vivo phenotypes of both isoflurane-sensitive and -resistant mice indicates that inhibition of mitochondrial complex I is a primary mechanism of action of volatile anesthetics., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Symmetric, progressive, necrotizing lesions in the brainstem are a defining feature of Leigh syndrome (LS). A mechanistic understanding of the pathogenesis of these lesions has been elusive. Here, we report that leukocyte proliferation is causally involved in the pathogenesis of LS. Depleting leukocytes with a colony-stimulating factor 1 receptor inhibitor disrupted disease progression, including suppression of CNS lesion formation and a substantial extension of survival. Leukocyte depletion rescued diverse symptoms, including seizures, respiratory center function, hyperlactemia, and neurologic sequelae. These data reveal a mechanistic explanation for the beneficial effects of mTOR inhibition. More importantly, these findings dramatically alter our understanding of the pathogenesis of LS, demonstrating that immune involvement is causal in disease. This work has important implications for the mechanisms of mitochondrial disease and may lead to novel therapeutic strategies.

Background: If anaesthetics cause permanent cognitive deficits in some children, the implications are enormous, but the molecular causes of anaesthetic-induced neurotoxicity, and consequently possible therapies, are still debated. Anaesthetic exposure early in development can be neurotoxic in the invertebrate Caenorhabditis elegans causing endoplasmic reticulum (ER) stress and defects in chemotaxis during adulthood. We screened this model organism for compounds that alleviated neurotoxicity, and then tested these candidates for efficacy in mice., Methods: We screened compounds for alleviation of ER stress induction by isoflurane in C. elegans assayed by induction of a green fluorescent protein (GFP) reporter. Drugs that inhibited ER stress were screened for reduction of the anaesthetic-induced chemotaxis defect. Compounds that alleviated both aspects of neurotoxicity were then blindly tested for the ability to inhibit induction of caspase-3 by isoflurane in P7 mice., Results: Isoflurane increased ER stress indicated by increased GFP reporter fluorescence (240% increase, P<0.001). Nine compounds reduced induction of ER stress by isoflurane by 90-95% (P<0.001 in all cases). Of these compounds, tetraethylammonium chloride and trehalose also alleviated the isoflurane-induced defect in chemotaxis (trehalose by 44%, P=0.001; tetraethylammonium chloride by 23%, P<0.001). In mouse brain, tetraethylammonium chloride reduced isoflurane-induced caspase staining in the anterior cortical (-54%, P=0.007) and hippocampal regions (-46%, P=0.002)., Discussion: Tetraethylammonium chloride alleviated isoflurane-induced neurotoxicity in two widely divergent species, raising the likelihood that it may have therapeutic value. In C. elegans, ER stress predicts isoflurane-induced neurotoxicity, but is not its cause., (Copyright © 2021 British Journal of Anaesthesia. Published by Elsevier Ltd. All rights reserved.)

Dr. Anne Marie Lynn (1949-present), Professor Emeritus of Anesthesiology, Pain Medicine, and Pediatrics at the University of Washington, Seattle, was one of the most influential women in pediatric anesthesiology of her generation. Dr. Lynn embodies the spirit of discovery and advancement that have created the practice of pediatric anesthesiology as we know it today. A pioneer in pain medicine pharmacology, particularly morphine and ketorolac, her research transformed pediatric anesthesia, pediatric pain medicine, and pediatric intensive care medicine. Through her journal articles, book chapters, national and international lectures, mentoring of residents, fellows, and faculty, and leadership in the Society for Pediatric Anesthesia, she inspired a generation of women and men physicians by demonstrating that gender should not be a barrier to undertaking roles once only held only by men. In 2017, for her many contributions, she was awarded the Society for Pediatric Anesthesia's Myron Yaster lifetime achievement award., (© 2021 John Wiley & Sons Ltd.)

Background: Children with mitochondrial disease undergo anesthesia for a wide array of surgical procedures. However, multiple medications used for their perioperative care can affect mitochondrial function. Defects in function of the mitochondrial electron transport chain (ETC) can lead to a profound hypersensitivity to sevoflurane in children. We studied the sensitivities to sevoflurane, during mask induction and maintenance of general anesthesia, in children presenting for muscle biopsies for diagnosis of mitochondrial disease., Methods: In this multicenter study, 91 children, aged 6 months to 16 years, presented to the operating room for diagnostic muscle biopsy for presumptive mitochondrial disease. General anesthesia was induced by a slow increase of inhaled sevoflurane concentration. The primary end point, end-tidal (ET) sevoflurane necessary to achieve a bispectral index (BIS) of 60, was recorded. Secondary end points were maximal sevoflurane used to maintain a BIS between 40 and 60 during the case, and maximum and minimum heart rate and blood pressures. After induction, general anesthesia was maintained according to the preferences of the providers directing the cases. Primary data were analyzed comparing data from patients with complex I deficiencies to other groups using nonparametric statistics in SPSS v.27., Results: The median sevoflurane concentration to reach BIS of 60 during inductions (ET sevoflurane % [BIS = 60]) was significantly lower for patients with complex I defects (0.98%; 95% confidence interval [CI], 0.5-1.4) compared to complex II (1.95%; 95% CI, 1.2-2.7; P < .001), complex III (2.0%; 95% CI, 0.7-3.5; P < .001), complex IV (2.0%; 95% CI, 1.7-3.2; P < .001), and normal groups (2.2%; 95% CI, 1.8-3.0; P < .001). The sevoflurane sensitivities of complex I patients did not reach significance when compared to patients diagnosed with mitochondrial disease but without an identifiable ETC abnormality (P = .172). Correlation of complex I activity with ET sevoflurane % (BIS = 60) gave a Spearman's coefficient of 0.505 (P < .001). The differences in sensitivities between groups were less during the maintenance of the anesthetic than during induction., Conclusions: The data indicate that patients with complex I dysfunction are hypersensitive to sevoflurane compared to normal patients. Hypersensitivity was less common in patients presenting with other mitochondrial defects or without a mitochondrial diagnosis., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 International Anesthesia Research Society.)

Volatile anesthetics (VAs) are widely used in medicine, but the mechanisms underlying their effects remain ill-defined. Though routine anesthesia is safe in healthy individuals, instances of sensitivity are well documented, and there has been significant concern regarding the impact of VAs on neonatal brain development. Evidence indicates that VAs have multiple targets, with anesthetic and non-anesthetic effects mediated by neuroreceptors, ion channels, and the mitochondrial electron transport chain. Here, we characterize an unexpected metabolic effect of VAs in neonatal mice. Neonatal blood β-hydroxybutarate (β-HB) is rapidly depleted by VAs at concentrations well below those necessary for anesthesia. β-HB in adults, including animals in dietary ketosis, is unaffected. Depletion of β-HB is mediated by citrate accumulation, malonyl-CoA production by acetyl-CoA carboxylase, and inhibition of fatty acid oxidation. Adults show similar significant changes to citrate and malonyl-CoA, but are insensitive to malonyl-CoA, displaying reduced metabolic flexibility compared to younger animals., Competing Interests: JS, AF, RB, KS, JS, AP, GS, KP, SJ, HW, BJ, PM, MS No competing interests declared, SJ Reviewing editor, eLife, (© 2021, Stokes et al.)

Leigh Syndrome (LS) is a mitochondrial disorder defined by progressive focal neurodegenerative lesions in specific regions of the brain. Defects in NDUFS4, a subunit of complex I of the mitochondrial electron transport chain, cause LS in humans; the Ndufs4 knockout mouse (Ndufs4(KO)) closely resembles the human disease. Here, we probed brain region-specific molecular signatures in pre-symptomatic Ndufs4(KO) to identify factors which underlie focal neurodegeneration. Metabolomics revealed that free amino acid concentrations are broadly different by region, and glucose metabolites are increased in a manner dependent on both region and genotype. We then tested the impact of the mTOR inhibitor rapamycin, which dramatically attenuates LS in Ndufs4(KO), on region specific metabolism. Our data revealed that loss of Ndufs4 drives pathogenic changes to CNS glutamine/glutamate/α-ketoglutarate metabolism which are rescued by mTOR inhibition Finally, restriction of the Ndufs4 deletion to pre-synaptic glutamatergic neurons recapitulated the whole-body knockout. Together, our findings are consistent with mTOR inhibition alleviating disease by increasing availability of α-ketoglutarate, which is both an efficient mitochondrial complex I substrate in Ndufs4(KO) and an important metabolite related to neurotransmitter metabolism in glutamatergic neurons., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Routine general anesthesia is considered to be safe in healthy individuals. However, pre-clinical studies in mice, rats, and monkeys have repeatedly demonstrated that exposure to anesthetic agents during early post-natal periods can lead to acute neurotoxicity. More concerning, later-life defects in cognition, assessed by behavioral assays for learning and memory, have been reported. Although the potential for anesthetics to damage the neonatal brain is well-documented, the clinical significance of the pre-clinical models in which damage is induced remains quite unclear. Here, we systematically evaluate critical physiological parameters in post-natal day 7 neonatal mice exposed to 1.5% isoflurane for 2-4 hours, the most common anesthesia induced neurotoxicity paradigm in this animal model. We find that 2 or more hours of anesthesia exposure results in dramatic respiratory and metabolic changes that may limit interpretation of this paradigm to the clinical situation. Our data indicate that neonatal mouse models of AIN are not necessarily appropriate representations of human exposures., Competing Interests: The authors have declared that no competing interests exist.

What We Already Know About This Topic: In mice, restriction of loss of the mitochondrial complex I gene Ndufs4 to glutamatergic neurons confers a profound hypersensitivity to volatile anesthetics.Astrocytes are crucial to glutamatergic synapse functioning during excitatory transmission., What This Article Tells Us That Is New: In a tamoxifen-activated astrocyte-specific Ndufs4(KO) mouse, the induction EC50s for tail clamp in both isoflurane and halothane were similar between the control and astrocyte-specific Ndufs4(KO) mice at 3 weeks after 4-hydroxy tamoxifen injection. However, the emergent concentrations in both anesthetics for the astrocyte-specific Ndufs4(KO) mice were half that of the controls.Similarly, the induction EC50s for loss of righting reflex were similar between the control and astrocyte-specific Ndufs4(KO) mice; concentrations for regain of righting reflex in both anesthetics for the astrocyte-specific Ndufs4(KO) mice were much less than the control.Thus, mitochondrial complex I function within astrocytes is essential for normal emergence from anesthesia., Background: In mice, restriction of loss of the mitochondrial complex I gene Ndufs4 to glutamatergic neurons confers a profound hypersensitivity to volatile anesthetics similar to that seen with global genetic knockout of Ndufs4. Astrocytes are crucial to glutamatergic synapse functioning during excitatory transmission. Therefore, the authors examined the role of astrocytes in the anesthetic hypersensitivity of Ndufs4(KO)., Methods: A tamoxifen-activated astrocyte-specific Ndufs4(KO) mouse was constructed. The specificity of the astrocyte-specific inducible model was confirmed by using the green fluorescent protein reporter line Ai6. Approximately 120 astrocyte-specific knockout and control mice were used for the experiments. Mice were anesthetized with varying concentrations of isoflurane or halothane; loss of righting reflex and response to a tail clamp were determined and quantified as the induction and emergence EC50s. Because norepinephrine has been implicated in emergence from anesthesia and astrocytes respond to norepinephrine to release gliotransmitters, the authors measured norepinephrine levels in the brains of control and knockout Ndufs4 animals., Results: The induction EC50s for tail clamp in both isoflurane and halothane were similar between the control and astrocyte-specific Ndufs4(KO) mice at 3 weeks after 4-hydroxy tamoxifen injection (induction concentration, EC50(ind)-isoflurane: control = 1.27 ± 0.12, astrocyte-specific knockout = 1.21 ± 0.18, P = 0.495; halothane: control = 1.28 ± 0.05, astrocyte-specific knockout = 1.20 ± 0.05, P = 0.017). However, the emergent concentrations in both anesthetics for the astrocyte-specific Ndufs4(KO) mice were less than the controls for tail clamp; (emergence concentration, EC50(em)-isoflurane: control = 1.18 ± 0.10, astrocyte-specific knockout = 0.67 ± 0.11, P < 0.0001; halothane: control = 1.08 ± 0.09, astrocyte-specific knockout = 0.59 ± 0.12, P < 0.0001). The induction EC50s for loss of righting reflex were also similar between the control and astrocyte-specific Ndufs4(KO) mice (EC50(ind)-isoflurane: control = 1.02 ± 0.10, astrocyte-specific knockout = 0.97 ± 0.06, P = 0.264; halothane: control = 1.03 ± 0.05, astrocyte-specific knockout = 0.99 ± 0.08, P = 0.207). The emergent concentrations for loss of righting reflex in both anesthetics for the astrocyte-specific Ndufs4(KO) mice were less than the control (EC50(em)-isoflurane: control = 1.0 ± 0.07, astrocyte-specific knockout = 0.62 ± 0.12, P < 0.0001; halothane: control = 1.0 ± 0.04, astrocyte-specific KO = 0.64 ± 0.09, P < 0.0001); N ≥ 6 for control and astrocyte-specific Ndufs4(KO) mice. For all tests, similar results were seen at 7 weeks after 4-hydroxy tamoxifen injection. The total norepinephrine content of the brain in global or astrocyte-specific Ndufs4(KO) mice was unchanged compared to control mice., Conclusions: The only phenotype of the astrocyte-specific Ndufs4(KO) mouse was a specific impairment in emergence from volatile anesthetic-induced general anesthesia. The authors conclude that normal mitochondrial function within astrocytes is essential for emergence from anesthesia.

Mitochondrial diseases represent a significant clinical challenge. Substantial efforts have been devoted to identifying therapeutic strategies for mitochondrial disorders, but effective interventions have remained elusive. Recently, we reported attenuation of disease in a mouse model of the human mitochondrial disease Leigh syndrome through pharmacological inhibition of the mechanistic target of rapamycin (mTOR). The human mitochondrial disorder MELAS/MIDD (Mitochondrial Encephalopathy with Lactic Acidosis and Stroke-like Episodes/Maternally Inherited Diabetes and Deafness) shares many phenotypic characteristics with Leigh syndrome. MELAS/MIDD often leads to organ failure and transplantation and there are currently no effective treatments. To examine the therapeutic potential of mTOR inhibition in human mitochondrial disease, four kidney transplant recipients with MELAS/MIDD were switched from calcineurin inhibitors to mTOR inhibitors for immunosuppression. Primary fibroblast lines were generated from patient dermal biopsies and the impact of rapamycin was studied using cell-based end points. Metabolomic profiles of the four patients were obtained before and after the switch. pS6, a measure of mTOR signaling, was significantly increased in MELAS/MIDD cells compared to controls in the absence of treatment, demonstrating mTOR overactivation. Rapamycin rescued multiple deficits in cultured cells including mitochondrial morphology, mitochondrial membrane potential, and replicative capacity. Clinical measures of health and mitochondrial disease progression were improved in all four patients following the switch to an mTOR inhibitor. Metabolomic analysis was consistent with mitochondrial function improvement in all patients., (Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

What We Already Know About This Topic: WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Knockout of the mitochondrial protein Ndufs4 (Ndufs4[KO]) in mice causes hypersensitivity to volatile anesthetics but resistance to ketamine. The authors hypothesized that electrocorticographic changes underlying the responses of Ndufs4(KO) to volatile anesthetics and to ketamine would be similar in mutant and control mice., Methods: Electrocorticographic recordings at equipotent volatile anesthetic concentrations were compared between genotypes. In separate studies, control and cell type-specific Ndufs4(KO) mice were anesthetized with intraperitoneal ketamine to determine their ED50s., Results: Ndufs4 (KO) did not differ from controls in baseline electrocorticography (N = 5). Compared to baseline, controls exposed to isoflurane (EC50) lost power (expressed as mean baseline [µV/Hz]; mean isoflurane [µV/Hz]) in delta (2.45; 0.50), theta (1.41; 0.16), alpha (0.23; 0.05), beta (0.066; 0.016), and gamma (0.020; 0.005) frequency bands (N = 5). Compared to baseline, at their isoflurane EC50, Ndufs4(KO) maintained power in delta (1.08; 1.38), theta (0.36; 0.26), and alpha (0.09; 0.069) frequency bands but decreased in beta (0.041; 0.023) and gamma (0.020; 0.0068) frequency bands (N = 5). Similar results were seen for both genotypes in halothane. Vesicular glutamate transporter 2 (VGLUT2)-specific Ndufs4(KO) mice were markedly resistant to ketamine (ED50; 125 mg/kg) compared to control mice (ED50; 75 mg/kg; N = 6). At their respective ED95s for ketamine, mutant (N = 5) electrocorticography spectra showed a decrease in power in the beta (0.040; 0.020) and gamma (0.035; 0.015) frequency bands not seen in controls (N = 7)., Conclusions: Significant differences exist between the electrocorticographies of mutant and control mice at equipotent doses for volatile anesthetics and ketamine. The energetic state specifically of excitatory neurons determines the behavioral response to ketamine.

Background: The mechanisms of action of volatile anaesthetics are unclear. Volatile anaesthetics selectively inhibit complex I in the mitochondrial respiratory chain. Mice in which the mitochondrial complex I subunit NDUFS4 is knocked out [Ndufs4(KO)] either globally or in glutamatergic neurons are hypersensitive to volatile anaesthetics. The volatile anaesthetic isoflurane selectively decreases the frequency of spontaneous excitatory events in hippocampal slices from Ndufs4(KO) mice., Methods: Complex I inhibition by isoflurane was assessed with a Clark electrode. Synaptic function was measured by stimulating Schaffer collateral fibres and recording field potentials in the hippocampus CA1 region., Results: Isoflurane specifically inhibits complex I dependent respiration at lower concentrations in mitochondria from Ndufs4(KO) than from wild-type mice. In hippocampal slices, after high frequency stimulation to increase energetic demand, short-term synaptic potentiation is less in KO compared with wild-type mice. After high frequency stimulation, both Ndufs4(KO) and wild-type hippocampal slices exhibit striking synaptic depression in isoflurane at twice the 50% effective concentrations (EC 50 ). The pattern of synaptic depression by isoflurane indicates a failure in synaptic vesicle recycling. Application of a selective A 1 adenosine receptor antagonist partially eliminates isoflurane-induced short-term depression in both wild-type and Ndufs4(KO) slices, implicating an additional mitochondria-dependent effect on exocytosis. When mitochondria are the sole energy source, isoflurane completely eliminates synaptic output in both mutant and wild-type mice at twice the (EC 50 ) for anaesthesia., Conclusions: Volatile anaesthetics directly inhibit mitochondrial complex I as a primary target, limiting synaptic ATP production, and excitatory vesicle endocytosis and exocytosis., (Copyright © 2018 British Journal of Anaesthesia. Published by Elsevier Ltd. All rights reserved.)

It is difficult to study the genetics and molecular mechanisms of anesthesia in humans. Fortunately, the genetic approaches in model organisms can, and have, led to profound insights as to the targets of anesthetics. In turn, the organization of these putative targets into meaningful pathways has begun to elucidate the mechanisms of action of these agents. However, it is important to first appreciate the strengths, and limitations, of genetic approaches to understand the anesthetic action. Here we compare the commonly used genetic model organisms, various anesthetic endpoints, and different modes of genetic screens. Coupled with the more specific data presented in subsequent chapters, this chapter places those results in a framework with which to analyze the discoveries across organisms and eventually extend the resulting models to humans., (© 2018 Elsevier Inc. All rights reserved.)

Knockout of the mitochondrial complex I protein, NDUFS4, profoundly increases sensitivity of mice to volatile anesthetics. In mice carrying an Ndufs4lox/lox gene, adeno-associated virus expressing Cre recombinase was injected into regions of the brain postulated to affect sensitivity to volatile anesthetics. These injections generated otherwise phenotypically wild type mice with region-specific, postnatal inactivation of Ndufs4, minimizing developmental effects of gene loss. Sensitivities to the volatile anesthetics isoflurane and halothane were measured using loss of righting reflex (LORR) and movement in response to tail clamp (TC) as endpoints. Knockdown (KD) of Ndufs4 in the vestibular nucleus produced resistance to both anesthetics for movement in response to TC. Ndufs4 loss in the central and dorsal medial thalami and in the parietal association cortex increased anesthetic sensitivity to both TC and LORR. Knockdown of Ndufs4 only in the parietal association cortex produced striking hypersensitivity for both endpoints, and accounted for half the total change seen in the global KO (Ndufs4(KO)). Excitatory synaptic transmission in the parietal association cortex in slices from Ndufs4(KO) animals was hypersensitive to isoflurane compared to control slices. We identified a direct neural circuit between the parietal association cortex and the central thalamus, consistent with a model in which isoflurane sensitivity is mediated by a thalamic signal relayed through excitatory synapses to the parietal association cortex. We postulate that the thalamocortical circuit is crucial for maintenance of consciousness and is disrupted by the inhibitory effects of isoflurane/halothane on mitochondria.

Mitochondria are best known for harboring pathways involved in ATP synthesis through the tricarboxylic acid cycle and oxidative phosphorylation. Major advances in understanding these roles were made with C aenorhabditis elegans mutants affecting key components of the metabolic pathways. These mutants have not only helped elucidate some of the intricacies of metabolism pathways, but they have also served as jumping off points for pharmacology, toxicology, and aging studies. The field of mitochondria research has also undergone a renaissance, with the increased appreciation of the role of mitochondria in cell processes other than energy production. Here, we focus on discoveries that were made using C. elegans , with a few excursions into areas that were studied more thoroughly in other organisms, like mitochondrial protein import in yeast. Advances in mitochondrial biogenesis and membrane dynamics were made through the discoveries of novel functions in mitochondrial fission and fusion proteins. Some of these functions were only apparent through the use of diverse model systems, such as C . elegans Studies of stress responses, exemplified by mitophagy and the mitochondrial unfolded protein response, have also benefitted greatly from the use of model organisms. Recent developments include the discoveries in C. elegans of cell autonomous and nonautonomous pathways controlling the mitochondrial unfolded protein response, as well as mechanisms for degradation of paternal mitochondria after fertilization. The evolutionary conservation of many, if not all, of these pathways ensures that results obtained with C. elegans are equally applicable to studies of human mitochondria in health and disease., (Copyright © 2017 by the Genetics Society of America.)

Mitochondrial dysfunction can increase oxidative stress and extend lifespan in Caenorhabditis elegans. Homeostatic mechanisms exist to cope with disruptions to mitochondrial function that promote cellular health and organismal longevity. Previously, we determined that decreased expression of the cytosolic pentose phosphate pathway (PPP) enzyme transaldolase activates the mitochondrial unfolded protein response (UPRmt) and extends lifespan. Here we report that transaldolase (tald-1) deficiency impairs mitochondrial function in vivo, as evidenced by altered mitochondrial morphology, decreased respiration, and increased cellular H2O2 levels. Lifespan extension from knockdown of tald-1 is associated with an oxidative stress response involving p38 and c-Jun N-terminal kinase (JNK) MAPKs and a starvation-like response regulated by the transcription factor EB (TFEB) homolog HLH-30. The latter response promotes autophagy and increases expression of the flavin-containing monooxygenase 2 (fmo-2). We conclude that cytosolic redox established through the PPP is a key regulator of mitochondrial function and defines a new mechanism for mitochondrial regulation of longevity.

Introduction: Neurotoxicity induced by early developmental exposure to volatile anesthetics is a characteristic of organisms across a wide range of species, extending from the nematode C. elegans to mammals. Prevention of anesthetic-induced neurotoxicity (AIN) will rely upon an understanding of its underlying mechanisms. However, no forward genetic screens have been undertaken to identify the critical pathways affected in AIN. By characterizing such pathways, we may identify mechanisms to eliminate isoflurane induced AIN in mammals., Methods: Chemotaxis in adult C. elegans after larval exposure to isoflurane was used to measure AIN. We initially compared changes in chemotaxis indices between classical mutants known to affect nervous system development adding mutants in response to data. Activation of specific genes was visualized using fluorescent markers. Animals were then treated with rapamycin or preconditioned with isoflurane to test effects on AIN., Results: Forty-four mutations, as well as pharmacologic manipulations, identified two pathways, highly conserved from invertebrates to humans, that regulate AIN in C. elegans. Activation of one stress-protective pathway (DAF-2 dependent) eliminates AIN, while activation of a second stress-responsive pathway (endoplasmic reticulum (ER) associated stress) causes AIN. Pharmacologic inhibition of the mechanistic Target of Rapamycin (mTOR) blocks ER-stress and AIN. Preconditioning with isoflurane prior to larval exposure also inhibited AIN., Discussion: Our data are best explained by a model in which isoflurane acutely inhibits mitochondrial function causing activation of responses that ultimately lead to ER-stress. The neurotoxic effect of isoflurane can be completely prevented by manipulations at multiple points in the pathways that control this response. Endogenous signaling pathways can be recruited to protect organisms from the neurotoxic effects of isoflurane., (Copyright © 2016 Elsevier Inc. All rights reserved.)

An enigma of modern medicine has persisted for over 150 years. The mechanisms by which volatile anesthetics (VAs) produce their effects (loss of consciousness, analgesia, amnesia, and immobility) remain an unsolved mystery. Many attractive putative molecular targets have failed to produce a significant effect when genetically tested in whole-animal models [1-3]. However, mitochondrial defects increase VA sensitivity in diverse organisms from nematodes to humans [4-6]. Ndufs4 knockout (KO) mice lack a subunit of mitochondrial complex I and are strikingly hypersensitive to VAs yet resistant to the intravenous anesthetic ketamine [7]. The change in VA sensitivity is the largest reported for a mammal. Limiting NDUFS4 loss to a subset of glutamatergic neurons recapitulates the VA hypersensitivity of Ndufs4(KO) mice, while loss in GABAergic or cholinergic neurons does not. Baseline electrophysiologic function of CA1 pyramidal neurons does not differ between Ndufs4(KO) and control mice. Isoflurane concentrations that anesthetize only Ndufs4(KO) mice (0.6%) decreased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) only in Ndufs4(KO) CA1 neurons, while concentrations effective in control mice (1.2%) decreased sEPSC frequencies in both control and Ndufs4(KO) CA1 pyramidal cells. Spontaneous inhibitory postsynaptic currents (sIPSCs) were not differentially affected between genotypes. The effects of isoflurane were similar on evoked field excitatory postsynaptic potentials (fEPSPs) and paired pulse facilitation (PPF) in KO and control hippocampal slices. We propose that CA1 presynaptic excitatory neurotransmission is hypersensitive to isoflurane in Ndufs4(KO) mice due to the inhibition of pre-existing reduced complex I function, reaching a critical reduction that can no longer meet metabolic demands., Competing Interests: The authors declare no competing financial interests., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Lack of NDUFS4, a subunit of mitochondrial complex I (NADH:ubiquinone oxidoreductase), causes Leigh syndrome (LS), a progressive encephalomyopathy. Knocking out Ndufs4, either systemically or in brain only, elicits LS in mice. In patients as well as in KO mice distinct regions of the brain degenerate while surrounding tissue survives despite systemic complex I dysfunction. For the understanding of disease etiology and ultimately for the development of rationale treatments for LS, it appears important to uncover the mechanisms that govern focal neurodegeneration., Results: Here we used the Ndufs4(KO) mouse to investigate whether regional and temporal differences in respiratory capacity of the brain could be correlated with neurodegeneration. In the KO the respiratory capacity of synaptosomes from the degeneration prone regions olfactory bulb, brainstem and cerebellum was significantly decreased. The difference was measurable even before the onset of neurological symptoms. Furthermore, neither compensating nor exacerbating changes in glycolytic capacity of the synaptosomes were found. By contrast, the KO retained near normal levels of synaptosomal respiration in the degeneration-resistant/resilient "rest" of the brain. We also investigated non-synaptic mitochondria. The KO expectedly had diminished capacity for oxidative phosphorylation (state 3 respiration) with complex I dependent substrate combinations pyruvate/malate and glutamate/malate but surprisingly had normal activity with α-ketoglutarate/malate. No correlation between oxidative phosphorylation (pyruvate/malate driven state 3 respiration) and neurodegeneration was found: Notably, state 3 remained constant in the KO while in controls it tended to increase with time leading to significant differences between the genotypes in older mice in both vulnerable and resilient brain regions. Neither regional ROS damage, measured as HNE-modified protein, nor regional complex I stability, assessed by blue native gels, could explain regional neurodegeneration., Conclusion: Our data suggests that locally insufficient respiration capacity of the nerve terminals may drive focal neurodegeneration.

To understand primary mitochondrial disease, we utilized a complex I-deficient Caenorhabditis elegans mutant, gas-1. These animals strongly upregulate the expression of gst-14 (encoding a glutathione S-transferase). Knockdown of gst-14 dramatically extends the lifespan of gas-1 and increases hydroxynonenal (HNE) modified mitochondrial proteins without improving complex I function. We observed no change in reactive oxygen species levels as measured by Mitosox staining, consistent with a potential role of GST-14 in HNE clearance. The upregulation of gst-14 in gas-1 animals is specific to the pharynx. These data suggest that an HNE-mediated response in the pharynx could be beneficial for lifespan extension in the context of complex I dysfunction in C. elegans. Thus, whereas HNE is typically considered damaging, our work is consistent with recent reports of its role in signaling, and that in this case, the signal is pro-longevity in a model of mitochondrial dysfunction., Competing Interests: The authors declare no competing financial interests., (Published by Elsevier Ireland Ltd.)

Mitochondria play an important role in numerous diseases as well as normative aging. Severe reduction in mitochondrial function contributes to childhood disorders such as Leigh Syndrome, whereas mild disruption can extend the lifespan of model organisms. The Caenorhabditis elegans isp-1 gene encodes the Rieske iron-sulfur protein subunit of cytochrome c oxidoreductase (complex III of the electron transport chain). The partial loss of function allele, isp-1(qm150), leads to several pleiotropic phenotypes. To better understand the molecular mechanisms of ISP-1 function, we sought to identify genetic suppressors of the delayed development of isp-1(qm150) animals. Here we report a series of intragenic suppressors, all located within a highly conserved six amino acid tether region of ISP-1. These intragenic mutations suppress all of the evaluated isp-1(qm150) phenotypes, including developmental rate, pharyngeal pumping rate, brood size, body movement, activation of the mitochondrial unfolded protein response reporter, CO2 production, mitochondrial oxidative phosphorylation, and lifespan extension. Furthermore, analogous mutations show a similar effect when engineered into the budding yeast Rieske iron-sulfur protein Rip1, revealing remarkable conservation of the structure-function relationship of these residues across highly divergent species. The focus on a single subunit as causal both in generation and in suppression of diverse pleiotropic phenotypes points to a common underlying molecular mechanism, for which we propose a "spring-loaded" model. These observations provide insights into how gating and control processes influence the function of ISP-1 in mediating pleiotropic phenotypes including developmental rate, movement, sensitivity to stress, and longevity.

Single-gene mutations that disrupt mitochondrial respiratory chain function in Caenorhabditis elegans change patterns of protein expression and metabolites. Our goal was to develop useful molecular fingerprints employing adaptable techniques to recognize mitochondrial defects in the electron transport chain. We analyzed mutations affecting complex I, complex II, or ubiquinone synthesis and discovered overarching patterns in the response of C. elegans to mitochondrial dysfunction across all of the mutations studied. These patterns are in KEGG pathways conserved from C. elegans to mammals, verifying that the nematode can serve as a model for mammalian disease. In addition, specific differences exist between mutants that may be useful in diagnosing specific mitochondrial diseases in patients., (Copyright © 2014 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Simple multicellular animal model systems are central to studying the complex mechanisms underlying a bewildering array of diseases involving dysfunctional mitochondria. Mutant nuclear- and mitochondrial-encoded subunits of the Caenorhabditis elegans mitochondrial respiratory chain (MRC) have been investigated, including GAS-1, NUO-1, NUO-6, MEV-1, SDHB-1, CLK-1, ISP-1, CTB-1, and ATP-2. These, as well as proteins that modify the MRC indirectly, have been studied on the molecular, cellular, and organismal levels through the variety of experimental approaches that are readily achievable in C. elegans. In C. elegans, MRC dysfunction can mimic signs and symptoms observed in human patients with primary mitochondrial disorders, such as neuromuscular deficits, developmental delay, altered anesthetic sensitivity, and increased lactate levels. Antioxidant dietary supplements, coenzyme Q substitutes, and flavin cofactors have been explored as potential therapeutic strategies. Furthermore, mutants with altered longevity have proved useful for probing the contributions of bioenergetics, reactive oxygen species, and stress responses to the process of aging. C. elegans will undoubtedly continue to provide a useful system in which to explore unanswered questions in mitochondrial biology and disease.

Mutations affecting mitochondrial complex I, a multi-subunit assembly that couples electron transfer to proton pumping, are the most frequent cause of heritable mitochondrial diseases. However, the mechanisms by which complex I dysfunction results in disease remain unclear. Here, we describe a Drosophila model of complex I deficiency caused by a homoplasmic mutation in the mitochondrial-DNA-encoded NADH dehydrogenase subunit 2 (ND2) gene. We show that ND2 mutants exhibit phenotypes that resemble symptoms of mitochondrial disease, including shortened lifespan, progressive neurodegeneration, diminished neural mitochondrial membrane potential and lower levels of neural ATP. Our biochemical studies of ND2 mutants reveal that complex I is unable to efficiently couple electron transfer to proton pumping. Thus, our study provides evidence that the ND2 subunit participates directly in the proton pumping mechanism of complex I. Together, our findings support the model that diminished respiratory chain activity, and consequent energy deficiency, are responsible for the pathogenesis of complex-I-associated neurodegeneration., (© 2014. Published by The Company of Biologists Ltd.)

How an organism ages is a question that has fascinated biologists, and the elderly, for centuries. One useful approach to understanding complex processes such as aging is to study genetic variation in model organisms such as the nematode, Caenorhabditis elegans. Classical mutant strains and RNAi screens have demonstrated that mitochondrial function is a major factor affecting longevity. Recent advances in the biochemical methods for studying mitochondrial functions have extended the usefulness of C. elegans for deciphering the molecular mechanisms by which mitochondria determine lifespan. Defects of all complexes in the mitochondrial respiratory chain have been described and have varied effects on lifespan. The phenotypes of these mutants indicate that the locality, production rate, and/or steady-state level of reactive oxygen species (ROS) is a defining lifespan-determining factor in these mutants. Mutants of enzymes involved in ROS scavenging have also been described, such as mitochondrial superoxide dismutases, and reveal a complex connection between ROS and lifespan. Energy balance, transcriptional signaling pathways, stress tolerance, and metabolic restructuring are also tied to ROS, and may also play roles in the mutants' altered lifespans. In this review, we discuss how findings with C. elegans genetics extend our understanding of the contributions of the mitochondrial respiratory chain and ROS to the process of aging., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Anesthetics used in infants and children are implicated in the development of neurocognitive disorders. Although propofol induces neuroapoptosis in developing brain, the underlying mechanisms require elucidation and may have an energetic basis. We studied substrate utilization in immature swine anesthetized with either propofol or isoflurane for 4 hours. Piglets were infused with 13-Carbon-labeled glucose and leucine in the common carotid artery to assess citric acid cycle (CAC) metabolism in the parietal cortex. The anesthetics produced similar systemic hemodynamics and cerebral oxygen saturation by near-infrared spectroscopy. Compared with isoflurane, propofol depleted ATP and glycogen stores. Propofol decreased pools of the CAC intermediates, citrate, and α-ketoglutarate, while markedly increasing succinate along with decreasing mitochondrial complex II activity. Propofol also inhibited acetyl-CoA entry into the CAC through pyruvate dehydrogenase, while promoting glycolytic flux with marked lactate accumulation. Although oxygen supply appeared similar between the anesthetic groups, propofol yielded a metabolic phenotype that resembled a hypoxic state. Propofol impairs substrate flux through the CAC in the immature cerebral cortex. These impairments occurred without systemic metabolic perturbations that typically accompany propofol infusion syndrome. These metabolic abnormalities may have a role in the neurotoxity observed with propofol in the vulnerable immature brain.

Mitochondrial dysfunction contributes to numerous health problems, including neurological and muscular degeneration, cardiomyopathies, cancer, diabetes, and pathologies of aging. Severe mitochondrial defects can result in childhood disorders such as Leigh syndrome, for which there are no effective therapies. We found that rapamycin, a specific inhibitor of the mechanistic target of rapamycin (mTOR) signaling pathway, robustly enhances survival and attenuates disease progression in a mouse model of Leigh syndrome. Administration of rapamycin to these mice, which are deficient in the mitochondrial respiratory chain subunit Ndufs4 [NADH dehydrogenase (ubiquinone) Fe-S protein 4], delays onset of neurological symptoms, reduces neuroinflammation, and prevents brain lesions. Although the precise mechanism of rescue remains to be determined, rapamycin induces a metabolic shift toward amino acid catabolism and away from glycolysis, alleviating the buildup of glycolytic intermediates. This therapeutic strategy may prove relevant for a broad range of mitochondrial diseases.

The processes that control aging remain poorly understood. We have exploited mutants in the nematode, Caenorhabditis elegans, that compromise mitochondrial function and scavenging of reactive oxygen species (ROS) to understand their relation to lifespan. We discovered unanticipated roles and interactions of the mitochondrial superoxide dismutases (mtSODs): SOD-2 and SOD-3. Both SODs localize to mitochondrial supercomplex I:III:IV. Loss of SOD-2 specifically (i) decreases the activities of complexes I and II, complexes III and IV remain normal; (ii) increases the lifespan of animals with a complex I defect, but not the lifespan of animals with a complex II defect, and kills an animal with a complex III defect; (iii) induces a presumed pro-inflammatory response. Knockdown of a molecule that may be a pro-inflammatory mediator very markedly extends lifespan and health of certain mitochondrial mutants. The relationship between the electron transport chain, ROS, and lifespan is complex, and defects in mitochondrial function have specific interactions with ROS scavenging mechanisms. We conclude that mtSODs are embedded within the supercomplex I:III:IV and stabilize or locally protect it from reactive oxygen species (ROS) damage. The results call for a change in the usual paradigm for the interaction of electron transport chain function, ROS release, scavenging, and compensatory responses., (© 2013 the Anatomical Society and John Wiley & Sons Ltd.)

Mitochondrial disease, once thought to be a rare clinical entity, is now recognized as an important cause of a wide range of neurologic, cardiac, muscle, and endocrine disorders . The incidence of disorders of the respiratory chain alone is estimated to be about 1 per 4-5000 live births, similar to that of more well-known neurologic diseases . High-energy requiring tissues are uniquely dependent on the energy delivered by mitochondria and therefore have the lowest threshold for displaying symptoms of mitochondrial disease. Thus, mitochondrial dysfunction most commonly affects function of the central nervous system, the heart and the muscular system . Mutations in mitochondrial proteins cause striking clinical features in those tissues types, including encephalopathies, seizures, cerebellar ataxias, cardiomyopathies, myopathies, as well as gastrointestinal and hepatic disease. Our knowledge of the contribution of mitochondria in causing disease or influencing aging is expanding rapidly . As diagnosis and treatment improve for children with mitochondrial diseases, it has become increasingly common for them to undergo surgeries for their long-term care. In addition, often a muscle biopsy or other tests needing anesthesia are required for diagnosis. Mitochondrial disease represents probably hundreds of different defects, both genetic and environmental in origin, and is thus difficult to characterize. The specter of possible delayed complications in patients caused by inhibition of metabolism by anesthetics, by remaining in a biochemically stressed state such as fasting/catabolism, or by prolonged exposure to pain is a constant worry to physicians caring for these patients. Here, we review the considerations when caring for a patient with mitochondrial disease., (© 2013 John Wiley & Sons Ltd.)

Mitochondrial respiratory dysfunction is linked to the pathogenesis of multiple diseases, including heart failure, but the specific mechanisms for this link remain largely elusive. We modeled the impairment of mitochondrial respiration by the inactivation of the Ndufs4 gene, a protein critical for complex I assembly, in the mouse heart (cKO). Although complex I-supported respiration decreased by >40%, the cKO mice maintained normal cardiac function in vivo and high-energy phosphate content in isolated perfused hearts. However, the cKO mice developed accelerated heart failure after pressure overload or repeated pregnancy. Decreased NAD(+)/NADH ratio by complex I deficiency inhibited Sirt3 activity, leading to an increase in protein acetylation and sensitization of the permeability transition in mitochondria (mPTP). NAD(+) precursor supplementation to cKO mice partially normalized the NAD(+)/NADH ratio, protein acetylation, and mPTP sensitivity. These findings describe a mechanism connecting mitochondrial dysfunction to the susceptibility to diseases and propose a potential therapeutic target., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Mounting evidence from animal studies shows that anesthetic exposure in early life leads to apoptosis in the developing nervous system. This loss of neurons has functional consequences in adulthood. Clinical retrospective reviews have suggested that multiple anesthetic exposures in early childhood are associated with learning disabilities later in life as well. Despite much concern about this phenomenon, little is known about the mechanism by which anesthetics initiate neuronal cell death. Caenorhabditis elegans, a powerful genetic animal model, with precisely characterized neural development and cell death pathways, affords an excellent opportunity to study anesthetic-induced neurotoxicity. We hypothesized that exposing the nematode to volatile anesthetics early in life would induce neuron cell death, producing a behavioral defect that would be manifested in adulthood., Methods: After synchronization and hatching, larval worms were exposed to volatile anesthetics at their 95% effective concentration for 4 hours. On day 4 of life, exposed and control worms were tested for their ability to sense and move to an attractant (i.e., to chemotax). We determined the rate of successful chemotaxis using a standardized chemotaxis index., Results: Wild-type nematodes demonstrated striking deficits in chemotaxis indices after exposure to isoflurane (ISO) or sevoflurane (SEVO) in the first larval stage (chemotaxis index: untreated, 85 ± 2; ISO, 52 ± 2; SEVO, 47 ± 2; P < 0.05 for both exposures). The mitochondrial mutant gas-1 had a heightened effect from the anesthetic exposure (chemotaxis index: untreated, 71 ± 2; ISO, 29 ± 12; SEVO, 24 ± 13; P < 0.05 for both exposures). In contrast, animals unable to undergo apoptosis because of a mutation in the pathway that mediates programmed cell death (ced-3) retained their ability to sense and move toward an attractant (chemotaxis index: untreated, 76 ± 10; ISO, 73 ± 9; SEVO, 76 ± 10). Furthermore, we discovered that the window of greatest susceptibility to anesthetic neurotoxicity in nematodes occurs in the first larval stage after hatching (L1). This coincides with a period of neurogenesis in this model. All values are means ± SD., Conclusion: These data indicate that anesthetics affect neurobehavior in nematodes, extending the range of phyla in which early exposure to volatile anesthetics has been shown to cause functional neurological deficits. This implies that anesthetic-induced neurotoxicity occurs via an ancient underlying mechanism. C elegans is a tractable model organism with which to survey an entire genome for molecules that mediate the toxic effects of volatile anesthetics on the developing nervous system.

One hundred and sixty five years have passed since the first documented use of volatile anesthetics to aid in surgery, but we have yet to understand the underlying mechanism of action of these drugs. There is no question that, in vitro, volatile anesthetics can affect the function of numerous neuronal and non-neuronal proteins. In fact, volatile anesthetics are capable of binding such diverse proteins as albumin and bacterial luciferase. The promiscuity of volatile anesthetic binding makes it difficult to determine which proteins are modulated by anesthetics to cause the state of anesthesia. Consequently, despite a great deal of in vitro data, the fundamental physiological process that volatile anesthetics perturb to effect neuronal silencing is not yet identified. Recently, data has increasingly indicated that membrane leak channels may play a role in the anesthetic response. Here we comment on the use of optogenetics to further support such a model.

Anesthetics are in routine use, yet the mechanisms underlying their function are incompletely understood. Studies in vitro demonstrate that both GABA(A) and NMDA receptors are modulated by anesthetics, but whole animal models have not supported the role of these receptors as sole effectors of general anesthesia. Findings in C. elegans and in children reveal that defects in mitochondrial complex I can cause hypersensitivity to volatile anesthetics. Here, we tested a knockout (KO) mouse with reduced complex I function due to inactivation of the Ndufs4 gene, which encodes one of the subunits of complex I. We tested these KO mice with two volatile and two non-volatile anesthetics. KO and wild-type (WT) mice were anesthetized with isoflurane, halothane, propofol or ketamine at post-natal (PN) days 23 to 27, and tested for loss of response to tail clamp (isoflurane and halothane) or loss of righting reflex (propofol and ketamine). KO mice were 2.5 - to 3-fold more sensitive to isoflurane and halothane than WT mice. KO mice were 2-fold more sensitive to propofol but resistant to ketamine. These changes in anesthetic sensitivity are the largest recorded in a mammal.