Interferon-gamma contributes to disease progression in the Ndufs4(-/-) model of Leigh syndrome.

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.

Potassium Leak Channels and Mitochondrial Complex I Interact in Glutamatergic Interneurons of the Mouse Spinal Cord.

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.

Glutamine metabolism in diseases associated with mitochondrial dysfunction.

Mitochondrial dysfunction can arise from genetic defects or environmental exposures and impact a wide range of biological processes. Among these are metabolic pathways involved in glutamine catabolism, anabolism, and glutamine-glutamate cycling. In recent years, altered glutamine metabolism has been found to play important roles in the pathologic consequences of mitochondrial dysfunction. Glutamine is a pleiotropic molecule, not only providing an alternate carbon source to glucose in certain conditions, but also playing unique roles in cellular communication in neurons and astrocytes. Glutamine consumption and catabolic flux can be significantly altered in settings of genetic mitochondrial defects or exposure to mitochondrial toxins, and alterations to glutamine metabolism appears to play a particularly significant role in neurodegenerative diseases. These include primary mitochondrial diseases like Leigh syndrome (subacute necrotizing encephalopathy) and MELAS (mitochondrial myopathy with encephalopathy, lactic acidosis, and stroke-like episodes), as well as complex age-related neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Pharmacologic interventions targeting glutamine metabolizing and catabolizing pathways appear to provide some benefits in cell and animal models of these diseases, indicating glutamine metabolism may be a clinically relevant target. In this review, we discuss glutamine metabolism, mitochondrial disease, the impact of mitochondrial dysfunction on glutamine metabolic processes, glutamine in neurodegeneration, and candidate targets for therapeutic intervention.

Impact of dietary ketosis on volatile anesthesia toxicity in a model of Leigh syndrome.

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.

TREK-1 and TREK-2 Knockout Mice Are Not Resistant to Halothane or Isoflurane.

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.

Volatile anaesthetic toxicity in the genetic mitochondrial disease Leigh syndrome.

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 (O2 100%) or mock (air) exposures (lifespan after 30-min exposures ∗P<0.05 for isoflurane 0.4% vs air or vs O2, ∗∗P<0.005 for isoflurane 0.6% vs air or O2; 60-min exposures ∗∗P<0.005 for isoflurane 0.2% vs air, ∗P<0.05 for isoflurane 0.2% vs O2). 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% O2 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.

Tetraethylammonium chloride reduces anaesthetic-induced neurotoxicity in Caenorhabditis elegans and mice.

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.

Mitochondrial Function and Anesthetic Sensitivity in the Mouse Spinal Cord.

BACKGROUND: Ndufs4 knockout (KO) mice are defective in mitochondrial complex I function and hypersensitive to inhibition of spinal cord-mediated response to noxious stimuli by volatile anesthetics. It was hypothesized that, compared to wild-type, synaptic or intrinsic neuronal function is hypersensitive to isoflurane in spinal cord slices from knockout mice. METHODS: Neurons from slices of the vestibular nucleus, central medial thalamus, and spinal cord from wild-type and the global Ndufs4 knockout were patch clamped. Unstimulated synaptic and intrinsic neuronal characteristics were measured in response to isoflurane. Norfluoxetine was used to block TREK channel conductance. Cholinergic cells were labeled with tdTomato. RESULTS: All values are reported as means and 95% CIs. Spontaneous synaptic activities were not different between the mutant and control. Isoflurane (0.6%; 0.25 mM; Ndufs4[KO] EC95) increased the holding current in knockout (ΔHolding current, 126 pA [95% CI, 99 to 152 pA]; ΔHolding current P < 0.001; n = 21) but not wild-type (ΔHolding current, 2 7 pA [95% CI, 9 to 47 pA]; ΔHolding current, P = 0.030; n = 25) spinal cord slices. Knockout and wild-type ΔHolding currents were significantly different (P < 0.001). Changes comparable to those in the knockout were seen in the wild type only in 1.8% (0.74 mM) isoflurane (ΔHolding current, 72 pA [95% CI, 43 to 97 pA]; ΔHolding current, P < 0.001; n = 13), the control EC95. Blockade of action potentials indicated that the increased holding current in the knockout was not dependent on synaptic input (ΔHolding current, 154 pA [95% CI, 99 to 232 pA]; ΔHolding current, P = 0.506 compared to knockout without blockade; n = 6). Noncholinergic neurons mediated the increase in holding current sensitivity in Ndufs4 knockout. The increased currents were blocked by norfluoxetine. CONCLUSIONS: Isoflurane increased an outwardly rectifying potassium current in ventral horn neurons of the Ndufs4(KO) mouse at a concentration much lower than in controls. Noncholinergic neurons in the spinal cord ventral horn mediated the effect. Presynaptic functions in Ndufs4(KO) slices were not hypersensitive to isoflurane. These data link anesthetic sensitivity, mitochondrial function, and postsynaptic channel activity.

Anesthetic Hypersensitivity in a Case-Controlled Series of Patients With Mitochondrial Disease.

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.

Regional metabolic signatures in the Ndufs4(KO) mouse brain implicate defective glutamate/α-ketoglutarate metabolism in mitochondrial disease.

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.

mTOR inhibitors may benefit kidney transplant recipients with mitochondrial diseases.

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.

Mitochondrial Function in Astrocytes Is Essential for Normal Emergence from Anesthesia in Mice.

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.

Anesthetics Have Different Effects on the Electrocorticographic Spectra of Wild-type and Mitochondrial Mutant Mice.

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.

Tether mutations that restore function and suppress pleiotropic phenotypes of the C. elegans isp-1(qm150) Rieske iron-sulfur protein.

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.

Early developmental exposure to volatile anesthetics causes behavioral defects in Caenorhabditis elegans.

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.

Mitochondrial DNA copy number and oxidative DNA damage in placental tissues from gestational diabetes and control pregnancies: a pilot study.

BACKGROUND: Available evidence supports the role of reactive oxygen species in the pathogenesis of placental insufficiency, gestational diabetes mellitus (GDM), and other pregnancy complications. Abnormal placental mitochondrial function resulting from reactive oxygen species may also be an important precedent of adverse perinatal outcomes. METHODS: We investigated the association of placental oxidative stress with placental mitochondrial DNA (mtDNA) copy number, an indicator of placental mitochondrial density and possible mitochondrial dysfunction, using samples collected from GDM cases and controls. 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidative DNA damage, was measured in placentas of 19 GDM cases and 21 controls using a competitive immunoassay. Placental mtDNA copy number was determined using real-time quantitative PCR. Bivariate and multivariable linear regression procedures were used to evaluate associations of these two biomarkers. RESULTS: Placental DNA oxidation was positively associated with mtDNA copy number in both GDM and control placentas. After adjusting for maternal age, pre-pregnancy body mass index and gestational age at delivery, mtDNA copy number increased (beta = 67.0; 95% CI 27.8 - 106.2, p = 0.001) for every 0.1 ng/microg increase of placental 8-OHdG among GDM cases and controls. CONCLUSIONS: These cross sectional data suggest an association of placental mtDNA copy number with oxidative stress. The consequences of placental oxidative stress and mitochondrial dysfunction on the course and outcomes of pregnancy remain to be elucidated in larger prospective studies.

A primordial target: Mitochondria mediate both primary and collateral anesthetic effects of volatile anesthetics.

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).

Peripheral macrophages drive CNS disease in the Ndufs4(-/-) model of Leigh syndrome.

Subacute necrotizing encephalopathy, or Leigh syndrome (LS), is the most common pediatric presentation of genetic mitochondrial disease. LS is a multi-system disorder with severe neurologic, metabolic, and musculoskeletal symptoms. The presence of progressive, symmetric, and necrotizing lesions in the brainstem are a defining feature of the disease, and the major cause of morbidity and mortality, but the mechanisms underlying their pathogenesis have been elusive. Recently, we demonstrated that high-dose pexidartinib, a CSF1R inhibitor, prevents LS CNS lesions and systemic disease in the Ndufs4(-/-) mouse model of LS. While the dose-response in this study implicated peripheral immune cells, the immune populations involved have not yet been elucidated. Here, we used a targeted genetic tool, deletion of the colony-stimulating Factor 1 receptor (CSF1R) macrophage super-enhancer FIRE (Csf1rΔFIRE), to specifically deplete microglia and define the role of microglia in the pathogenesis of LS. Homozygosity for the Csf1rΔFIRE allele ablates microglia in both control and Ndufs4(-/-) animals, but onset of CNS lesions and sequalae in the Ndufs4(-/-), including mortality, are only marginally impacted by microglia depletion. The overall development of necrotizing CNS lesions is not altered, though microglia remain absent. Finally, histologic analysis of brainstem lesions provides direct evidence of a causal role for peripheral macrophages in the characteristic CNS lesions. These data demonstrate that peripheral macrophages play a key role in the pathogenesis of disease in the Ndufs4(-/-) model.

Caenorhabditis elegans UCP4 protein controls complex II-mediated oxidative phosphorylation through succinate transport.

The novel uncoupling proteins (UCP2-5) are implicated in the mitochondrial control of oxidant production, insulin signaling, and aging. Attempts to understand their functions have been complicated by overlapping expression patterns in most organisms. Caenorhabditis elegans nematodes are unique because they express only one UCP ortholog, ceUCP4 (ucp4). Here, we performed detailed metabolic analyzes in genetically modified nematodes to define the function of the ceUCP4. The knock-out mutant ucp4 (ok195) exhibited sharply decreased mitochondrial succinate-driven (complex II) respiration. However, respiratory coupling and electron transport chain function were normal in ucp4 mitochondria. Surprisingly, isolated ucp4 mitochondria showed markedly decreased succinate uptake. Similarly, ceUCP4 inhibition blocked succinate respiration and import in wild type mitochondria. Genetic and pharmacologic inhibition of complex I function was selectively lethal to ucp4 worms, arguing that ceUCP4-regulated succinate transport is required for optimal complex II function in vivo. Additionally, ceUCP4 deficiency prolonged lifespan in the short-lived mev1 mutant that exhibits complex II-generated oxidant production. These results identify a novel function for ceUCP4 in the regulation of complex II-based metabolism through an unexpected mechanism involving succinate transport.