Altered Anesthetic Sensitivity of Mice Lacking Ndufs4, a Subunit of Mitochondrial Complex I

RDP and AQ were supported by the Howard Hughes Medical Institute (HHMI). AQ was a recipient of MICINN postdoctoral mobility program fellowship from the Spanish Ministerio de Ciencia e Innovación. PGM and MMS were supported by National Institutes of Health (NIH) grant GM58881. These studies were also supported in part by the Mitochondrial Research Guild. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Anesthetics are in routine use, yet the mechanisms underlying their function are incompletely understood. Studies in vitro demonstrate that both GABAA 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

Leukocytes mediate disease pathogenesis in the Ndufs4(KO) mouse model of Leigh syndrome

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

Mechanisms underlying neonate-specific metabolic effects of volatile anesthetics

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

Subcomplex Iλ Specifically Controls Integrated Mitochondrial Functions in Caenorhabditis elegans

Complex I dysfunction is a common, heterogeneous cause of human mitochondrial disease having poorly understood pathogenesis. The extensive conservation of complex I composition between humans and Caenorhabditis elegans permits analysis of individual subunit contribution to mitochondrial functions at both the whole animal and mitochondrial levels. We provide the first experimentally-verified compilation of complex I composition in C. elegans, demonstrating 84% conservation with human complex I. Individual subunit contribution to mitochondrial respiratory capacity, holocomplex I assembly, and animal anesthetic behavior was studied in C. elegans by RNA interference-generated knockdown of nuclear genes encoding 28 complex I structural subunits and 2 assembly factors. Not all complex I subunits directly impact respiratory capacity. Subcomplex Iλ subunits along the electron transfer pathway specifically control whole animal anesthetic sensitivity and complex II upregulation, proportionate to their relative impairment of complex I-dependent oxidative capacity. Translational analysis of complex I dysfunction facilitates mechanistic understanding of individual gene contribution to mitochondrial disease. We demonstrate that functional consequences of complex I deficiency vary with the particular subunit that is defective

Region-Specific Defects of Respiratory Capacities in the Ndufs4(KO) Mouse Brain.

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.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.Our data suggests that locally insufficient respiration capacity of the nerve terminals may drive focal neurodegeneration

Metabolic capacity of synaptosomes.

<p>A: Synaptosomes were fuelled with glucose plus pyruvate and mitochondrial respiration was maximized by uncoupling with the protonophore FCCP. Oxygen consumption rates (OCR) were corrected for non-mitochondrial oxygen consumption. Note, the degeneration-prone regions of the KO: olfactory bulb (OB), brainstem (BS) and cerebellum (CB),—but not the resilient “rest” of the brain (R) -, have significantly lower capacities to respire than those of the controls. B: Glycolytic capacity was assessed as the external acidification rate (ECAR) measured after blocking oxidative phosphorylation and stimulating ATP turnover with 4-aminopyridine. Note, for any given brain region the capacity for glycolysis was fairly stable between genotypes and with age. The only significant (α = 5%) change observed was a decrease with age in the controls (*) from cerebellum. The number of biological repeats is stated inside the bars. * denotes significant (α = 5%) differences after Holm-Bonferroni correction for multiple comparisons.</p