Complex I dependent capacity for oxidative phosphorylation of isolated (non-synaptic) mitochondria.

<p>(A) Maximal ADP-stimulated respiration (state 3) of mitochondria from whole brain fuelled with complex I dependent electron donor substrates. Note, while increase of oxphos capacity with age only reached statistical significance for controls powered with glutamate/malate, it becomes significant (3-way ANOVA P<0.001) for the aggregate of all substrates and all genotypes. (B,C,D) State 3 respiration for mitochondria isolated from brain stem (BS), cerebellum (CB) and remainder of brain (R). The complex I dependent electron donor substrates were pyruvate + malate (B), glutamate + malate (C) and α-ketoglutarate + malate (D). Olfactory bulb was excluded from both whole brain and R preparations. * denotes significant (α = 0.05) differences after Holm's correction for multiple pairwise comparisons.</p

Stability of complex I is not increased in KO mitochondria from resilient "rest" brain.

<p>Blue Native PAGE of mitochondrial proteins from brainstem (BS) and “rest”brain (R) were electrophoresed under conditions which leave wildtype mitochondrial supercomplexes intact (A). Purpelish diaphorase activity staining revealed bands containing the NAD binding site of complex I (black arrow heads). All proteins were nonspecifically counterstained aqua blue with Coomassie Blue. Select landmark bands are labeled: supercomplexes containing complex I III and IV (I III IV), incomplete supercomplex (I III), solitary complex I (I), ATP synthetase (V), a low molecular weight fragment of complex I (I fgmt). (Strong diaphorase activity at grey arrow heads may be dihydrolipoamid DH). Densitometry results (B): Activity stain for the indicated complex I containing bands was normalized against the protein stain of the complex V band (serving as an internal loading control).</p

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

Markers for synaptosomes and mitochondria in synaptosomal preparations.

<p>Western blots were simultaneously probed for synaptophysin (red) and the ATPase-α (green). A blot with 3 independent brainstem (BS) synaptosome preparations for each combination of genotype and age group is shown as an example (A). 14 additional blots (not shown) were evaluated to cover samples from this and the other brain regions. The same “rest” brain (R) synaptosome preparation (Reference) was present on each blot for normalization. Densitometry results for synaptophysin (B) and ATPase-α (C): The signal of each band was normalized to the reference signal from the same blot. Data are mean ± SD, N = 3 independent biological samples for each condition with 3 technical repeats per sample.</p

Capacity for oxidative phosphorylation (state 3 respiration) of isolated non-synaptic mitochondria by brain region and age group.

<p>A: Complex I-dependent state 3 respiration with pyruvate plus malate remained constant with age in the KO but tended to increase in the controls leading to significant capacity shortfalls in older KO mice in all brain regions tested: brain stem (BS), cerebellum (CB) and remainder of the brain excluding olfactory bulb (R). B: With complex I dependent electron transport blocked by rotenone, the capacity for state 3 respiration via complex II increased with age in the controls reaching significance in CB and R. In the KO capacity did not significantly increase with age but was already elevated in the young cohort. The number of biological replicates is stated inside the bars. * denotes significant changes at significance level (α = 5%) after Holm-Bonferroni correction for multiple comparisons.</p

HNE damage to mitochondrial protein from vulnerable regions in KO is not elevated compared to unaffected controls.

<p>Western blots with mitochondrial protein were probed for a covalent modification caused by HNE, a reactive product of ROS-triggered lipid peroxidation. Overall damage was assessed by densitometry of whole lanes. Data were normalized to reference samples which were present on each blot. Note that damage in the degeneration prone regions brainstem (BS) and cerebellum (CB) of the KO does not exceed the damage in the respective regions from controls. * denotes significant difference after Holm Bonferroni correction at the α = 5% level.</p

HNE-damage to synaptosomal protein from vulnerable regions of the KO does not exceed damage in controls.

<p>ROS damage to synaptosomes was assessed by SDS PAGE Western blots probed for HNE reaction products. A blot (A) with 12 independent brainstem samples is shown as an example. (See supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148219#pone.0148219.s003" target="_blank">S3 Fig</a>. for the full set.) The positions of the 3 most immunoreactive bands are labeled (HNE) showing an unidentified band at 53kD band as the predominant reaction product. Non-specific bands (non-spec.) were independent of the primary antibody. Densitometry results (B) for all brain regions: For each sample the 53kD signal was normalized to the reference sample (“Ref”), an identical aliquot of which was run on each blot. The number of biological replicates is stated at the bottom of each bar. * denotes significant (α = 5%) differences after Holm-Bonferroni correction for multiple comparisons.</p

EC₅₀s for isoflurane (ISO) and halothane (HAL) to cause immobility in response to tail pinch.

<p>Dark bars show the EC₅₀s for wild-type (WT) mice (n = 10, ISO; n = 6, HAL); open bars show the values for the <i>Ndufs4</i> KO mice (n = 10, ISO; n = 6, HAL). Error bars show the standard deviations. The values for the KO animals were significantly different from those for WT with a p<0.01.</p

The dose response curves for WT and <i>Ndufs4</i> KO mice for LORR after intraperitoneal propofol.

<p>Dose-response curves were generated using the percentage of mice that presented LORR at the measured concentrations (n = 5–7 per group for each injection dose). The values for the KO animals were significantly different from those for WT (p<0.01).</p

Regional knockdown of NDUFS4 implicates a thalamocortical circuit mediating anesthetic sensitivity

<div><p>Knockout of the mitochondrial complex I protein, NDUFS4, profoundly increases sensitivity of mice to volatile anesthetics. In mice carrying an <i>Ndufs4</i>^lox/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 <i>Ndufs4</i>, 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 <i>Ndufs4</i> in the vestibular nucleus produced resistance to both anesthetics for movement in response to TC. <i>Ndufs4</i> loss in the central and dorsal medial thalami and in the parietal association cortex increased anesthetic sensitivity to both TC and LORR. Knockdown of <i>Ndufs4</i> only in the parietal association cortex produced striking hypersensitivity for both endpoints, and accounted for half the total change seen in the global KO (<i>Ndufs4(KO)</i>). Excitatory synaptic transmission in the parietal association cortex in slices from <i>Ndufs4(KO)</i> 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.</p></div