Conventional T (Tcon) cells and Foxp3(+) T-regulatory (Treg) cells are thought to have differing metabolic requirements, but little is known of mitochondrial functions within these cell populations in vivo. In murine studies, we found that activation of both Tcon and Treg cells led to myocyte enhancer factor 2 (Mef2)-induced expression of genes important to oxidative phosphorylation (OXPHOS). Inhibition of OXPHOS impaired both Tcon and Treg cell function compared to wild-type cells but disproportionally affected Treg cells. Deletion of Pgc1α or Sirt3, which are key regulators of OXPHOS, abrogated Treg-dependent suppressive function and impaired allograft survival. Mef2 is inhibited by histone/protein deacetylase-9 (Hdac9), and Hdac9 deletion increased Treg suppressive function. Hdac9(-/-) Treg showed increased expression of Pgc1α and Sirt3, and improved mitochondrial respiration, compared to wild-type Treg cells. Our data show that key OXPHOS regulators are required for optimal Treg function and Treg-dependent allograft acceptance. These findings provide a novel approach to increase Treg function and give insights into the fundamental mechanisms by which mitochondrial energy metabolism regulates immune cell functions in vivo.

Rationale:Persons with obstructive sleep apnea may have significant residual hypersomnolence, despite therapy. Long-term hypoxia/ reoxygenation events in adult mice, simulating oxygenation patterns of moderate-severe sleep apnea, result in lasting hypersomnolence, oxidative injury, and proinflammatory responses in wakeactive brain regions. We hypothesized that long-term intermittent hypoxia activates brain NADPH oxidase and that this enzyme serves as a critical source of superoxide in the oxidation injury and in hypersomnolence. Objectives: We sought to determine whether long-term hypoxia/ reoxygenation events in mice result in NADPH oxidase activation and whether NADPH oxidase is essential for the proinflammatory response and hypersomnolence. Methods: NADPH oxidase gene and protein responses were measured in wake-active brain regions in wild-type mice exposed to long-term hypoxia/reoxygenation. Sleep and oxidative and proinflammatory responses were measured in adult mice either devoid of NADPH oxidase activity (gp91 phox -null mice) or in which NADPH oxidase activity was systemically inhibited with apocynin osmotic pumps throughout hypoxia/reoxygenation. Main Results: Long-term intermittent hypoxia increased NADPH oxidase gene and protein responses in wake-active brain regions. Both transgenic absence and pharmacologic inhibition of NADPH oxidase activity throughout long-term hypoxia/reoxygenation conferred resistance to not only long-term hypoxia/reoxygenation hypersomnolence but also to carbonylation, lipid peroxidation injury, and the proinflammatory response, including inducible nitric oxide synthase activity in wake-active brain regions. Conclusions: Collectively, these findings strongly support a critical role for NADPH oxidase in the lasting hypersomnolence and oxidative and proinflammatory responses after hypoxia/reoxygenation patterns simulating severe obstructive sleep apnea oxygenation, highlighting the potential of inhibiting NADPH oxidase to prevent oxidation-mediated morbidities in obstructive sleep apnea.Keywords: intermittent hypoxia; non-REM sleep; oxidation; peroxynitrite Obstructive sleep apnea (OSA) with daytime hypersomnolence is present in at least 2 to 4% of adults in developed countries (1). This disorder manifests as repeated events of sleep statedependent reductions in upper airway dilator motoneuronal activity, with consequent upper airway occlusions and oxyhemoglobin desaturations, each terminating with abrupt arousal and reoxygenation (2). The hypoxia and reoxygenation events may occur as frequently as once every minute of sleep. Despite therapy to alleviate OSA events, many individuals with OSA have residual sleepiness (3, 4). Mechanisms of the residual hypersomnolence in persons with OSA are not understood, but severity of hypoxemia in OSA predicts, in part, the severity of hypersomnolence (5, 6).Long-term intermittent hypoxia (LTIH) in mice, modeling the patterns of hypoxia/reoxygenation observed in moderate to severe sleep apnea, results in protracted hypersomnolence (7,8) and h...

Summary Background Despite seventeen decades of continuous clinical use, the neuronal mechanisms through which volatile anesthetics act to produce unconsciousness remain obscure. One emerging possibility is that anesthetics exert their hypnotic effects by hijacking endogenous arousal circuits. A key sleep-promoting component of this circuitry is the ventrolateral preoptic nucleus (VLPO), a hypothalamic region containing both state-independent neurons and neurons that preferentially fire during natural sleep. Results Using c-Fos immunohistochemistry as a biomarker for antecedent neuronal activity, we show that isoflurane and halothane increase the number of active neurons in the VLPO, but only when mice are sedated or unconscious. Destroying VLPO neurons produces an acute resistance to isoflurane-induced hypnosis. Electrophysiological studies prove that the neurons depolarized by isoflurane belong to the subpopulation of VLPO neurons responsible for promoting natural sleep, while neighboring non-sleep-active VLPO neurons are unaffected by isoflurane. Finally, we show that this anesthetic-induced depolarization is not solely due to a presynaptic inhibition of wake-active neurons as previously hypothesized, but rather is due to a direct postsynaptic effect on VLPO neurons themselves arising from the closing of a background potassium conductance. Conclusions Cumulatively, this work demonstrates that anesthetics are capable of directly activating endogenous sleep-promoting networks and that such actions contribute to their hypnotic properties.

Modern society enables a shortening of sleep times, yet long-term consequences of extended wakefulness on the brain are largely unknown. Essential for optimal alertness, locus ceruleus neurons (LCns) are metabolically active neurons that fire at increased rates across sustained wakefulness. We hypothesized that wakefulness is a metabolic stressor to LCns and that, with extended wakefulness, adaptive mitochondrial metabolic responses fail and injury ensues. The nicotinamide adenine dinucleotide-dependent deacetylase sirtuin type 3 (SirT3) coordinates mitochondrial energy production and redox homeostasis. We find that brief wakefulness upregulates SirT3 and antioxidants in LCns, protecting metabolic homeostasis. Strikingly, mice lacking SirT3 lose the adaptive antioxidant response and incur oxidative injury in LCns across brief wakefulness. When wakefulness is extended for longer durations in wild-type mice, SirT3 protein declines in LCns, while oxidative stress and acetylation of mitochondrial proteins, including electron transport chain complex I proteins, increase. In parallel with metabolic dyshomeostasis, apoptosis is activated and LCns are lost. This work identifies mitochondrial stress in LCns upon wakefulness, highlights an essential role for SirT3 activation in maintaining metabolic homeostasis in LCns across wakefulness, and demonstrates that extended wakefulness results in reduced SirT3 activity and, ultimately, degeneration of LCns.