Iron chelation by deferiprone does not rescue the Niemann-Pick Disease Type C1 mouse model.

Niemann-Pick Disease Type C (NP-C) is a fatal lysosomal storage disorder with progressive neurodegeneration. In addition to the characteristic cholesterol and lipid overload phenotype, we previously found that altered metal homeostasis is also a pathological feature. Increased brain iron in the Npc1-/- mouse model of NP-C may potentially contribute to neurodegeneration, similar to neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Deferiprone (DFP) is a brain penetrating iron chelator that has demonstrated effectiveness in preventing neurological deterioration in Parkinson's disease clinical trials. Therefore, we hypothesized that DFP treatment, targeting brain iron overload, may have therapeutic benefits for NP-C. Npc1-/- mice were assigned to four experimental groups: (1) pre-symptomatic (P15) + 75 mg/kg DFP; (2) pre-symptomatic (P15) + 150 mg/kg DFP; (3) symptomatic (P49) + 75 mg/kg DFP; (4) symptomatic (P49) + 150 mg/kg DFP. Our study found that in Npc1-/- mice, DFP treatment did not offer any improvement over the expected disease trajectory and median lifespan. Moreover, earlier treatment and higher dose of DFP resulted in adverse effects on body weight and onset of ataxia. The outcome of our study indicated that, despite increased brain iron, Npc1-/- mice were vulnerable to pharmacological iron depletion, especially in early life. Therefore, based on the current model, iron chelation therapy is not a suitable treatment option for NP-C.

Keratinocyte Proline-Rich Protein Deficiency in Atopic Dermatitis Leads to Barrier Disruption.

Atopic dermatitis is a common inflammatory skin disease caused by the interaction of genetic and environmental factors. By allelic copy number analysis at missense single-nucleotide polymorphisms on 26 genes with copy number variation, we identified a significant association between atopic dermatitis and human KPRP. Human KPRP expression, which was localized to the upper granular layer of epidermis, was significantly decreased in atopic dermatitis compared with normal skin. KPRP was histologically colocalized with loricrin and was mainly detected in cytoskeleton fractions of human keratinocytes. To further investigate the role of KPRP in skin, Kprp-knockout mice were generated. Heterozygous knockout (Kprp+/-) mice exhibited reduced KPRP expression to level a similar to that of human AD lesional skin. Kprp+/- mice showed abnormal desmosome structure and detachment of lower layers of the stratum corneum. Percutaneous inflammation by topical application of croton oil or oxazolone was enhanced, and epicutaneous immunization with ovalbumin induced a high level of IgE in Kprp+/- mice. Our study, started from allelic copy number analysis in human AD, identified the importance of KPRP, the decrease of which leads to barrier dysfunction.

Regulator of calcineurin 1 differentially regulates TLR-dependent MyD88 and TRIF signaling pathways.

Toll-like receptors (TLRs) recognize the conserved molecular patterns in microorganisms and trigger myeloid differentiation primary response 88 (MyD88) and/or TIR-domain-containing adapter-inducing interferon-ß (TRIF) pathways that are critical for host defense against microbial infection. However, the molecular mechanisms that govern TLR signaling remain incompletely understood. Regulator of calcineurin-1 (RCAN1), a small evolutionarily conserved protein that inhibits calcineurin phosphatase activity, suppresses inflammation during Pseudomonas aeruginosa infection. Here, we define the roles for RCAN1 in P. aeruginosa lipopolysaccharide (LPS)-activated TLR4 signaling. We compared the effects of P. aeruginosa LPS challenge on bone marrow-derived macrophages from both wild-type and RCAN1-deficient mice and found that RCAN1 deficiency increased the MyD88-NF-κB-mediated cytokine production (IL-6, TNF and MIP-2), whereas TRIF-interferon-stimulated response elements (ISRE)-mediated cytokine production (IFNß, RANTES and IP-10) was suppressed. RCAN1 deficiency caused increased IκBα phosphorylation and NF-κB activity in the MyD88-dependent pathway, but impaired ISRE activation and reduced IRF7 expression in the TRIF-dependent pathway. Complementary studies of a mouse model of P. aeruginosa LPS-induced acute pneumonia confirmed that RCAN1-deficient mice displayed greatly enhanced NF-κB activity and MyD88-NF-κB-mediated cytokine production, which correlated with enhanced pulmonary infiltration of neutrophils. By contrast, RCAN1 deficiency had little effect on the TRIF pathway in vivo. These findings demonstrate a novel regulatory role of RCAN1 in TLR signaling, which differentially regulates MyD88 and TRIF pathways.

Preventive Effects of Vitamin C on Diethylnitrosamine-induced Hepatotoxicity in Smp30 Knockout Mice.

Vitamin C (L-ascorbic acid) is well known as a free radical scavenger that protects cells against damage from oxidative stress. Herein, we investigated the effects of vitamin C against diethylnitrosamine (DEN)-induced hepatotoxicity. Male wild-type (C57BL/6) and senescence marker protein-30 (Smp30) knockout (KO) mice were used and divided in the following four groups: WT group (n=15): Wild-type (WT) mice fed vitamin C-free diet with tap water; WV group (n=14): WT mice fed vitamin C-free diet with water supplemented with 1.5 g/kg vitamin C; KT group (n=12): Smp30 KO mice fed vitamin C-free diet with tap water; and KV group (n=13): Smp30 KO mice fed vitamin C-free diet with water supplemented with 1.5 g/kg vitamin C. A single intraperitoneal injection of DEN (5 mg/kg body weight) was injected in the second week during the experimental period. Mice were sacrificed after 17 weeks of treatment to investigate the effect of dietary vitamin C on DEN-induced hepatotoxicity. The results showed that vitamin C significantly increased the mean lifespan (p<0.05) in the WT, WV and KV groups compared with the KT group. The serum concentrations of γ-glutamyl transpeptidase, alanine aminotransferase, and aspartate aminotransferase did not significantly differ among groups. The WT group exhibited significantly more acute cellular swelling accompanied by centrilobular necrosis, focal lymphocyte infiltration, and eosinophilic intracytoplasmic inclusion bodies as compared with the WV and KV groups, suggesting that vitamin C had a hepatoprotective effect. Dysplastic, large, and binucleated hepatocytes were also observed in the WT group, but these pathological signs were absent from the WV and KV groups. Our experimental evidence suggests that vitamin C supplementation in Smp30 KO mice was effective for the treatment of DEN-induced hepatotoxicity.

Effect of Melilotus extract on lung injury via the upregulation of tumor necrosis factor-α-induced protein-8-like 2 in septic mice.

As a Traditional Chinese Medicine, Melilotus extracts have been reported to function as an anti­inflammatory agent, antioxidant and inhibitor of capillary permeability. The present study aimed to identify the mechanisms by which Melilotus interferes with inflammation­associated and oxidative stress pathways during sepsis. An animal model of cecal ligation­perforation (CLP)­induced sepsis was established. Two hours prior to surgery, animals in the treatment group were administered 25 mg/kg Melilotus extract tablets and subsequently every 8 h. At 24 h post­administration, pathological modifications in lung tissue and expression levels of tumor necrosis factor­α­induced protein­8­like 2 (TIPE2) expression, nuclear factor (NF)­κB, toll­like receptor 4 (TLR4), heme oxygenase­1 (HO­1), inhibitor of κB kinase (IκB), pro­inflammatory mediators (interleukin­6 and tumor necrosis factor­α), myeloperoxidase (MPO), malondialdehyde (MDA) and superoxide dismutase (SOD), were examined. The results showed that Melilotus extract had a marked effect on the pathological manifestation of lung tissue and lung inflammatory response, the upregulation of TIPE2, HO­1 and IκB expression, and the inhibition of TLR4 and NF­κB activities. In addition, following treatment with Melilotus extract, the model animals demonstrated decreased levels of MPO and MDA as well as increased levels of SOD. In conclusion, these results indicated that Melilotus extract may be a potential therapeutic agent for the treatment of CLP­induced lung injury, the mechanism of which proceeded via inflammation­ and oxidation­associated pathways by increasing TIPE2 expression.

Reduced brown adipose tissue thermogenesis during environmental interactions in transgenic rats with ataxin-3-mediated ablation of hypothalamic orexin neurons.

Thermogenesis in brown adipose tissue (BAT) contributes to substantial increases in body temperature evoked by threatening or emotional stimuli. BAT thermogenesis also contributes to increases in body temperature that occur during active phases of the basic rest-activity cycle (BRAC), as part of normal daily life. Hypothalamic orexin-synthesizing neurons influence many physiological and behavioral variables, including BAT and body temperature. In conscious unrestrained animals maintained for 3 days in a quiet environment (24-26°C) with ad libitum food and water, we compared temperatures in transgenic rats with ablation of orexin neurons induced by expression of ataxin-3 (Orx_Ab) with wild-type (WT) rats. Both baseline BAT temperature and baseline body temperature, measured at the onset of BRAC episodes, were similar in Orx_Ab and WT rats. The time interval between BRAC episodes was also similar in the two groups. However, the initial slopes and amplitudes of BRAC-related increases in BAT and body temperature were reduced in Orx_Ab rats. Similarly, the initial slopes and amplitudes of the increases in BAT temperatures induced by sudden exposure to an intruder rat (freely moving or confined to a small cage) or by sudden exposure to live cockroaches were reduced in resident Orx_Ab rats. Constriction of the tail artery induced by salient alerting stimuli was also reduced in Orx_Ab rats. Our results suggest that orexin-synthesizing neurons contribute to the intensity with which rats interact with the external environment, both when the interaction is "spontaneous" and when the interaction is provoked by threatening or salient environmental events.

ENA-A actimineral resource A extends lifespan associated with antioxidant mechanism in SMP30 knockout mice.

ENA-actimineral resource A (ENA-A) is an alkaline mineral water and has a few biological activities such as antioxidant activity. The aim of this study was to examine the effects of ENA-A on lifespan in mice using senescence marker protein-30 knockout mice. The present study had groups of 18-week-old mice (n = 24), 26-week-old mice (n = 12), and 46-week-old mice (n = 20). Each differently aged mice group was divided into three subgroups: a control group, a 5 % ENA-A-treated group, and a 10 % ENA-A-treated group. Mice in the 18-week-old group were treated with vitamin C drinking water 1.5 g/L. However, the mice in the 26-week-old and 46-week-old groups were not treated with vitamin C. The experiments were done for 18 weeks. All vitamin C-treated mice were alive at week 18 (100% survival rate). In the non-vitamin C group, the 10% ENA-A-treated mice were alive at week 18. The control and 5% ENA-A-treated mice died by week 15. As expected, vitamin C was not detected in the non-vitamin C-treated group. However, vitamin C levels were increased in an ENA-A dose-dependent manner in the vitamin C-treated group. In the TUNEL assay, a number of positive hepatocytes significantly decreased in an ENA-A dose-dependent manner. Periodic acid Schiff positive hepatocytes were significantly increased in an ENA-A dose-dependent manner. In addition, the expression level of CuZnSOD was increased by the ENA-A treatment. These data suggest that the intake of ENA-A has a critical role in the anti-aging mechanism and could be applied toward the lifespans of humans.

[Diagnostic and therapeutic update in narcolepsy]. / Actualizacion diagnostica y terapeutica en narcolepsia.

Narcolepsy is an emblematic, unique disease within sleep disorders that is characterised by excessive daytime sleepiness, cataplexy and other abnormal manifestations of REM sleep. In the last 14 years truly spectacular progress has been made in our knowledge of this disease, since the discovery of its cause, i.e. a loss of the hypothalamic neurons that synthesise hypocretin, a previously unknown neurotransmitter, and its probable aetiopathogenic mechanisms, i.e. an autoimmune process in a patient with very precise immunological characteristics - a specific type of HLA and a specific type of T-cell receptor. The cause of this autoimmune process remains unknown. The definitive treatment - the administration of hypocretin, which is the substance missing in the organism - is still unavailable, but there are powerful drugs for treating its main symptoms, the sleepiness and the cataplexy. Some of these are classic compounds (methylphenidate, clomipramine), while others are more recent (modafinil, venlafaxine, sodium oxybate), but together they allow many patients to experience significant improvements. Lack of knowledge about the disease, both on the part of patients and their relatives as well as physicians, is the reason for the great delay in its diagnosis, with even more dramatic consequences when the disease begins in infancy.

Sleepiness that cannot be overcome: narcolepsy and cataplexy.

Narcolepsy-cataplexy syndrome is characterized by excessive daytime sleepiness, cataplexy, sleep paralysis, hypnagogic hallucinations and disturbed nocturnal sleep. It is strongly associated with the genetic marker, human leucocyte antigen (HLA) DQB1*06:02. A deficit in the endogenous hypocretin/orexin system due to neuronal degeneration in the lateral hypothalamus, induced by an autoimmune-mediated process, is the primary pathophysiology associated with the human disease. The important finding of an association with hypocretin genes in animal models of narcolepsy has led to the establishment of cerebrospinal fluid hypocretin measurements as a new diagnostic test for human narcolepsy. This is a fascinating story of translation of basic science research into clinical practice in sleep medicine during the past decade. Recent advances have shed light on the associations between respiratory medicine and narcolepsy-cataplexy research. The first is that upper airway infections, including H1N1 and/or streptococcal infections, may initiate or reactivate an immune response that leads to loss of hypocretin-secreting cells and narcolepsy in genetically susceptible individuals. The second is that an increased incidence of sleep disordered breathing among narcoleptic subjects may relate to the impairment of central control of breathing, linked to hypocretin deficiency or carriage of HLADQB1*06:02, in animals and human subjects with narcolepsy, respectively, indicating neural dysfunction in an area where respiratory and sleep-wake systems are closely interrelated.

Role of orexin in the central regulation of glucose and energy homeostasis.

Hypothalamic orexin neurons are known to regulate sleep/wake stability, feeding behavior, emotions, autonomic nerve activity, and whole-body energy metabolism. In addition, emerging evidence indicates that orexin contributes to central regulation of glucose homeostasis. Intriguingly, central administration of orexin is reported to cause blood glucose-elevating effect or blood glucose-lowering effect in rodents, depending on the experimental conditions. Here we reviewed the recent reports regarding the mode and mechanism of actions of orexin on these two opposing effects, and discuss the functional significance for the maintenance of glucose homeostasis. The fact that orexin exhibits biphasic effects on autonomic nerve activity and lipolysis suggests that orexin dually regulates the glucose appearance. In fact, orexin neurons are activated not only depending on the demand for glucose but also according to a circadian rhythm in the suprachiasmatic nucleus. The excited orexin neurons appear to alter the sympathetic or parasympathetic outflow to the periphery, and modulate the glucose production and utilization. Furthermore, deficiency of orexin action, particularly reduction of orexin 2 receptor-signaling, disrupts the mechanism for protection against insulin resistance associated with aging or induced by chronic high fat feeding in mice. Taken together, hypothalamic orexin system may manage multiple tasks to coordinate the interconnection among the arousal, feeding, circadian, and glucose homeostasis pathways.

Highly specific role of hypocretin (orexin) neurons: differential activation as a function of diurnal phase, operant reinforcement versus operant avoidance and light level.

Hypocretin (Hcrt) cell loss is responsible for narcolepsy, but Hcrt's role in normal behavior is unclear. We found that Hcrt knock-out mice were unable to work for food or water reward during the light phase. However, they were unimpaired relative to wild-type (WT) mice when working for reward during the dark phase or when working to avoid shock in the light or dark phase. In WT mice, expression of Fos in Hcrt neurons occurs only in the light phase when working for positive reinforcement. Expression was seen throughout the mediolateral extent of the Hcrt field. Fos was not expressed when expected or unexpected unearned rewards were presented, when working to avoid negative reinforcement, or when given or expecting shock, even though these conditions elicit maximal electroencephalogram (EEG) arousal. Fos was not expressed in the light phase when light was removed. This may explain the lack of light-induced arousal in narcoleptics and its presence in normal individuals. This is the first demonstration of such specificity of arousal system function and has implications for understanding the motivational and circadian consequences of arousal system dysfunction. The current results also indicate that comparable and complementary specificities must exist in other arousal systems.

Hypothalamus, hypocretins/orexin, and vigilance control.

The hypothalamus has re-emerged as a key regulator of sleep and wakefulness, shifting the focus away from the brainstem and thalamocortical systems (ascending reticular activating systems). Several new sleep control systems in the hypothalamus and their interaction with the circadian pacemaker in the suprachiasmatic nucleus have been identified recently. More recently, deficiency of the hypothalamic peptide, hypocretin/orexin, has been found to be the major pathophysiological factor in human narcolepsy-cataplexy, the sleep disorder characterized by excessive daytime sleepiness and rapid eye movement sleep abnormalities. The results from a series of experiments suggest that the hypocretin system is involved in the maintenance of wakefulness and stabilizes the vigilance states. The hypocretin system also plays a role in the link between sleep and other fundamental hypothalamic functions, such as the regulation of food intake, metabolism, hormone release, and temperature. Sleep deprivation is known to alter hormone release, increase body temperature, stimulate appetite, and activate the sympathetic nervous system. Sleep control systems within the hypothalamus may therefore be closely integrated with homeostatic systems needed for survival. In this chapter, the role of the hypothalamus in vigilance control is discussed, with a particular emphasis on the hypocretins/orexin system.

Nor-ursodeoxycholic acid reverses hepatocyte-specific nemo-dependent steatohepatitis.

BACKGROUND: Hepatocyte-specific NEMO/NF-κB deleted mice (NEMO(Δhepa)) develop spontaneous non-alcoholic steatohepatitis (NASH). Free fatty acids and bile acids promote DR5 expression. TRAIL/NK cell-mediated activation of TRAIL-R2/DR5 plays an important role during acute injury in NEMO(Δhepa) mice. AIM: To inhibit the progression of NASH in the absence of hepatocyte-NEMO/NF-kB signaling. METHODS: NEMOf/f and NEMO(Δhepa) mice were fed with a low-fat diet, and with two anticholestatic diets; UDCA and NorUDCA. The impact of these treatments on the progression of NASH was evaluated. RESULTS: We show that high expression of DR5 in livers from NEMO(Δhepa) mice is accompanied by an abundant presence of bile acids (BAs), misregulation of BA transporters and significant alteration of lipid metabolism-related genes. Additionally, mice lacking NEMO in hepatocytes spontaneously showed ductular response at young age. Unexpectedly, feeding of NEMO(Δhepa) mice with low-fat diet failed to improve chronic liver injury. Conversely, anti-cholestatic treatment with nor-ursodeoxycholic acid (NorUDCA), but not with ursodeoxycholic acid (UDCA), led to a significant attenuation of liver damage in NEMO(Δhepa) mice. The strong therapeutic effect of NorUDCA relied on a significant downregulation of LXR-dependent lipogenesis and the normalisation of BA metabolism through mechanisms involving cross-talk between Cyp7a1 and SHP. This was associated with the significant improvement of liver histology, NEMO(Δhepa)/NorUDCA-treated mice showed lower apoptosis and reduced CyclinD1 expression, indicating attenuation of the compensatory proliferative response to hepatocellular damage. Finally, fibrosis and ductular reaction markers were significantly reduced in NorUDCA-treated NEMO(Δhepa) mice. CONCLUSIONS: Overall, our work demonstrates the contribution of bile acids metabolism to the progression of NASH in the absence of hepatocyte-NF-kB through mechanisms involving DR5-apoptosis, inflammation and fibrosis. Our work suggests a potential therapeutic effect of NorUDCA in attenuating the progression of NASH.

[Connectomics of orexin-producing neurons: analysis using mouse models].

Orexin A and orexin B (also known as hypocretin 1 and hypocretin 2) are hypothalamic neuropeptides that we discovered thirteen years ago. Initially, these peptides were recognized as regulators of feeding behavior. Subsequently, several studies suggested that orexin deficiency causes narcolepsy in humans and other mammalian species, highlighting roles of this hypothalamic neuropeptide in the regulation of sleep and wakefulness. Studies of efferent and afferent systems of orexin-producing neurons have shown that the orexin neuronal system has close interactions with systems that regulate emotion, energy homeostasis, reward, and arousal. These observations suggest that orexin neurons are involved in sensing the body's external and internal environments, and regulate vigilance states accordingly.

Hypothalamic modulation of breathing.

Hypothalamus has long been known to be involved in the regulation of breathing. For example, many neurons are activated by hypoxia and hypercapnia and stimulation to the hypothalamus increases respiration. However, precise characters of these neurons have not well understood until recently presumably because hypothalamus is a heterogeneous structure intermingly containing many kind of neurotransmitters. The situation has dramatically changed by a discovery of hypothalamic neuropeptide orexin in 1998 and subsequent development of orexin-knockout mice in 1999. Here I summarize our recent discovery of the possible contribution of orexin to the vigilance-state-dependent adjustment of central respiratory regulation. Orexin-deficient mice show an attenuated hypercapnic ventilatory response during the awake but not during the sleep period, whereas basal ventilation remained normal, irrespective of the vigilance state. Orexin supplementation remedied the defect, and the administration of an orexin receptor antagonist to wild-type mice mimicked the abnormality. Hypercapnic stimulation activated orexinergic neurons in the wild-type mice. Orexin-deficient mice also showed frequent sleep apneas and loss of repetitive intermittent hypoxia-induced ventilatory and phrenic long-term facilitation. Hence, it is possible that the orexin system is one of the essential modulators required for coordinating the circuits controlling respiration and behavior.

Hypocretin/orexin and narcolepsy: new basic and clinical insights.

Narcolepsy is a chronic sleep disorder, characterized by excessive daytime sleepiness (EDS), cataplexy, sleep paralysis and hypnagogic hallucinations. Both sporadic (95%) and familial (5%) forms of narcolepsy exist in humans. The major pathophysiology of human narcolepsy has been recently discovered based on the discovery of narcolepsy genes in animals; the genes involved in the pathology of the hypocretin/orexin ligand and its receptor. Mutations in hypocretin-related genes are rare in humans, but hypocretin ligand deficiency is found in a large majority of narcolepsy with cataplexy. Hypocretin ligand deficiency in human narcolepsy is probably due to the post-natal cell death of hypocretin neurones. Although a close association between human leucocyte antigen (HLA) and human narcolepsy with cataplexy suggests an involvement of autoimmune mechanisms, this has not yet been proved. Hypocretin deficiency is also found in symptomatic cases of narcolepsy and EDS with various neurological conditions, including immune-mediated neurological disorders, such as Guillain-Barre syndrome, MA2-positive paraneoplastic syndrome and neuromyelitis optica (NMO)-related disorder. The findings in symptomatic narcoleptic cases may have significant clinical relevance to the understanding of the mechanisms of hypocretin cell death and choice of treatment option. The discoveries in human cases lead to the establishment of the new diagnostic test of narcolepsy (i.e. low cerebrospinal fluid hypocretin-1 levels for 'narcolepsy with cataplexy' and 'narcolepsy due to medical condition'). As a large majority of human narcolepsy patients are ligand deficient, hypocretin replacement therapy may be a promising new therapeutic option, and animal experiments using gene therapy and cell transplantations are in progress.

Orexin neurons in the hypothalamus mediate cardiorespiratory responses induced by disinhibition of the amygdala and bed nucleus of the stria terminalis.

We previously showed that the defense response elicited by stressors was attenuated in prepro-orexin knockout mice and in orexin neuron-ablated mice, and we proposed that orexin serves as a master switch within multiple efferent pathways that mediate the defense response. In this study we sought to determine whether excitation of the amygdala (AMG) or the bed nucleus of stria terminalis (BNST) activates orexin-containing neurons and whether those neurons are essential in eliciting cardiorespiratory responses to the stimulus. In urethane-anesthetized mice, the GABA-A receptor antagonist bicuculline was microinjected into the AMG or BNST and blood pressure, heart rate, and respiration were measured. Injection of bicuculline in either site induced long-lasting dose-dependent cardiorespiratory excitation in wild-type mice. In contrast, mice in which orexin neurons had been ablated demonstrated no such response after activation of the AMG and an attenuated response after activation of the BNST. Double immunohistochemical staining for orexin and c-Fos, an indicator of neural activation, revealed that an injection of bicuculline induced significantly larger numbers of orexin positive neurons that expressed c-Fos in the perifornical/dorsomedial hypothalamus (58.2+/-6.4% into AMG and 66.4+/-6.6% into BNST, n=3 each) than did vehicle (18.2+/-4.4% into AMG and 28.3+/-2.1% into BNST). Disinhibition to the BNST induced widespread expression of c-Fos not only in orexin-containing neurons but also other neurons in the hypothalamus. We conclude that orexin-containing neurons in the medial hypothalamus mediate at least a part of AMG- and BNST-induced cardiorespiratory responses.

Orexin signaling mediates the antidepressant-like effect of calorie restriction.

During periods of reduced food availability, animals must respond with behavioral adaptations that promote survival. Despite the fact that many psychiatric syndromes include disordered eating patterns as a component of the illness, little is known about the neurobiology underlying behavioral changes induced by short-term calorie restriction. Presently, we demonstrate that 10 d of calorie restriction, corresponding to a 20-25% weight loss, causes a marked antidepressant-like response in two rodent models of depression and that this response is dependent on the hypothalamic neuropeptide orexin (hypocretin). Wild-type mice, but not mice lacking orexin, show longer latency to immobility and less total immobility in the forced swim test after calorie restriction. In the social defeat model of chronic stress, calorie restriction reverses the behavioral deficits seen in wild-type mice but not in orexin knock-out mice. Additionally, chronic social defeat stress induces a prolonged reduction in the expression of prepro-orexin mRNA via epigenetic modification of the orexin gene promoter, whereas calorie restriction enhances the activation of orexin cells after social defeat. Together, these data indicate that orexin plays an essential role in mediating reduced depression-like symptoms induced by calorie restriction.

Age-related insulin resistance in hypothalamus and peripheral tissues of orexin knockout mice.

AIMS/HYPOTHESIS: Orexin/hypocretin is a hypothalamic neuropeptide that regulates motivated behaviours, such as feeding and arousal, and, importantly, is also involved in energy homeostasis. The aim of this study was to reveal the role of orexin in the regulation of insulin sensitivity for glucose metabolism. METHODS: Orexin knockout mice fasted overnight underwent oral glucose tolerance testing and insulin tolerance testing. The impact of orexin deficiency on insulin signalling was studied by Western blotting to measure levels of Akt phosphorylation and its upstream and downstream molecules in the hypothalamus, muscle and liver in orexin knockout mice. RESULTS: We found that orexin deficiency caused the age-related development of impaired glucose tolerance and insulin resistance in both male mice without obesity and female mice with mild obesity, fed a normal chow diet. When maintained on a high-fat diet, these abnormalities became more pronounced exclusively in female orexin knockout mice that developed severe obesity. Insulin signalling through Akt was disrupted in peripheral tissues of middle-aged (9-month-old) but not young adult (2-to-3-month-old) orexin knockout mice fed a normal chow diet. Moreover, basal levels of hypothalamic Akt phosphorylation were abnormally elevated in orexin knockout mice at every age studied, and insulin stimulation failed to increase the level of phosphorylation. Similar abnormalities were observed with respect to GSK3beta phosphorylation in the hypothalamus and peripheral tissues of middle-aged orexin knockout mice. CONCLUSIONS/INTERPRETATION: Our results demonstrate a novel role for orexin in hypothalamic insulin signalling, which is likely to be responsible for preventing the development of peripheral insulin resistance with age.

Neural substrates of awakening probed with optogenetic control of hypocretin neurons.

The neural underpinnings of sleep involve interactions between sleep-promoting areas such as the anterior hypothalamus, and arousal systems located in the posterior hypothalamus, the basal forebrain and the brainstem. Hypocretin (Hcrt, also known as orexin)-producing neurons in the lateral hypothalamus are important for arousal stability, and loss of Hcrt function has been linked to narcolepsy. However, it is unknown whether electrical activity arising from Hcrt neurons is sufficient to drive awakening from sleep states or is simply correlated with it. Here we directly probed the impact of Hcrt neuron activity on sleep state transitions with in vivo neural photostimulation, genetically targeting channelrhodopsin-2 to Hcrt cells and using an optical fibre to deliver light deep in the brain, directly into the lateral hypothalamus, of freely moving mice. We found that direct, selective, optogenetic photostimulation of Hcrt neurons increased the probability of transition to wakefulness from either slow wave sleep or rapid eye movement sleep. Notably, photostimulation using 5-30 Hz light pulse trains reduced latency to wakefulness, whereas 1 Hz trains did not. This study establishes a causal relationship between frequency-dependent activity of a genetically defined neural cell type and a specific mammalian behaviour central to clinical conditions and neurobehavioural physiology.