A rare form of narcolepsy (HLA-DR2-) shows possible association with (functionally relevant) alpha-interferon gene polymorphisms

Narcolepsy is a neuropsychiatric disease caused by complex disturbance of sleep regulation. The main symptoms comprise daytime sleepiness and cataplexy. Although the aetiology remains unclear so far, narcolepsy is genetically characterized by strong linkage to the human leukocyte antigen complex as more than 90% of the patients are typed HLA-DR2+. Recently, it has become apparent that the orexin (hypocretin) neurotransmitter system plays a key role in the pathogenesis of the disease. Canine narcolepsy is caused by mutations in the orexin receptor 2 gene, and narcoleptic patients show specifically decreased cerebrospinal fluid orexin levels. Decreased promotor activity of the prepro-orexin gene is caused by binding of alpha-interferon in vitro. To investigate the possible role of IFNA gene polymorphisms in the pathogenesis of narcolepsy, we have genotyped two single nucleotide polymorphisms in IFNA genes as well as a neighbouring microsatellite. No association was evident in the prevalent DR2+ group. Yet, the IFNA10 single nucleotide polymorphisms and the IFNA microsatellite are associated with the DR2- patient group. Thus, the pathogenetic role of interferons needs to be defined in DR2- narcolepsy.     

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.     

Merkel cells, a new localization of prepro-orexin and orexin receptors

Orexins (OXA and OXB) are peptides derived from a common precursor called prepro-orexin. They act through G-protein receptors named orexin 1 receptor (OX(1)R) and orexin 2 receptor (OX(2)R). Orexins were first demonstrated in neurons of the lateral hypothalamus and found to be related to the control of food intake. However, it has been shown that they are widely distributed in both the nervous system and peripheral tissues, including endocrine organs such as the pituitary and adrenal glands. Merkel cells are neuroendocrine cells situated in the epidermis, tactile hairs and oral mucosa, and act as mechanoreceptors. Up to the present, various neuropeptides have been detected in these cells. The aim of the present study was to detect the presence of prepro-orexin and orexin receptors (OX(1)R and OX(2)R) in porcine Merkel cells using immunohistochemistry. Prepro-orexin was expressed in the cytoplasm of Merkel cells in the skin of the pig snout. Immunoreactivity for prepro-orexin was more intense in the mature side of the cell, where the dense-cored granules are accumulated. Epidermal nerve terminals associated with Merkel cells and dermal nerve fibres showed no immunostaining. Both orexin receptors (OX(1)R and OX(2)R) were also demonstrated in the cytoplasm of Merkel cells of pig snout skin. The finding of orexins and their receptors in Merkel cells suggests that they have an autocrine function. Further studies are needed to ascertain the significance of this function.     

Orexins: from neuropeptides to energy homeostasis and sleep/wake regulation

The neuropeptides orexin A and orexin B (also called hypocretin 1 and 2) were recently discovered by a "reverse pharmacology" approach as ligands for two previously orphan G protein coupled receptors: orexin receptors 1 and 2. Neurons producing orexins are located exclusively in the lateral hypothalamic area but project broadly to various parts of the brain, and they have been implicated in the control of energy homeostasis and arousal maintenance. The orexin receptors are also broadly expressed in the central nervous system. Murine and canine models suggest that defective signaling in the orexin system is responsible for the sleep/wake disorder narcolepsy. Although narcoleptic patients rarely have genetic defects in the orexin system, they lack these neuropeptides in the brain and cerebrospinal fluid, indicating that human narcolepsy is an orexin deficiency syndrome in the majority of cases. A connection between sleep/wake regulation and energy homeostasis is hypothesized with orexin neuropeptides as a molecular link.     

Localization of orexin B and orexin-2 receptor in the rat epididymis

The peptides orexin A (OXA) and orexin B (OXB) derived from the proteolytic cleavage of a common precursor molecule, prepro-orexin, were originally described in the rat hypothalamus. Successively, they have been found in many other brain regions as well as in peripheral organs of mammals and other less evolved animals. The widespread localization of orexins accounts for the multiple activities that they exert in the body, including the regulation of energy homeostasis, feeding, metabolism, sleep and arousal, stress, addiction, and cardiovascular and endocrine functions. Both OXA and OXB peptides bind to two G-coupled receptors, orexin-1 (OX1R) and orexin-2 (OX2R) receptor, though with different binding affinity. Altered expression/activity of orexins and their receptors has been associated with a large number of human diseases. Though at present evidence highlighted a role for orexins and cognate receptors in mammalian reproduction, their central and/or local effects on gonadal functions remain poorly known. Here, we investigated the localization of OXB and OX2R in the rat epididymis. Immunohistochemical staining of sections from caput, corpus and cauda segments of the organ showed intense signals for both OXB and OX2R in the principal cells of the lining epithelium, while no staining was detected in the other cell types. Negative results were obtained from immunohistochemical analysis of hypothalamic and testicular tissues from OX2R knock-out mice (OX2R^?/?) and OX1R/OX2R double knock-out (OX1R^?/?; OX2R^?/?) mice, thus demonstrating the specificity of the rabbit polyclonal anti-OX2R antibody used in our study. On contrary, the same antibody clearly showed the presence of OX2R in sections from hypothalamus and testis of normal mice and rats which are well known to express the receptor. Thus, our results provide the first definite evidence for the immunohistochemical localization of OXB and OX2R in the principal cells of rat epididymis.     

Promotion of sleep by suvorexant-a novel dual orexin receptor antagonist

Orexins/hypocretins are key neuropeptides responsible for regulating central arousal and reward circuits. Two receptors respond to orexin signaling, orexin 1 receptor (OX(1)R) and orexin 2 receptor (OX(2)R) with partially overlapping nervous system distributions. Genetic studies suggest orexin receptor antagonists could be therapeutic for insomnia and other disorders with disruptions of sleep and wake. Suvorexant (MK-4305) is a potent, selective, and orally bioavailable antagonist of OX(1)R and OX(2)R currently under clinical investigation as a novel therapy for insomnia. Examination of Suvorexant in radioligand binding assays using tissue from transgenic rats expressing the human OX(2)R found nearly full receptor occupancy (>90%) at plasma exposures of 1.1 μM. Dosed orally Suvorexant significantly and dose-dependently reduced locomotor activity and promoted sleep in rats (10, 30, and 100 mg/kg), dogs (1 and 3 mg/kg), and rhesus monkeys (10 mg/kg). Consistent cross-species sleep/wake architecture changes produced by Suvorexant highlight a unique opportunity to develop dual orexin antagonists as a novel therapy for insomnia.     

Interactions of the orexin/hypocretin neurones and the histaminergic system

Histaminergic and orexin/hypocretin systems are components in the brain wake‐promoting system. Both are affected in the sleep disorder narcolepsy, but the role of histamine in narcolepsy is unclear. The histaminergic neurones are activated by the orexin/hypocretin system in rodents, and the development of the orexin/hypocretin neurones is bidirectionally regulated by the histaminergic system in zebrafish. This review summarizes the current knowledge of the interactions of these two systems in normal and pathological conditions in humans and different animal models.     

大鼠发情周期下丘脑中食欲素及其受体的变化

目的 观察大鼠发情周期各期下丘脑中食欲素及其受体(OX1R、OX2R)表达的变化,探讨食欲素对发情周期的可能调节作用.方法 通过竞争性逆转录多聚酶链式反应 (competitive RT-PCR)方法检测大鼠下丘脑中食欲素前体(Prepro-Orexin)、食欲素-A、OXIR和OX2R在发情周期各期的表达.结果 发情前期的OX1R mRNA表达水平明显高于发情后期,而prepro-OX和OX2R mRNA的表达各期间没有明显的不同.结论 食欲素可能通过结合OX1R调节促性腺激素释放激素和/或黄体生成素的分泌而参与排卵的发生.     

Prepro-hypocretin (prepro-orexin) expression is unaffected by short-term sleep deprivation in rats and mice

The hypocretin/orexin ligand-receptor system has recently been implicated in the sleep disorder narcolepsy. During the dark (active) period, null mutants of the prepro-orexin (prepro-hypocretin) gene have cataplectic attacks and increased levels of both rapid eye movement (REM) and non-REM (NREM) sleep. Intracerebroventricular injection of one of the encoded neuropeptides, orexin-A, early in the light period increases wakefulness and reduces REM sleep in the rat, suggesting that this system may be involved in the normal regulation of sleep and wakefulness. To further test this hypothesis, we measured hypocretin (hcrt) mRNA levels by both Northern hybridization and Taqman analysis in mouse and rat hypothalamus after short-term (6 h) sleep deprivation (SD) and 2-4 hours after recovery from SD. Although our SD procedures effectively induced a sleep debt and increased c-fos mRNA expression in the cortex and hypothalamus as described by other investigators, we found that hcrt mRNA levels were not significantly changed in either species either after SD or after recovery from SD. If the hcrt system is involved in normal regulation of sleep and wakefulness, longer periods of SD may be necessary to affect hcrt mRNA levels or changes may occur at the protein rather than mRNA level. Alternatively, this system may also be involved in another function that counterbalances any SD-induced changes in hcrt mRNA levels.     

Genetic association,seasonal infections and autoimmune basis of narcolepsy

In recent years, a growing number of potential autoimmune disorders affecting neurons in the central nervous system have been identified, including narcolepsy. Narcolepsy is a lifelong sleep disorder characterized by excessive daytime sleepiness with irresistible sleep attacks, cataplexy (sudden bilateral loss of muscle tone), hypnagogic hallucinations, and abnormalities of Rapid Eye Movement sleep. Narcolepsy is generally a sporadic disorder and is caused by the loss of hypocretin (orexin)-producing neurons in the hypothalamus region of the brain. Studies have established that more than 90% of patients have a genetic association with HLA DQB1*06:02. Genome-wide association analysis shows a strong association between narcolepsy and polymorphisms in the TCRα locus and weaker associations within TNFSF4 (also called OX40L), Cathepsin H and the P2RY11-DNMT1 (purinergic receptor subtype P2Y₁₁ to DNMT1, a DNA methytransferase) loci, suggesting an autoimmune basis. Mutations in DNMT1 have also been reported to cause narcolepsy in association with a complex neurological syndrome, suggesting the importance of DNA methylation in the pathology. More recently, narcolepsy was identified in association with seasonal streptococcus, H1N1 infections and following AS03-adjuvanted pH1N1 influenza vaccination in Northern Europe. Potential immunological pathways responsible for the loss of hypocretin producing neurons in these cases may be molecular mimicry or bystander activation. Specific autoantibodies or T cells cross-reactive with hypocretin neurons have not yet been identified, however, thus narcolepsy does not meet Witebsky's criteria for an autoimmune disease. As the brain is not an easily accessible organ, mechanisms of disease initiation and progression remain a challenge to researchers.     

Adenosine inhibits activity of hypocretin/orexin neurons by the A1 receptor in the lateral hypothalamus: a possible sleep-promoting effect

Neurons in the lateral hypothalamus (LH) that contain hypocretin/orexin have been established as important promoters of arousal. Deficiencies in the hypocretin/orexin system lead to narcolepsy. The inhibition of hypocretin/orexin neurons by sleep-promoting neurotransmitters has been suggested as one part of the sleep regulation machinery. Adenosine has been identified as a sleep promoter and its role in sleep regulation in the basal forebrain has been well documented. However, the effect of adenosine on arousal-promoting hypocretin/orexin neurons has not been addressed, despite recent evidence that immunocytochemical visualization of adenosine receptors was detected in these neurons. In this study, we examined the hypothesis that adenosine inhibits the activity of hypocretin/orexin neurons by using electrophysiological methods in brain slices from mice expressing green fluorescent protein in hypocretin/orexin neurons. We found that adenosine significantly attenuated the frequency of action potentials without a change in membrane potential in hypocretin/orexin neurons. The adenosine-mediated inhibition arises from depression of excitatory synaptic transmission to hypocretin/orexin neurons because adenosine depresses the amplitude of evoked excitatory postsynaptic potential and the frequency of spontaneous and miniature excitatory postsynaptic currents in these neurons. At the cell body of the hypocretin/orexin neurons, adenosine inhibits voltage-dependent calcium currents without the induction of GIRK current. The inhibitory effect of adenosine is dose dependent, pertussis toxin sensitive, and mediated by A1 receptors. In summary, our data suggest that in addition to its effect in the basal forebrain, adenosine exerts its sleep-promoting effect in the LH by inhibition of hypocretin/orexin neurons.     

Validation of antibodies for neuroanatomical localization of the P2Y11 receptor in macaque brain

Focus on the purinergic receptor P2Y₁₁ has increased following the finding of an association between the sleep disorder narcolepsy and a genetic variant in P2RY11 causing decreased gene expression. Narcolepsy is believed to arise from an autoimmune destruction of the hypothalamic neurons that produce the neuropeptide hypocretin/orexin. It is unknown how a decrease in expression of P2Y₁₁ might contribute to an autoimmune reaction towards the hypocretin neurons and the development of narcolepsy. To advance narcolepsy research it is therefore extremely important to determine the neuroanatomical localization of P2Y₁₁ in the brain with particular emphasis on the hypocretin neurons. In this article we used western blot, staining of blood smears, and flow cytometry to select two antibodies for immunohistochemical staining of macaque monkey brain. Staining was seen in neuron-like structures in cortical and hypothalamic regions. Rats do not have a gene orthologue to the P2Y₁₁ receptor and therefore rat brain was used as negative control tissue. The chromogenic signal observed in macaque monkey brain in neurons was not considered reliable, because the antibodies stained rat brain in a similar distribution pattern. Hence, the neuroanatomical localization of the P2Y₁₁ receptor remains undetermined due to the lack of specific P2Y₁₁ antibodies for brain immunohistochemistry.     

Sleep disorders,obesity, and aging: The role of orexin

The hypothalamic neuropeptides orexin A and B (hypocretin 1 and 2) are important homeostatic mediators of central control of energy metabolism and maintenance of sleep/wake states. Dysregulation or loss of orexin signaling has been linked to narcolepsy, obesity, and age-related disorders. In this review, we present an overview of our current understanding of orexin function, focusing on sleep disorders, energy balance, and aging, in both rodents and humans. We first discuss animal models used in studies of obesity and sleep, including loss of function using transgenic or viral-mediated approaches, gain of function models using exogenous delivery of orexin receptor agonist, and naturally-occurring models in which orexin responsiveness varies by individual. We next explore rodent models of orexin in aging, presenting evidence that orexin loss contributes to age-related changes in sleep and energy balance. In the next section, we focus on clinical importance of orexin in human obesity, sleep, and aging. We include discussion of orexin loss in narcolepsy and potential importance of orexin in insomnia, correlations between animal and human studies of age-related decline, and evidence for orexin involvement in age-related changes in cognitive performance. Finally, we present a summary of recent studies of orexin in neurodegenerative disease. We conclude that orexin acts as an integrative homeostatic signal influencing numerous brain regions, and that this pivotal role results in potential dysregulation of multiple physiological processes when orexin signaling is disrupted or lost.     

Insomnia following hypocretin2-saporin lesions of the substantia nigra

The neuropeptide hypocretin, also known as orexin, has been implicated in waking since its deletion leads to the sleep disorder narcolepsy. Hypocretin neurons project to major arousal areas, and in an effort to determine which region is responsible for the changes in sleep-wake architecture we have developed the neurotoxin hypocretin2-saporin, which lesions hypocretin receptor bearing neurons. Here, in rats, we investigate the effects of hypocretin2-saporin lesions of the substantia nigra and ventral tegmental area in the regulation of sleep and wakefulness. Bilateral injection of hypocretin2-sap into both the ventral tegmental area and substantia nigra (92 and 184 ng/microl, 0.25 microl in the ventral tegmental area and 0.5 microl in the substantia nigra) or into the substantia nigra alone (184 ng/microl, 0.5 microl) produced insomnia. The insomnia seemed to be associated with a large increase in locomotion on days 4 and 6 postinjection, as hyperactivity and stereotypic movements were consistently observed on the video recordings in all lesioned rats. In these rats, a nearly complete loss of both tyrosine hydroxylase and neuron-specific nuclear protein (neuronal nuclei) immunoreactive cells in the substantia nigra as well as diminution of tyrosine hydroxylase-immunoreactive fibers in the caudate putamen was found. Following bilateral injection of hypocretin2-sap at a lower concentration (46 ng/microl, 0.25 microl in the ventral tegmental area and 0.5 microl in the substantia nigra), very little reduction in the number of tyrosine hydroxylase- and neuronal nuclei-immunoreactive neurons and only a temporary increase in wakefulness (17.4% increase during light-off period on day 6 postinjection) were observed. Ventral tegmental area lesions (184 ng/mul of hypocretin2-sap, 0.25 microl, bilateral injections) did not produce significant changes in sleep, although most of the tyrosine hydroxylase- and neuronal nuclei-immunoreactive neurons in the ventral tegmental area were destroyed. Insomnia following hypocretin2-sap lesions of the substantia nigra could be secondary to increased motor activity resulting from reduction of tonic inhibitory control by the substantia nigra.     

Cerebrospinal fluid histamine levels are decreased in patients with narcolepsy and excessive daytime sleepiness of other origin

Histaminergic neurons of the hypothalamic tuberomammillary nucleus constitute a major wake‐promoting system. In animals, cerebrospinal fluid (CSF) histamine levels are increased during wakefulness and after sleep deprivation and decreased during sleep. An involvement of the histamine system in human disorders has not, to our knowledge, been reported. We measured hypocretin‐1 and histamine levels in the lumbar CSF of 28 patients with and without excessive daytime sleepiness (EDS) as assessed by the Epworth Sleepiness Scale (ESS). There were 10 patients with EDS (ESS > 10, mean ESS = 14). Diagnoses included narcolepsy (n = 4), idiopathic hypersomnia (n = 2), sleep apnoea (n = 2) and multiple sclerosis (n = 2). Three patients were treated with stimulants. Their mean CSF histamine was 258 ± 159 PM. There were 18 patients without EDS (ESS < 9, mean ESS = 5). No patients were treated with stimulants. Their mean CSF histamine was significantly higher (624 ± 481 PM, P = 0.007). There was a significant inverse correlation (r = ?0.48, P = 0.02) between ESS and both CSF histamine and hypocretin‐1 levels. These observations suggest that narcolepsy and EDS of other origin are associated in humans with lower CSF histamine levels and therefore with a reduced activity of the wake‐promoting histaminergic neuronal system.     

妊娠、分娩以及泌乳期大鼠下丘脑中食欲素及其受体表达的变化

目的 观察大鼠下丘脑中食欲素(Orexin)及其受体(OX1R、OX2R)在妊娠、分娩以及泌乳期表达的变化,以探讨Orexin与生殖功能之间的关系.方法 通过竞争性逆转录多聚酶链式反应(Competitive RT-PCR)和免疫组织化学方法测定大鼠下丘脑中Orexin前体(Prepro-OX)、Orexin-A、OX1R和OX2R在妊娠、分娩以及泌乳期的表达.结果 Orexin-A和OX1R阳性神经元主要分别存在于大鼠下丘脑外侧区(LHA)以及妊娠和泌乳期大鼠的下丘脑室旁核 (PVN)和视上核(SON).泌乳第1d的Prepro-Orexin mRNA和OX1R的表达水平明显高于妊娠后期和泌乳期.OX2R的表达在生殖各时期之间没有明显改变.结论 Orexin可能参与大鼠泌乳早期的生殖功能调节,下丘脑的PVN及SON可能是其重要的作用部位.     

Pattern of hypocretin (orexin) soma and axon loss,and gliosis,in human narcolepsy

Human narcolepsy is correlated with a greatly reduced number of hypocretin (orexin) containing neurons and axons, and an elevated level of hypothalamic gliosis. We now report that the percentage loss of Hcrt cells and percentage elevation of GFAP staining are variable across forebrain and brain-stem nuclei, and are maximal in the posterior and tuberomammillary hypothalamic region. Regional gliosis and percent loss of hypocretin axons in narcoleptics are not correlated with regional hypocretin cell soma density in normals or with regional percent soma loss in narcoleptics. Rather they are independently and strongly correlated with the regional density of hypocretin axons and the message density for hypocretin receptor 2, as quantified in the rat. These results are consistent with the hypotheses that the loss of hypocretin function in narcolepsy results from a cytotoxic or immunologically mediated attack focused on hypocretin receptor 2 or an antigen anatomically linked to hypocretin receptor 2, and that this process is intensified in regions of high axonal density.     

Progressive dopamine and hypocretin deficiencies in Parkinson’s disease: is there an impact on sleep and wakefulness?

Sleep–wake disturbances are frequent in patients with Parkinson’s disease, but prospective controlled electrophysiological studies of sleep in those patients are surprisingly sparse, and the pathophysiology of sleep–wake disturbances in Parkinson’s disease remains largely elusive. In particular, the impact of impaired dopaminergic and hypocretin (orexin) signalling on sleep and wakefulness in Parkinson’s disease is still unknown. We performed a prospective, controlled electrophysiological study in patients with early and advanced Parkinson’s disease, e.g. in subjects with presumably different levels of dopamine and hypocretin cell loss. We compared sleep laboratory tests and cerebrospinal fluid levels with hypocretin‐deficient patients with narcolepsy with cataplexy, and with matched controls. Nocturnal sleep efficiency was most decreased in advanced Parkinson patients, and still lower in early Parkinson patients than in narcolepsy subjects. Excessive daytime sleepiness was most severe in narcolepsy patients. In Parkinson patients, objective sleepiness correlated with decrease of cerebrospinal fluid hypocretin levels, and repeated hypocretin measurements in two Parkinson patients revealed a decrease of levels over years. This suggests that dopamine and hypocretin deficiency differentially affect sleep and wakefulness in Parkinson’s disease. Poorer sleep quality is linked to dopamine deficiency and other disease‐related factors. Despite hypocretin cell loss in Parkinson’s disease being only partial, disturbed hypocretin signalling is likely to contribute to excessive daytime sleepiness in Parkinson patients.     

The effect of age on prepro-orexin gene expression and contents of orexin A and B in the rat brain

Orexin A and B (hypocretin 1 and 2) are hypothalamic peptides, which are synthesized in the lateral hypothalamus. Orexins participate in the regulation energy balance, food intake, vigilance and several endocrine and autonomic functions. The widespread projections of the orexin neurons suggest that they may have a role in coordination of different brain activities. The effects of ageing on the orexin system have not been studied previously. Prepro-orexin gene expression in the lateral hypothalamus, and the contents of orexin A and B peptides in the lateral hypothalamus and hypothalamus were measured in young, middle-aged and old (3, 12 and 24 months) rats. In the course of ageing, the expression of the prepro-orexin gene and the levels of orexin A and B decreased; the main decrease occurred by 12 months. Sleep deprivation for 6h increased slightly the expression of prepro-orexin gene in young rats. Deterioration of the orexin system may play a role in the phenomenon associated with aging, e.g. decreased consolidation of vigilance states, endocrine changes and dysfunctions of autonomic nervous system.