Identifying the First Sleep-Related Genes

For this article, Jennifer Fisher Wilson interviewed Emmanuel Mignot, Center for Narcolepsy, department of psychiatry, Stanford University School of Medicine, in Stanford, Calif., and Masashi Yanagisawa, Howard Hughes Medical Institute investigator, department of molecular genetics, University of Texas Southwestern Medical Center at Dallas. Data from the Web of Science (ISI, Philadelphia) show that Hot Papers are cited 50 to 100 times more often than the average paper of the same type and age. L. Lin, J. Faraco, R. Li, H. Kadotani, W. Rogers, X.Y. Lin, X.H. Qiu, P.J. de Jong, S. Nishino, E. Mignot, "The sleep disorder canine narcolepsy is caused by a mutation in the hypocretin (orexin) receptor 2 gene," Cell, 98[3]:365-76 Aug. 6, 1999. (Cited in 179 papers) R.M. Chemelli, J.T. Willie, C.M. Sinton, J.K. Elmquist, T. Scammell, C. Lee, J.A. Richardson, S.C. Williams, Y.M. Xiong, Y. Kisanuki, T.E. Fitch, M. Nakazato, R.E. Hammer, C.B. Saper, M. Yanagisawa, "Narcolepsy in orexin knockout mice: Molecular genetics of sleep regulation," Cell, 98[4]:437-51, Aug. 20, 1999. (Cited in 179 papers) Courtesy of Masashi Yanagisawa Masashi Yanagisawa (right) with the orexin knockout mice (top) used for his research on narcolepsy. Severe daytime sleepiness, inability to stay awake or sleep for extended periods of time, cataplexy (muscle atonia triggered by emotions such as laughing or anger), and an inability to move on sleep onset or awakening: These are all signs of narcolepsy, an uncommon, disabling neurological disorder that affects about one in every 2,000 people. Narcolepsy has long fascinated sleep researchers because it is the only known neurological disorder that affects the generation and organization of sleep. In 1999, scientists of these Hot Papers identified the genes that cause narcolepsy, opening the door to understanding sleep at a molecular level. The findings marked the first time that a sleep gene was isolated, and led directly to understanding narcolepsy's pathology. The genetic pathway--involving a neuropeptide called orexin or hypocretin, and its receptor gene--was identified in two, nearly simultaneous, publications in Cell. A group at Stanford University discovered the receptor gene, and a group at University of Texas Southwestern Medical Center discovered the ligand gene. "The papers essentially confirmed one another. That's very important. It's rare to get an instant confirmation of a finding like this," says psychiatrist Emmanuel Mignot, of the Center for Narcolepsy at Stanford University School of Medicine and the senior investigator on the receptor gene paper. The two groups came to their findings using completely different approaches. The Stanford group, using the positional cloning approach, pinpointed that mutations in an Orexin receptor gene caused canine narcolepsy. The Texas group developed orexin knockout mice and observed in them narcoleptic-like sleep and behavioral abnormalities. "The beauty here is that two independent groups studied the gene from totally different directions using different approaches and different species. It's amazing that ultimately we published such similar findings so closely together," says molecular scientist Masashi Yanagisawa, a Howard Hughes Medical Institute investigator at University of Texas Southwestern Medical Center and the senior investigator on the gene ligand paper. Orexin From a Narcolepsy Perspective Mignot, who has worked in narcolepsy research for 15 years, isolated the orexin receptor after searching much of that time for the cause of narcolepsy in canine models. Mignot and his group conducted lineage studies on three breeds of dogs--Doberman pinschers, Labradors, and Dachshunds--in which the disorder is transmitted as an autosomal recessive trait called canarc-1. After cloning the canine narcolepsy gene, the researchers identified the gene mutation that encodes the receptor for orexin receptor 2, a novel neuropeptide that causes narcolepsy. A few months after publishing the canine isolation findings, the group followed with another report confirming the gene's role in human narcolepsy in which measurements of cerebrospinal fluid in narcoleptic patients showed that they had no orexins.1 Postmortem mutation studies of human narcoleptic patients confirmed that the brains lacked orexin-containing cells. "That was the first evidence that humans have hypocretin deficiency that causes this disorder," Mignot says. The gene has exerted a strong influence on the field of sleep research. Although orexin is so far only specifically implicated in narcolepsy, many researchers consider it possible that the gene is involved in all sleep regulation. Conclusive studies have not yet proven the gene's physiological effect, but all signs point to this, according to Mignot. "It fits very well for being a mastermind for controlling a lot of cell groups that we know are significantly changing during sleep," Mignot says. He lists support for the theory in the findings of the phenotype for knockout mice, the strong sleep-inhibiting effects of orexin injections into the cerebrospinal fluid, and neuroanatomical findings that orexin acts on monoaminergic cell groups known to change in the sleep cycle. Currently, Mignot is working on these physiological experiments and trying to find other genes in the sleep pathway. He's also pursuing how narcolepsy actually occurs. Human cases are sporadic and almost never genetically inherited. Research has associated human leucocyte antigen (HLA) with the disorder, suggesting autoimmunity. The working hypothesis is that a specific HLA allele targets and knocks out orexin in the brain. Orexin from a Molecular Genetics Perspective Yanagisawa's group also continues to pursue experiments related to narcolepsy. Originally, they did not expect that their work would relate to sleep disorders at all. When they discovered orexin in 1998, they thought that it was implicated in feeding behavior. Yanagisawa's group and another group independently identified the same neuropeptide system in 1998--calling it, respectively, orexin and hypocretin.2,3 One paper identified orexin using a fancy differential PCR technique while the Yanagisawa group used brute-force biochemical purification, based on ligand binding to cell lines expressing selected orphan G-protein coupled receptors. The findings, the basis for an earlier Hot Paper article,4 indicated orexin upregulation during times of hunger. When Yanagisawa started to conduct experiments with knockout mice lacking orexin, he expected that the mice would exhibit disturbances in their appetite. Mice feed primarily at night, so experiments included infrared videotape observation. It was in reviewing these tapes that Yanagisawa and his colleagues suddenly realized that the knockout mice slept at inappropriate times, exhibiting a cataplexic type of narcolepsy. "That was completely unexpected. This initial behavior attack was so striking that we wanted to diagnose it," says Yanagisawa. Measurement of brain waves using EEG immediately revealed that the sleep pattern was disturbed, and the findings implicated narcolepsy, he adds. Although the role of orexin in narcolepsy is now proven, Yanagisawa still believes that the molecule also plays a role in eating behavior and appetite control. His current, central hypothesis is that a regulatory mechanism in the brain links nutritional status to sleep and wakefulness regulation, and orexin might be that mechanism. The hypothesis ties into the narcolepsy findings, he says. "Everyone knows that when hungry, animals are more vigilant. And when satiated, they are more sleepy. This applies to humans as well," Yanagisawa says. It might be, he adds, that when orexin is present and acting normally in the brain, it controls these adaptive responses linking hunger and wakefulness, but when it is completely absent in the brain, the abnormality manifests itself as narcolepsy. Yanagisawa also continues to pursue sleep-related research. In particular, he recently published a report on a new transgenic mouse in which the orexin promoter drives the expression of a neurotoxin that postnatally destroys orexin-producing neurons.5 The mice exhibit the same narcolepsy phenotype as the orexin knockout mice. The opportunity that orexin offers for developing a fundamental cure for narcolepsy is interesting, too, Yanagisawa notes. The idea is to make an orally active orexin mimic, or orexin receptor agonist. The orexin receptor is a G-protein coupled receptor (GPCR), making it particularly amenable in drug discovery because of its small size and structure, he says. Scientists are hopeful that orexin will lead the way for not only narcolepsy therapy, but also for other sleep disorder therapies. Interest is high particularly because sleep disorders, such as restless leg syndrome, insomnia, and sleep apnea, are common in humans, and only symptomatic treatment is available. Yanagisawa predicts that an orexin-related treatment for narcolepsy could be available within the next 10 years. Jennifer Fisher Wilson (jfwilson@snip.net) is a contributing editor for The Scientist. References 1. S. Nishino et al., "Hypocretin (orexin) deficiency in human narcolepsy," Lancet, 355:39-40, 2000. 2. T. Sakurai et al., "Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior," Cell, 92:573-85, 1998. 3. L. de Lecea et al, "The hypocretins: hypothalamus-specific peptides with neuroexcitatory activity," Proceedings of the National Academy of Sciences, 95:322-7, 1998. 4. J. Kling, "A role for reverse pharmacology: orexin discovery benefits two fields of research," The Scientist, 14[6]:18, March 20, 2000. 5. J. Hara et al., "Genetic ablation of orexin neurons in mice results in narcolepsy, hypophagia, and obesity," Neuron, 30[2]:345-54, May 2001.

Identifying the First Sleep-Related Genes

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