Genetic And Molecular Mysteries Of Sleep Are Keeping Researchers Alert

SIDEBAR : Sleep Research Resources Some consider sleep an unavoidable nuisance; others, a sweet indulgence. For the most part, though, we take our slumber for granted, rarely considering why we spend a hefty chunk of our lives unconscious. But for sleep researchers, that question represents a supreme mystery. Exactly what purpose sleep serves, as well as how the body regulates sleeping and waking, remain largely unknown. Behavioral scientists and physiologists have pursued these questions for decades, laying the groundwork for more recent recruits to sleep scholarship: cell and molecular biologists, geneticists, and neuroscientists, among others. Much like their counterparts who study the molecular origins of hunger and obesity, these new sleep researchers expect to gain novel insights into this basic biological drive. But at this early stage, ambiguous and conflicting evidence often adds to the field's inherent mystery. "I think the function of sleep will ultimately be determined by molecular biology, by identifying differences between sleeping and waking brain cells," observes Priyattam Shiromani, an associate professor of psychology at Harvard University. He says he expects such efforts will reinforce long-standing notions that sleep is restorative and will reveal cellular factors that regulate sleep and wakefulness. That knowledge could prove valuable to the more than 40 million Americans who suffer from sleep problems, according to the National Center on Sleep Disorders Research (NCSDR). Situated within the National Heart, Lung and Blood Institute, the center was established in 1993 to support and coordinate research and public health efforts in the sleep-research area. According to NCSDR, sleep disorders, sleep deprivation, and sleepiness add more than $15 billion to the United States' health care bill. Lost worker productivity and accidents would likely increase that amount, but actual costs have never been calculated. TURNING IN: James Kiley cites efforts to take sleep studies to the molecular level. "We think molecular and genetic approaches to understanding sleep function and the mechanisms that control it are ripe for research right now," says the center's director, James Kiley. Accordingly, the National Institutes of Health recently announced a request for applications to fund research on the molecular and genetic basis of sleep and sleep disorders. NIH has set aside $3.2 million for such projects in fiscal year 1997, Kiley reports. "The emphasis is to take research on sleep, which is now conducted largely on organs or whole animals, and transfer it to the cellular, molecular, and biochemical level." Sleep Research Society (SRS) and American Sleep Disorders Association (ASDA) 1610 14th St., N.W., Suite 300, Rochester, Minn. 55901 (507) 287-6006 % Fax: (507) 287-6008 ASDA E-mail: asda@millcomm.com SRS Web site: http://bisleep.medsch.ucla.edu/SRS/srs_main.html ASDA Web site: http://www.wisc.edu/asda 600 members (SRS); 2,700 members (ASDA) Presidents: J. Christian Gillin (SRS), David P. White (ASDA) Executive Director: Jerome Barrett (ASDA) Journal: SLEEP (published by ASDA and SRS) (http://www-leland.stanford.edu/dept/sleep/journal) National Sleep Foundation 1367 Connecticut Ave., N.W., Suite 200, Washington, D.C. 20036 (202) 785-2300 - Fax: (202) 785-2880 E-mail: natsleep@haven.ios.com President: Lorraine Wearley Associate Director: Reid Blank Newsletter (for physicians): Sleep Medicine Alert Web site: http://www.websciences.org/nsf/ Additional Journals: Journal of Sleep Research Blackwell Science Ltd. P.O. Box 88, Oxford OX2 OEL, England Phone: 011-44-1865-206206 - Fax: 011-44-1865-721205 Sleep Research Brain Information Center P.O. Box 951746, UCLA School of Medicine Los Angeles, Calif. 90095 (310) 825-3417 - Fax: (310) 206-3499 E-mail: kstewart@ucla.edu or mchase@ucla.edu web site: http://www.bisleep.medsch.ucla.edu Internet Site: Additional information on the National Center on Sleep Disorders Research can be obtained through the National Institutes of Health's home page: http://nhlbi.nih.gov/nhlbi/nhlbi.htm. One sleep disorder that appears particularly amenable to molecular genetic studies is narcolepsy, a disturbance in the normal pattern of brain activity during sleep. NCSDR estimates that 250,000 Americans have narcolepsy-many also suffer from Parkinson's disease or multiple sclerosis. However, not all of those who are afflicted exhibit the disorder's most dramatic symptom: episodic loss of muscle control triggered by sudden emotional reactions. Most feel overwhelmingly sleepy during the day and risk falling asleep while driving or working. Studies of familial narcolepsy in dogs, as well as in people, may provide the first clues to the cause of this debilitating and apparently complex disorder. Moreover, Kiley observes, "anything we do to better understand the genetic basis of a sleep disorder will tell us something about the nature of normal sleep." Two distinct regulators appear to influence sleep behavior. Circadian rhythms-alternating cycles of light and dark that control biological clocks (R. Lewis, The Scientist, Dec. 11, 1995, page 14)-dictate the time of day that most animals sleep. But researchers also suspect that a homeostatic (feedback) process governs the amount and depth of sleep. Such a mechanism would explain why the longer people go without sleep, the drowsier they become-and the longer and more deeply they're likely to snooze when they finally catch up. "There is a circadian regulator of sleep in [the part of the brain known as] the suprachiasmatic nucleus," explains Shiromani. If those neurons are destroyed in experimental animals, he continues, they still sleep as much as ever, though not in a daylight-determined pattern. "It has long been suspected that there might be a sleep-inducing factor built up during wakefulness that dissipates during sleep," Shiromani says. "What is it? We don't know." In an initial attempt to reveal molecular differences between sleeping and waking neurons, Shiromani and colleagues in the laboratory of Clifford Saper, a professor in Harvard's department of neurology (J. Sherin et al., Science, 271:216-9, 1996), monitored the expression of a protein, c-Fos, in certain neural clusters known to be electrically active during sleep. In sleeping animals, these active cells expressed high levels of the c-Fos protein compared with quiescent neurons, the researchers discovered. Now that they've identified at least one sleep-regulated gene, Shiromani and coworkers plan to examine mice that lack the c-Fos gene in order to study the protein's physiological role in sleep-active neurons. In contrast to the idea that sleep-inducing substances accumulate in wakeful brain cells, some researchers theorize that an energy deficit triggers slumber. A recent paper in Progress in Neurobiology (J. Benington, H.C. Heller, 45:347-60, 1995) suggests that the depletion of glycogen, the brain's only source of stored energy, signals cells to sleep. Glycogen stores are limited and possibly depleted during waking hours in particularly active regions of the brain, according to author Joel Benington, now an assistant professor of biology at St. Bonaventure University in St. Bonaventure, N.Y. He and coauthor H. Craig Heller, a professor of biology at Stanford University who was Benington's postdoctoral adviser when the paper was written, speculated that the neurotransmitter adenosine-which is elevated when cellular glycogen stores are depleted-could be the messenger that sends brain cells to sleep. "Adenosine makes neurons less responsive, so it could make sleep even deeper after sleep loss," Benington observes. Moreover, he says, caffeine blocks adenosine receptors, which he says are located in "all the right places" in the brain to control slow-wave sleep, the dreamless slumber that increases in intensity following sleep deprivation. Based on this suggestive evidence, the researchers are investigating whether glycogen reserves are, in fact, depleted in the waking brain. The idea that the brain produces sleep-inducing substances is hardly new. Early in this century, two French physiologists, Rene Legendre and Henri Pieron, injected cerebrospinal fluid from sleep-deprived dogs into others that were well-rested, prompting them to fall into a deep, prolonged slumber (S. Inoue, Biology of Sleep Substances, Boca Raton, Fla., CRC Press, 1989). The researchers never successfully isolated the somnogenic compound. Recently, however, scientists at the Scripps Research Institute revisited these early experiments. Thanks to advances in analytical chemistry, they were able to isolate an intriguing molecule-a fatty acid primary amide they call oleamide-from the cerebrospinal fluid of sleep-deprived cats (B. Cravatt et al., Science, 268:1506-9, 1995). The researchers chemically synthesized the substance and reported that it induced sleep when injected into rats. They also discovered an enzyme that breaks down oleamide in vivo and found that synthetic inhibitors of this enzyme produce the same effects as oleamide when administered to animals. "We're interested in [oleamides] for a variety of reasons," notes Steven Henriksen, an associate professor at Scripps and one of the authors of the paper. For one thing, he says, "these lipids appear far more pharmacologically selective than current therapeutics for insomnia." People frequently become desensitized to or dependent on sleeping pills because the drugs act directly to block or enhance neurotransmitter receptors, according to Henriksen. He speculates that oleamide may act indirectly to modify receptors and thus may prove a safer soporific. Other researchers, however, have failed to reproduce the sleep-inducing effects of oleamide in rats. "For whatever reason, we just didn't see the same effects [from oleamide] on sleep as the Scripps researchers," reports Dale Edgar, director of the Sleep and Circadian Neurobiology Laboratory and an associate professor of psychiatry and behavioral sciences at the Stanford University School of Medicine. He and coworkers-as well as a second independent research group at the Janssen Research Foundation in Beerse, Belgium-tested oleamide prepared by the R.W. Johnson Pharmaceutical Research Institute in Raritan, N.J. The institute is a subsidiary of New Brunswick, N.J.-based Johnson and Johnson, then the holder of the patent for the compound. Both groups got similarly negative results (C. Dugovic et al., Journal of Sleep Research, 5 [Suppl.1]: 54, 1996). In order to avoid these apparent problems with oleamide and thereby produce a promising drug candidate, Scripps researchers are developing and testing chemical analogs of the inhibitor of the oleamide-destroying enzyme. "Excessive daytime sleepiness [due to insomnia] has contributed to a host of disasters, from Chernobyl to major oil spills," Henriksen notes. "Anything that would promote normal, healthy sleep without side effects would be welcome." Immunological Functions CONNECTION: James Krueger explores the link btween sleepiness and the immune response. Another class of sleep-inducing substances came to light about 10 years ago, when physiologists began probing the common experience of sleepiness at the onset of influenza and other infections. "We soon discovered that the immune response to infection directly affects sleep," recalls James Krueger, a professor of physiology at the University of Tennessee in Memphis. Krueger and others found that a host of molecules previously recognized as immunomodulators, including interleukin-1, tumor necrosis factor (TNF), and other cytokines, were also produced and recognized by receptors in the brain, where they were shown to induce sleep (J. Krueger, Neuroimmunomodulation, 1:100-9, 1994). Levels of these somnogenic molecules were shown to rise in response to a variety of foreign stimuli, such as peptidoglycans from bacterial cell walls and double-stranded RNA from viruses. Recently, Krueger reports, researchers in his laboratory have discovered that mice lacking one of two TNF receptors-which renders the animals unresponsive to the cytokine-sleep less than control mice do. "It's surprising that they fail to compensate developmentally" for these changes, Krueger observes. NO DOZE: Carol Everson studies sleep deprivation in rats. Just as immune modulators appear to influence sleep, so sleep-or the lack of it-seems to affect the immune system. For example, Krueger's Tennessee colleague Carol Everson, an assistant professor of physiology, has shown that after prolonged sleep deprivation, rats' immune systems appear to become suppressed. The animals ultimately die of systemic infections (C. Everson, Behavioural Brain Research, 69:43-54, 1995). Rats deprived of basic needs such as food or water-or of sleep-show few overt signs of gross pathology until near death, she explains. Eventually, however, something goes obviously wrong, and that something may provide clues to the early consequences of deprivation. "We're still not certain if sleep serves a direct immunological function, or whether sleep deprivation affects hormonal and neurological systems that eventually lead to decreased resistance to infection," explains Everson. To resolve this dilemma, she is carefully studying the time course of sleep deprivation in rats, tracing the path by which the deadly microorganisms overcome their sleep-deprived host. In doing so, she says, "we're trying to understand the timing, location, and mechanisms of local immunosuppression." UP ALL NIGHT: Harvey Moldofsky investigates sleep deprivation. Chronically sleep-deprived humans appear to suffer from a lesser version of these rodents' fate, according to Harvey Moldofsky, director of the Center for Sleep and Chronobiology at the University of Toronto. For example, he says, nonrestorative sleep is intimately linked with fibromyalgia (chronic fatigue syndrome). "The sleeping, waking brain appears to serve as a conductor for a the body's peripheral functions. The immune and endocrine systems, as well as thermoregulation-they're all interconnected," Moldofsky maintains. "If you deprive someone of sleep, these systems become disregulated and the body becomes vulnerable to disease." Links between sleep and the immune system are also being explored by scientists studying the genetic origins of narcolepsy. Like insulin-dependent diabetes or multiple sclerosis, narcolepsy in humans appears to result from a combination of multigenic and environmental influences, according to Emmanuel Mignot, director of Stanford University's Center for Narcolepsy and an assistant professor of psychiatry and behavioral sciences. However, a clear linkage marker for narcolepsy has been identified in dogs, which, unlike humans, inherit narcolepsy as a simple autosomal recessive trait. The marker is a DNA segment highly homologous to a known human immunoglobulin switch, which suggests that the disorder might be a form of autoimmune disease, Mignot observes. While narcolepsy runs in some families, for the most part it appears to arise sporadically. Mignot and coworkers are taking a two-pronged approach to tracing the genetic roots of the disorder. To identify as many narcolepsy-related genes as possible, they are conducting linkage analyses of families with several affected members. They are also attempting to use the canine narcolepsy marker to isolate its potentially influential human homolog. NARCOLEPSY STUDIES: Jerome Siegel seeks a marker in dogs. Equally obscure as the genetic roots of narcolepsy are the physiological changes that underlie its symptoms. Last year, however, Jerome Siegel, chief of neurobiological research at Sepulveda Veterans Affairs Medical Center in Sepulveda, Calif., and a professor of psychiatry at the University of California, Los Angeles, reported the first evidence of narcolepsy-related neural damage (Society for Neuroscience Abstracts, 21:1235, 1995). Examining the brains of young narcoleptic Doberman pinschers, Siegel and coworkers found signs of significant axonal degeneration in areas known to be important to emotional and motor control. "No one had previously seen signs of neurodegeneration in either human or animal narcoleptics," Siegel says, adding that these differences between narcoleptic and normal brains could be observed for only a limited period in young animals. With time, the debris that results from this early damage gets metabolized, obscuring all traces of pathology in older animals, he reports. The only way researchers will be able to document the occurrence of a similarly transient process in humans is by identifying a biochemical marker for the cell type or component lost in narcolepsy that can be detected in older brains, according to Siegel. "If we can find some kind of marker that works in dogs," he explains, "maybe we can look in humans and see the same kind of damage there." As with most molecular sleep research, these explorations of narcolepsy at the cellular level are in their infancy. Current studies of the genetic bases of sleep disorders resemble research on cystic fibrosis about 10 years ago-before a gene was identified-comments NIH's Kiley. "We have lots of tools and techniques-and many good questions-to investigate sleep at the molecular level," he states. "At the moment, there's relatively little going on. But with appropriate attention, we expect an explosion of new knowledge." Alison Mack is a freelance science and medical writer based in Wilmington, Del.

Genetic And Molecular Mysteries Of Sleep Are Keeping Researchers Alert

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