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When researchers turned off the gene Lhx1 in the SCN of mouse embryos,
some fell into a pattern of two to three separate cycles of sleep and activity per day,
in contrast to the single daily cycle found in normal mice, while others' rhythms
were completely disorganized.
Protein crucial to circadian rhythms found in mice
Johns Hopkins research reports they have identified a protein essential to the formation of the tiny brain region in mice that coordinates sleep-wake cycles and other circadian rhythms.
By disabling the gene for that key protein in test animals, the scientists were able to home in on the mechanism by which that brain region, known as the suprachiasmatic nucleus or SCN, becomes the body's master clock while the embryo is developing.
The results of their experiments, reported April 2 in Cell:Neuron are an important step toward better management of the bad effects of disruptive sleep habits on shift workers, as well as treatment for people with other sleep disorders.
"Shift workers tend to have higher rates of diabetes, obesity, depression and cancer. Many researchers think that's somehow connected to their irregular circadian rhythms, and thus to the SCN. Our new research will help us and other researchers isolate the specific impacts of the SCN on mammalian health."
Seth Blackshaw, PhD, associate professor, Department of Neuroscience and the Institute for Cell Engineering, Johns Hopkins University School of Medicine
Blackshaw explains that every cell in the body has its own "clock" that regulates aspects such as its rate of energy use. The SCN is the master clock that synchronizes these individual timekeepers so that people feel sleepy at night and alert during the day, are hungry at mealtimes, and are prepared for the energy influx that hits fat cells after eating.
"A unique property of the SCN is that if its cells are grown in a dish, they quickly synchronize their clocks with each another."
Seth Blackshaw, PhD
But while evidence like this gave researchers an idea of the SCN's importance, they hadn't completely figured out it performed differently from that of the body's other clocks, or from other parts of the brain.
The Johns Hopkins team looked for ways to knock down SCN function by targeting and disabling certain genes that disrupt the formation of the SCN clock. They analyzed which genes were active in different areas of developing mouse brains to identify only those "turned on" in the SCN. One of the "hits" was Lhx1, a member of a family of genes which affect development through the controll of other genes.
When the researchers turned off Lhx1 in the SCN of mouse embryos, the adult mice lacked distinctive biochemical signatures found in the SCN of normal mice.
The genetically modified mice behaved differently, too. Some fell into a pattern of two to three separate cycles of sleep and activity per day, in contrast to the single daily cycle found in normal mice, while others' rhythms were completely disorganized, said Blackshaw. Though an SCN is present in mutant mice, it communicates poorly with clocks elsewhere in the body.
Blackshaw expects that mutant mice will prove a useful tool in finding if disrupted signaling from the SCN actually leads to the health problems experienced by shift workers. And if so, how this might happen. Although mouse models do not correlate fully to human disease, their biochemical and genetic makeup are closely aligned.
Blackshaw's team also plans to continue studying the biochemical chain of events surrounding the Lhx1 protein to determine what it switches on or off and in turn, what turns Lhx1 on. Those genes could be at the root of inherited sleep disorders, Blackshaw says, and the proteins they make could prove useful as starting points for the development of new drugs to treat insomnia and even jet lag.
•WAKE acts in arousal-promoting clock cells (LNvs) to regulate timing of sleep onset
•WAKE levels cycle in these LNv cells, and WAKE expression depends on CLOCK function
•WAKE acts downstream of CLOCK to upregulate RDL, a GABAA receptor
•WAKE reduces excitability of LNvs at dusk to promote the switch from wake to sleep
How the circadian clock regulates the timing of sleep is poorly understood. Here, we identify a Drosophila mutant, wide awake (wake), that exhibits a marked delay in sleep onset at dusk. Loss of WAKE in a set of arousal-promoting clock neurons, the large ventrolateral neurons (l-LNvs), impairs sleep onset. WAKE levels cycle, peaking near dusk, and the expression of WAKE in l-LNvs is Clock dependent. Strikingly, Clock and cycle mutants also exhibit a profound delay in sleep onset, which can be rescued by restoring WAKE expression in LNvs. WAKE interacts with the GABAA receptor Resistant to Dieldrin (RDL), upregulating its levels and promoting its localization to the plasma membrane. In wake mutant l-LNvs, GABA sensitivity is decreased and excitability is increased at dusk. We propose that WAKE acts as a clock output molecule specifically for sleep, inhibiting LNvs at dusk to promote the transition from wake to sleep.
Other authors on the paper were Joseph L. Bedont, Tara A. LeGates, Mardi S. Byerly, Hong Wang, Jianfei Hu, Alan C. Rupp, Jiang Qian, G. William Wong and Samer Hattar, all of Johns Hopkins, and Emily A. Slat and Erik D. Herzog of Washington University, Saint Louis.
This study was supported by the Johns Hopkins Brain Science Institute and the National Institute of Mental Health (grant number 63104).
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