In comparison to other body systems, the brain remains as largely uncharted territory, with scientists only beginning to plumb its depths. Moreover, while sleep is essential for life and we spend one-third of our lives doing it, the function of sleep and the neurophysiology that regulates it remain a longstanding mystery within biology. Yet now, an international team of investigators from the University of Tsukuba, Japan and the University of Texas (UT) Southwestern Medical Center has just identified the first two core genes that regulate the amount of deep sleep and dreaming—a key development they believe will lead to the discovery of a network of related genes controlling sleep.
The researchers utilized an unbiased genetic screen for sleep defects in mice, which yielded two interesting mutants that they appropriately named Sleepy—which sleeps excessively—and Dreamless—which lacks rapid eye movement (REM) sleep. Instead of beginning with a hypothesis about specific genes that might be involved, the researchers introduced random genetic mutations in more than 8000 mice and screened them using electroencephalography (EEG) to determine which ones had abnormal sleep as a result of the genetic perturbations.
“To be able to screen 8000 mice is something that most people would say is too much work,” noted HHMI investigator and co-senior study author Joseph Takahashi, Ph.D., chairman of neuroscience with the O'Donnell Brain Institute at UT Southwestern Medical Center. Dr. Takahashi explained that each mouse had to be surgically wired for the EEG readings, among other steps. “Technically, this project was very challenging.”
“The barrier in the past has been that it's a very laborious process,” Dr. Takahashi added. “To do a genetic screen, you should be prepared to screen thousands of animals before you find something interesting, and most people are just not willing to measure EEGs in thousands of mice.”
The findings from this new study were published recently in Nature in an article entitled “Forward-Genetics Analysis of Sleep in Randomly Mutagenized Mice.”
The Sleepy and Dreamless mutations were identified and subsequently mapped to locations within the mouse genome. Sleepy mice, which need approximately one-third more sleep than normal mice, carry a mutation in the Sik3 kinase gene. Because Sik3 kinase can phosphorylate many proteins, it is likely to be involved in many signaling pathways, which makes it trickier to characterize.
“We noticed that Sleepy mutants showed an exaggerated response to sleep deprivation,” remarked lead study author Hiromasa Funato, Ph.D., principal investigator at the International Institute for Integrative Sleep Medicine at the University of Tsukuba. “Examining the brains of sleep-deprived mice revealed changes in the phosphorylation of amino acids within the SIK3 protein. These changes were disturbed by the Sik3 mutation in Sleepy mice, which is why they have an increased sleep need.”
Alternatively, Dreamless mice, which have reduced REM sleep, carry a mutation in a sodium channel. Understanding the effects of the dreamless mutation was more straightforward. The mutation increases the conductivity of a leaky sodium channel that was previously known to regulate neuronal excitability.
“This mutant pedigree, named Dreamless, carries a mutation in the Nalcn gene, which encodes an ion channel thought to control neuronal excitability,” explained study co-author Chika Miyoshi, Ph.D., assistant professor at the International Institute for Integrative Sleep Medicine. “The Dreamless mutation caused increased ion conductance through the channel and increased activity of REM-terminating neurons, which is compatible with REM sleep instability.”
Interestingly, genes related to Sik3 in Drosophila and nematode worms were also shown to be involved in regulating the amount of sleep-like behaviors in these animals, while a mutation in a Nalcn-related Drosophila gene was previously found to control a circadian change in neuronal excitability. These conserved roles in invertebrate genes show the importance of managing sleep in all animals.
“This research is just the beginning. We believe that these two genes are the first of many that regulate sleep,” said Dr.Takahashi.
“We hope that the discovery of these essential genes is just the beginning of our long journey into the black box of sleep regulation,” concluded co-senior study author Masashi Yanagisawa, Ph.D., Professor in the International Institute for Integrative Sleep Medicine, University of Tsukuba and department of molecular genetics at UT Southwestern Medical Center. “It is amazing that we know almost nothing about the simple question of what is 'sleepiness' physically in our brain. We will start from these genes and try to solve the great mystery.”