Genetic Analysis of Sleep
by Amanda Crocker and Amita Sehgal, Howard Hughes Medical Institute, Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia
Almost 20 years ago, the gene underlying fatal familial insomnia was discovered, and first suggested the concept that a single gene can regulate sleep. In the two decades since, there have been many advances in the field of behavioral genetics, but it is only in the past 10 years that the genetic analysis of sleep has emerged as an important discipline.
Major findings include the discovery of a single gene underlying the sleep disorder narcolepsy, and identification of loci that make quantitative contributions to sleep characteristics. The sleep field has also expanded its focus from mammalian model organisms to Drosophila, zebrafish, and worms, which is allowing the application of novel genetic approaches.
Researchers have undertaken large-scale screens to identify new genes that regulate sleep, and are also probing questions of sleep circuitry and sleep function on a molecular level. As genetic tools continue to be refined in each model organism, the genes that support a specific function in sleep will become more apparent. Thus, while our understanding of sleep still remains rudimentary, rapid progress is expected from these recently initiated studies.
The recognition that sleep may be regulated by conserved genetic mechanisms has not yet led to a unified understanding of it. A closely related process—the generation of circadian rhythms—is now explained on the basis of a universal model, largely because of mechanistic studies done in phylogentically very diverse organisms.
If there is a specific neurotransmitter for sleep, it is still hypothetical. Thus, sleep does not appear to be controlled by a singe locus or dedicated genes. It is better understood as a broad system-wide phenomenon.
Hypotheses for sleep include somatic theories (healing of the body and other endocrine functions), cellular metabolic theories (removal of reactive oxidative species and energy replenishment), brain-specific functions such as synaptic plasticity (in adults, this would underlie memory consolidation), or synaptic downscaling.
Genetics provides a new way to address the regulation and function of sleep. While for the past 20 years genetics has been used primarily to verify lesion and pharmacological studies through targeted gene approaches, it can now be used to probe more intricate questions in sleep.
Identification of genes required for sleep homeostasis
The big question remains: Why do we sleep? There is now the growing sense that the function of sleep may fall out of its molecular analysis. Since few sleep-regulating molecules are known, studies are under way to identify novel genes required for sleep. These studies include forward genetic screens as well as genetic manipulation of candidate genes, by focusing on changes in sleep amount as a readout of sleep homeostasis.
Sleep and metabolism
There have long been theories that sleep is important for metabolism (Benington and Heller 1995). This is supported by the potential role for adenosine, and by reports showing associations between glycogen levels and sleep. In addition, there appears to be anatomic overlap in the regulation of sleep and metabolism.
More recently, genes important for dealing with cellular stress have been implicated in sleep regulation. Through both differential expression profiles and targeted gene approaches, the gene Bip is implicated as a sleep-promoting factor. Bip is important for the unfolded protein response in the endoplasmic reticulum (ER), and is up-regulated following periods of sleep deprivation in mice (Cirelli et al. 2005b). In addition, flies with altered Bip levels show changes in their homeostatic response to sleep deprivation.
Genes important for synaptic modulation
One of the current hypotheses for why we sleep is that it allows for, or even promotes, synaptic downscaling (Tononi and Cirelli 2006). This hypothesis is based on the presumption that, during wakefulness, the interaction of animals with their environment leads to the strengthening of some synapses, while others remain the same. It postulates that synaptic downscaling during sleep promotes efficiency in terms of energy and space, while maintaining the relative ratios of the strength of synapses. This hypothesis has been supported in recent years by differential expression studies of genes whose expression changes with sleep/wake state.
Genes involved in learning and memory
In both mice and flies, many genes important for learning and memory have been targeted for sleep analysis. These include, but are not limited to, CREB, protein kinase A (PKA), cAMP, ERK, cGMP, and some of the ion channels.
Genetics can tell us a lot about what sleep does for organisms, but the potential of this approach has only just started to be recognized in the sleep field. With the generation of conditional and anatomically restricted knockouts (or knock-ins) in mice, we are on the verge of answering many questions.
These include determining the roles of adenosine and BDNF in sleep and memory. In flies, anatomically and/or temporally restricted expression of sleep-regulating transgenes has already been performed.
These approaches have provided great insight into the role of specific signaling pathways in sleep. In the future, this technology will be used to rescue sleep mutants in a region-specific manner, although some of these mutations, such as in ion channels, may turn out to have global effects that cannot be rescued in specific areas.
However, the real power of the fly, worm, and fish models lies in their amenability to unbiased genetic screens. With a process like sleep, about which little is known, we suggest that the best approach is one that is not associated with any preconceived assumptions,
At this point, there is no evidence that a single gene, or subset of genes, acting in a specific subset of neurons is responsible for sleep.
It is more likely that sleep is a network phenomenon. It is also likely that there will be many hypotheses for why we sleep and strong evidence for each, since many of the neurotransmitters and signaling pathways that keep us awake serve other functions.
For instance, orexin is apparently involved in both feeding behavior and maintaining wakefulness. Sleep deprivation results in several impaired processes, some of which may turn out to reflect consequences of increased wakefulness rather than indicating an actual function of sleep.
With the advancement of new genetic tools, it is likely that we will soon see experiments directly testing some of these hypotheses, such as cellular metabolic function and synaptic scaling.
From the data discussed in this review, it is likely that sleep is important for overall homeostatic regulation of the entire organism, possibly down to within-the-cell homeostasis.
It is clear that sleep is a very basic process, and that studying it in model organisms will provide significant insight into why we sleep. In general, advances in genetics in all model organisms will provide a wealth of knowledge for the sleep field in the coming years.
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