Issue Date: June 4, 2012
Picking Apart Our Circadian Clock
Frequent travelers are only too familiar with the fact that our bodies keep track of time. We may be able to travel across multiple time zones in just a few hours, but our clock keeps ticking to the time at home. On our first days in the new time zone, we feel disoriented and sleep deprived.
These effects are manifestations of our circadian clock, the body’s internal machinery that tracks time in approximately 24-hour cycles. The human circadian clock is controlled by about 20 genes that are turned on and off in a tightly orchestrated, cycling feedback loop. To tick to a new time zone—that is, to get over jet lag—this circadian machinery needs to witness a few daylight and dark cycles.
The clock may be a bane to shift workers and travelers—not to mention blind people who can’t perceive the light needed to tune their clock to Earth’s 24-hour cycle—but our circadian clock does confer a variety of advantages. One of the most prominent is metabolic efficiency, which the clock promotes by timing activities with day-night rhythms, such as feeling sleepy at the end of the day.
When we are asleep at night, we don’t need to supply our stomachs with digestive enzymes or our muscles with the chemical energy to move around. Instead, the body diverts energy resources to our immune system for upkeep and to the brain for memory storage. “Even a modest increase in efficiency—but in every cell of the body—can make a big difference overall to an organism,” says Joseph Takahashi, at the University of Texas Southwestern Medical Center, whose lab discovered and cloned several mammalian-clock genes.
The circadian clock has long been accepted as a factor in human sleep cycles. Now researchers are waking up to the fact that it also plays an important role in health and disease. Addiction, infertility, obesity, diabetes, cancer, and heart disease are just a few of the major medical problems that can be connected to circadian rhythm dysfunction.
Long ignored by the pharmaceutical industry—except now and again as a possible target for sleep medication—our circadian clock is primed to be a major player in drug discovery for a wide range of diseases. It has potential impact on “every protein target we work on,” says Timothy M. Willson, director of chemical biology at GlaxoSmithKline.
All living organisms on Earth have an internal clock that ticks to the light cycle of the planet’s rotation. The circadian rhythm helps an organism anticipate and “prepare for the different events that will happen during the day,” explains Thomas Burris, who studies the clock at Scripps Research Institute in Jupiter, Fla. Plants, for example, have long been known to use the circadian clock to anticipate sunrise and rotate their leaves to the east. Early this year, Rice University researchers found that the Arabidopsis plant clock makes sure chemical defense molecules are produced just before dawn, in preparation for caterpillar attacks, which typically occur in the morning (Proc. Natl. Acad. Sci. USA, DOI: 10.1073/pnas.1116368109). Expending the energy to make the defense molecules all night long would be wasteful. In humans, the circadian clock orchestrates myriad biological functions such as glucose metabolism and visual processing by the retina, as well as the daily rise and fall of blood pressure and body temperature.
The first biological clocks likely evolved 2.5 billion years ago, just after the Great Oxidation Event, the environmental change that first exposed microbes on the planet to harmful, daily fluxes of reactive oxygen species in the atmosphere. Akhilesh B. Reddy, a circadian scientist at Cambridge University, and Andrew J. Millar from the University of Edinburgh have found that peroxiredoxin proteins cycle through oxidation and reduction with a 24-hour rhythm, which could have afforded microbes protection from cycling concentrations of reactive oxygen species (Nature, DOI: 10.1038/nature11088). They’ve also found functioning vestiges of this clock in all organisms, from bacteria to humans, but to date their research has not determined what role, if any, peroxiredoxin proteins play in modern-day circadian rhythms.
Instead, all organisms have evolved a way to keep time by means of a cycling feedback loop of gene transcription and translation. The genes involved are different in plants, insects, animals, birds, bacteria, and fungi. This parallel development of different clocks that use a similar genetic-feedback-loop mechanism is an example of convergent evolution—like wings, which operate similarly in birds and insects but evolved independently. Having a circadian clock is so useful that all organisms evolved a way to coordinate biological processes with the daily sunrise and sunset.
Because our body and much of its metabolism are tuned to this sunlight cycle, it should come as no surprise that long-term disruption of this cycle will have serious effects, and not just on sleeping patterns. Epidemiologists have long noted that “people with disruption of the clock—shift workers such as nurses and flight attendants—all have higher risk of cancer, metabolic syndrome, obesity, and depression,” says Paolo Sassone-Corsi, a chronobiologist at the University of California, Irvine. “Disruption of the clock brings dramatic consequences.”
Proving a causal relationship between such conditions and circadian rhythm disruption, however, has been hard when so many variables may be at play. Last month, Northwestern University’s Fred W. Turek and coworkers published a paper making an explicit link in mice between shift-work-like schedules and poor pregnancy outcomes, such as reproductive abnormalities and miscarriage (PLoS One, DOI: 10.1371/journal.pone.0037668). The study “may have important implications for the reproductive health of female shift workers, women with circadian rhythm sleep disorders, and/or women with disturbed circadian rhythms for other reasons,” the authors note.
In March of this year, researchers reported the first molecular mechanism, based on the circadian clock, explaining the longtime medical observation that heart attacks occur more frequently in the early-morning waking hours than at any other time of the day. The team found that the circadian clock modulates the expression of a protein called KLF15, which in turn controls the expression of an important component of a potassium ion channel required to keep hearts beating rhythmically (Nature, DOI: 10.1038/nature10852). Deficiency or excess of KLF15 causes arrhythmic heart rates, abnormal repolarization, and enhanced vulnerability to heart attacks. Why precisely the clock modulates KLF15’s expression in this way is unknown, but the Case Western Reserve University scientists who made the discovery think the work is a first step in “future efforts to prevent or treat cardiac arrhythmias by modulating the circadian clock” with drugs or behavior.
More than reproduction and heart health, the majority of work connecting the circadian clock to disease has focused on energy metabolism, obesity, and diabetes. “The clock can anticipate that the animal is going to be fasting during the night and eating during the morning,” Burris explains. For example, during the day, the clock tells the body not to release as much glucose from energy stores because it can get energy from meals. But, he says, “in the nighttime, when you are fasting and need to have glucose, the clock induces gluconeogenesis from the liver. If you have gluconeogenesis induced at the wrong time, you will probably get diabetes.
“The problem now is that we are living a very different lifestyle than what we evolved as humans to live. So there is a lot of desynchronization in what the clock is expecting in terms of feeding and fasting versus what we actually do—midnight snacks, for example. We evolved as hunter-gatherers. If you think of the scale of evolution, a lot of things like farming are recent developments. And the even more modern development of food being available at any whim, especially high-calorie food, is really a big issue” for our energy metabolism cycles, Burris notes. “If you have a fatty meal when you are producing enzymes for releasing fat rather than breaking it down, then that fat is going to be shuttled into places it shouldn’t go.”
Modern circadian research has its roots in the 1950s and 1960s in Germany and the U.S. By the 1970s, researchers had discovered that the circadian clock’s mission control in mammals is a portion of the brain called the suprachiasmatic nucleus (SCN). The tiny, rice-grain-sized region of the brain sits near the optic nerve and receives light and dark input from the retina. This information keeps gene transcription and translation in the 20,000 or so cells of the SCN in phase with the 24-hour cycle of Earth’s rotation. Without this light-dark input, people’s circadian rhythms will tick to individual, typically longer-than-24-hour cycles.
Researchers long thought that the SCN was the sole place in the human body that kept time, but they couldn’t have been more wrong, Takahashi says. “Now we’d say the SCN is like the conductor of a symphony” of clocks throughout the body, he says. Every cell is a timekeeping instrument that contributes to the body’s overall rhythm, with the SCN at the helm.
To discover that every cell keeps time, circadian biologists needed to know the genes behind the circadian rhythm and to note that they were being expressed universally in the body. By the late 1980s, researchers had cloned the first circadian genes in fungi and fruit flies, but it took until the 1990s before cloning of mammalian-clock genes began to catch up.
By 1998, molecular biologists had begun to tabulate a basic list of mammalian-clock genes. That’s when Ueli Schibler at the University of Geneva and his colleagues grew rat liver and fibroblast cells in a dish and discovered that they were expressing clock genes just as the SCN does.
“It was like, ‘Whoa!’ ” says Sassone-Corsi. “We realized it’s not just the central clock in the SCN that directs behavior in sleep-wake cycles, but there are individual clocks in every tissue. People started to discover that there’s a clock expressed in liver, heart, skin, fat, pancreas, stem cells, you name it.”
“As our ability to detect gene expression gets better, we see that even more genes are under circadian control,” Takahashi says. “About 10 to 15% of the expressed genes in any tissue are controlled by the circadian clock. But that’s only in one tissue. If you look at other tissues, those sets of genes [under clock control] are different. That means if you were to look at enough tissues, the majority of genes could be under circadian regulation.”
In the past couple of years, fundamental research on circadian rhythms has expanded from genetics to biochemistry. It’s not that chemists haven’t been interested in the field. In fact they’ve made seminal contributions. In the 1970s, for example, J. Woodland Hastings, now at Harvard University, discovered that bioluminescence in marine organisms is due to circadian production and destruction of bioluminescent proteins.
But the past two decades of work to unravel the clock’s genetic underpinnings has now set the stage for a closer look at the clock’s chemistry—a phase that has begun in earnest.
Researchers are finding that clock proteins are heavily and transiently phosphorylated, acetylated, and ubiquitinated, Sassone-Corsi says. His group is also looking at the epigenetics of clock control, in particular the role of histone acetyltransferases.
Researchers such as Takahashi and UC San Diego’s Steve A. Kay are increasingly screening compound libraries for small molecules that tweak circadian clock proteins. In 2009, Kay called for more screening to find “compounds that potently affect the clock function” to help iron out the basic biochemistry and to serve as drug leads (Chem. Biol., DOI: 10.1016/j.chembiol.2009.09.002). To date, a few dozen such molecules have been found, most of which extend or contract the period of the clock cycle. In early 2012, Burris and his colleagues found a small molecule called SR9011 that targets clock proteins and also decreases obesity in mice that eat too much (Nature, DOI: 10.1038/nature11030).
Researchers are also trying to solve the structures of clock proteins, which “has proven to be extremely difficult in many cases,” says Jay Dunlap, a Dartmouth Medical School researcher who studies the circadian clock in fungi. Structures of cyanobacteria clock proteins have been solved, but researchers know the three-dimensional form of only a handful of proteins from fungi, flies, and mammals—although this is starting to change. Last year, researchers announced the first structure of a full-length mammalian-clock protein, called cryptochrome, in Nature (DOI: 10.1038/nature10618). And last week, Takahashi and his colleagues published the first crystal structure of an important mouse protein-protein complex that, they note, “is a starting point for understanding at an atomic level the mechanism driving the mammalian circadian clock” (Science, DOI: 10.1126/science/1222804).
Drug developers are also starting to pay more attention to circadian biology than they have in the past because the clock directly encroaches on obesity, infertility, cancer, heart disease, and many other hot-topic medical conditions, Takahashi says.
But more fundamentally, pharma researchers are also realizing that the circadian clock orchestrates the timing of protein production, and proteins are typically what drugs target, GSK’s Willson says. The timing of protein synthesis should give drug developers pause for thought, he adds. Both in clinical trials and in early animal work, the pharmaceutical industry has mostly ignored the circadian rhythms of protein production. What if a potential blockbuster drug were relegated to the dustbin in error because it had been tested on animals or in humans at a time when the target wasn’t even there? Or perhaps the failed drug lead was tested at a time when off-target proteins were present in high amounts, leading to severe side effects that could be avoided if the drug were taken at a different time of day?
Willson says he initially got interested in chronobiology as a source of new drugs. “But then as I talked to some of the leading academics in the field, I began to realize that the bigger application of all of that fundamental biology was on existing drugs,” Willson says. “I still think there are interesting new targets, but clearly the concept of when you dose your drugs is something we should all be thinking about with drug development.” For example, mice are nocturnal—the opposite of humans, for whom they are supposedly proxies. Willson has instituted a lab at GSK where infrared-goggle-wearing technicians test drugs on rodents in the dark during their active period to correspond with the human active period. This is just one of several ways he’s trying to accommodate circadian rhythms in the drug discovery process.
As the wider role of the circadian clock hits home for biologists and drug developers alike, circadian researchers are working out the details of biology’s fourth dimension. “What we’re going to do with time and medicine at the beginning of the 21st century is what Einstein did with time and physics at the beginning of the 20th century,” Northwestern’s Turek says. “What we’ve got now is just the tip of the iceberg.”
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