Researchers obtain key insights into how the internal body clock is tuned

Researchers at UT Southwestern Medical Center have found a new way that internal body clocks are regulated by a type of molecule known as long non-coding RNA.

The internal body clocks, called circadian clocks, regulate the daily “rhythms” of many bodily functions, from waking and sleeping to body temperature and hunger. They are largely “tuned” to a 24-hour cycle that is influenced by external cues such as light and temperature.

“Although we know that long non-coding RNAs are abundant in many organisms, what they do in the body, and how they do it, has not been clear so far,” said Dr. Yi Liu, Professor of Physiology. “Our work establishes a role for long non-coding RNAs in ‘tuning’ the circadian clock, but also shows how they control gene expression.”

Determining how circadian clocks work is crucial to understanding several human diseases, including sleep disorders and depression in which the clock malfunctions. The influence of a functional clock is evident in the reduced performance of shift workers and the jet lag felt by long-distance travellers.

Dr. Liu and his team were able to learn more about the circadian rhythms by studying model systems involving the bread mold, Neurospora crassa. The researchers found that the expression of a clock gene named frequency (frq) is controlled by a long non-coding RNA named qrf (frq backwards) − an RNA molecule that is complementary, or antisense, to frq. Unlike normal RNA molecules, qrf does not encode a protein, but it can control whether and how much frq protein is produced.

Specifically, qrf RNA is produced in response to light, and can then interfere with the production of the frq protein. In this way, qrf can “re-set” the circadian clock in a light-dependent way. This regulation works both ways: frq can also block the production of qrf. This mutual inhibition ensures that the frq and qrf RNA molecules are present in opposite “phases” of the clock and allows each RNA to oscillate robustly. Without qrf, normal circadian rhythms are not sustained, indicating that the long non-coding RNA is required for clock functions.

The findings are published online in the journal Nature.

“We anticipate a similar mode of action may operate in other organisms because similar RNAs have been found for clock genes in mice. In addition, such RNAs may also function in other biological processes because of their wide presence in genomes,” said Dr. Liu, who is the Louise W. Kahn Scholar in Biomedical Research.

UT Southwestern investigators are leaders in unraveling the gene networks underlying circadian clocks and have shown that most body organs, such as the pancreas and liver, have their own internal clocks, and that virtually every cell in the human body contains a clock. It now appears that the clocks and clock-related genes – some 20 such genes have been identified – affect virtually all of the cells’ metabolic pathways, from blood sugar regulation to cholesterol production.

Other UT Southwestern researchers involved in the latest findings include Dr. Zhihong Xue, Qiaohong Ye, Dr. Juchen Yang and Dr. Guanghua Xiao. Support for this research included grants from the National Institutes of Health, the Welch Foundation, the Cancer Prevention Research Institute of Texas, and the Biotechnology and Biological Sciences Research Council.

“This study adds to an important body of work that has shown the ubiquity of a circadian clock across species, including humans, and its role in metabolic regulation in cells, organs, and organisms,” said Dr. Michael Sesma, Program Director in the Division of Genetics and Developmental Biology at the of the National Institutes of Health’s National Institute of General Medical Sciences, which partially funded the research. “These new results from Dr. Liu and his colleagues also extend beyond understanding the function of an anti-sense RNA in the fine tuning of a cell’s daily rhythm; they provide an example of the means by which anti-sense transcription likely regulates other key molecular and physiological processes in cells and organisms.”

(Image: Fotolia)

(Image caption: A peptide responsible for cell communication in the brain, Vip (green) is reduced in the brains of mice that have little or no Lhx1 (right). Credit: Salk Institute for Biological Studies)

Single gene controls jet lag

Scientists at the Salk Institute for Biological Studies have identified a gene that regulates sleep and wake rhythms.

The discovery of the role of this gene, called Lhx1, provides scientists with a potential therapeutic target to help night-shift workers or jet lagged travelers adjust to time differences more quickly. The results, published in eLife, can point to treatment strategies for sleep problems caused by a variety of disorders.

“It’s possible that the severity of many dementias comes from sleep disturbances,” says Satchidananda Panda, a Salk associate professor who led the research team. “If we can restore normal sleep, we can address half of the problem.”

Every cell in the body has a “clock” – an abundance of proteins that dip or rise rhythmically over approximately 24 hours. The master clock responsible for establishing these cyclic circadian rhythms and keeping all the body’s cells in sync is the suprachiasmatic nucleus (SCN), a small, densely packed region of about 20,000 neurons housed in the brain’s hypothalamus.

More so than in other areas of the brain, the SCN’s neurons are in close and constant communication with one another. This close interaction, combined with exposure to light and darkness through vision circuits, keeps this master clock in sync and allows people to stay on essentially the same schedule every day. The tight coupling of these cells also helps make them collectively resistant to change. Exposure to light resets less than half of the SCN cells, resulting in long periods of jet lag.

In the new study, researchers disrupted the light-dark cycles in mice and compared changes in the expression of thousands of genes in the SCN with other mouse tissues. They identified 213 gene expression changes that were unique to the SCN and narrowed in on 13 of these that coded for molecules that turn on and off other genes. Of those, only one was suppressed in response to light: Lhx1.

“No one had ever imagined that Lhx1 might be so intricately involved in SCN function,” says Shubhroz Gill, a postdoctoral researcher and co-first author of the paper. Lhx1 is known for its role in neural development: it’s so important, that mice without the gene do not survive. But this is the first time it has been identified as a master regulator of light-dark cycle genes.

By recording electrical activity in the SCN of animals with reduced amounts of the Lhx1 protein, the researchers saw that the SCN neurons weren’t in sync with one another, despite appearing rhythmic individually.

“It was all about communication–the neurons were not talking to each other without this molecule,” says Ludovic Mure, a postdoctoral researcher and an author on the paper. A next step in the work will be to understand exactly how Lhx1 affects the expression of genes that creates this synchronicity.

Studying a mouse version of jet lag–an 8-hour shift in their day-night cycle–the scientists found that those with little or no Lhx1 readjusted much faster to the shift than normal mice. This suggests that because these neurons are less in sync with one another, they are more easily able to shift to a new schedule, though it is difficult for them to maintain that schedule, Panda says.

These mice also exhibited reduced activity of certain genes, including one that creates vasoactive intestinal peptide or Vip, a molecule that has important roles in development and as a hormone in the intestine and blood. In the brain, Vip affects cell communication, but nobody had known that Lhx1 regulated it until now, Panda says. Interestingly, the team also found that adding Vip restored cell synchrony in the SCN.

“This approach helped us to close that knowledge gap and show that Vip is a very important protein, at least for SCN,” Panda says. “It can compensate for the loss of Lhx1.”

On the other hand, cutting back on Vip could be another way to treat jet lag. Vip could be an even easier drug target compared with Lhx1 because Vip is secreted from cells rather than inside cells, Panda says. “If we find a drug that will block the Vip receptor or somehow break down Vip, then maybe that will help us reset the clock much faster,” he adds.

The new results take the group a step closer to their goal of creating cell regenerative therapies that restore the SCN and ameliorate sleep problems. The scientists have made their gene expression data available through a searchable web interface at http://scn.salk.edu, giving other researchers a handy way to explore the effect of light and dark in genes in the SCN and other tissues.

Genes discovered linking circadian clock with eating schedule

For most people, the urge to eat a meal or snack comes at a few, predictable times during the waking part of the day. But for those with a rare syndrome, hunger comes at unwanted hours, interrupts sleep and causes overeating.

Now, Salk scientists have discovered a pair of genes that normally keeps eating schedules in sync with daily sleep rhythms, and, when mutated, may play a role in so-called night eating syndrome. In mice with mutations in one of the genes, eating patterns are shifted, leading to unusual mealtimes and weight gain. The results were published in Cell Reports today.

"We really never expected that we would be able to decouple the sleep-wake cycle and the eating cycle, especially with a simple mutation," says senior study author Satchidananda Panda, an associate professor in Salk’s Regulatory Biology Laboratory. "It opens up a whole lot of future questions about how these cycles are regulated."

More than a decade ago, researchers discovered that individuals with an inherited sleep disorder often carry a particular mutation in a protein called PER2. The mutation is in an area of the protein that can be phosphorylated—the ability to bond with a phosphate chemical that changes the protein’s function. Humans have three PER, or period, genes, all thought to play a role in the daily circadian clock and all containing the same phosphorylation spot.

The Salk scientists joined forces with a Chinese team led by Ying Xu of Nanjing University to test whether mutations in the equivalent area of PER1 would have the same effect as those in PER2 that caused the sleep disorder. So they bred mice to lack the mouse period genes, and added in a human PER1 or PER2 with a mutation in the phosphorylation site. As expected, mice with a mutated PER2 had sleep defects, dozing off earlier than usual. The same wasn’t true for PER1 mutations though.

"In the mice without PER1, there was no obvious defect in their sleep-wake cycles," says Panda. "Instead, when we looked at their metabolism, we suddenly saw drastic changes."

Mice with the PER1 phosphorylation defects ate earlier than other mice—causing them to wake up and snack before their sleep cycle was over—and ate more food throughout their normal waking period. When the researchers looked at the molecular details of the PER1 protein, they found that the mutated PER1 led to lower protein levels during the sleeping period, higher levels during the waking period, and a faster degradation of protein whenever it was produced by cells.

Panda and his colleagues hypothesize that normally, PER1 and PER2 are kept synchronized since they have identical phosphorylation sites—they are turned on and off at the same times, keeping sleep and eating cycles aligned. But a mutation in one of the genes could break this link, and cause off-cycle eating or sleeping.

"For a long time, people discounted night eating syndrome as not real," says Panda. "These results in mice suggest that it could actually be a genetic basis for the syndrome." The researchers haven’t yet tested, however, whether any humans with night eating syndrome have mutations in PER1.

When Panda and Xu’s team restricted access to food, providing it only at the mice’s normal meal times, they found that even with a genetic mutation in PER1, mice could maintain a normal weight. Over a 10-week follow-up, these mice—with a PER1 mutation but timed access to food—showed no differences to control animals. This tells the researchers that the weight gain caused by PER1 is entirely caused by meal mistiming, not other metabolic defects.

Next, they hope to study exactly how PER1 controls appetite and eating behavior—whether its molecular actions work through the liver, fat cells, brain or other organs.

For resetting circadian rhythms, neural cooperation is key

Fruit flies are pretty predictable when it comes to scheduling their days, with peaks of activity at dawn and dusk and rest times in between. Now, researchers reporting in the Cell Press journal Cell Reports on April 17th have found that the clusters of brain cells responsible for each of those activity peaks—known as the morning and evening oscillators, respectively—don’t work alone. For flies’ internal clocks to follow the sun, cooperation is key.

"Without proper synchronization, circadian clocks are useless or can even be deleterious to organisms," said Patrick Emery from the University of Massachusetts Medical School. "In addition, most organisms have to detect changes in day length to adapt their rhythms to seasons.

"Our work clearly shows that light is detected by individual neurons that then communicate with each other to properly define the phase of circadian behavior," he added. "This emphasizes the importance of neural interaction in the generation of properly phased circadian rhythms."

In the brains of Drosophila fruit flies, there are approximately 150 circadian neurons, explained Emery and coauthor Yong Zhang, including a small group of morning oscillators that promote activity early in the day and another group of evening oscillators that promote activity later. Morning oscillators also set the pace of molecular rhythms in other parts of the brain, and hence the phase of circadian behavior. Scientists had thought they did this by relying heavily on their own sensitivity to light—what Emery calls “cell-autonomous photoreception.” Indeed, these cells do express fruit flies’ dedicated photoreceptor Cryptochrome (CRY). But recent evidence suggested that something was missing from that simple view.

In the new study, the researchers manipulated CRY’s ability to function through another clock component, known as JET (short for Jetlag), in different circadian neurons and watched what happened. The studies show that light detection by the morning oscillators isn’t enough to keep flies going about their business in a timely way. They need those evening oscillators too.

JET’s role is bigger than expected as well. In addition to enabling cell-autonomous light sensing, the protein also allows distinct circadian neurons to talk to each other in rapid fashion after light exposure, although the researchers don’t yet know how.

The new model also suggests that flies and mammals have more similarities than had been appreciated when it comes to synchronizing their activities to the sun, the researchers say. In mammals, specific neurons of the circadian pacemaker of the brain (known as the Suprachiasmatic Nucleus or SCN) receive light input from the retina. Those cells then communicate with pacemaker neurons, which resets the circadian network as a whole.

Chrono, the last piece of the circadian clock puzzle?

In an article published today in PLOS Biology, researchers from the RIKEN Brain Science Institute in Japan report the identification of Chrono, a gene involved in the regulation of the body clock in mammals and that might be a key component of the body’s response to stress.

The circadian clock is like an orchestra with many conductors

You’ve switched to the night shift and your weight skyrockets, or you wake at 7 a.m. on weekdays but sleep until noon on weekends—a social jet lag that can fog your Saturday and Sunday.

Life runs on rhythms driven by circadian clocks, and disruption of these cycles is associated with serious physical and emotional problems, says Orie Shafer, a University of Michigan assistant professor of molecular, cellular and developmental biology.

Now, new findings from Shafer and U-M doctoral student Zepeng Yao challenge the prevailing wisdom about how our body clocks are organized, and suggest that interactions among neurons that govern circadian rhythms are more complex than originally thought.

Yao and Shafer looked at the circadian clock neuron network in fruit flies, which is functionally similar to that of mammals, but at only 150 clock neurons is much simpler. Previously, scientists thought that a master group of eight clock neurons acted as pacemaker for the remaining 142 clock neurons—think of a conductor leading an orchestra—thus imposing the rhythm for the fruit fly circadian clock. It is thought that the same principle applies to mammals.

Interactions among clock neurons determine the strength and speed of circadian rhythms, Yao says. So, when researchers genetically changed the clock speeds of only the group of eight master pacemakers they could examine how well the conductor alone governed the orchestra. They found that without the environmental cues, the orchestra didn’t follow the conductor as closely as previously thought.

Some of the fruit flies completely lost sense of time, and others simultaneously demonstrated two different sleep cycles, one following the group of eight neurons and the other following some other set of neurons.

"The finding shows that instead of the entire orchestra following a single conductor, part of the orchestra is following a different conductor or not listening at all," Shafer said.

The findings suggest that instead of a group of master pacemaker neurons, the clock network consists of many independent clocks, each of which drives rhythms in activity. Shafer and Yao suspect that a similar organization will be found in mammals, as well.

"A better understanding of the circadian clock mechanisms will be critical for attempts to alleviate the adverse effects associated with circadian disorders," Yao said.

Disrupting the circadian clock through shift work is associated with diabetes, obesity, stress, heart disease, mood disorders and cancer, among other disorders, Yao says. The International Agency for Research on Cancer classified shift work that disrupts circadian rhythms as a human carcinogen equal to cancer-causing ultraviolet radiation.

Scientists wake up to causes of sleep disruption in Alzheimer’s disease

Being awake at night and dozing during the day can be a distressing early symptom of Alzheimer’s disease, but how the disease disrupts our biological clocks to cause these symptoms has remained elusive.

Now, scientists from Cambridge have discovered that in fruit flies with Alzheimer’s the biological clock is still ticking but has become uncoupled from the sleep-wake cycle it usually regulates. The findings – published in Disease Models & Mechanisms – could help develop more effective ways to improve sleep patterns in people with the disease.

People with Alzheimer’s often have poor biological rhythms, something that is a burden for both patients and their carers. Periods of sleep become shorter and more fragmented, resulting in periods of wakefulness at night and snoozing during the day. They can also become restless and agitated in the late afternoon and early evening, something known as ‘sundowning’.

Biological clocks go hand in hand with life, and are found in everything from single celled organisms to fruit flies and humans. They are vital because they allow organisms to synchronise their biology to the day-night changes in their environments.

Until now, however, it has been unclear how Alzheimer’s disrupts the biological clock. According to Dr Damian Crowther of Cambridge’s Department of Genetics, one of the study’s authors: “We wanted to know whether people with Alzheimer’s disease have a poor behavioural rhythm because they have a clock that’s stopped ticking or they have stopped responding to the clock.”

The team worked with fruit flies – a key species for studying Alzheimer’s. Evidence suggests that the A-beta peptide, a protein, is behind at least the initial stages of the disease in humans. This has been replicated in fruit flies by introducing the human gene that produces this peptide.

Taking a group of healthy flies and a group with this feature of Alzheimer’s, the researchers studied sleep-wake patterns in the flies, and how well their biological clocks were working.

They measured sleep-wake patterns by fitting a small infrared beam, similar to movement sensors in burglar alarms, to the glass tubes housing the flies. When the flies were awake and moving, they broke the beam and these breaks in the beam were counted and recorded.

To study the flies’ biological clocks, the researchers attached the protein luciferase – an enzyme that emits light – to one of the proteins that forms part of the biological clock. Levels of the protein rise and fall during the night and day, and the glowing protein provided a way of tracing the flies’ internal clock.

"This lets us see the brain glowing brighter at night and less during the day, and that’s the biological clock shown as a glowing brain. It’s beautiful to be able to study first hand in the same organism the molecular working of the clock and the corresponding behaviours," Dr Crowther said.

They found that healthy flies were active during the day and slept at night, whereas those with Alzheimer’s sleep and wake randomly. Crucially, however, the diurnal patterns of the luciferase-tagged protein were the same in both healthy and diseased flies, showing that the biological clock still ticks in flies with Alzheimer’s.

"Until now, the prevailing view was that Alzheimer’s destroyed the biological clock," said Crowther.

"What we have shown in flies with Alzheimer’s is that the clock is still ticking but is being ignored by other parts of the brain and body that govern behaviour. If we can understand this, it could help us develop new therapies to tackle sleep disturbances in people with Alzheimer’s."

Dr Simon Ridley, Head of Research at Alzheimer’s Research UK, who helped to fund the study, said: “Understanding the biology behind distressing symptoms like sleep problems is important to guide the development of new approaches to manage or treat them. This study sheds more light on the how features of Alzheimer’s can affect the molecular mechanisms controlling sleep-wake cycles in flies.

"We hope these results can guide further studies in people to ensure that progress is made for the half a million people in the UK with the disease."

Computer models help decode cells that sense light without seeing

Researchers have found that the melanopsin pigment in the eye is potentially more sensitive to light than its more famous counterpart, rhodopsin, the pigment that allows for night vision.

For more than two years, the staff of the Laboratory for Computational Photochemistry and Photobiology (LCPP) at Ohio’s Bowling Green State University (BGSU), have been investigating melanopsin, a retina pigment capable of sensing light changes in the environment, informing the nervous system and synchronizing it with the day/night rhythm. Most of the study’s complex computations were carried out on powerful supercomputer clusters at the Ohio Supercomputer Center (OSC).

The research recently appeared in the Proceedings of the National Academy of Sciences USA, in an article edited by Arieh Warshel, Ph.D., of the University of Southern California. Warshel and two other chemists received the 2013 Nobel Prize in Chemistry for developing multiscale models for complex chemical systems, the same techniques that were used in conducting the BGSU study, “Comparison of the isomerization mechanisms of human melanopsin and invertebrate and vertebrate rhodopsins.”

“The retina of vertebrate eyes, including those of humans, is the most powerful light detector that we know,” explains Massimo Olivucci, Ph.D., a research professor of Chemistry and director of LCPP in the Center for Photochemical Sciences at BGSU. “In the human eye, light coming through the lens is projected onto the retina where it forms an image on a mosaic of photoreceptor cells that transmits information from the surrounding environment to the brain’s visual cortex. In extremely poor illumination conditions, such as those of a star-studded night or ocean depths, the retina is able toperceive intensities corresponding to only a few photons, which are indivisible units of light. Such extreme sensitivity is due to specialized photoreceptor cells containing a light sensitive pigment called rhodopsin.”

For a long time, it was assumed that the human retina contained only photoreceptor cells specialized in dim-light and daylight vision, according to Olivucci. However, recent studies revealed the existence of a small number of intrinsically photosensitive nervous cells that regulate non-visual light responses. These cells contain a rhodopsin-like protein named melanopsin, which plays a role in the regulation of unconscious visual reflexes and in the synchronization of the body’s responses to the dawn/dusk cycle, known as circadian rhythms or the “body clock,” through a process known as photoentrainment.

The fact that the melanopsin density in the vertebrate retina is 10,000 times lower than that of rhodopsin density, and that, with respect to the visual photoreceptors, the melanopsin-containing cells capture a million-fold fewer photons, suggests that melanopsin may be more sensitive than rhodopsin. The comprehension of the mechanism that makes this extreme light sensitivity possible appears to be a prerequisite to the development of new technologies.

Both rhodopsin and melanopsin are proteins containing a derivative of vitamin A, which serves as an “antenna” for photon detection. When a photon is detected, the proteins are set in an activated state, through a photochemical transformation, which ultimately results in a signal being sent to the brain. Thus, at the molecular level, visual sensitivity is the result of a trade-off between two factors: light activation and thermal noise. It is currently thought that light-activation efficiency (i.e., the number of activation events relative to the total number of detected photons) may be related to its underlying speed of chemical transformation. On the other hand, the thermal noise depends on the number of activation events triggered by ambient body heat in the absence of photon detection.

“Understanding the mechanism that determines this seemingly amazing light sensitivity of melanopsin may open up new pathways in studying the evolution of light receptors in vertebrate and, in turn, the molecular basis of diseases, such as “seasonal affecting disorders,” Olivucci said. “Moreover, it provides a model for developing sub-nanoscale sensors approaching the sensitivity of a single-photon.”

For this reason, the LCPP group – working together with Francesca Fanelli, Ph.D., of Italy’s Università di Modena e Reggio Emilia – has used the methodology developed by Warshel and his colleagues to construct computer models of human melanopsin, bovine rhodopsin and squid rhodopsin. The models were constructed by BGSU research assistant Samer Gozem, Ph.D., BGSU visiting graduate student Silvia Rinaldi, who now has completed his doctorate, and visiting research assistant Federico Melaccio, Ph.D. – both visiting from Italy’s Università di Siena. The models were used to study the activation of the pigments and show that melanopsin light activation is the fastest, and its thermal activation is the slowest, which was expected for maximum light sensitivity.

The computer models of human melanopsin, and bovine and squid rhodopsins, provide further support for a theory reported by the LCPP group in the September 2012 issue of Science Magazine which explained the correlation between thermal noise and perceived color, a concept first proposed by the British neuroscientist Horace Barlow in 1957. Barlow suggested the existence of a link between the color of light perceived by the sensor and its thermal noise and established that the minimum possible thermal noise is achieved when the absorbing light has a wavelength around 470 nanometers, which corresponds to blue light.

“This wavelength and corresponding bluish color matches the wavelength that has been observed and simulated in the LCPP lab,” said Olivucci. “In fact, our calculations also indicate that a shift from blue to even shorter wavelengths (i.e. indigo and violet) will lead to an inversion of the trend and an increase of thermal noise towards the higher levels seen for a red color. Therefore, melanopsin may have been selected by biological evolution to stand exactly at the border between two opposite trends to maximize light sensitivity.”