Key Protein is Linked to Circadian Clocks, Helps Regulate Metabolism

Inside each of us is our own internal timing device. It drives everything from sleep cycles to metabolism, but the inner-workings of this so-called “circadian clock” are complex, and the molecular processes behind it have long eluded scientists. But now, researchers at the Gladstone Institutes have discovered how one important protein falls under direct instructions from the body’s circadian clock. Furthermore, they uncover how this protein regulates fundamental circadian processes—and how disrupting its normal function can throw this critical system out of sync.

In the latest issue of the Journal of Neuroscience, Gladstone Investigator Katerina Akassoglou, PhD, and her team reveal in animal models how the production of the p75 neurotrophin receptor (p75NTR) protein oscillates in time with the body’s natural circadian clock—and how these rhythmic oscillations help regulate vital metabolic functions. This discovery underscores the widespread importance of p75NTR by offering insight into how the circadian clock helps maintain the body’s overall metabolic health.

Virtually every organism on the planet—from bacteria to humans—has a circadian clock, a biological timing mechanism that oscillates with a period of about 24 hours and is coordinated with the cycle of day and night. And while it runs independent of external cues, it is influenced by the rhythms of light, temperature and food availability. Intriguingly, recent studies have also found a link between circadian clocks and metabolism.

“Important metabolic functions are also heavily influenced by circadian clocks, which is why activities such as chronic night-shift work—which can cause a misalignment of this clock—increase one’s risk for metabolic and autoimmune diseases such as obesity, Type 2 diabetes, cancer and multiple sclerosis,” said Dr. Akassoglou. Dr. Akassoglou is also a professor of neurology at the University of California, San Francisco, (UCSF) with which Gladstone is affiliated. “In this study, we pinpointed p75NTR as an important molecular ‘link’ between circadian clocks and metabolic health.”

Originally, p75NTR was only thought to be active in the nervous system. Later studies found it to be active in many cell types throughout the body, suggesting that it impacts a variety of biological functions. Last year, Gladstone researchers discovered that p75NTR was present in the liver and in fat cells, and that it regulates glucose levels in the blood—an important metabolic process. Since these findings uncovered a link between p75NTR and metabolism, the research team tested—first in a petri dish and then in animal models—whether there was also a link between p75NTR and the circadian clock.

The team focused on two genes called Clock and Bmal1. These so-called “circadian regulator genes,” and others like them, are found throughout the body. Their activity controls the body’s circadian clock. The researchers wanted to see if there was a connection between these circadian genes and p75NTR.

“Our initial experiments revealed such a connection,” recalls Gladstone Postdoctoral Fellow Bernat Baeza-Raja, PhD, the paper’s lead author. “In individual cells, we saw that p75NTR production was controlled by Clock and Bmal1, which bind directly to the gene that codes for the p75NTR and start production of the protein.”

But perhaps even more important than how p75NTR was produced was when. The team found that p75NTR production, like the circadian clock genes themselves, oscillated in a 24-hour cycle—in sync with the cells’ natural circadian rhythm. Experiments in mouse models further supported these findings.

And when the team genetically modified a group of mice so that it lacked the circadian Clock gene, everything else fell out of sync. The circadian oscillation of p75NTR production was disrupted, and p75NTR levels dropped.

However, what was most fascinating, say the researchers, was how a drop in p75NTR levels then affected a variety of circadian clock systems. Specifically, the regular oscillations of other circadian genes in the brain and the liver became disrupted, as well as genes known to regulate glucose and lipid metabolism.

“The finding that a loss of p75NTR affected circadian and metabolic systems is strong evidence that this protein is intricately tied to both,” said Life Sciences Institute Director Alan Saltiel, PhD, who is also a professor at the University of Michigan and was not involved in the study. “It will be fascinating to see what additional insight Dr. Akassoglou and her team will uncover as they continue to examine the role of p75NTR in circadian clocks and metabolic function.”

“While these findings reveal p75NTR to be an important link between circadian clocks and metabolism, the system is complex, and there are likely other factors at play,” said Dr. Akassoglou. “We are currently working to identify the relationship between the circadian clock, metabolism and the immune system, so that one day we could develop therapies to treat diseases influenced by circadian clock disruption—including not only obesity and diabetes, but also potentially multiple sclerosis and even Alzheimer’s disease.”

(Image: Brain Treatment Center)

Study Shows How the Nanog Protein Promotes Growth of Head and Neck Cancer

A new study led by researchers at The Ohio State University Comprehensive Cancer Center – Arthur G. James Cancer Hospital and Richard J. Solove Research Institute (OSUCCC–James) has identified a biochemical pathway in cancer stem cells that is essential for promoting head and neck cancer.

The study shows that a protein called Nanog, which is normally active in embryonic stem cells, promotes the growth of cancer stem cells in head and neck cancer. The findings provide information essential for designing novel targeted drugs that might improve the treatment of head and neck cancer.

Normally, Nanog helps healthy embryonic stem cells maintain their undifferentiated, uncommitted (i.e., pluripotent) state. But recent evidence suggests that Nanog promotes tumor growth by stimulating the proliferation of cancer stem cells.

“This study defines a signaling axis that is essential for head and neck cancer progression, and our findings show that this axis may be disrupted at three key steps,” says principal investigator Quintin Pan, PhD, associate professor of otolaryngology at the OSUCCC – James. “Targeted drugs that are designed to inhibit any or all of these three steps might greatly improve the treatment of head and neck cancer.”

The findings were published in a recent issue of the journal Oncogene.

Specifically, the study shows that an enzyme called “protein kinase C-epsilon” (PKCepsilon) adds energy-packing phosphate groups to the Nanog molecule. This phosphorylation of Nanog stabilizes and activates the molecule.

It also triggers a series of events: Two Nanog molecules bind together, and these are joined by a third “co-activating” molecule called p300. This molecular complex then binds to the promoter region of a gene called Bmi1, an event that increases the expression of the gene. This, in turn, stimulates proliferation of cancer stem cells.

“Our work shows that the PKCepsilon/Nanog/Bmi1 signaling axis is essential to promote head and neck cancer,” Pan says. “And it provides initial evidence that the development of inhibitors that block critical points in this axis might yield a potent collection of targeted anti-cancer therapeutics that could be valuable for the treatment of head and neck cancer.”

(Image: Gray’s Anatomy of the Human Body)

Fiber-optic pen helps see inside brains of children with learning disabilities

For less than $100, University of Washington researchers have designed a computer-interfaced drawing pad that helps scientists see inside the brains of children with learning disabilities while they read and write.

The device and research using it to study the brain patterns of children will be presented June 18 at the Organization for Human Brain Mapping meeting in Seattle. A paper describing the tool, developed by the UW’s Center on Human Development and Disability, was published this spring in Sensors, an online open-access journal. “Scientists needed a tool that allows them to see in real time what a person is writing while the scanning is going on in the brain,” said Thomas Lewis, director of the center’s Instrument Development Laboratory. “We knew that fiber optics were an appropriate tool. The question was, how can you use a fiber-optic device to track handwriting?”

To create the system, Lewis and fellow engineers Frederick Reitz and Kelvin Wu hollowed out a ballpoint pen and inserted two optical fibers that connect to a light-tight box in an adjacent control room where the pen’s movement is recorded. They also created a simple wooden square pad to hold a piece of paper printed with continuously varying color gradients. The custom pen and pad allow researchers to record handwriting during functional magnetic resonance imaging, or fMRI, to assess behavior and brain function at the same time.Other researchers have developed fMRI-compatible writing devices, but “I think it does something similar for a tenth of the cost,” Reitz said of the UW system. By using supplies already found in most labs (such as a computer), the rest of the supplies – pen, fiber optics, wooden pad and printed paper – cost less than $100.The device connects to a computer with software that records every aspect of the handwriting, from stroke order to speed, hesitations and liftoffs. Understanding how these physical patterns correlate with a child’s brain patterns can help scientists understand the neural connections involved.

Researchers studied 11- and 14-year-olds with either dyslexia or dysgraphia, a handwriting and letter-processing disorder, as well as children without learning disabilities. Subjects looked at printed directions on a screen while their heads were inside the fMRI scanner. The pen and pad were on a foam pad on their laps.

Subjects were given four-minute blocks of reading and writing tasks. Then they were asked to simply think about writing an essay (they later wrote the essay when not using the fMRI). Just thinking about writing caused many of the same brain responses as actual writing would.

“If you picture yourself writing a letter, there’s a part of the brain that lights up as if you’re writing the letter,” said Todd Richards, professor of radiology and principal investigator of the UW Integrated Brain Imaging Center. “When you imagine yourself writing, it’s almost as if you’re actually writing, minus the motion problems.”

Richards and his staff are just starting to analyze the data they’ve collected from about three dozen subjects, but they have already found some surprising results.

“There are certain centers and neural pathways that we didn’t necessarily expect” to be activated, Richards said. “There are language pathways that are very well known. Then there are other motor pathways that allow you to move your hands. But how it all connects to the hand and motion is still being understood.”

Besides learning disorders, the inexpensive pen and pad also could help researchers study diseases in adults, especially conditions that cause motor control problems, such as stroke, multiple sclerosis and Parkinson’s disease.

“There are several diseases where you cannot move your hand in a smooth way or you’re completely paralyzed,” Richards said. “The beauty is it’s all getting recorded with every stroke, and this device would help us to study these neurological diseases.”

Not all reading disabilities are dyslexia

A common reading disorder goes undiagnosed until it becomes problematic, according to the results of five years of study by researchers at Vanderbilt’s Peabody College of education and human development in collaboration with the Kennedy Krieger Institute/Johns Hopkins School of Medicine. Results of the study were recently published online by the National Institutes of Health.

Dyslexia, a reading disorder in which a child confuses letters and struggles with sounding out words, has been the focus of much reading research.

But that’s not the case with the lesser known disorder Specific Reading Comprehension Deficits or S-RCD, in which a child reads successfully but does not sufficiently comprehend the meaning of the words, according to lead investigator Laurie Cutting, Patricia and Rodes Hart Chair at Peabody.

“S-RCD is like this: I can read Spanish, because I know what sounds the letters make and how the words are pronounced, but I couldn’t tell you what the words actually mean,” Cutting said. “When a child is a good reader, it’s assumed their comprehension is on track. But 3 to 10 percent of those children don’t understand most of what they’re reading. By the time the problem is recognized, often closer to third or fourth grade, the disorder is disrupting their learning process.”

Researchers have been able to pinpoint brain activity and understand its role in dyslexia, but no functional magnetic resonance imaging or fMRI studies, until now, have examined the neurobiological profile of those who exhibit poor reading comprehension despite intact word-level abilities.

Neuroimaging of children showed that the brain function of those with S-RCD while reading is quite different and distinct from those with dyslexia. Those with dyslexia exhibited abnormalities in a specific region in the occipital-temporal cortex, a part of the brain that is associated with successfully recognizing words on a page.

But those with S-RCD did not show abnormalities in this region, instead showing specific abnormalities in regions typically associated with memory.

“It may be that these individuals have a whole different neurobiological signature associated with how they read that is not efficient for supporting comprehension,” Cutting said. “We want to understand the different systems that support reading and see which ones help different types of difficulties, and how we can target the cognitive systems that support those skills.”

The study, an ongoing 10-year effort supported by National Institutes of Health grant No. M01-RR000052, has enrolled more than 300 children to date.

The discerning fruit fly: Linking brain-cell activity and behavior in smell recognition

Behind the common expression “you can’t compare apples to oranges” lies a fundamental question of neuroscience: How does the brain recognize that apples and oranges are different? A group of neuroscientists at Cold Spring Harbor Laboratory (CSHL) has published new research that provides some answers.

In the fruit fly, the ability to distinguish smells lies in a region of the brain called the mushroom body (MB). Prior research has demonstrated that the MB is associated with learning and memory, especially in relation to the sense of smell, also known as olfaction.

CSHL Associate Professor Glenn Turner and colleagues have now mapped the activity of brain cells in the MB, in flies conditioned to have Pavlovian behavioral responses to different odors. Their results, outlined in a paper published today by the Journal of Neuroscience, suggest that the activity of a remarkably small number of neurons — as few as 25 — is required to be able to distinguish between different odors.

They also found that a similarly small number of nerve cells are involved in grouping alike odors. This means, for instance, that “if you’ve learned that oranges are good, the smell of a tangerine will also get you thinking about food,” says Robert Campbell, a postdoctoral researcher in the Turner lab and lead author on the new study.

These intriguing new findings are part of a broad effort in contemporary neuroscience to determine how the brain, easily the most complex organ in any animal, manages to make a mass of raw sensory data intelligible to the individual — whether a person or a fly — in order to serve as a basis for making vital decisions.

Looking closely at Kenyon cells

The neurons in the fly MB are known as Kenyon cells, named after their discoverer, the neuroscientist Frederick Kenyon, who was the first person to stain and visualize individual neurons in the insect brain. Kenyon cells receive sensory inputs from organs that perceive smell, taste, sight and sound. This confluence of sensory input in the MB is important for memory formation, which comes about through a linking of different types of information.

Kenyon cells make up only about 4% of the entire fly brain and are extremely sensitive to inputs triggered by odors, in which only two connections between neurons, called synapses, separate them from the receptor cells at the “front end” of the olfactory system.

But in contrast to other regions of the brain, such as the vertebrate hippocampus, the sensory responses in the MB are few in number and relatively weak. It is the sparseness of the signals in the Kenyon cell neurons that makes studying memory formation in flies so promising to Turner and his team. “We set out to learn if these signals were really informative to the animal’s learning and memory with regard to smell,” Turner says.

In particular, Turner’s group wanted to see if they could link these signals with actual behavior in flies. The team used an imaging technique that allowed them to view the responses of over 100 Kenyon cells at a time and, importantly, quantify their results. They found that even the very sparse responses in these cells that are triggered by odors provide a large amount of information about odor identity. Turner suspects the very selectiveness of the response helps in the accurate formation and recall of memories.

When the researchers used two odors blended together in a series of increasingly similar concentrations, they found that two very similar smells could be distinguished as a result of the activity of as few as 25 Kenyon cells. This correlated well with the behavior of the flies: when brain activity suggested the flies had difficulty discerning the odors, their behavior also showed they could not choose between them.

The activity of these cells also accounts for flies’ ability to discern novel odors and group them together. This was determined in a “generalization” test, in which the degree to which flies learned a generalized aversion to unfamiliar test odors could be predicted based upon the relatively similar activity patterns of Kenyon cells that the odors induced.

“Being able to do this type of ‘mind-reading’ means we really understand what signals these activity patterns are sending,” says Turner. Ultimately, he and colleagues hope to be able to relate their findings in the fly brain with the operation of the brain in mammals.

Quality of waking hours determines ease of falling asleep

The quality of wakefulness affects how quickly a mammal falls asleep, UT Southwestern Medical Center researchers report in a study that identifies two proteins never before linked to alertness and sleep-wake balance.

“This study supports the idea that subjective sleepiness is influenced by the quality of experiences right before bedtime. Are you reluctantly awake or excited to be awake?” said Dr. Masashi Yanagisawa, professor of molecular genetics and a Howard Hughes Medical Institute investigator at UT Southwestern. He is principal author of the study published online in May in the Proceedings of the National Academy of Sciences.

Co-author Dr. Robert Greene, UT Southwestern professor of psychiatry and a physician at the Dallas VA Medical Center, said the study is unique in showing that the need for sleep (called sleep homeostasis) can be separated from wakefulness both behaviorally and biochemically, meaning the two processes can now be studied individually.

“Two of the great mysteries in neuroscience are why do we sleep and what is sleep’s function? Separating sleep need from wakefulness and identifying two different proteins involved in these steps represents a fundamental advance,” he said.

If borne out by further research, this study could lead to new ways of assessing and possibly treating sleep disorders, perhaps by focusing more attention on the hours before bedtime because the quality of wakefulness has a profound effect on sleep, Dr. Yanagisawa said.

The experiment featured three groups of mice with virtually identical genes. The control group slept and woke at will and followed the usual mouse pattern of sleeping during the day and being awake at night. The two test groups were treated the same and had the same amount of sleep delay – six hours – but they were kept awake in different ways, said lead author Dr. Ayako Suzuki, a postdoctoral researcher who works in the laboratories of both Dr. Yanagisawa and Dr. Greene.

The first test group’s sleep was delayed by a series of cage changes. Mice are intensely curious, so each cage change was followed by an hour spent vigorously exploring the new surroundings. This behavior would roughly correspond to teenagers voluntarily delaying bedtime with a new and stimulating event like a rock concert or video game.

Researchers kept the second group awake as gently as possible, usually by waving a hand in front of the cage or tapping it lightly whenever the mice appeared to be settling down to sleep. That test group would more resemble parents reluctantly staying awake awaiting a child’s return from a concert.

Both test groups experienced the same amount of sleep deprivation, but their reactions to the different forms of alertness were striking, Dr. Yanagisawa said. In one test, the cage-changing group took longer to fall asleep than the gentle-handling group even though an analysis of their brain waves indicated equal amounts of sleep need in both test groups.

“The need to sleep is as high in the cage-changing group as in the gentle-handling group, but the cage-changers didn’t feel sleepy at all. Their time to fall asleep was nearly the same as the free-sleeping, well-rested control group,” he said.

The researchers identified two proteins that affected these responses, each linked to different aspects of sleep:  phosphorylated dynamin 1 levels were linked to how long it took to fall asleep, while phosphorylated N-myc downstream regulated gene 2 protein levels tracked the amount of sleep deprivation and corresponded to the well-known brain-wave measure of sleep need, they report.

“The two situations are different biochemically, which is a novel finding,” Dr. Yanagisawa said, adding, “These proteins are completely new to sleep research and have never before been linked to sleep need and wakefulness.”

From an evolutionary perspective, an arousal mechanism that adapts to environmental stimuli is crucial because sleeping on a rigid schedule could be dangerous. “Animals, including humans, must be able to keep themselves at least temporarily alert, say during a natural disaster,” he said.

(Image: Robert Manella / Getty Images)

Voices may not trigger brain’s reward centers in children with autism

In autism, brain regions tailored to respond to voices are poorly connected to reward-processing circuits, according to a new study by scientists at the Stanford University School of Medicine.

The research could help explain why children with autism struggle to grasp the social and emotional aspects of human speech. “Weak brain connectivity may impede children with autism from experiencing speech as pleasurable,” said Vinod Menon, PhD, senior author of the study, published online June 17 in Proceedings of the National Academy of Sciences. Menon is a professor of psychiatry and behavioral sciences at Stanford and a member of the Child Health Research Institute at Lucile Packard Children’s Hospital.

"The human voice is a very important sound; it not only conveys meaning but also provides critical emotional information to a child," said Daniel Abrams, PhD, a postdoctoral scholar in psychiatry and behavioral sciences who was the study’s lead author. Insensitivity to the human voice is a hallmark of autism, Abrams said, adding, "We are the first to show that this insensitivity may originate from impaired reward circuitry in the brain."

The study focused on children with a high-functioning form of autism. They had IQ scores in the normal range and could speak and read, but had difficulty holding a back-and-forth conversation or understanding emotional cues in another person’s voice.

The scientists compared functional magnetic resonance imaging brain scans from 20 of these children with scans from 19 typically developing children, paying particular attention to a portion of the brain that responds selectively to the sound of human voices. Prior research has shown that adults with autism had low voice-selective cortex activity in response to speech. But until this study by Menon and his colleagues, no one had looked at connections between the voice-selective cortex and other brain regions in individuals with autism.

The new study found that in children with a high-functioning form of autism, the voice-selective cortex on the left side of the brain was weakly connected to the nucleus accumbens and the ventral tegmental area — brain structures that release dopamine in response to rewards. The voice-selective cortex on the right side of the brain, which specializes in detecting vocal cues such as intonation and pitch, was weakly connected to the amygdala, which processes emotional cues.

The weaker these connections in children with autism, the worse their communication deficits, the study showed. The researchers were able to predict the children’s scores on the verbal portion of a standard test of autism severity by looking at the degree of impairment in these brain connections.

The findings may help to validate some autism therapies already in use, said co-author Jennifer Phillips, PhD, a clinical associate professor of psychiatry and behavioral sciences at Stanford who also treats children with autism at Packard Children’s. For instance, pivotal-response training aims to increase social use of language in children who can speak some words but who usually do not talk to others.

"Pivotal-response training goes after ways to naturally motivate kids to start using language and other forms of social interaction," Phillips said. Future studies could test whether brain connections leading from voice to reward centers are strengthened by autism therapies, she added.

The findings also help resolve a long-standing debate about why individuals with autism show less-than-normal interest in human voices. The team investigated two competing theories to explain the phenomenon: that individuals with autism have a deficit in their social motivation, or, alternatively, that they have sensory-processing deficits which impair their ability to fully hear human voices. The new study found normal connections between voice-selective cortex and primary auditory brain regions in children with high-functioning autism, suggesting that these children do not have sensory-processing deficits.

The next steps for researchers include studying the consequences of the weak voice-to-reward circuit in autism. “It is likely that children with autism don’t attend to voices because they are not rewarding or emotionally interesting, impacting the development of their language and social communication skills,” Menon said. “We have discovered an aberrant brain circuit underlying a core deficit in autism; our findings may aid the development of new treatments for this disorder.”

(Image: Getty Images)