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Proteomic Study Shows that Sleep Loss May Affect Synapse Formation Needed for Memory

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NOTE: This story has been updated from a previous version, which contained an error about the molecule adenosine.

A new proteomic study by scientists at Harvard Medical School shows that rats deprived of sleep for six hours exhibited changes in cytoskeletal and synaptic proteins that play an important role in forming the synapses required for memory, attention, and cognition.

The study, which is believed to be the first to use proteomic techniques to look at sleep deprivation, is noteworthy because understanding the molecular effects of sleep deprivation could have important applications for humans, who typically spend one-third of their lives sleeping.

"At any one time, probably 10 to 20 percent of people have difficulty sleeping," noted Robert McCarley, a leader of the study, which was published Nov. 4 in the online version of the Journal of Neuroscience Research. "Plus, people with depression often have severe sleep difficulty. There may be symptoms of depression that relate to sleep deprivation. There are a lot of disorders that impact on sleep, and once we find out what sleep is doing, we can better manage what disorders we have."

Radhika Basheer, an assistant professor at Harvard and the first author of the study, said that the study indicates that there is a lot of synaptic plasticity during sleep deprivation.

"One of the consequences of sleep deprivation is loss of memory, cognition, and attention — all of which require synaptic reinforcements," she said. "This is evidence that, yes, synaptic proteins seem to be changing with sleep deprivation."


"I was surprised that most of the high-abundance proteins from cytoskeleton seemed to be showing such remarkable changes. I was expecting some, but I didn't expect that with my first examination of proteins, all [of them] would be associated with cytoskeletal and synaptic functions."

Previously, Basheer and McCarley had studied the effects of a molecule called adenosine on sleep. As organisms expend energy, adenosine triphosphate is used up, and broken down to adenosine. The resulting increased level of extracellular adenosine in the basal forebrain induces sleepiness, Basheer explained.

"It's a relatively short-term mechanism," said McCarley, who is a professor of psychiatry at Harvard Medical School and the head of the neuroscience laboratory at the VA Healthcare System. "However, if you restrict an individual's sleep over several nights, you get accumulation effects of sleep deprivation. This accumulation can't be accounted for by the simple feedback loop of adenosine. The changes that cause alterations over a long time probably have to be related to changes in transcription, translation and protein production."

To study these more cumulative effects of sleep deprivation, McCarley, Basheer, and their colleagues compared the proteome of the basal forebrains from rats that had been sleep deprived to the proteome of normal rat basal forebrains.

Rats are nocturnal animals. Normal laboratory rats that are kept in darkness for 12 hours and light for 12 hours sleep for 65 or 70 percent of the time that the lights are on.

To deprive rats of sleep, the researchers played with them gently during the day for six hours. If a sleep-deprived rat began to sit down in one place and curl up, a researcher would tap the cage or brush the rat gently awake, or introduce an interesting object into the cage. Rats were also connected to an electroencephalogram to monitor if they were going into slow-wave sleep mode.

At the end of the six hours of sleep deprivation, the rats were sacrificed, and the basal forebrains were harvested for proteomic analysis. The researchers extracted proteins from the samples and ran them out on 2D electrophoresis gels. They compared those gels with gels containing proteins from the basal forebrains of normal rats that had not been sleep deprived.

Among 969 protein spots that were compared, the researchers found 89 spots that showed more than two-fold differences in expression between the sleep deprived rats and undisturbed controls. Of those 89, 11 showed greater than three-fold differences in expression.

The researchers extracted the proteins that showed over three-fold differences in expression from the gels, then identified them using MALDI mass spectrometry.

"I was surprised that most of the high-abundance proteins from cytoskeleton seemed to be showing such remarkable changes," said Basheer. "I was expecting some, but I didn't expect that with my first examination of proteins, all [of them] would be associated with cytoskeletal and synaptic functions."

Proteins identified included tubulin, a protein involved in forming microtubules that make up the cytoskeleton; neuromodulin, a protein responsible for cytoskeletal filamentous-actin stabilization; Rho A GTPase, a protein involved in cytoskeletal remodeling; and SNP25b, a protein involved in synaptogenesis and axonal growth.

Identification of other proteins that showed less than three-fold differences in expression is ongoing, Basheer said.

"I can't tell you the identity of those proteins now, but we think they would not just be cytoskeletal proteins," she said.

To study more precisely the changes in synapses due to sleep deprivation, the researchers would like in the future to specifically extract synapses from the rats' basal forebrains and to analyze their proteomes, Basheer said. The researchers are also planning on doing studies of more long-term sleep deprivation.

McCarley said that his research team is not planning to do any studies on sleep deprivation in humans because the studies would require working at night, which could result in sleep deprivation for the researchers themselves.

"It's hard," said McCarley. "People sleep at night and we work during the day. And proteomic [sleep] studies in humans are not really possible. You can't sample the brain, and you can't get access to brain proteins through the blood. You can look at energy [use] through magnetic resonance spectrospcopy, but not proteins."

— Tien-Shun Lee ([email protected])

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