Image courtesy of Antalique Tran
Sleep has long been recognized as an essential mammalian function that provides crucial time for the brain to recover. Learning and memory are bolstered by the restorative capacity of sleep to change the strength, form, and adaptability of synapses, the structures which facilitate electrical and chemical signaling between neurons, the cells of the brain and nervous system. Sleep loss, meanwhile, impairs general cognitive ability and function.
Though the power of sleep to recharge the brain is well-accepted, the mechanisms underlying the need for sleep and its cyclic nature are less clear. Most experts generally believe that two principal forces interact to regulate sleep. One is the body’s natural chemical rhythms known as the circadian clock. Among other profound effects on physiological processes, the circadian clock is the internal factor responsible for maintaining the body’s preferred natural sleep phase—nighttime for humans and other diurnal species. The other component of sleep regulation is known as the homeostat, or the recurring pressure to fall asleep based on time spent awake and the quality of previous sleep cycles—something we recognize as fatigue. While these two mechanisms are acknowledged as the sources of sleep’s regulatory and fortifying power, the way in which they interact remains poorly understood.
The recent neuroscientific insights of a European team of researchers, however, could go a long way toward unraveling this mystery and more precisely defining the interdependence between circadian and homeostatic factors in driving localized control of sleep processes in the brain. Led by Professors Steven Brown and Shiva Tyagarajan of the University of Zurich and Professor Maria Robles of the Ludwig Maximilian University of Munich, the work examines the area of the brain responsible for learning, memory, and other cognitive procedures affected by sleep loss in order to model synaptic function across an entire 24-hour wake and sleep cycle.
mRNA transport responds to circadian tendencies
The alteration of specific synaptic proteins has previously been linked to induction of sleep pressure in the brain. This process is separated into two distinct steps, known as transcription and translation. Transcription is the first step in gene expression, whereby a segment of DNA is copied into RNA, a class of molecule that serves as the template for protein production. This copy can then be transported to other sites, including synapses, as messenger RNA (mRNA). The next step is translation, where proteins are created based on the RNA template. Once these phases have been completed, some of the new proteins undergo phosphorylation, the molecular attachment of a phosphate group that activates enzyme function. In the course of conducting their research, the investigators determined that these different stages of the protein modification process, essential to sleep cycling, are regulated by separate procedures, and that circadian and homeostatic effects are more pronounced at various points in the process. “We hope that it will lead both to a better knowledge of why we sleep in the first place, and how this sleep is locally regulated,” Brown said.
The investigators collected four mouse forebrains every four hours over a 24-hour period to analyze the rhythmic oscillations of mRNAs in synapses and in the forebrain as a whole over the course of the entire sleep and wake cycle. The control group of mice was kept on a schedule consisting of alternating twelve-hour periods of light and dark laboratory conditions to simulate daytime and nighttime. The researchers found more oscillations in mRNA in the synapses than in the entire forebrain—revealing mRNA delivery is responsible for such transcriptional oscillations in synapses.
Since mRNA oscillation intensity heightens around the transitions between the lights-on and lights-off periods analogous to dawn and dusk, investigators sought to confirm the hypothesis that such oscillation is circadian-driven. They compared three groups of mice under different laboratory conditions: one group followed the simulated day-night schedule, one was kept in constant darkness, and the third was composed of mice with their circadian clocks genetically disabled. Results indicated stark differences in oscillatory patterns for mice lacking functioning circadian regulators, indicating that the buildup of transcriptional oscillations around such pivotal times is generated by circadian effects.
In order to isolate the influences on transcription brought about by circadian and homeostatic rhythms, mice were deprived of sleep for six hours prior to euthanasia. Levels of sleep pressure are indicated by amplitudes of electroencephalogram oscillations. Testing over twenty-four hours with sustained, elevated sleep pressure revealed little difference in amplitude compared to normal circadian phases, suggesting that sleep pressure is not the key factor in controlling synaptic mRNA oscillations.
A correlation between phosphorylation and sleep pressure
Having guessed that post-transcriptional oscillations are brought on by processes involving protein modification rather than those involving mRNA transport following this data, the investigators pivoted to explore which sleep-regulating method most directly contributes to their presence. Mice were once more kept on alternating twelve-hour schedules and euthanized every four hours. The team observed that, similar to mRNAs, phosphorylated proteins’ oscillations cluster primarily around simulated dawn and dusk, indicating the role of sleep and wake pressure in shaping synaptic phosphorylation processes. Further analysis of kinases, the enzymes that perform phosphorylation, revealed that the types of kinases present at dawn are associated with excitatory synaptic function while those present at dusk are inhibitory. Such insights reflect the buildup of sleep and wake pressure, strengthening the claim that the homeostat is largely responsible for protein cycling independent of time of day.
To support these discoveries, the team deprived the mice of sleep for four hours prior to collecting brain samples and measured phosphorylation. Whereas one fourth of the effect of circadian rhythms on mRNA were preserved under sleep deprivation, a mere two percent of oscillating phosphorylated proteins remained. According to Brown, this dissimilar behavior under the same conditions was a profound moment in the research process. “The fact that synaptic RNA and protein abundance responded differently to sleep deprivation made us realize that different steps in protein production might be under different control,” he said.
Sleep functions better-defined than ever
The mapping of the synaptic phosphorylation process across an entire sleep-wake cycle gives experts a clearer understanding than ever before of how circadian rhythms and homeostatic pressures interact. The results indicate that circadian cycles transport synaptic mRNAs most heavily at dawn and dusk prior to the first stages of protein modification, while the phosphorylation that follows is modulated by sleep pressure and homeostatic regulation of the body. As characterized by neuroscientist Robert Greene of the University of Texas Southwestern Medical Center, the brain prepares itself with anticipatory circadian tendencies but only follows through on such preparation as the result of need-based sleep or wake pressure.
Though the insights offer exciting delineation between the two interdependent processes that govern sleep processes, they remain largely theoretical. Brown emphasized that the specific molecular mechanisms underlying synaptic functions are yet to be elucidated, and that his team’s discoveries are merely puzzle pieces in fully explaining the crucial role of sleep’s restorative properties in daily function. Nonetheless, they confirm long-suspected procedural connections and leave little doubt regarding the direction of future research. “Even though they were done in mice, the findings have implications for promising avenues of research, drawing the focus straight to events at the synapse,” said neuroscientist Akhilesh B. Reddy of the University of Pennsylvania. “This is just the tip of the iceberg.”
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Willingham, E. (2019). Sleep Deprivation Shuts Down Production of Essential Brain Proteins. Scientific American.