Research Article

Sleep-wake cycles drive daily dynamics of synaptic phosphorylation

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Science  11 Oct 2019:
Vol. 366, Issue 6462, eaav3617
DOI: 10.1126/science.aav3617

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Sleep-wake cycles at mouse synapses

Analysis of the transcriptome, proteome, and phosphoproteome at synapses in the mouse brain during daily sleep-wake cycles reveals large dynamic changes (see the Perspective by Cirelli and Tononi). Noya et al. found that almost 70% of transcripts showed changes in abundance during daily circadian cycles. Transcripts and proteins associated with synaptic signaling accumulated before the active phase (dusk for these nocturnal animals), whereas messenger RNAs and protein associated with metabolism and translation accumulated before the resting phase. Brüning et al. found that half of the 2000 synaptic phosphoproteins quantified showed changes with daily activity-rest cycles. Sleep deprivation abolished nearly all (98%) of these phosphorylation cycles at synapses.

Science, this issue p. eaav2642, p. eaav3617; see also p. 189

Structured Abstract


Globally, circadian clock–driven protein phosphorylation is a key mechanism to temporally compartmentalize biological processes across the day in peripheral tissues. In the brain, phosphorylation of several proteins has been reported to correlate with sleep pressure, itself regulated in a circadian fashion. Locally in neurons, phosphorylation plays a critical role in the regulation of synaptic function by allowing rapid modulation of protein activity and could thus dynamically scale synaptic strength in response to circadian or sleep-driven cues. Understanding the magnitude and origin of phosphorylation dynamics within synaptic proteins on a system level would be of great value to mechanistically assess synaptic function deficiencies and to understand temporal contributions to brain pathologies.


Little is known about whether and how global phosphorylation signaling in synapses is shaped in a time-dependent manner. To comprehensively characterize phosphorylation rhythms in the synaptic compartment driven by circadian and sleep-wake–dependent cues, we biochemically isolated synaptoneurosomes from mouse forebrain and analyzed them with advanced mass spectrometry–based proteomics.


Of more than 8000 phosphopeptides in almost 2000 proteins accurately quantified in the synaptoneurosome compartment across 24 hours, 30% oscillated in abundance. The phases of rhythmic phosphopeptides were distributed in two major clusters, corresponding to the transition from wake to sleep at dawn and sleep to wake at dusk. In addition to important synaptic constituents such as ion channels, receptors, and scaffolds, a large number of kinases were among the synaptic proteins modulated by phosphorylation in a time-dependent manner. More than half of the detected phosphorylated kinases in synapses show rhythmic phosphorylation at one or more residues. Predictive and experimental data demonstrate that widespread dynamic regulation of kinase activity is a core phospho-dependent functional process at synapses. Together, our data uncover molecular processes in synapses whose activity is temporally gated by phosphorylation, such as synaptic inhibition at dawn and excitation at dusk. We further assessed circadian- and sleep-driven signals by interfering with the sleep-wake cycle by applying 4 hours of sleep deprivation at different times of the day. Sleep deprivation resulted in the loss of 98% of rhythmic phosphorylation in forebrain synapses but left circadian cycles unaffected in a core of 41 phosphoproteins that function in synaptic transport and scaffolding.


This global rhythmic phosphoproteome of isolated synaptoneurosomes reveals a major reorganization of the synaptic molecular compartment concomitant with changes in activity. Our results indicate that phospho-dependent activation of kinases in response to sleep and wake is a core driver of these synaptic phosphorylation dynamics. Together, our data point toward an association of synaptic potentiation with wakefulness (activity) and down-scaling with sleep (rest). High sleep pressure induced through sleep deprivation almost completely abrogates both peaks of daily phosphorylation cycles in synapses. We thus hypothesize that modulation of phosphorylation-mediated synaptic signaling could be a key driver underlying sleep- and wake-dependent mechanisms to regulate synaptic homeostasis and function.

Daily dynamics of global phosphorylation in forebrain synaptoneurosomes under basal and sleep-deprived conditions.

(A) Quantitative phosphoproteomics analyses of isolated synaptoneurosomes from rested and sleep-deprived mice around the clock. (B and C) More than 30% of phosphorylations in many synaptic components and numerous kinases cycle daily with peaks at the sleep-wake-sleep transitions. (D and E) Sleep deprivation abolished 98% of all phosphorylation cycles in synaptoneurosomes.


The circadian clock drives daily changes of physiology, including sleep-wake cycles, through regulation of transcription, protein abundance, and function. Circadian phosphorylation controls cellular processes in peripheral organs, but little is known about its role in brain function and synaptic activity. We applied advanced quantitative phosphoproteomics to mouse forebrain synaptoneurosomes isolated across 24 hours, accurately quantifying almost 8000 phosphopeptides. Half of the synaptic phosphoproteins, including numerous kinases, had large-amplitude rhythms peaking at rest-activity and activity-rest transitions. Bioinformatic analyses revealed global temporal control of synaptic function through phosphorylation, including synaptic transmission, cytoskeleton reorganization, and excitatory/inhibitory balance. Sleep deprivation abolished 98% of all phosphorylation cycles in synaptoneurosomes, indicating that sleep-wake cycles rather than circadian signals are main drivers of synaptic phosphorylation, responding to both sleep and wake pressures.

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