The forebrain synaptic transcriptome is organized by clocks but its proteome is driven by sleep

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 Circadian changes in abundance of transcripts and proteins are monitored at mouse synapses. INTRODUCTION Temporally consolidated behaviors such as sleep normally occur in synchrony with endogenous circadian rhythms and both have been reported to contribute to global daily oscillations of transcription in brain. Neurons have further adapted specialized means to traffic RNA into distant dendritic and axonal arbors, where it is locally translated. Together, these mechanisms allow coordination of physiology with environmental needs. RATIONALE About 6% of the forebrain transcriptome oscillates in a time-of-day–dependent manner, and it has been proposed that this oscillation is mostly driven by the sleep-wake state to enable daily changes in synaptic structure and function. In turn, such synaptic scaling is thought to form a critical feature of the sleep-wake process. Given the highly local capacity for synaptic remodeling, an essential missing link in this argument is the effects of circadian clocks and sleep pressure upon messenger RNA (mRNA) and related protein abundance at synapses themselves. To address this question, we examined daily rhythms of transcript and protein abundance in transcriptome and proteome of synapses from the mouse forebrain, using biochemically purified synaptoneurosomes isolated across the 24-hour day both at normal sleep pressure and at constant high sleep pressure. RESULTS Notably, 67% of synaptic mRNAs showed circadian oscillations, with a mean amplitude of about twofold. Further, 93% of these oscillating transcripts were exclusively rhythmic in synaptoneurosomes, suggesting an entirely posttranscriptional origin for synaptic mRNA oscillations. This observation was supported by single-molecule fluorescence in situ hybridization. Rhythmic synaptic transcripts formed two distinct waves, anticipating either dawn or dusk, and both required a functional circadian clock. These two waves showed completely different functional signatures: synaptic signaling preceded the active phase, whereas metabolism and translation preceded the resting phase. Comprehensive circadian characterization of the synaptic proteome demonstrated the functional relevance of this temporal gating for synaptic function and energy homeostasis. Overall, the oscillations of 75% of synaptic proteins were concomitant with their rhythmic transcripts, indicating a key role for local synaptic translation. Under conditions of high sleep pressure, one-fourth of mRNAs remained identically circadian, and most preserved some degree of circadian rhythmicity. In contrast, no substantial circadian rhythm could be detected in any protein when sleep pressure was constantly high. CONCLUSION Examining the dynamics of mRNAs in the synaptic landscape revealed the largest proportion of circadian transcripts in any tissue, cell, or organelle described to date. These synaptic oscillations are controlled posttranscriptionally and the daily dynamics of transcripts and their related proteins clearly delineate different cellular modes between sleep and wake. Our study provides insight into the connectivity between sleep and circadian rhythms and suggests an elegant paradigm whereby a molecular clock provisions synapses with mRNAs before dawn and dusk, which are later translated in response to activity-rest cycles. Circadian clocks regulate synaptic mRNAs but sleep and wake regulate their proteins. (A) Workflow: Forebrain synaptoneurosomes were isolated across the day at low and high sleep pressure. (B) Synaptic transcripts can maintain circadian rhythmicity under high sleep pressure (C) but protein rhythms are completely abolished. (D) Gene ontology highlights the complete temporal segregation of predusk (top) and predawn (bottom) synaptic function. Neurons have adapted mechanisms to traffic RNA and protein into distant dendritic and axonal arbors. Taking a biochemical approach, we reveal that forebrain synaptic transcript accumulation shows overwhelmingly daily rhythms, with two-thirds of synaptic transcripts showing time-of-day–dependent abundance independent of oscillations in the soma. These transcripts formed two sharp temporal and functional clusters, with transcripts preceding dawn related to metabolism and translation and those anticipating dusk related to synaptic transmission. Characterization of the synaptic proteome around the clock demonstrates the functional relevance of temporal gating for synaptic processes and energy homeostasis. Unexpectedly, sleep deprivation completely abolished proteome but not transcript oscillations. Altogether, the emerging picture is one of a circadian anticipation of messenger RNA needs in the synapse followed by translation as demanded by sleep-wake cycles.