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RESEARCH ◥ of this temporal gating for synaptic function RESEARCH ARTICLE SUMMARY and energy homeostasis. Overall, the oscil- lations of 75% of synaptic proteins were con- NEUROSCIENCE comitant with their rhythmic transcripts, indicating a key role for local synaptic tran- The forebrain synaptic transcriptome is organized by slation. Under conditions of high sleep pres- ◥ sure, one-fourth of mRNAs clocks but its proteome is driven by sleep ON OUR WEBSITE remained identically circa- Read the full article dian, and most preserved Sara B. Noya, David Colameo, Franziska Brüning, Andrea Spinnler, Dennis Mircsof, Lennart Opitz, at http://dx.doi. some degree of circadian Matthias Mann, Shiva K. Tyagarajan*, Maria S. Robles*, Steven A. Brown* org/10.1126/ rhythmicity. In contrast, science.aav2642 no substantial circadian .................................................. rhythm could be detected INTRODUCTION: Temporally consolidated beha- protein abundance in transcriptome and pro- in any protein when sleep pressure was con- viors such as sleep normally occur in synchrony teome of synapses from the mouse forebrain, stantly high. with endogenous circadian rhythms and both using biochemically purified synaptoneuro- have been reported to contribute to global daily somes isolated across the 24-hour day both at CONCLUSION: Examining the dynamics of oscillations of transcription in brain. Neurons normal sleep pressure and at constant high mRNAs in the synaptic landscape revealed the have further adapted specialized means to sleep pressure. largest proportion of circadian transcripts in traffic RNA into distant dendritic and axonal any tissue, cell, or organelle described to date. Downloaded from arbors, where it is locally translated. Together, RESULTS: Notably, 67% of synaptic mRNAs These synaptic oscillations are controlled post- these mechanisms allow coordination of physi- showed circadian oscillations, with a mean transcriptionally and the daily dynamics of ology with environmental needs. amplitude of about twofold. Further, 93% transcripts and their related proteins clearly of these oscillating transcripts were exclu- delineate different cellular modes between RATIONALE: About 6% of the forebrain tran- sively rhythmic in synaptoneurosomes, sug- sleep and wake. Our study provides insight scriptome oscillates in a time-of-day–dependent gesting an entirely posttranscriptional origin into the connectivity between sleep and cir- http://science.sciencemag.org/ manner, and it has been proposed that this os- for synaptic mRNA oscillations. This observa- cadian rhythms and suggests an elegant para- cillation is mostly driven by the sleep-wake state tion was supported by single-molecule fluores- digm whereby a molecular clock provisions to enable daily changes in synaptic structure cence in situ hybridization. Rhythmic synaptic synapses with mRNAs before dawn and dusk, and function. In turn, such synaptic scaling is transcripts formed two distinct waves, antici- which are later translated in response to activity- thought to form a critical feature of the sleep- pating either dawn or dusk, and both required rest cycles.▪ wake process. Given the highly local capacity a functional circadian clock. These two waves for synaptic remodeling, an essential missing showed completely different functional signa- link in this argument is the effects of circadian tures: synaptic signaling preceded the active clocks and sleep pressure upon messenger RNA phase, whereas metabolism and translation *Corresponding author. Email: [email protected] (mRNA) and related protein abundance at preceded the resting phase. Comprehensive (S.A.B.); [email protected] (M.S.R.); [email protected] (S.T.) on November 7, 2019 synapses themselves. To address this question, circadian characterization of the synaptic pro- Cite this article as S. B. Noya et al., Science 366, we examined daily rhythms of transcript and teome demonstrated the functional relevance eaav2642 (2019). DOI: 10.1126/science.aav2642 A High Throughput D Sequencing Synaptic LC-MS/MS transmission Ion transport Enrichment 2.4 Relative d power Circadian 2.0 Synaptic 1.6 L TP Sleep 1.2 NMDA Fold change Day Night 0.8 receptor Synapse organization 4 16 Learning Ca+ homeostasis B 2 mRNA Expression Metabolic process Response to oxidative stress Redox Enrichment 0 process Circadian Synaptic -2 Transcriptome Translation 40S assembly Day Night Fold change C max b-oxidation 2.5 10 Protein levels 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 Proteome pressure. (B) Synaptic transcripts can maintain circadian rhythmicity under high sleep pressure (C) but min protein rhythms are completely abolished. (D) Gene ontology highlights the complete temporal Day Night segregation of predusk (top) and predawn (bottom) synaptic function. Noya et al., Science 366, 200 (2019) 11 October 2019 1of1 RESEARCH ◥ a circadian fashion, which are then translated RESEARCH ARTICLE in response to sleep-wake cycles. NEUROSCIENCE The forebrain synaptic transcriptome shows pervasive daily rhythmicity The forebrain synaptic transcriptome is organized by To generate a time-resolved map of the synaptic transcriptome from the mouse forebrain, we clocks but its proteome is driven by sleep purified synaptoneurosomes using biochemical fractionation with discontinuous Percoll gra- Sara B. Noya1, David Colameo1*, Franziska Brüning2,4, Andrea Spinnler1, Dennis Mircsof1, dients (22). Synaptoneurosomes represent axo- Lennart Opitz3, Matthias Mann4,5, Shiva K. Tyagarajan1†, Maria S. Robles2†, Steven A. Brown1† nal nerve terminals (cytoplasm, synaptic vesicles, mitochondria, and cytoskeleton) and attached Neurons have adapted mechanisms to traffic RNA and protein into distant dendritic and postsynaptic structures. We collected fore- axonal arbors. Taking a biochemical approach, we reveal that forebrain synaptic transcript brains every 4 hours across the day in biological accumulation shows overwhelmingly daily rhythms, with two-thirds of synaptic transcripts triplicates under natural conditions of light showing time-of-day–dependent abundance independent of oscillations in the soma. These and dark (LD) and identified mRNAs by high- transcripts formed two sharp temporal and functional clusters, with transcripts preceding dawn throughput sequencing in the forebrain homo- related to metabolism and translation and those anticipating dusk related to synaptic genates and the purified synaptoneurosomes transmission. Characterization of the synaptic proteome around the clock demonstrates the (Fig. 1A). After normalization and threshold- functional relevance of temporal gating for synaptic processes and energy homeostasis. ing (see materials and methods), we identified Downloaded from Unexpectedly, sleep deprivation completely abolished proteome but not transcript oscillations. 14,073 unique transcripts. These transcripts over- Altogether, the emerging picture is one of a circadian anticipation of messenger RNA needs in lapped almost completely with those identi- the synapse followed by translation as demanded by sleep-wake cycles. fied in the hippocampal neuropil (23), a highly projection-enriched brain region (Fig. 1B), as well as with other similar transcriptomes of cell-autonomous circadian clock based mRNA processing, polyadenylation, and trans- forebrain synapses (24) (fig. S1A). In addition, http://science.sciencemag.org/ upon feedback loops of transcription and lation have appeared (9–11). Complicating mat- quantitative polymerase chain reaction demon- translation functions in nearly every cell ters still further, circadian oscillations in mRNA strated enrichment of known synaptic mRNAs A of the mammalian body and influences levels can also be driven by temporally consol- and depletion of nuclear ones (fig. S1B). These most aspects of physiology. In the brain, idated behaviors. In liver, timing of food in- measures confirmed the validity of our bio- the most obvious manifestation of circadian take contributes to rhythmic transcription (12), chemical approach. Reasoning that a fold change control is the consolidation of sleep into day and in cortex, sleep-wake cycles play a major of ≥1.5 in synaptic versus forebrain abundance or night. In synchrony with sleep and wake, role (3). should represent a reasonable criterion with daily rhythmic oscillations also occur in a sub- Relative to all other cell types, neurons pre- which to identify a specific synaptic element, stantial fraction of the brain transcriptome, sent a special case because mRNA is distrib- we found 3104 unique synaptically enriched ranging from 3 to 4% in retina, cerebellum, uted to potentially distant compartments such mRNAs (Fig. 1B and table S1). Gene Ontology brainstem, and hypothalamus, to 8 to 10% in as axonal terminals and postsynaptic spines. (GO) analysis corroborated the overwhelming on November 7, 2019 cortex and suprachiasmatic nuclei (1–4). These Although the axodendritic arbor contains only predominance of synaptic annotations among oscillations are likely driven in part by rhythmic ~10% of total transcripts (13), a large literature these transcripts, as well as their stepwise en- assembly and disassembly of transcription demonstrates that mRNAs are actively trans- richment across the analytical steps of the complexes and chromatin modifiers orches- ported to neurites (14–16). Downstream, local procedure (Fig. 1C). trated by circadian