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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 Sleep deprivation in mice disrupts phosphorylation cycles at synapses. 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.

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. 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.

Significance Learning mechanisms rely on plasticity properties of excitatory synapses and an activity-dependent rewiring of excitatory networks. Inhibitory synapses also display plasticity properties, but it remains unknown whether and how excitatory activity and plasticity can affect the organization of inhibitory networks. Here we show that synaptic and neuronal activity directly regulates the number and function of perisomatic inhibitory synapses through a mechanism that involves the phosphorylation of gephyrin by the enzyme calcium/calmodulin-dependent protein kinase II. The results identify a homeostatic mechanism through which cell activity can continuously adjust its excitation/inhibition balance. Maintaining a proper balance between excitation and inhibition is essential for the functioning of neuronal networks. However, little is known about the mechanisms through which excitatory activity can affect inhibitory synapse plasticity. Here we used tagged gephyrin, one of the main scaffolding proteins of the postsynaptic density at GABAergic synapses, to monitor the activity-dependent adaptation of perisomatic inhibitory synapses over prolonged periods of time in hippocampal slice cultures. We find that learning-related activity patterns known to induce N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation and transient optogenetic activation of single neurons induce within hours a robust increase in the formation and size of gephyrin-tagged clusters at inhibitory synapses identified by correlated confocal electron microscopy. This inhibitory morphological plasticity was associated with an increase in spontaneous inhibitory activity but did not require activation of GABA A receptors. Importantly, this activity-dependent inhibitory plasticity was prevented by pharmacological blockade of Ca ²⁺/calmodulin-dependent protein kinase II (CaMKII), it was associated with an increased phosphorylation of gephyrin on a site targeted by CaMKII, and could be prevented or mimicked by gephyrin phospho-mutants for this site. These results reveal a homeostatic mechanism through which activity regulates the dynamics and function of perisomatic inhibitory synapses, and they identify a CaMKII-dependent phosphorylation site on gephyrin as critically important for this process.

Neurons are highly compartmentalized cells with tightly controlled subcellular protein organization. While brain transcriptome, connectome and global proteome maps are being generated, system-wide analysis of temporal protein dynamics at the subcellular level are currently lacking. Here, we perform a temporally-resolved surfaceome analysis of primary neuron cultures and reveal dynamic surface protein clusters that reflect the functional requirements during distinct stages of neuronal development. Direct comparison of surface and total protein pools during development and homeostatic synaptic scaling demonstrates system-wide proteostasis-independent remodeling of the neuronal surface, illustrating widespread regulation on the level of surface trafficking. Finally, quantitative analysis of the neuronal surface during chemical long-term potentiation (cLTP) reveals fast externalization of diverse classes of surface proteins beyond the AMPA receptor, providing avenues to investigate the requirement of exocytosis for LTP. Our resource (neurosurfaceome.ethz.ch) highlights the importance of subcellular resolution for systems-level understanding of cellular processes. Cell surface proteins contribute to neuronal development and activity-dependent synaptic plasticity. Here, the authors perform a time-resolved surfaceome analysis of developing primary neurons and in response to homeostatic synaptic scaling and chemical long-term potentiation (cLTP), revealing surface proteome remodeling largely independent of global proteostasis.

Postsynaptic scaffolding proteins ensure efficient neurotransmission by anchoring receptors and signaling molecules in synapse-specific subcellular domains. In turn, posttranslational modifications of scaffolding proteins contribute to synaptic plasticity by remodeling the postsynaptic apparatus. Though these mechanisms are operant in glutamatergic synapses, little is known about regulation of GABAergic synapses, which mediate inhibitory transmission in the CNS. Here, we focused on gephyrin, the main scaffolding protein of GABAergic synapses. We identify a unique phosphorylation site in gephyrin, Ser270, targeted by glycogen synthase kinase 3β (GSK3β) to modulate GABAergic transmission. Abolishing Ser270 phosphorylation increased the density of gephyrin clusters and the frequency of miniature GABAergic postsynaptic currents in cultured hippocampal neurons. Enhanced, phosphorylation-dependent gephyrin clustering was also induced in vitro and in vivo with lithium chloride. Lithium is a GSK3β inhibitor used therapeutically as mood-stabilizing drug, which underscores the relevance of this posttranslational modification for synaptic plasticity. Conversely, we show that gephyrin availability for postsynaptic clustering is limited by Ca 2+ -dependent gephyrin cleavage by the cysteine protease calpain-1. Together, these findings identify gephyrin as synaptogenic molecule regulating GABAergic synaptic plasticity, likely contributing to the therapeutic action of lithium.

GABA A receptors (GABA A Rs) mediate the majority of fast inhibitory neurotransmission in the brain via synergistic association with the postsynaptic scaffolding protein gephyrin and its interaction partners. However, unlike their counterparts at glutamatergic synapses, gephyrin and its binding partners lack canonical protein interaction motifs; hence, the molecular basis for gephyrin scaffolding has remained unclear. In this study, we identify and characterize two new posttranslational modifications of gephyrin, SUMOylation and acetylation. We demonstrate that crosstalk between SUMOylation, acetylation and phosphorylation pathways regulates gephyrin scaffolding. Pharmacological intervention of SUMO pathway or transgenic expression of SUMOylation-deficient gephyrin variants rescued gephyrin clustering in CA1 or neocortical neurons of Gabra2 -null mice, which otherwise lack gephyrin clusters, indicating that gephyrin SUMO modification is an essential determinant for scaffolding at GABAergic synapses. Together, our results demonstrate that concerted modifications on a protein scaffold by evolutionarily conserved yet functionally diverse signalling pathways facilitate GABAergic transmission. Gephyrin is a cytoplasmic scaffolding protein that selectively forms postsynaptic scaffolds at GABAergic and glycinergic synapses. Here the authors characterize regulatory mechanisms determining gephyrin scaffolding and GABAA receptor synaptic transmission that involve acetylation, SUMOylation and phosphorylation.

Gephyrin is the main scaffolding protein at inhibitory postsynaptic sites, and its clusters are the signaling hubs where several molecular pathways converge. Post-translational modifications (PTMs) of gephyrin alter GABA A receptor clustering at the synapse, but it is unclear how this affects neuronal activity at the circuit level. We assessed the contribution of gephyrin PTMs to microcircuit activity in the mouse barrel cortex by slice electrophysiology and in vivo two-photon calcium imaging of layer 2/3 (L2/3) pyramidal cells during single-whisker stimulation. Our results suggest that, depending on the type of gephyrin PTM, the neuronal activities of L2/3 pyramidal neurons can be differentially modulated, leading to changes in the size of the neuronal population responding to the single-whisker stimulation. Furthermore, we show that gephyrin PTMs have their preference for selecting synaptic GABA A receptor subunits. Our results identify an important role of gephyrin and GABAergic postsynaptic sites for cortical microcircuit function during sensory stimulation.

Neuroscience currently requires the use of antibodies to study synaptic proteins, where antibody binding is used as a correlate to define the presence, plasticity, and regulation of synapses. Gephyrin is an inhibitory synaptic scaffolding protein used to mark GABAergic and glycinergic postsynaptic sites. Despite the importance of gephyrin in modulating inhibitory transmission, its study is currently limited by the tractability of available reagents. Designed Ankyrin Repeat Proteins (DARPins) are a class of synthetic protein binder derived from diverse libraries by in vitro selection and tested by high-throughput screening to produce specific binders. In order to generate a functionally diverse toolset for studying inhibitory synapses, we screened a DARPin library against gephyrin mutants representing both phosphorylated and dephosphorylated states. We validated the robust use of anti-gephyrin DARPin clones for morphological identification of gephyrin clusters in rat neuron culture and mouse brain tissue, discovering previously overlooked clusters. This DARPin-based toolset includes clones with heterogenous gephyrin binding modes that allowed for identification of the most extensive gephyrin interactome to date and defined novel classes of putative interactors, creating a framework for understanding gephyrin’s nonsynaptic functions. This study demonstrates anti-gephyrin DARPins as a versatile platform for studying inhibitory synapses in an unprecedented manner.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

GABAergic inhibitory neurotransmission contributes to diverse aspects of brain development and adult plasticity, including the expression of complex cognitive processes. This is afforded for in part by the dynamic adaptations occurring at inhibitory synapses, which show great heterogeneity both in terms of upstream signaling and downstream effector mechanisms. Single-particle tracking and live imaging have revealed that complex receptor-scaffold interactions critically determine adaptations at GABAergic synapses. Super-resolution imaging studies have shown that protein interactions at synaptic sites contribute to nano-scale scaffold re-arrangements through post-translational modifications (PTMs), facilitating receptor and scaffold recruitment to synaptic sites. Additionally, plasticity mechanisms may be affected by the protein composition at individual synapses and the type of pre-synaptic input. This mini-review article examines recent discoveries of plasticity mechanisms that are operational within GABAergic synapses and discusses their contribution towards functional heterogeneity in inhibitory neurotransmission.