Quantitative phosphoproteomic analysis of the molecular substrates of sleep need
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion
ORGANISM(S): Mus Musculus (ncbitaxon:10090)
SUBMITTER: 
Qinghua Liu
PROVIDER: MSV000081865 | MassIVE | Sat Dec 23 22:55:00 GMT 2017
SECONDARY ACCESSION(S): PXD008558
REPOSITORIES: MassIVE
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Project description:Sleep and wake have global effects on brain physiology, from molecular changes1-4 and neuronal activities to synaptic plasticity3-7. Sleep-wake homeostasis is maintained by the generation of a sleep need that accumulates during waking and dissipates during sleep8-11. Here we investigate the molecular basis of sleep need using quantitative phosphoproteomic analysis of the sleep-deprived and Sleepy mouse models of increased sleep need. Sleep deprivation induces cumulative phosphorylation of the brain proteome, which dissipates during sleep. Sleepy mice, owing to a gain-of-function mutation in the Sik3 gene 12 , have a constitutively high sleep need despite increased sleep amount. The brain proteome of these mice exhibits hyperphosphorylation, similar to that seen in the brain of sleep-deprived mice. Comparison of the two models identifies 80 mostly synaptic sleep-need-index phosphoproteins (SNIPPs), in which phosphorylation states closely parallel changes of sleep need. SLEEPY, the mutant SIK3 protein, preferentially associates with and phosphorylates SNIPPs. Inhibition of SIK3 activity reduces phosphorylation of SNIPPs and slow wave activity during non-rapid-eye-movement sleep, the best known measurable index of sleep need, in both Sleepy mice and sleep-deprived wild-type mice. Our results suggest that phosphorylation of SNIPPs accumulates and dissipates in relation to sleep need, and therefore SNIPP phosphorylation is a molecular signature of sleep need. Whereas waking encodes memories by potentiating synapses, sleep consolidates memories and restores synaptic homeostasis by globally downscaling excitatory synapses4-6. Thus, the phosphorylation-dephosphorylation cycle of SNIPPs may represent a major regulatory mechanism that underlies both synaptic homeostasis and sleep-wake homeostasis.
| S-EPMC6350790 | biostudies-literature
Project description:Every day, we sleep for a third of the day. Sleep is important for cognition, brain waste clearance, metabolism, and immune responses. Homeostatic regulation of sleep is maintained by progressively rising sleep need during wakefulness, which then dissipates during sleep. The molecular mechanisms governing sleep are largely unknown. Here, we used a combination of single-cell RNA sequencing and cell-type specific proteomics to interrogate the molecular and functional underpinnings of sleep. Different cell-types in the brain regions show similar transcriptional response to sleep need whereas sleep deprivation changes overall expression indicative of altered antigen processing, synaptic transmission and cellular metabolism in brainstem, cortex and hypothalamus, respectively. Increased sleep need enhances expression of transcription factor Sox2, Mafb, and Zic1 in brainstem; Hlf, Cebpb and Sox9 in cortex, and Atf3, Fosb and Mef2c in hypothalamus. Results from cell-type proteome analysis suggest that sleep deprivation changes abundance of proteins in cortical neurons indicative of altered synaptic vesicle cycles and glucose metabolism whereas in astrocytes it alters the abundance of proteins associated with fatty acid degradation. Similarly, phosphoproteomics of each cell type demonstrates large shifts in site-specific protein phosphorylation in neurons and astrocytes of sleep deprived mice. Our results indicate that sleep deprivation regulates transcriptional, translational and post-translational responses in a cell-specific manner and advances our understanding of the cellular and molecular mechanisms that govern sleep-wake homeostasis in mammals.
2022-10-13 | PXD018334 | Pride
Project description:Homeostatic scaling is a global form of synaptic plasticity used by neurons to adjust overall synaptic weight and maintain neuronal firing rates while protecting information coding. While homeostatic scaling has been demonstrated in vitro, a clear physiological function of this plasticity type has not been defined. Sleep is an essential process that modifies synapses to support cognitive functions such as learning and memory. Evidence suggests that information coding during wake drives synapse strengthening which is offset by weakening of synapses during sleep .Here we use biochemical fractionation, proteomics and in vivo two-photon imaging to characterize wide-spread changes in synapse composition in mice through the wake/sleep cycle. We find that during the sleep phase, synapses are weakened through dephosphorylation and removal of synaptic AMPA-type glutamate receptors (AMPARs) driven by the immediate early gene Homer1a and signaling from group I metabotropic glutamate receptors (mGluR1/5), consistent with known mechanisms of homeostatic scaling-down in vitro. Further, we find that these changes are important in the consolidation of contextual memories. While Homer1a gene expression is driven by neuronal activity during wake, Homer1a protein targeting to synapses serves as an integrator of arousal and sleep need through signaling by the wake-promoting neuromodulator noradrenaline (NA) and sleep-promoting modulator adenosine. During sleep or periods of increased sleep need Homer1a enters synapses where it remodels mGluR1/5 signaling complexes to promote AMPAR removal. Thus, we have characterized widespread changes occurring at synapses through the wake/sleep cycle and demonstrated that known mechanisms of homeostatic scaling-down previously demonstrated only in vitro are active in the brain during sleep to remodel synapses, contributing to memory consolidation.
2017-02-13 | PXD004537 | Pride
Project description:Sleep need accumulates during waking and dissipates during sleep to maintain sleep homeostasis (process S). Besides regulation of daily sleep amount, a hallmark of process S is the homeostatic sleep regulation: sleep loss causes increased amount and intensity of subsequent recovery sleep. The central regulators of process S and specific brain regions that govern sleep homeostasis in mammals remain unclear. Here, we report that enhanced calcineurin activity in the mouse brain neurons sharply increases non-rapid eye movement sleep (NREMS) to average ~17-h/day. Knockout of calcineurin in adult mouse brain diminishes baseline NREMS to average ~4-h/day, but also abolishes recovery NREMS owing to inability to accumulate sleep need during wakefulness. Calcineurin promotes baseline NREMS in both excitatory and inhibitory neurons and by antagonizing PKA and activating SIK3 via S551 dephosphorylation. Moreover, calcineurin is specifically required in the locus coeruleus-noradrenergic (LCNA) neurons for the transcriptomic and homeostatic responses to sleep loss. While chemo-/optogenetic activation of LCNA neurons increases the amount and intensity of NREMS, ablation and inhibition of LCNA neurons diminish recovery NREMS following sleep deprivation. These results establish calcineurin as a central regulator of process S and identify LC as a potential sleep need center that governs NREMS homeostasis in mice.
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2018-09-24 | PXD009374 | Pride
Project description:Extended periods of waking result in physiological impairments in humans, rats, and flies. Sleep homeostasis, the increase in sleep observed following sleep loss, is believed to counter the negative effects of prolonged waking by restoring vital biological processes that are degraded during sleep deprivation. Sleep homeostasis, as with other behaviors, is influenced by both genes and environment. We report here that during periods of starvation, flies remain spontaneously awake but, in contrast to sleep deprivation, do not accrue any of the negative consequences of prolonged waking. Specifically, the homeostatic response and learning impairments that are a characteristic of sleep loss are not observed following prolonged waking induced by starvation. To identify the genes responsible for the protective effects of starvation we conducted transcription profiling of sleep deprived flies that accrue sleep debt compared to starved siblings that do not. Genes involved in lipid metabolism were highly enriched in our dataset of 84 differentially regulated transcripts. Follow up genetic studies established that 6 genes involved in lipid metabolism strongly influence sleep homeostasis. Two of these genes, brummer (bmm) and Lipid storage droplet 2 (Lsd2), are in the same lipolysis pathway but exert antagonistic effects on lipid storage. bmm mutant flies have excess fat stores and display a large homeostatic response following sleep deprivation. In contrast, Lsd2 mutant flies, which phenocopy aspects of starvation as measured by low triglyceride stores, do not exhibit a homeostatic response following sleep loss. Importantly, Lsd2 mutant flies are not learning impaired after sleep deprivation. These results provide the first genetic evidence, to our knowledge, that lipid metabolism plays an important role in regulating the homeostatic response and can protect against neuronal impairments induced by prolonged waking. Two-condition experiments: sleep deprived vs starved. RNA from 8 biological replicates for each condition was pooled in groups of 2 to create 4 samples. Each of the 4 samples is run in duplicate with untreated circadian matched controls.
2014-04-15 | E-GEOD-18550 | biostudies-arrayexpress
Project description:The molecular mechanisms governing sleep are largely unknown. Here, we used a combination of single-cell RNA sequencing to interrogate the molecular and functional underpinnings of sleep. Different cell types in three important brain regions for sleep (brainstem, cortex and hypothalamus) had a similar transcriptional response to sleep need, with a large proportion of cells changing during recovery sleep. In contrast, sleep deprivation regulated expression of different functions in each brain region. This includes antigen processing, synaptic transmission and cellular metabolism in brainstem, cortex and hypothalamus, respectively. Increased sleep need enhances expression of the transcription factors Sox2, Mafb, and Zic1 in brainstem; Hlf, Cebpb and Sox9 in cortex, and Atf3, Fosb and Mef2c in hypothalamus. In turn, these transcription factors regulate downstream gene expression during sleep deprivation and recovery. In cortex, we also interrogated the proteome of two major cell types: neurons and astrocytes. We found surprising functional overlap of proteins that mediate vesicle and neurotransmitter transport in both cell types. In contrast, other functions were specific to each cell type.
2022-08-24 | GSE137665 | GEO
Project description:Different mammalian species vary greatly in their daily sleep quota, ranging from 2-4 hours in giraffes to 20-22 hours in koalas and bats. In humans, the sleep quantity and quality of individuals are governed by genetic factors and exhibit age-dependent variations. However, the molecular pathways and effector mechanisms that regulate daily sleep need in mammals remain unknown. Here, using an adult brain chimeric (ABC)-expression/knockout (KO) system for somatic genetics analysis of sleep in adult mice, we report that gain-of-function of histone deacetylases HDAC4/5 significantly reduces, whereas loss-of-function of HDAC4/5 increases daily non-rapid eye movement sleep (NREMS) amount and delta power–two key indicators of sleep need. Similarly, ABC-expression of cAMP-response element binding protein (CREB) or A-CREB, an inhibitor of transcriptional activity of CREB, decreases or increases NREMS amount and delta power, respectively. A combination of genetic and transcriptomic analysis reveals that HDAC4 functions in tandem with CREB in both transcriptional and sleep regulation. Consistent with their functions downstream of LKB1-SIK3 kinase cascade, ABC-expression of HDAC4/5CA or CREB rescues hypersomnia of Sik3E13∆/+ mice, whereas ABC-expression of SIK3/SLP-ST221E or HDAC(4+5)VP16 rescues insomnia of ABC-Lkb1KO mice. Taken together, these results identify LKB1-SIK3-HDAC4/5-CREB as the first major molecular pathway for transcriptional regulation of daily sleep need in mammals.
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Project description:Extended periods of waking result in physiological impairments in humans, rats, and flies. Sleep homeostasis, the increase in sleep observed following sleep loss, is believed to counter the negative effects of prolonged waking by restoring vital biological processes that are degraded during sleep deprivation. Sleep homeostasis, as with other behaviors, is influenced by both genes and environment. We report here that during periods of starvation, flies remain spontaneously awake but, in contrast to sleep deprivation, do not accrue any of the negative consequences of prolonged waking. Specifically, the homeostatic response and learning impairments that are a characteristic of sleep loss are not observed following prolonged waking induced by starvation. To identify the genes responsible for the protective effects of starvation we conducted transcription profiling of sleep deprived flies that accrue sleep debt compared to starved siblings that do not. Genes involved in lipid metabolism were highly enriched in our dataset of 84 differentially regulated transcripts. Follow up genetic studies established that 6 genes involved in lipid metabolism strongly influence sleep homeostasis. Two of these genes, brummer (bmm) and Lipid storage droplet 2 (Lsd2), are in the same lipolysis pathway but exert antagonistic effects on lipid storage. bmm mutant flies have excess fat stores and display a large homeostatic response following sleep deprivation. In contrast, Lsd2 mutant flies, which phenocopy aspects of starvation as measured by low triglyceride stores, do not exhibit a homeostatic response following sleep loss. Importantly, Lsd2 mutant flies are not learning impaired after sleep deprivation. These results provide the first genetic evidence, to our knowledge, that lipid metabolism plays an important role in regulating the homeostatic response and can protect against neuronal impairments induced by prolonged waking.
2014-04-15 | GSE18550 | GEO
Project description:A molecular pathway for transcriptional regulation of daily sleep need in mice
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