L1 - SLEEP: P- HYPOTHESIS OF SLEEP. Ode et al. 2020. Frontiers Psychol

Question 1

P-dependent control of the sleep-wake cycle - intro

Answer 1

• researchers are conducting genetic studies to identify candidate genes in the molecular signalling pathway responsible for sleep homeostasis control in mammals
• want to find genetic manipualtiosn that alter animal’s baseline sleep duration, SWA, and response to sleep deprivation –> validating whether candidate gene product controls sleep involves confirming changes in activity of candidate protein in sleep-wake cycle or sleep-deprived state
• recent studies highlight the importance of kinases in inducing sleep; investigations also focus in the P state of kinases along w/ sleep-wake cycle

Question 2

CAMKIIa/b (calcium/calmodulin dependent protein kinase)

Answer 2

• identified as a sleep-promoting kinase through reverse genetic screening in mice using CRISPR/Cas9
• KO of CAMKIIa/b in embryos = significant decrease in sleep duration –> suggests role in promoting sleep, but specific role needs further clarification
• both isoforms are expressed in neurons & atrocytes
• CAMKII has a unique self-regulatory mechanism involving auto-P at T286/287 which keeps the kinase active even in the absence of calcium/ calmodulin
• studies show increased P at T286/287 after sleep deprivation or during awake/dark phase, suggesting upregulation of CAMKII activity during increases sleep need conditions
• CAMKII is known for its crucial role in synaptic plasticity, with auto-P at multiple residues regulating synaptic strength; the specific roles of these auto-P events in regulating sleep duration remains unknown

Question 3

SIK3 (salt-inducible kinase 3)

Answer 3

• sleep promoting kinase in mammals –> discovered through large scale forward genetic screening, identifying the “sleepy” mutant w/ prolonged NREMS, due to exon skipping of SIK3 (suggested its a gain of function mutation in SIK3)
• SIK3 activity is regulated by P –> CRISPR-mediated mutagenesis confirmed role of this P-site (particularly S551) in promoting NREMS
• similar mutations in other SIK family proteins also promoted NREMS, suggesting a conserved role in sleep regulation across species; however, the exact role of S551 remains unclear
• C. elegans & drosophila support the sleep-promoting role of SIK3, further highlighting its evolutionary conservation
• however, further studies, incl SIK3 KO are needed to clarify the role of SIK3 and its P-dependent regulation in sleep duration

Question 4

ERK (extracellular-signal-regulated kinase)

Answer 4

• sleep-promoting kinase, initially observed in drosophila, where active form of ERK increases sleep duration, and ERK inhibition reduces sleep
• embryonic mice KO & conditional KO of ERK2 in cortical neurons, both lead to decreased NREMS
• administration of ERK inhibitors also decreases NREMS
• P ERK1/2 levels are higher in arousal state than NREMS

Question 5

the P hypothesis

Answer 5

• kinase-centric view of sleep need –> suggesting that protein activities modified by P serve as a molecular signature of sleep need
• overall, sleep-promoting kinases serve as key molecules linking cellular signalling pathways to the induction of sleep
• the regulation of different timescales in sleep control presents a challenge, as P occurs rapidly, but accumulation of sleep happens more slowly
• multi-site P, a core mechanism of ERK & SIK3, may encode slower dynamics by creating time delays and altering protein structures
• circadian clock proteins like PER & FRQ undergo progessive multisite P within a 24-hr period, suggesting that P dynamics can act as a timer for biological processes
• in the context of sleep control, identifying critical substrate proteins & P residues for sleep-promoting kinases is crucial
• the kinases likely affect neuronal firing patterns, possibly through modulating ion channels & pumps in the cortex
• they may directly influence neuronal excitability by modulating enzymatic & cellular transport systems that control neurotransmitter levels

Question 6

conclusion - P hypothesis

Answer 6

• CAMKII, SIK3, ERK, have been identified as sleep-inducing kinases through genetic analyses
• phosphoproteomic investigations have revealed that the P status of synaptic proteins is strongly influenced by the sleep-wake cycle
• P hypothesis proposes that protein P plays a key role in sleep regulation
• while primarily investigated in mouse models, the conservation of protein P across species suggests that this hypothesis may apply to sleep regulation in other organisms incl. humans