SLEEP L2: Zhou et al. 2022. Nature - LKB1/SIK3

Question 1

intro

Answer 1

• LKB1 protein activates the AMP-activated protein kinase family (AMPK), including SIK3
• mutations in SIK3 lead to increased NREM sleep and altered brain activity patterns
  -SIK3 regulates HDACs, which control the activity of TFs involved in various physiological processes incl. sleep regulation (remove acetyl from Lys, so +, so stronger interaction, so less accessible to T)
  -using AAV-mediated somatic genetics, researchers identified the LKB1-SIK3-HDAC4/5-CREB pathway as a key regulator of sleep duration in mice

Question 2

LKB1-SIK3 inhibits HDAC4/5 to regulate sleep

Answer 2

• study used adult brain chimeric (ABC) KO of LKB1 using recombinant AAV virus to deliver Cre recombinase across mouse brain neurons
• immunostaining confirmed significant reduction of LKB1 expression in neurons of ABC-LKB1 KO mice
• KO mice exhibited decrease in daily NREM sleep & decreased NREM delta power, particularly during dark phase
• also showed moderate reduction in REM sleep during light phase
• reduced P of SIK3 at T221 (downstream target of LKB1) was observed in cortical lysates of KO mice
• also found reduced P of HDAC4 at Ser245, and increased nuclear translocation of HDAC4 in cortex of KO mice, indicating HDAC4 activation due to SIK3 inactivation
  -fusion proteins interfering w/transcriptional activities of HDAC4/5 fully restored NREM sleep amount and delta power in KO mice, suggesting that LKB1-SIK3 pathway regulates sleep by suppressing HDAC4/5 activity

Question 3

ABC HDAC4/5 KO increases NREM sleep

Answer 3

-triple target of CRISPR was used to achieve KO of HDAC4, 5, or both in mouse brains –> resulted in higher NREMS amount and delta power in mice deficient in both compared to single KO mice
- behavioural tests showed that ABC-HDAC4 KO mice appeared healthy and normal, indicating that sleep phenotype observed was likely not a secondary effect of blindness or behavioural abnormalities

Question 4

ABC-HDAC4/5 (CN) reduces NREMs

Answer 4

• CN = constitutively nuclear-localised
• HDAC4/5 are catalytically inactive, and recruit the NCoR-SMRT-HDAC3 complex to regulate TFs like FOXO
• mutation disrupting HDAC4’s association with this complex abolished its ability to reduce NREMS, suggesting role for HDAC4 in recruiting the complex to regulate sleep quantity in mice

Question 5

HDAC4/5 are regulated by sleep-wake cycle

Answer 5

• mice have a diurnal sleeping pattern, sleeping more at day than night
• P of HDAC4/5 at S245 decreased gradually during light phase and increased during dark phase
• subcellular fractionation showed increased nuclear translocation of HDAC5 in cortex and hypothalamus during dark phase
• increased S245 P and reduced nuclear:cytoplasmic ratios of HDAC4/5 were observed in the cortex following sleep deprivation; however, no significant changes were observed in HDAC4/5 distribution in hypothalamus after sleep deprivation
• KO or constitutive nuclear expression of HDAC4 did not impair the homeostatic response to sleep deprivation, indicating that HDAC4 is dispensable in the process

Question 6

HDAC4/5 regulates sleep in posterior hypothalamus

Answer 6

• to pinpoint where in the mouse brain HDAC4/5 regulate sleep, researchers conducted stereotactic injections of AAV in the thalamus, PO area, or posterior hypothalamus
• no sleep phenotype was observed in thalamus or PO, regions known to be involved in sleep regulation
• EEG/EMG recordings confirmed that gain and loss of function of HDAC4 in posterior hypothalamus neurons had opposing effects on NREMS amount, w/o significant effect on NREMS delta power or REMS amount
• results indicate that HDAC4/5 specifically regulate NREMS amount in the posterior hypothalamus
• moreover, KD of LKB1, SIK3, or HDAC4/5 did not alter circadian rhythm in U2OS cells suggesting that the pathway regulates sleep amount w/o altering the circadian clock

Question 7

HDAC4 and CREB coregulate NREMS

Answer 7

• HDAC4/5 regulate activities of TFs such as MEF2, OXO and CREB
• ABC expression of WT, constitutively active, or dominant-negative mutant of MEF2A/C/C, FOXO1/3, did not alter sleep quantity in mice
• expression of CREB or inhibition of CREB transcriptional activity significantly decreased/increased NREMS respectively
  -CREB but not its inhibitor, moderately reduced REMS, consistent w/previous reports

Question 8

discussion

Answer 8

-the proposed model for transcriptional regulation of daily sleep amount in mice suggests that LKB1 activates SIK3, leading to increased NREMS amount and delta power, by suppressing HDAC4/5 activities through phosphorylation and cytoplasmic sequestration
-P of HDAC4/5 rises during dark phase and declines during light phase, tracking sleep need accumulation and dissipation
-deP enables HDAC4/5 translocation to the nucleus, where they cooperate w/CREB and other factors to reduce NREMS by transcriptional regulation of sleep and wake-promoting genes

-HDAC4 is implicated in regulating NREMS amount in the posterior hypothalamus, suggesting an unexplored brain center for sleep quantity and regulation
- complements findings on SIK3-HDAC4 signalling in hypothalamical and cortical neurons regulating NREMS amount and delta power

-AAV-mediated somatic genetics analysis facilitates rapid identification of new sleep regulatory genes and efficient dissection of molecular pathways of sleep regulation in mice