SIK3-HDAC4 in the suprachiasmatic nucleus regulates the timing of arousal at the dark onset and circadian period in mice.

Mammals exhibit circadian cycles of sleep and wakefulness under the control of the suprachiasmatic nucleus (SCN), such as the strong arousal phase-locked to the beginning of the dark phase in laboratory mice. Here, we demonstrate that salt-inducible kinase 3 (SIK3) deficiency in gamma-aminobutyric acid (GABA)-ergic neurons or neuromedin S (NMS)-producing neurons delayed the arousal peak phase and lengthened the behavioral circadian cycle under both 12-h light:12-h dark condition (LD) and constant dark condition (DD) without changing daily sleep amounts. In contrast, the induction of a gain-of-function mutant allele of Sik3 in GABAergic neurons exhibited advanced activity onset and a shorter circadian period. Loss of SIK3 in arginine vasopressin (AVP)-producing neurons lengthened the circadian cycle, but the arousal peak phase was similar to that in control mice. Heterozygous deficiency of histone deacetylase (HDAC) 4, a SIK3 substrate, shortened the circadian cycle, whereas mice with HDAC4 S245A, which is resistant to phosphorylation by SIK3, delayed the arousal peak phase. Phase-delayed core clock gene expressions were detected in the liver of mice lacking SIK3 in GABAergic neurons. These results suggest that the SIK3-HDAC4 pathway regulates the circadian period length and the timing of arousal through NMS-positive neurons in the SCN.

Kinase signalling in excitatory neurons regulates sleep quantity and depth.

Progress has been made in the elucidation of sleep and wakefulness regulation at the neurocircuit level1,2. However, the intracellular signalling pathways that regulate sleep and the neuron groups in which these intracellular mechanisms work remain largely unknown. Here, using a forward genetics approach in mice, we identify histone deacetylase 4 (HDAC4) as a sleep-regulating molecule. Haploinsufficiency of Hdac4, a substrate of salt-inducible kinase 3 (SIK3)3, increased sleep. By contrast, mice that lacked SIK3 or its upstream kinase LKB1 in neurons or with a Hdac4S245A mutation that confers resistance to phosphorylation by SIK3 showed decreased sleep. These findings indicate that LKB1-SIK3-HDAC4 constitute a signalling cascade that regulates sleep and wakefulness. We also performed targeted manipulation of SIK3 and HDAC4 in specific neurons and brain regions. This showed that SIK3 signalling in excitatory neurons located in the cerebral cortex and the hypothalamus positively regulates EEG delta power during non-rapid eye movement sleep (NREMS) and NREMS amount, respectively. A subset of transcripts biased towards synaptic functions was commonly regulated in cortical glutamatergic neurons through the expression of a gain-of-function allele of Sik3 and through sleep deprivation. These findings suggest that NREMS quantity and depth are regulated by distinct groups of excitatory neurons through common intracellular signals. This study provides a basis for linking intracellular events and circuit-level mechanisms that control NREMS.

Mice Lacking Cerebellar Cortex and Related Structures Show a Decrease in Slow-Wave Activity With Normal Non-REM Sleep Amount and Sleep Homeostasis.

In addition to the well-known motor control, the cerebellum has recently been implicated in memory, cognition, addiction, and social behavior. Given that the cerebellum contains more neurons than the cerebral cortex and has tight connections to the thalamus and brainstem nuclei, it is possible that the cerebellum also regulates sleep/wakefulness. However, the role of the cerebellum in sleep was unclear, since cerebellar lesion studies inevitably involved massive inflammation in the adjacent brainstem, and sleep changes in lesion studies were not consistent with each other. Here, we examine the role of the cerebellum in sleep and wakefulness using mesencephalon- and rhombomere 1-specific Ptf1a conditional knockout (Ptf1a cKO) mice, which lack the cerebellar cortex and its related structures, and exhibit ataxic gait. Ptf1a cKO mice had similar wake and non-rapid eye movement sleep (NREMS) time as control mice and showed reduced slow wave activity during wakefulness, NREMS and REMS. Ptf1a cKO mice showed a decrease in REMS time during the light phase and had increased NREMS delta power in response to 6 h of sleep deprivation, as did control mice. Ptf1a cKO mice also had similar numbers of sleep spindles and fear memories as control mice. Thus, the cerebellum does not appear to play a major role in sleep-wake control, but may be involved in the generation of slow waves.

Loss of the conserved PKA sites of SIK1 and SIK2 increases sleep need.

Although sleep is one of the most conserved behaviors, the intracellular mechanism regulating sleep/wakefulness remains unknown. We recently identified a protein kinase, SIK3, as a sleep-regulating molecule. Mice that lack a well-conserved protein kinase A (PKA) phosphorylation site, S551, showed longer non-rapid eye movement (NREM) sleep and increased NREMS delta density. S551 of SIK3 is conserved in other members of the SIK family, such as SIK1 (S577) and SIK2 (S587). Here, we examined whether the PKA phosphorylation sites of SIK1 and SIK2 are involved in sleep regulation by generating Sik1S577A and Sik2S587A mice. The homozygous Sik1S577A mice showed a shorter wake time, longer NREMS time, and higher NREMS delta density than the wild-type mice. The heterozygous and homozygous Sik2S587A mice showed increased NREMS delta density. Both the Sik1S577A and Sik2S587A mice exhibited proper homeostatic regulation of sleep need after sleep deprivation. Despite abundant expression of Sik1 in the suprachiasmatic nucleus, the Sik1S577A mice showed normal circadian behavior. Although Sik2 is highly expressed in brown adipose tissue, the male and female Sik2S587A mice that were fed either a chow or high-fat diet showed similar weight gain as the wild-type littermates. These results suggest that PKA-SIK signaling is involved in the regulation of sleep need.

Forward-genetics analysis of sleep in randomly mutagenized mice.

Sleep is conserved from invertebrates to vertebrates, and is tightly regulated in a homeostatic manner. The molecular and cellular mechanisms that determine the amount of rapid eye movement sleep (REMS) and non-REMS (NREMS) remain unknown. Here we identify two dominant mutations that affect sleep and wakefulness by using an electroencephalogram/electromyogram-based screen of randomly mutagenized mice. A splicing mutation in the Sik3 protein kinase gene causes a profound decrease in total wake time, owing to an increase in inherent sleep need. Sleep deprivation affects phosphorylation of regulatory sites on the kinase, suggesting a role for SIK3 in the homeostatic regulation of sleep amount. Sik3 orthologues also regulate sleep in fruitflies and roundworms. A missense, gain-of-function mutation in the sodium leak channel NALCN reduces the total amount and episode duration of REMS, apparently by increasing the excitability of REMS-inhibiting neurons. Our results substantiate the use of a forward-genetics approach for studying sleep behaviours in mice, and demonstrate the role of SIK3 and NALCN in regulating the amount of NREMS and REMS, respectively.

Identification of mutations through dominant screening for obesity using C57BL/6 substrains.

The discovery of leptin substantiated the usefulness of a forward genetic approach in elucidating the molecular network regulating energy metabolism. However, no successful dominant screening for obesity has been reported, which may be due to the influence of quantitative trait loci between the screening and counter strains and the low fertility of obese mice. Here, we performed a dominant screening for obesity using C57BL/6 substrains, C57BL/6J and C57BL/6N, with the routine use of in vitro fertilization. The screening of more than 5000 mutagenized mice established two obese pedigrees in which single nucleotide substitutions in Mc4r and Sim1 genes were identified through whole-exome sequencing. The mutation in the Mc4r gene produces a premature stop codon, and the mutant SIM1 protein lacks transcriptional activity, showing that the haploinsufficiency of SIM1 and MC4R results in obesity. We further examined the hypothalamic neuropeptide expressions in the mutant pedigrees and mice with diet-induced obesity, which showed that each obesity mouse model has distinct neuropeptide expression profiles. This forward genetic screening scheme is useful and applicable to any research field in which mouse models work.

Cardiopulmonary arrest due to persistent coronary spasm in a young woman. Are we properly diagnosing vasospastic angina?

Coronary spasm is a risk factor for acute myocardial infarction and sudden cardiac death. This is a case of a young female patient with cardiopulmonary arrest induced by coronary spasm on arrival at our hospital. There has been no case that prolonged spontaneous attack was confirmed in multi-vessels. This case demonstrates that persistent coronary spasm is lethal and an important cause of cardiopulmonary arrest even in young people. It is extremely important to detect patients with coronary spasm before the ischemic events associated with cardiopulmonary arrest occur.

Upper gastrointestinal bleeding in patients receiving dual antiplatelet therapy after coronary stenting.

OBJECTIVE: We investigated the risk of upper gastrointestinal (UGI) bleeding and the protective effect of concomitant anti-secretory drugs during dual antiplatelet therapy administered following implantation of drug-eluting stents (DES) for coronary heart disease. Because proton pump inhibitors (PPIs) are reported to decrease the platelet inhibitory effects of clopidogrel, we also assessed cardiovascular outcomes in patients taking thienopyridine derivatives with or without anti-secretory drug. METHODS: We retrospectively analyzed 243 patients, who underwent DES implantation between January 2006 and December 2007 and were receiving dual anti-platelet therapy post-surgery. The main outcome measurement was the presence of UGI bleeding. Cardiovascular outcomes were assessed by follow-up coronary angiography (CAG) findings. Data were collected from medical records. RESULTS: Eight cases of UGI bleeding were observed during the follow-up period, none of whom were taking anti-secretory drugs. Among the 243 cases, 108 cases were taking anti-secretory drugs: a PPI (67 cases), and an H2 receptor antagonist (41 cases). No UGI bleeding was observed among patients who were taking concomitant anti-secretory drugs. The 1- and 2-year cumulative incidences of UGI bleeding among patients who were not taking anti-secretory drugs were 4.5% and 9.2%, respectively. When CAG findings were compared between patients not taking any anti-secretory drug, taking PPI, or taking H2RA, significantly more stenotic lesions of the coronary artery were observed in the PPI-treatment group. CONCLUSION: Concomitant use of an anti-secretory agent was associated with a reduced risk of UGI bleeding. Use of PPI may be associated with an attenuation of the effect of dual antiplatelet therapy.

Provoked coronary spasm predicts adverse outcome in patients with acute myocardial infarction: a novel predictor of prognosis after acute myocardial infarction.

OBJECTIVES: This study was conducted to examine the relationship between provoked coronary spasm and clinical course in patients with acute myocardial infarction (AMI). BACKGROUND: Coronary spasm has a pathogenetic role in the occurrence of AMI and progressive atherosclerosis. There is no report that focused on the prognostic significance of provoked coronary spasm in AMI patients. METHODS: Our group investigated 240 consecutive patients who underwent spasm-provocation tests using acetylcholine after AMI. Coronary spasm was defined as a transient total or subtotal occlusion of the luminal diameter. The patients were divided into 2 groups (positive group: n = 174, negative group: n = 66). RESULTS: The clinical courses of the 2 groups were compared at long-term follow up (mean, 43 months). Major adverse cardiac events (death, acute coronary syndrome, or revascularization) occurred in 82 patients (47.1%) in the positive group and 18 patients (27.3%) in the negative group (p = 0.0055). The frequency of major adverse cardiac event-free survival was significantly lower in the positive group than in the negative group (p = 0.0018). Provoked coronary spasm was a significant independent predictor of poor prognosis. CONCLUSIONS: Provoked coronary spasm predicts adverse outcome in AMI patients.

Time-frequency analysis of QRS complex with wavelet transform in patients with triple-vessel disease.

OBJECTIVE: Significant Q-wave is sometimes invisible in the patients with triple vessel disease (TVD) even though TVD is a serious coronary heart disease. We offer the preliminary method to analyze the time-frequency profile of QRS in TVD patients. METHODS: Electrocardiograms (ECG) band-pass filtered through 50 to 300Hz were recorded from the persons without heart diseases (Normal group; n=24), the patients with single vessel disease (SVD group; n=12) and TVD (TVD group; n=12) and saved into PC. For each subject, the time-frequency powers of ECG (lead II) were calculated by the continuous wavelet transform (CWT) with 40 frequency bands. They were integrated during QRS to get the integrated time-frequency powers (ITFP) for all the frequency bands. RESULTS: The ITFP at lower frequency range (90 Hz or less) were smaller in SVD and TVD groups, compared with normal group. The ITFP at higher frequency range (120 to 350 Hz) were larger in patients with recurrent heart failure due to TVD. The increase in ITFP at wider frequency bands was seen with and without significant Q waves. CONCLUSION: The present results that the increase in higher frequency power in TVD with recurrent heart failure may indicate the severity of myocardial damage, regardless of significant Q-wave.