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.

Efficient induction of proximity-dependent labelling by biotin feeding in BMAL1-BioID knock-in mice.

Proximity-dependent biotin identification (BioID) is a useful method to identify unknown protein-protein interactions. Few reports have described genetically engineered knock-in mouse models for in vivo BioID. Thus, little is known about the proper method for biotin administration and which tissues are applicable. Here, we established a BioID knock-in mouse model of Brain and Muscle ARNT-Like 1 (BMAL1) and the BirA biotin ligase with R118G mutation (BirA*). The BMAL1-BioID mouse model was used to investigate the effect of biotin diet feeding on protein biotinylation in several tissues. The BMAL1-BirA* fusion protein-retained proper intracellular localization of BMAL1 and binding to CLOCK protein in HEK293T cells. A biotin labelling assay in mouse embryonic fibroblasts revealed the protein biotinylation activity of BMAL1-BirA* expressed in knock-in mouse cells depending on biotin supplementation. Lastly, feeding a 0.5% biotin diet for 7 days induced protein biotinylation in the brain, heart, testis and liver of BMAL1-BioID mice without adverse effects on spermatogenesis. In the kidney, the biotin diet increased biotinylated protein levels in BMAL1-BioID and control mice, suggesting the existence of endogenous biotinylation activity. These results provide valuable information to optimize the in vivo BioID procedure.

Enhancing endogenous adenosine A2A receptor signaling induces slow-wave sleep without affecting body temperature and cardiovascular function.

Insomnia is one of the most common sleep problems with an estimated prevalence of 10%-15% in the general population. Although adenosine A2A receptor (A2AR) agonists strongly induce sleep, their cardiovascular effects preclude their use in treating sleep disorders. Enhancing endogenous A2AR signaling, however, may be an alternative strategy for treating insomnia, because adenosine levels in the brain accumulate during wakefulness. In the present study, we found that 3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzoic acid, denoted A2AR positive allosteric modulator (PAM)-1, enhanced adenosine signaling at the A2AR and induced slow wave sleep (SWS) without affecting body temperature in wild-type male mice after intraperitoneal administration, whereas the SWS-inducing effect of this benzoic acid derivative was abolished in A2AR KO mice. In contrast to the A2AR agonist CGS 21680, the A2AR PAM-1 did not affect blood pressure or heart rate. These findings indicate that enhancing A2AR signaling promotes SWS without cardiovascular effects. Therefore, small molecules that allosterically modulate A2ARs could help people with insomnia to fall asleep.

Nrf2 Improves Leptin and Insulin Resistance Provoked by Hypothalamic Oxidative Stress.

The relationship between loss of hypothalamic function and onset of diabetes mellitus remains elusive. Therefore, we generated a targeted oxidative-stress murine model utilizing conditional knockout (KO) of selenocysteine-tRNA (Trsp) using rat-insulin-promoter-driven-Cre (RIP-Cre). These Trsp-KO (TrspRIPKO) mice exhibit deletion of Trsp in both hypothalamic cells and pancreatic ß cells, leading to increased hypothalamic oxidative stress and severe insulin resistance. Leptin signals are suppressed, and numbers of proopiomelanocortin-positive neurons in the hypothalamus are decreased. In contrast, Trsp-KO mice (TrspIns1KO) expressing Cre specifically in pancreatic ß cells, but not in the hypothalamus, do not display insulin and leptin resistance, demonstrating a critical role of the hypothalamus in the onset of diabetes mellitus. Nrf2 (NF-E2-related factor 2) regulates antioxidant gene expression. Increased Nrf2 signaling suppresses hypothalamic oxidative stress and improves insulin and leptin resistance in TrspRIPKO mice. Thus, Nrf2 harbors the potential to prevent the onset of diabetic mellitus by reducing hypothalamic oxidative damage.

Renoprotective effect of vasopressin v2 receptor antagonist tolvaptan in Dahl rats with end-stage heart failure.

Tolvaptan is a highly selective and orally effective arginine vasopressin V2 receptor antagonist, and is potentially useful for the treatment of heart failure (HF) patients. However, the renoprotective effect of long-term tolvaptan therapy and its underlying mechanisms remain unknown. We evaluated the effects of chronic treatment with tolvaptan on renal dysfunction, podocyte injury, inflammation, oxidative stress, Rho-kinase, epithelial-mesenchymal transition (EMT), and the extracellular signal-regulated protein kinase (ERK1/2) pathway in the renal cortex of Dahl salt-sensitive hypertensive (DS) rats with end-stage severe HF. DS and Dahl salt-resistant rats were fed a high-salt diet at 6 weeks of age. DS rats were treated with vehicle and tolvaptan (0.05% concentration in diet) from the age of 11 to 18 weeks. Vehicle-treated DS rats developed proteinuria, renal dysfunction, glomerulosclerosis, and interstitial fibrosis, which were ameliorated by tolvaptan without changing blood pressure. Decreased expression of nephrin and podocin and increased desmin-positive area in failing rats were restored by tolvaptan. Upregulation of NAD(P)H oxidase p22(phox), p47(phox), and gp91(phox), EMT markers such as transforming growth factor-ß1, vimentin, and fibronectin expression, and Rho-kinase and ERK1/2 phosphorylation in DS rats were significantly suppressed by tolvaptan. Tolvaptan administration resulted in significant inhibition of tumor necrosis factor-α and monocyte chemoattractant protein-1 expression, and nuclear factor-κB phosphorylation. We concluded that long-term tolvaptan therapy may improve renal dysfunction, glomerulosclerosis, podocyte injury, and inflammation associated with oxidative stress, as well as EMT, ERK, and the Rho-kinase pathway in the failing heart of DS rats. Thus, tolvaptan may be a therapeutic strategy for end-stage severe HF.

Regulatory roles for APJ, a seven-transmembrane receptor related to angiotensin-type 1 receptor in blood pressure in vivo.

APJ is a G-protein-coupled receptor with seven transmembrane domains, and its endogenous ligand, apelin, was identified recently. They are highly expressed in the cardiovascular system, suggesting that APJ is important in the regulation of blood pressure. To investigate the physiological functions of APJ, we have generated mice lacking the gene encoding APJ. The base-line blood pressure of APJ-deficient mice is equivalent to that of wild-type mice in the steady state. The administration of apelin transiently decreased the blood pressure of wild-type mice and a hypertensive model animal, a spontaneously hypertensive rat. On the other hand, this hypotensive response to apelin was abolished in APJ-deficient mice. This apelin-induced response was inhibited by pretreatment with a nitric-oxide synthase inhibitor, and apelin-induced phosphorylation of endothelial nitric-oxide synthase in lung endothelial cells from APJ-deficient mice disappeared. In addition, APJ-deficient mice showed an increased vasopressor response to the most potent vasoconstrictor angiotensin II, and the base-line blood pressure of double mutant mice homozygous for both APJ and angiotensin-type 1a receptor was significantly elevated compared with that of angiotensin-type 1a receptor-deficient mice. These results demonstrate that APJ exerts the hypotensive effect in vivo and plays a counterregulatory role against the pressor action of angiotensin II.

Hypothalamic orexin neurons regulate arousal according to energy balance in mice.

Mammals respond to reduced food availability by becoming more wakeful and active, yet the central pathways regulating arousal and instinctual motor programs (such as food seeking) according to homeostatic need are not well understood. We demonstrate that hypothalamic orexin neurons monitor indicators of energy balance and mediate adaptive augmentation of arousal in response to fasting. Activity of isolated orexin neurons is inhibited by glucose and leptin and stimulated by ghrelin. Orexin expression of normal and ob/ob mice correlates negatively with changes in blood glucose, leptin, and food intake. Transgenic mice, in which orexin neurons are ablated, fail to respond to fasting with increased wakefulness and activity. These findings indicate that orexin neurons provide a crucial link between energy balance and arousal.