Cortical parvalbumin neurons are responsible for homeostatic sleep rebound through CaMKII activation.

The homeostatic regulation of sleep is characterized by rebound sleep after prolonged wakefulness, but the molecular and cellular mechanisms underlying this regulation are still unknown. In this study, we show that Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent activity control of parvalbumin (PV)-expressing cortical neurons is involved in homeostatic regulation of sleep in male mice. Prolonged wakefulness enhances cortical PV-neuron activity. Chemogenetic suppression or activation of cortical PV neurons inhibits or induces rebound sleep, implying that rebound sleep is dependent on increased activity of cortical PV neurons. Furthermore, we discovered that CaMKII kinase activity boosts the activity of cortical PV neurons, and that kinase activity is important for homeostatic sleep rebound. Here, we propose that CaMKII-dependent PV-neuron activity represents negative feedback inhibition of cortical neural excitability, which serves as the distributive cortical circuits for sleep homeostatic regulation.

Distinct phosphorylation states of mammalian CaMKIIß control the induction and maintenance of sleep.

The reduced sleep duration previously observed in Camk2b knockout mice revealed a role for Ca2+/calmodulin-dependent protein kinase II (CaMKII)ß as a sleep-promoting kinase. However, the underlying mechanism by which CaMKIIß supports sleep regulation is largely unknown. Here, we demonstrate that activation or inhibition of CaMKIIß can increase or decrease sleep duration in mice by almost 2-fold, supporting the role of CaMKIIß as a core sleep regulator in mammals. Importantly, we show that this sleep regulation depends on the kinase activity of CaMKIIß. A CaMKIIß mutant mimicking the constitutive-active (auto)phosphorylation state promotes the transition from awake state to sleep state, while mutants mimicking subsequent multisite (auto)phosphorylation states suppress the transition from sleep state to awake state. These results suggest that the phosphorylation states of CaMKIIß differently control sleep induction and maintenance processes, leading us to propose a "phosphorylation hypothesis of sleep" for the molecular control of sleep in mammals.

Rapid and easy-to-use ES cell manipulation device with a small groove near culturing wells.

OBJECTIVE: Production of genetically modified mice including Knock-out (KO) or Knock-in (KI) mice is necessary for organism-level phenotype analysis. Embryonic stem cell (ESC)-based technologies can produce many genetically modified mice with less time without crossing. However, a complicated manual operation is required to increase the number of ESC colonies. Here, the objective of this study was to design and demonstrate a new device to easily find colonies and carry them to microwells. RESULTS: We developed a polydimethylsiloxane-based device for easy manipulation and isolation of ESC colonies. By introducing ESC colonies into the groove placed near culturing microwells, users can easily find, pick up and carry ESC colonies to microwells. By hydrophilic treatment using bovine serum albumin, 2-µL droplets including colonies reached the microwell bottom. Operation time using this device was shortened for both beginners (2.3-fold) and experts (1.5-fold) compared to the conventional colony picking operation. Isolated ESC colonies were confirmed to have maintained pluripotency. This device is expected to promote research by shortening the isolation procedure for ESC colonies or other large cells (e.g. eggs or embryos) and shortening training time for beginners as a simple sorter.

Fast and slow Ca2+-dependent hyperpolarization mechanisms connect membrane potential and sleep homeostasis.

Several lines of evidence indicate that the sleep-wake state of cortical neurons is regulated not only through neuronal projections from the lower brain, but also through the cortical neurons' intrinsic ability to initiate a slow firing pattern related to the slow-wave oscillation observed in electroencephalography of the sleeping brain. Theoretical modeling and experiments with genetic and pharmacological perturbation suggest that ion channels and kinases acting downstream of calcium signaling regulate the cortical-membrane potential and sleep duration. In this review, we introduce possible Ca2+-dependent hyperpolarization mechanisms in cortical neurons, in which Ca2+ signaling associated with neuronal excitation evokes kinase cascades, and the activated kinases modify ion channels or pumps to regulate the cortical sleep/wake firing mode.

Involvement of Ca(2+)-Dependent Hyperpolarization in Sleep Duration in Mammals.

The detailed molecular mechanisms underlying the regulation of sleep duration in mammals are still elusive. To address this challenge, we constructed a simple computational model, which recapitulates the electrophysiological characteristics of the slow-wave sleep and awake states. Comprehensive bifurcation analysis predicted that a Ca(2+)-dependent hyperpolarization pathway may play a role in slow-wave sleep and hence in the regulation of sleep duration. To experimentally validate the prediction, we generate and analyze 21 KO mice. Here we found that impaired Ca(2+)-dependent K(+) channels (Kcnn2 and Kcnn3), voltage-gated Ca(2+) channels (Cacna1g and Cacna1h), or Ca(2+)/calmodulin-dependent kinases (Camk2a and Camk2b) decrease sleep duration, while impaired plasma membrane Ca(2+) ATPase (Atp2b3) increases sleep duration. Pharmacological intervention and whole-brain imaging validated that impaired NMDA receptors reduce sleep duration and directly increase the excitability of cells. Based on these results, we propose a hypothesis that a Ca(2+)-dependent hyperpolarization pathway underlies the regulation of sleep duration in mammals.

Mammalian Reverse Genetics without Crossing Reveals Nr3a as a Short-Sleeper Gene.

The identification of molecular networks at the system level in mammals is accelerated by next-generation mammalian genetics without crossing, which requires both the efficient production of whole-body biallelic knockout (KO) mice in a single generation and high-performance phenotype analyses. Here, we show that the triple targeting of a single gene using the CRISPR/Cas9 system achieves almost perfect KO efficiency (96%-100%). In addition, we developed a respiration-based fully automated non-invasive sleep phenotyping system, the Snappy Sleep Stager (SSS), for high-performance (95.3% accuracy) sleep/wake staging. Using the triple-target CRISPR and SSS in tandem, we reliably obtained sleep/wake phenotypes, even in double-KO mice. By using this system to comprehensively analyze all of the N-methyl-D-aspartate (NMDA) receptor family members, we found Nr3a as a short-sleeper gene, which is verified by an independent set of triple-target CRISPR. These results demonstrate the application of mammalian reverse genetics without crossing to organism-level systems biology in sleep research.

Structure-function analysis of the EF-hand protein centrin-2 for its intracellular localization and nucleotide excision repair.

Centrin-2 is an evolutionarily conserved, calmodulin-related protein, which is involved in multiple cellular functions including centrosome regulation and nucleotide excision repair (NER) of DNA. Particularly to exert the latter function, complex formation with the XPC protein, the pivotal NER damage recognition factor, is crucial. Here, we show that the C-terminal half of centrin-2, containing two calcium-binding EF-hand motifs, is necessary and sufficient for both its localization to the centrosome and interaction with XPC. In XPC-deficient cells, nuclear localization of overexpressed centrin-2 largely depends on co-overexpression of XPC, and mutational analyses of the C-terminal domain suggest that XPC and the major binding partner in the centrosome share a common binding surface on the centrin-2 molecule. On the other hand, the N-terminal domain of centrin-2 also contains two EF-hand motifs but shows only low-binding affinity for calcium ions. Although the N-terminal domain is dispensable for enhancement of the DNA damage recognition activity of XPC, it contributes to augmenting rather weak physical interaction between XPC and XPA, another key factor involved in NER. These results suggest that centrin-2 may have evolved to bridge two protein factors, one with high affinity and the other with low affinity, thereby allowing delicate regulation of various biological processes.

Essential role of the adhesion receptor LFA-1 for T cell-dependent fulminant hepatitis.

Viral hepatitis affects more than 2 billion people worldwide. In particular, no effective treatment exists to abrogate death and liver damage in fulminant hepatitis. Activation of T cells is an initial and critical event in the pathogenesis of liver damage in autoimmune and viral hepatitis. The precise molecular mechanisms that induce T cell-mediated hepatocyte injury remain largely unclear. In mice, T cell-dependent hepatitis and acute liver damage can be modeled using ConA. In this study, we examined the role of the adhesion receptor LFA-1 in ConA-induced acute hepatic damage using LFA-1(-/-) (CD11a) mice. Massive liver cell apoptosis and metabolic liver damage were observed in LFA-1(+/+) mice following ConA injection. By contrast, LFA-1(-/-) mice were completely resistant to ConA-induced hepatitis and none of the LFA-1(-/-) mice showed any hepatic damage. Whereas activated hepatic T cells remained in the liver in LFA-1(+/+) mice, activated T cells were rapidly cleared from the livers of LFA-1(-/-) mice. Mechanistically, T cells from LFA-1(-/-) mice showed markedly reduced cytotoxicity toward liver cells as a result of impaired, activation-dependent adhesion. Importantly, adoptive transfer of hepatic T cells from LFA-1(+/+) mice, but not from LFA-1(-/-) mice, sensitized LFA-1(-/-) mice to ConA-induced hepatitis. Thus, LFA-1 expression on T cells is necessary and sufficient for T cell-mediated liver damage in vivo. These results provide the first genetic evidence on an adhesion receptor, LFA-1, that has a crucial role in fulminant hepatitis. These genetic data identify LFA-1 as a potential key target for the treatment of T cell-mediated hepatitis and the prevention of liver damage.