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.

Bioorthogonal chemical labeling of endogenous neurotransmitter receptors in living mouse brains.

Neurotransmitter receptors are essential components of synapses for communication between neurons in the brain. Because the spatiotemporal expression profiles and dynamics of neurotransmitter receptors involved in many functions are delicately governed in the brain, in vivo research tools with high spatiotemporal resolution for receptors in intact brains are highly desirable. Covalent labeling by chemical reaction (chemical labeling) of proteins without genetic manipulation is now a powerful method for analyzing receptors in vitro. However, selective target receptor labeling in the brain has not yet been achieved. This study shows that ligand-directed alkoxyacylimidazole (LDAI) chemistry can be used to selectively tether synthetic probes to target endogenous receptors in living mouse brains. The reactive LDAI reagents with negative charges were found to diffuse well over the whole brain and could selectively label target endogenous receptors, including AMPAR, NMDAR, mGlu1, and GABAAR. This simple and robust labeling protocol was then used for various applications: three-dimensional spatial mapping of endogenous receptors in the brains of healthy and disease-model mice; multi-color receptor imaging; and pulse-chase analysis of the receptor dynamics in postnatal mouse brains. Here, results demonstrated that bioorthogonal receptor modification in living animal brains may provide innovative molecular tools that contribute to the in-depth understanding of complicated brain functions.

Circadian ribosome profiling reveals a role for the Period2 upstream open reading frame in sleep.

Many mammalian proteins have circadian cycles of production and degradation, and many of these rhythms are altered posttranscriptionally. We used ribosome profiling to examine posttranscriptional control of circadian rhythms by quantifying RNA translation in the liver over a 24-h period from circadian-entrained mice transferred to constant darkness conditions and by comparing ribosome binding levels to protein levels for 16 circadian proteins. We observed large differences in ribosome binding levels compared to protein levels, and we observed delays between peak ribosome binding and peak protein abundance. We found extensive binding of ribosomes to upstream open reading frames (uORFs) in circadian mRNAs, including the core clock gene Period2 (Per2). An increase in the number of uORFs in the 5'UTR was associated with a decrease in ribosome binding in the main coding sequence and a reduction in expression of synthetic reporter constructs. Mutation of the Per2 uORF increased luciferase and fluorescence reporter expression in 3T3 cells and increased luciferase expression in PER2:LUC MEF cells. Mutation of the Per2 uORF in mice increased Per2 mRNA expression, enhanced ribosome binding on Per2, and reduced total sleep time compared to that in wild-type mice. These results suggest that uORFs affect mRNA posttranscriptionally, which can impact physiological rhythms and sleep.

Elucidation of the mechanism of amyloid A and transthyretin formation using mass spectrometry-based absolute quantification.

Amyloidosis is triggered by the truncation of amyloid precursor proteins, causing organ damages. While previous studies found the truncation of amyloid A (AA) and amyloid transthyretin (ATTR) occurs in C- and N-terminal, respectively, the detailed mechanism of the fibril formation remains unclear. Liquid chromatography mass spectrometry is usually applied for a qualitative purpose, and thus quantification of tryptic peptide residue is difficult. We therefore employed a mass spectrometry-based quantification by isotope-labeled cell-free (MS-QBIC) to analyze the truncation processes in amyloid fibrillogenesis of AA and ATTR using the formalin-fixed paraffin-embedded tissues of autopsy cases. In this study, the process of transthyretin from an 'early fibril state' consisting of full-length ATTR to a 'mature ATTR amyloid fibril' with a truncated low-amyloidogenic segment has been mathematically revealed. The amount of full-length ATTR was nine times higher than in mature fibers. Large cohort studies using MS-QBIC may shed light on the clinical significance of amyloid fibrils.

A growing understanding of the role of muscarinic receptors in the molecular pathology and treatment of schizophrenia.

Pre-clinical models, postmortem and neuroimaging studies all support a role for muscarinic receptors in the molecular pathology of schizophrenia. From these data it was proposed that activation of the muscarinic M1 and/or M4 receptor would reduce the severity of the symptoms of schizophrenia. This hypothesis is now supported by results from two clinical trials which indicate that activating central muscarinic M1 and M4 receptors can reduce the severity of positive, negative and cognitive symptoms of the disorder. This review will provide an update on a growing body of evidence that argues the muscarinic M1 and M4 receptors have critical roles in CNS functions that are dysregulated by the pathophysiology of schizophrenia. This realization has been made possible, in part, by the growing ability to visualize and quantify muscarinic M1 and M4 receptors in the human CNS using molecular neuroimaging. We will discuss how these advances have provided evidence to support the notion that there is a sub-group of patients within the syndrome of schizophrenia that have a unique molecular pathology driven by a marked loss of muscarinic M1 receptors. This review is timely, as drugs targeting muscarinic receptors approach clinical use for the treatment of schizophrenia and here we outline the background biology that supported development of such drugs to treat the disorder.

CCPG1 recognizes endoplasmic reticulum luminal proteins for selective ER-phagy.

The endoplasmic reticulum (ER) is a major cell compartment where protein synthesis, folding, and posttranslational modifications occur with assistance from a wide variety of chaperones and enzymes. Quality control systems selectively eliminate abnormal proteins that accumulate inside the ER due to cellular stresses. ER-phagy, that is, selective autophagy of the ER, is a mechanism that maintains or reestablishes cellular and ER-specific homeostasis through removal of abnormal proteins. However, how ER luminal proteins are recognized by the ER-phagy machinery remains unclear. Here, we applied the aggregation-prone protein, six-repeated islet amyloid polypeptide (6xIAPP), as a model ER-phagy substrate and found that cell cycle progression 1 (CCPG1), which is an ER-phagy receptor, efficiently mediates its degradation via ER-phagy. We also identified prolyl 3-hydroxylase family member 4 (P3H4) as an endogenous cargo of CCPG1-dependent ER-phagy. The ER luminal region of CCPG1 contains several highly conserved regions that we refer to as cargo-interacting regions (CIRs); these interact directly with specific luminal cargos for ER-phagy. Notably, 6xIAPP and P3H4 interact directly with different CIRs. These findings indicate that CCPG1 is a bispecific ER-phagy receptor for ER luminal proteins and the autophagosomal membrane that contributes to the efficient removal of aberrant ER-resident proteins through ER-phagy.

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.

An analysis modality for vascular structures combining tissue-clearing technology and topological data analysis.

The blood and lymphatic vasculature networks are not yet fully understood even in mouse because of the inherent limitations of imaging systems and quantification methods. This study aims to evaluate the usefulness of the tissue-clearing technology for visualizing blood and lymphatic vessels in adult mouse. Clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC) enables us to capture the high-resolution 3D images of organ- or area-specific vascular structures. To evaluate these 3D structural images, signals are first classified from the original captured images by machine learning at pixel base. Then, these classified target signals are subjected to topological data analysis and non-homogeneous Poisson process model to extract geometric features. Consequently, the structural difference of vasculatures is successfully evaluated in mouse disease models. In conclusion, this study demonstrates the utility of CUBIC for analysis of vascular structures and presents its feasibility as an analysis modality in combination with 3D images and mathematical frameworks.

Ubiquitination of phosphatidylethanolamine in organellar membranes.

The covalent conjugation of ubiquitin family proteins is a widespread post-translational protein modification. In the ubiquitin family, the ATG8 subfamily is exceptional because it is conjugated mainly to phospholipids. However, it remains unknown whether other ubiquitin family proteins are also conjugated to phospholipids. Here, we report that ubiquitin is conjugated to phospholipids, mainly phosphatidylethanolamine (PE), in yeast and mammalian cells. Ubiquitinated PE (Ub-PE) accumulates at endosomes and the vacuole (or lysosomes), and its level increases during starvation. Ub-PE is also found in baculoviruses. In yeast, PE ubiquitination is catalyzed by the canonical ubiquitin system enzymes Uba1 (E1), Ubc4/5 (E2), and Tul1 (E3) and is reversed by Doa4. Liposomes containing Ub-PE recruit the ESCRT components Vps27-Hse1 and Vps23 in vitro. Ubiquitin-like NEDD8 and ISG15 are also conjugated to phospholipids. These findings suggest that the conjugation to membrane phospholipids is not specific to ATG8 but is a general feature of the ubiquitin family.

A Novel Three-Dimensional Imaging System Based on Polysaccharide Staining for Accurate Histopathological Diagnosis of Inflammatory Bowel Diseases.

BACKGROUND & AIMS: Tissue-clearing and three-dimensional (3D) imaging techniques aid clinical histopathological evaluation; however, further methodological developments are required before use in clinical practice. METHODS: We sought to develop a novel fluorescence staining method based on the classical periodic acid-Schiff stain. We further attempted to develop a 3D imaging system based on this staining method and evaluated whether the system can be used for quantitative 3D pathological evaluation and deep learning-based automatic diagnosis of inflammatory bowel diseases. RESULTS: We successfully developed a novel periodic acid-FAM hydrazide (PAFhy) staining method for 3D imaging when combined with a tissue-clearing technique (PAFhy-3D). This strategy enabled clear and detailed imaging of the 3D architectures of crypts in human colorectal mucosa. PAFhy-3D imaging also revealed abnormal architectural changes in crypts in ulcerative colitis tissues and identified the distributions of neutrophils in cryptitis and crypt abscesses. PAFhy-3D revealed novel pathological findings including spiral staircase-like crypts specific to inflammatory bowel diseases. Quantitative analysis of crypts based on 3D morphologic changes enabled differential diagnosis of ulcerative colitis, Crohn's disease, and non-inflammatory bowel disease; such discrimination could not be achieved by pathologists. Furthermore, a deep learning-based system using PAFhy-3D images was used to distinguish these diseases The accuracies were excellent (macro-average area under the curve = 0.94; F1 scores = 0.875 for ulcerative colitis, 0.717 for Crohn's disease, and 0.819 for non-inflammatory bowel disease). CONCLUSIONS: PAFhy staining and PAFhy-3D imaging are promising approaches for next-generation experimental and clinical histopathology.

A hybrid open-top light-sheet microscope for versatile multi-scale imaging of cleared tissues.

Light-sheet microscopy has emerged as the preferred means for high-throughput volumetric imaging of cleared tissues. However, there is a need for a flexible system that can address imaging applications with varied requirements in terms of resolution, sample size, tissue-clearing protocol, and transparent sample-holder material. Here, we present a 'hybrid' system that combines a unique non-orthogonal dual-objective and conventional (orthogonal) open-top light-sheet (OTLS) architecture for versatile multi-scale volumetric imaging. We demonstrate efficient screening and targeted sub-micrometer imaging of sparse axons within an intact, cleared mouse brain. The same system enables high-throughput automated imaging of multiple specimens, as spotlighted by a quantitative multi-scale analysis of brain metastases. Compared with existing academic and commercial light-sheet microscopy systems, our hybrid OTLS system provides a unique combination of versatility and performance necessary to satisfy the diverse requirements of a growing number of cleared-tissue imaging applications.

Phosphorylation by casein kinase 2 enhances the interaction between ER-phagy receptor TEX264 and ATG8 proteins.

Selective autophagy cargos are recruited to autophagosomes primarily by interacting with autophagosomal ATG8 family proteins via the LC3-interacting region (LIR). The upstream sequence of most LIRs contains negatively charged residues such as Asp, Glu, and phosphorylated Ser and Thr. However, the significance of LIR phosphorylation (compared with having acidic amino acids) and the structural basis of phosphorylated LIR-ATG8 binding are not entirely understood. Here, we show that the serine residues upstream of the core LIR of the endoplasmic reticulum (ER)-phagy receptor TEX264 are phosphorylated by casein kinase 2, which is critical for its interaction with ATG8s, autophagosomal localization, and ER-phagy. Structural analysis shows that phosphorylation of these serine residues increases binding affinity by producing multiple hydrogen bonds with ATG8s that cannot be mimicked by acidic residues. This binding mode is different from those of other ER-phagy receptors that utilize a downstream helix, which is absent from TEX264, to increase affinity. These results suggest that phosphorylation of the LIR is critically important for strong LIR-ATG8 interactions, even in the absence of auxiliary interactions.

A design principle of spindle oscillations in mammalian sleep.

Neural oscillations are mainly regulated by molecular mechanisms and network connectivity of neurons. Large-scale simulations of neuronal networks have driven the population-level understanding of neural oscillations. However, cell-intrinsic mechanisms, especially a design principle, of neural oscillations remain largely elusive. Herein, we developed a minimal, Hodgkin-Huxley-type model of groups of neurons to investigate molecular mechanisms underlying spindle oscillation, which is synchronized oscillatory activity predominantly observed during mammalian sleep. We discovered that slowly inactivating potassium channels played an essential role in characterizing the firing pattern. The detailed analysis of the minimal model revealed that leak sodium and potassium channels, which controlled passive properties of the fast variable (i.e., membrane potential), competitively regulated the base value and time constant of the slow variable (i.e., cytosolic calcium concentration). Consequently, we propose a theoretical design principle of spindle oscillations that may explain intracellular mechanisms behind the flexible control over oscillation density and calcium setpoint.

The 103,200-arm acceleration dataset in the UK Biobank revealed a landscape of human sleep phenotypes.

SignificanceHuman sleep phenotypes are diversified by genetic and environmental factors, and a quantitative classification of sleep phenotypes would lead to the advancement of biomedical mechanisms underlying human sleep diversity. To achieve that, a pipeline of data analysis, including a state-of-the-art sleep/wake classification algorithm, the uniform manifold approximation and projection (UMAP) dimension reduction method, and the density-based spatial clustering of applications with noise (DBSCAN) clustering method, was applied to the 100,000-arm acceleration dataset. This revealed 16 clusters, including seven different insomnia-like phenotypes. This kind of quantitative pipeline of sleep analysis is expected to promote data-based diagnosis of sleep disorders and psychiatric disorders that tend to be complicated by sleep disorders.

A jerk-based algorithm ACCEL for the accurate classification of sleep-wake states from arm acceleration.

Arm acceleration data have been used to measure sleep-wake rhythmicity. Although several methods have been developed for the accurate classification of sleep-wake episodes, a method with both high sensitivity and specificity has not been fully established. In this study, we developed an algorithm, named ACceleration-based Classification and Estimation of Long-term sleep-wake cycles (ACCEL) that classifies sleep and wake episodes using only raw accelerometer data, without relying on device-specific functions. The algorithm uses a derivative of triaxial acceleration (jerk), which can reduce individual differences in the variability of acceleration data. Applying a machine learning algorithm to the jerk data achieved sleep-wake classification with a high sensitivity (>90%) and specificity (>80%). A jerk-based analysis also succeeded in recording periodic activities consistent with pulse waves. Therefore, the ACCEL algorithm will be a useful method for large-scale sleep measurement using simple accelerometers in real-world settings.

High-throughput Genetically Modified Animal Experiments Achieved by Next-generation Mammalian Genetics.

Animal models are essential tools for modern scientists to conduct biological experiments and investigate their hypotheses in vivo. However, for the past decade, raising the throughput of such animal experiments has been a great challenge. Conventionally, in vivo high-throughput assay was achieved through large-scale mutagen-driven forward genetic screening, which took years to find causal genes. In contrast, reverse genetics accelerated the causal gene identification process, but its throughput was also limited by 2 barriers, that is, the genome modification step and the time-consuming crossing step. Defined as genetics without crossing, next-generation genetics is able to produce gene-modified animals that can be analyzed at the founder generation (F0). This method is or can be accomplished through recent technological advances in gene editing and virus-based efficient gene modifications. Notably, next-generation genetics has accelerated the process of cross-species studies, and it will be a useful technique during animal experiments as it can provide genetic perturbation at an individual level without crossing. In this review, we begin by introducing the history of animal-based high-throughput analysis, with a specific focus on chronobiology. We then describe ways that gene modification efficiency during animal experiments was enhanced and why crossing remained a barrier to reaching higher efficiency. Moreover, we mention the Triple CRISPR as a critical technique for achieving next-generation genetics. Finally, we discuss the potential applications and limitations of next-generation mammalian genetics.

The circadian clock ticks in organoids.

Organoids are self-organizing in vitro 3D cultures that are histologically similar to a variety of human organs. A recent study by Rosselot et al (2021) shows that mature intestinal organoids possess species-specific circadian clocks similar to their respective in vivo context, suggesting organoids as promising platforms to study circadian medicine.

The role of calcium and CaMKII in sleep.

Sleep is an evolutionarily conserved phenotype shared by most of the animals on the planet. Prolonged wakefulness will result in increased sleep need or sleep pressure. However, its mechanisms remain elusive. Recent findings indicate that Ca2+ signaling, known to control diverse physiological functions, also regulates sleep. This review intends to summarize research advances in Ca2+ and Ca2+/calmodulin-dependent protein kinase II (CaMKII) in sleep regulation. Significant changes in sleep phenotype have been observed through calcium-related channels, receptors, and pumps. Mathematical modeling for neuronal firing patterns during NREM sleep suggests that these molecules compose a Ca2+-dependent hyperpolarization mechanism. The intracellular Ca2+ may then trigger sleep induction and maintenance through the activation of CaMKII, one of the sleep-promoting kinases. CaMKII and its multisite phosphorylation status may provide a link between transient calcium dynamics typically observed in neurons and sleep-wake dynamics observed on the long-time scale.