Kenta Sumiyama

Interleukin-33 (IL-33), an IL-1 family cytokine, is released during cell damage. However, the mechanism underlying IL-33 release is not fully understood. Here, we generate IL-33-mCherry-expressing mouse cell lines to monitor IL-33 release at single cell resolution. During necroptosis, SYTOX uptake that represents loss of membrane integrity, and IL-33-mCherry release occur simultaneously, indicating that Mlkl-mediated damage causes immediate membrane rupture. In contrast, some apoptotic and pyroptotic cells exhibit delayed IL-33-mCherry release relative to SYTOX uptake, whereas in other cells, both events occur simultaneously. IL-33-mCherry release is only modestly affected by loss of Gsdmd or Gsdme but is markedly reduced in Ninjurin-1 (Ninj1) knockout cells. These findings indicate that Gasdermins act upstream, whereas Ninj1-mediated membrane rupture represents the principal mechanism of IL-33 release. The delay in IL-33-mCherry release in some cells implies that the activation of Ninj1 occurs with variable timing, thus contributing to cell-to-cell heterogeneity.

Scleraxis (Scx) is a basic helix-loop-helix transcription factor predominantly expressed in tendons, ligaments, and their attachment sites, where it regulates maturation. Here, we report a novel Scx enhancer that drives tissue-specific expression. In transgenic reporter mice, a 5.3 kb downstream Scx enhancer (dSE) conferred robust, stable, and faithful reporter activity reproducing the endogenous Scx expression pattern. Within the dSE, we identified a 343 bp Scx enhancer (CSE), conserved from lobe-finned fishes to tetrapods, that recapitulates Scx expression in limbs. In developing limbs, CSE-deficient mice exhibited a marked reduction of endogenous Scx expression and a complete loss of the deltoid tuberosity (DT) supporting forelimb leverage, accompanied by attenuated BMP and TGFβ signaling, as in Scx-deficient mice. Precise enhancer activation within a particular developmental window was indispensable for DT formation. In contrast, tendon/ligament maturation retained prolonged plasticity, with Scx expression partially recovering later, likely through additional elements within the dSE. These findings uncover an essential role of the CSE in DT morphogenesis and reveal distinct temporal enhancer requirements for DT versus tendon/ligament formation, with additional enhancer dependence confined to tendons/ligaments.

Hematopoietic stem cells (HSCs) survive many types of cellular stress but often lose their regenerative and lymphopoietic capacities as a result. Such functional decline also occurs with age, and dysfunctional HSCs with impaired mitochondria accumulate during aging. However, the molecular link between HSC stress response and age-related functional decline remains poorly understood. Here we show that multiple stress responses converge on the RIPK3-MLKL axis to induce age-related changes in HSCs. The necroptosis effector MLKL is readily activated by inflammation and replication stress and accumulates in HSC mitochondria. Consequently, activated MLKL does not cause cell death but impairs HSC self-renewal and lymphoid differentiation. Such MLKL-mediated functional decline also occurs in HSCs during organismal aging, with activated MLKL primarily mediating age-related mitochondrial damage and reduced glycolytic flux. Collectively, our results establish the RIPK3-MLKL axis as a key mediator of HSC aging and identify a necroptosis-independent role of MLKL in mitochondrial damage.

A reduction in the number of neurons and an increased susceptibility to apoptosis are one of the characteristic features observed in Down syndrome (DS). However, the multifaceted actions of chromosomal aneuploidy hinder the elucidation of the underlying mechanism. Here, using neurons and astrocytes differentiated from patient-derived induced pluripotent stem cells (iPSCs), we aimed to clarify the neuropathology of DS by focusing on aneuploidy-associated stress and the dosage effects of specific genes. Human chromosomal trisomies 13, 18, and 21 exert a stress response on neurons, resulting in the accumulation of protein aggregates. In addition, DYRK1A overdosage in trisomy 21 astrocytes causes intrinsic activation of the NLRP3 inflammasome, which leads to the release of inflammatory cytokines. These dual actions reciprocally interact and enhance neuronal apoptosis in trisomy 21. Notably, correction of DYRK1A copy number successfully rescued apoptotic neural death in combination with a chemical chaperone treatment. Our study provides insights into the neuropathological mechanism of DS and its potential therapeutic strategy.

The metabolic, immune, and endocrine systems show profound seasonal changes in animals, including humans. In addition, morbidity from cardiovascular and psychiatric diseases is more severe and mortality rate is higher in winter. However, their molecular mechanisms remain unknown. Here we report the seasonal transcriptome of 80 tissues collected over 1 year from male and female rhesus macaques kept in a semi-natural outdoor environment. We find seasonal changes in plasma metabolites and hormones. Transcriptome analysis identifies sex differences in seasonally oscillating genes (SOGs) in all tissues studied, and we generate the web database ‘Non-Human Primate Seasonal Transcriptome Atlas (NHPSTA).’ Transcriptional regulatory network analysis, siRNA knockdown, and mutant mouse analyses reveal regulation of SOGs by GA-binding protein (GABP). We also demonstrate seasonal oscillations in the expression of disease risk factor genes and drug interacting genes. NHPSTA provides a molecular resource for seasonally regulated physiology and targets for therapeutic interventions for seasonally regulated diseases.

The phosphorylation of synaptic proteins is a significant biochemical reaction that controls the sleep–wake cycle in mammals^1,2,3. Protein phosphorylation in vivo is reversibly regulated by kinases and phosphatases. In this study, we investigate a pair of kinases and phosphatases that reciprocally regulate sleep duration. First, we perform a comprehensive screen of protein kinase A (PKA) and phosphoprotein phosphatase (PPP) family genes by generating 40 gene knockout mouse lines using prenatal and postnatal CRISPR targeting. We identify a regulatory subunit of PKA (Prkar2b), a regulatory subunit of protein phosphatase 1 (PP1; Pppr1r9b) and catalytic and regulatory subunits of calcineurin (also known as PP2B) (Ppp3ca and Ppp3r1) as sleep control genes. Using adeno-associated virus (AAV)-mediated stimulation of PKA and PP1–calcineurin activities, we show that PKA is a wake-promoting kinase, whereas PP1 and calcineurin function as sleep-promoting phosphatases. The importance of these phosphatases in sleep regulation is supported by the marked changes in sleep duration associated with their increased and decreased activities, ranging from approximately 17.3 h per day (PP1 expression) to 4.3 h per day (postnatal CRISPR targeting of calcineurin). Localization signals to the excitatory post-synapse are necessary for these phosphatases to exert their sleep-promoting effects. Furthermore, the wake-promoting effect of PKA localized to the excitatory post-synapse negated the sleep-promoting effect of PP1–calcineurin. These findings indicate that PKA and PP1–calcineurin have competing functions in sleep regulation at excitatory post-synapses.

Summary: Epidermal growth factor receptor ligands (EGFRLs) consist of seven proteins. In stark contrast to the amassed knowledge concerning the epidermal growth factor receptors themselves, the extracellular dynamics of individual EGFRLs remain elusive. Here, employing fluorescent probes and a tool for triggering ectodomain shedding of EGFRLs, we show that EREG, a low-affinity EGFRL, exhibits the most rapid and efficient activation of EGFR in confluent epithelial cells and mouse epidermis. In Madin-Darby canine kidney (MDCK) renal epithelial cells, EGFR- and ERK-activation waves propagate during collective cell migration in an ADAM17 sheddase- and EGFRL-dependent manner. Upon induction of EGFRL shedding, radial ERK activation waves were observed in the surrounding receiver cells. Notably, the low-affinity ligands EREG and AREG mediated faster and broader ERK waves than the high-affinity ligands. The integrity of tight/adherens junctions was essential for the propagation of ERK activation, implying that the tight intercellular spaces prefer the low-affinity EGFRL to the high-affinity ligands for efficient signal transmission. To validate this observation in vivo , we generated EREG-deficient mice expressing the ERK biosensor and found that ERK wave propagation and cell migration were impaired during skin wound repair. In conclusion, we have quantitatively demonstrated the distinctions among EGFRLs in shedding, diffusion, and target cell activation in physiological contexts. Our findings underscore the pivotal role of low-affinity EGFRLs in rapid intercellular signal transmission.

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.

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. We show here that Ca ²⁺/calmodulin-dependent protein kinase II (CaMKII)-dependent activity control of parvalbumin (PV)-expressing cortical neurons is involved in sleep homeostasis regulation. 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. 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.