Biology subjects

Miyata, S.

Publications and source records attributed to Miyata, S..

9 recordsLinked to original sources

bioRxiv2026

Disruption of CaMKII biomolecular condensation induces attention-deficit/hyperactivity disorder-like phenotypes

CaMKII is a multifunctional kinase essential for synaptic plasticity and memory formation. While its canonical role involves enzymatic phosphorylation, recent evidence suggests CaMKII also functions through liquid-liquid phase separation (LLPS) with substrate proteins, including GluN2B-containing NMDA receptors. However, the physiological significance remains unclear. Here, we generated CaMKII subtype knock-in (KI) mice carrying a point mutation (I205K) in the hydrophobic pocket, a key interface required for LLPS. These mice exhibited a complete loss of structural long-term potentiation (sLTP) despite normal spine morphology, marked hyperactivity and profound deficits in aversive memory formation. Atomoxetine, an approved attention-deficit/hyperactivity disorder (ADHD) treatment, ameliorated the hyperactive phenotype. Notably, we identified a patient carrying the I205N variant presenting with ADHD and mild intellectual disability, mirroring the behavioral features observed in I205K KI mice. Additional neurodevelopmental disorder-associated variants within the same hydrophobic pocket similarly disrupted LLPS in vitro. Molecular dynamics simulations revealed these variants destabilize the CaMKII-GluN2B interaction through distinct mechanisms that perturb the dynamic stability of the binding interface. These findings establish CaMKII-mediated phase separation as critical for linking synaptic molecular assembly to cognitive function and provide a unifying molecular basis for synaptic disorganization, hyperactivity, and memory deficits associated with neurodevelopmental disorders. SignificanceCaMKII is essential for synaptic plasticity and memory, yet the contribution of its non-catalytic functions, including liquid-liquid phase separation (LLPS), to brain function remains unclear. Here, we show that disrupting CaMKII LLPS with its synaptic partners impairs synaptic localization, abolishes LTP, and causes profound memory deficits in vivo. A knock-in mouse carrying the I205K mutation showed hyperactivity and impaired learning. This recapitulate the clinical phenotypes of a human patients with the I205N variant, which is associated with ADHD and intellectual disability. Together, these findings establish LLPS as a critical mechanism underlying CaMKII function in the brain and provide a disease-relevant framework for understanding synaptic dysfunction in neurodevelopmental disorders.

neuroscience↗

bioRxiv2025

Neuronal activity induces aggrecan expression to drive perineuronal net formation in cortical neurons

Synaptic plasticity, driven by activity-dependent changes in neuronal connectivity, underlies learning and memory. However, the mechanisms that sustain long-term synaptic stabilization are not well understood. Perineuronal nets (PNNs), which are lattice-like extracellular matrix structures that enwrap neurons, are thought to stabilize synapses. However, the mechanisms regulating their formation remain unclear. Here, we found that neuronal activity induces transcription of aggrecan, a core PNN component, which in turn promotes PNN assembly. In primary cultured mouse cortical neurons, pharmacological stimulation of neuronal activity robustly increased aggrecan expression, whereas activity blockade suppressed it. Among the PNN-related genes examined, aggrecan alone exhibited strong activity-dependent transcriptional regulation. Analysis of published in vivo datasets revealed selective upregulation of aggrecan in parvalbumin-positive interneurons following sensory stimulation. This regulation required calcium influx through voltage-gated calcium channels and was dependent on cAMP response element binding protein (CREB) signaling and chromatin remodeling. These findings reveal a novel molecular link between neuronal activity and PNN formation, providing insights into the mechanisms underlying the long-term stabilization of neural circuits.

neuroscience↗

bioRxiv2024

Development and Optimization of Genetic Manipulation Systems in Group I Clostridium botulinum

Clostridium botulinum causes the disease botulism, and nontoxigenic Clostridium sporogenes is closely related to group I C. botulinum. Despite its pathogenicity, C. botulinum remains poorly characterized. Genetic manipulation is critical for understanding bacterial physiology and disease. We compared the conjugal transformation efficiencies of seven strains, including group I C. botulinum (strains 62A, 7I03-H, Okra, Osaka05, 111) and C. sporogenes (strains JCM 1416T, ATCC 15579), and our results showed that few or no transformants were obtained in certain strains. In the present study, we demonstrate that our optimized protocol increases the efficiency of DNA transfer from E. coli donor cells to recipient strains. In addition, we developed a novel conjugal suicide vector pXMTL that contains xylose-inducible mazF as a counter-selection marker, and can be transferred into Clostridium spp. by conjugation. The allele-coupled exchange (ACE) system using pXMTL provides a rapid method for precise, markerless and scarless genome editing in group I C. botulinum and C. sporogenes. ImportanceGroup I C. botulinum and C. sporogenes exhibit low transformation efficiencies, and few or no transformants are yielded by some strains. In this study, we optimized the conjugation protocol to improve transformation efficiency. In addition, we developed a novel suicide vector pXMTL harboring a xylose-inducible mazF marker, and can be transferred into Clostridium spp. by conjugation. The combination of pXMTL and the optimized conjugation protocol provides a powerful tool for genetic manipulation of group I C. botulinum and C. sporogens.

microbiology↗

bioRxiv2023

CTRP7 as a Molecular Biomarker for Predicting Responsiveness to Pulmonary Vasodilators: Insights from Human and Animal Studies in Pulmonary Arterial Hypertension

BackgroundPulmonary arterial hypertension (PAH) is a life-threatening condition. Although pulmonary vasodilators have shown promise in managing PAH, the overall prognosis improvement is modest, partly because of the absence of a biomarker for guiding their selection. This study aimed to identify the molecular-based predictors of responsiveness to pulmonary vasodilators by combining human and basic studies. Methods and ResultsRNA sequencing was conducted on cultured pulmonary artery smooth muscle cells (PASMCs) from patients with and without PAH, identifying variations in 2,076 genes. The candidates were narrowed down to established clinical biomarkers, and their plasma levels were assessed in PAH (n = 123) and non-PAH patients (n = 25). C1q/TNF-related protein 7 (CTRP7) exhibited elevated expression in PASMCs (5.37 Log2 fold change, p < 0.01), and the plasma of patients with PAH compared to non-PAH (28.2 (9.3- 101.2) vs. 9.32 (4.6-18.6) ng/ml, p < 0.01). A plasma assessment revealed a significant correlation between CTRP7 and interleukin 6 (IL-6) levels (r = 0.544, p < 0.001). Chromatin immunoprecipitation demonstrated that IL-6 upregulated CTRP7 expression in PASMCs. Among pivotal factors, CTRP7 reduced prostacyclin analog receptor (PTGIR) expression through Rab5a-mediated internalization, resulting in diminished responsiveness to selexipag (a prostacyclin analog). Incorporating the human study, hypoxic PH mice demonstrated reduced PTGIR expression in the pulmonary arteries, which correlated with limited responses to selexipag treatment (low cardiac output and persistent pulmonary artery resistance), which was mitigated by silencing CTRP7 expression in the pulmonary arteries using adeno-associated virus 6. ConclusionsAmong the candidates with increased expression in patients with PAH based on RNA sequencing of PASMCs, CTRP7 demonstrated elevated plasma levels compared to non-PAH. CTRP7 regulated PTGIR internalization via inflammation and Rab5a, influencing responsiveness to selexipag. CTRP7 emerged as a compelling biomarker for predicting responsiveness to prostacyclin analogs, potentially advancing the development of innovative PAH treatment strategies. Novel and SignificanceO_ST_ABSWhat Is Known?C_ST_ABSO_LIPulmonary arterial hypertension (PAH) is a life-threatening vascular disease with limited treatment options. C_LIO_LIAlthough pulmonary vasodilators have shown promise in managing PAH, the overall improvement in prognosis is modest. C_LIO_LIFurther research is needed to understand the molecular features of pulmonary vasodilators and identify biomarkers for appropriate selection. C_LI What New Information Does This Article Contribute?O_LIC1q/TNF-related protein 7 (CTRP7) was found to be elevated in the pulmonary artery smooth muscle cells (PASMCs) and plasma of patients with pulmonary arterial hypertension (PAH). C_LIO_LIThis study established a connection between inflammation and pulmonary vascular responsiveness via CTRP7-mediated internalization and degradation of the prostacyclin analog receptor. C_LIO_LIThis study indicated the possibility of using plasma CTRP levels as a clinical biomarker to monitor pulmonary responsiveness, which can contribute to the development of novel treatment strategies for patients with PAH. C_LI Pulmonary arterial hypertension (PAH) is a life-threatening and enigmatic lung vascular disorder characterized by progressive pulmonary vascular remodeling and vasoconstriction. Despite significant advancements in the treatment of PAH using pulmonary vasodilators, some individuals display limited responses, primarily due to a lack of understanding of the molecular features of each pulmonary vasodilator and the absence of a biomarker for appropriate selection. The present study aimed to identify promising biomarkers for predicting a patients responsiveness to pulmonary vasodilators through a two-step approach, involving RNA sequencing of PASMCs and the measurement of plasma levels. CTRP7 emerged as a prominent biomarker, displaying increased expression in both PASMCs and plasma obtained from patients with PAH. Mechanistically, IL-6 transcriptionally modulated CTPR7, leading to the internalization and degradation of PTGIR, resulting in a reduced response to selexipag (prostacyclin analog) in PASMCs. Integrating the human studies, a hypoxic mouse model exhibited lower PTGIR expression in pulmonary arteries, correlating with limited responses to selexipag treatment, which were ameliorated by silencing CTRP7 expression in pulmonary arteries using adeno-associated virus 6 transduction. This study collectively demonstrates that CTRP7 emerges as a compelling biomarker for predicting responsiveness to prostacyclin analogs, with the potential to advance the development of innovative PAH treatment strategies.

molecular biology↗

bioRxiv2023

Drug metabolic activity as a selection factor for pluripotent stem cell-derived hepatic progenitor cells

As a metabolic organ, the liver plays a variety of roles, including detoxification. It has been difficult to obtain stable supplies of hepatocytes for transplantation and for accurate hepatotoxicity determination in drug discovery research. Human pluripotent stem cells, capable of unlimited self-renewal, may be a promising source of hepatocytes. In order to develop a stable supply of embryonic stem cell (ESC)-derived hepatocytes, we have purified human ESC-derived hepatic progenitor cells with exposure to cytocidal puromycin by using their ability to metabolize drugs. Hepatic progenitor cells stably proliferated at least 2^20-fold over 120 days, maintaining hepatic progenitor cell-like properties. High drug-metabolizing hepatic progenitor cells can be matured into liver cells by suppressing hepatic proliferative signals. The method we developed enables the isolation and proliferation of functional hepatic progenitors from human ESCs, thereby providing a stable supply of high-quality cell resources at high efficiency. Cells produced by this method may facilitate cell therapy for hepatic diseases and reliable drug discovery research.

cell biology↗

bioRxiv2022

Assembly of neuron- and radial glial cell-derived extracellular matrix molecules promotes radial migration of developing cortical neurons

Radial neuronal migration is a key neurodevelopmental event for proper cortical laminar organization. The multipolar-to-bipolar transition, a critical step in establishing neuronal polarity during radial migration, occurs in the subplate/intermediate zone (SP/IZ), a distinct region of the embryonic cerebral cortex. It has been known that the extracellular matrix (ECM) molecules are enriched in the SP/IZ. However, the molecular constitution and functions of the ECM formed in this region remain poorly understood. Here, we identified neurocan (NCAN) as a major chondroitin sulfate proteoglycan in the SP/IZ. NCAN binds to both radial glial cell-derived tenascin-C (TNC) and hyaluronan (HA), a large linear polysaccharide, forming a ternary complex of NCAN, TNC, and HA in the SP/IZ. Developing cortical neurons make contact with the ternary complex during migration. The enzymatic or genetic disruption of the ternary complex impairs radial migration by suppressing the multipolar-to-bipolar transition. Furthermore, both TNC and NCAN promoted the morphological maturation of cortical neurons in vitro. The present results provide evidence for the cooperative role of neuron- and radial glial cell-derived ECM molecules in cortical development.

neuroscience↗

bioRxiv2021

Drug metabolism activity is a Critical Intrinsic Selector for Hepatocytes to elongate cell lifespan by using a cell-killing antibiotic

Hepatocytes can be used to study the pathogenesis of liver diseases and drug discovery research. Human hepatocytes are, however, hardly expandable in vitro making it difficult to secure large numbers of cells from one donor. In this study, we aimed to establish an in vitro long-term culture system that enables stable proliferation and maintenance of human hepatocytes to ensure a constant supply. We purified human hepatocytes by selection with cytocidal puromycin, and cultured them for more than 60 population doublings over a span of more than 350 days. These results show that this simple culture system with usage of the cytocidal antibiotics enables efficient hepatocyte proliferation and is an effective method for generating a stable supply of hepatocytes for drug discovery research at a significant cost reduction.

cell biology↗

bioRxiv2020

Puromycin selection of cells with a high expression of the cytochrome P450 CYP3A4 gene activity from a patient with drug-induced liver injury (DILI) and their lifespan prolongation using a combination of CDK4R24C, cyclin D1 and TERT

Many drugs have the potential to induce the expression of drug-metabolizing enzymes, particularly cytochrome P450 3A4 (CYP3A4), in hepatocytes. Hepatocytes can accurately evaluate drug-mediated CYP3A4 induction as the gold standard for in vitro hepatic toxicology test, but their lot variation is an issue to be solved. Only a limited number of immortalized hepatocyte cells have been reported. In this study, we generated an immortalized cell expressing CYP3A4 from a patient with drug-induced liver injury (DILI). To generate DILI-derived cells with a high expression of CYP3A4, we employed a three-step approach: 1. Differentiation of DILI-induced pluripotent stem cells (DILI-iPSCs); 2. Immortalization of the differentiated cells; 3. Selection of the cells with puromycin. We hypothesize that cells with a high expression of cytochrome P450 genes can survive even after exposure to cytotoxic antibiotics because of high drug-metabolism activity. Puromycin, one of the cytotoxic antibiotics, was used in this study because of its rapid cytocidal effect at a low concentration. Phenotypic studies in vitro revealed that the puromycin-selected cells (HepaSM or SI cells) constitutively expressed the CYP3A4 gene at an extremely high level, and continued to proliferate at least up to 34 population doublings for more than 250 days. The expression profiles were independent of population doublings. Drug-mediated induction test revealed that the cells significantly increased CYP3A4 after exposure to rifampicin, suggesting that the immortalized cells would serve as another useful source for in vitro examination of drug metabolism and CYP3A4 induction.

cell biology↗

bioRxiv2020

Ammonia-based enrichment and long-term propagation of zone I hepatocyte-like cells

Zone I and zone III hepatocytes metabolize ammonia through urea cycle and drug by cytochrome P450, respectively. Ammonia has a cytotoxic effect, and can therefore be used as a selection agent for enrichment of hepatocytes. Besides, isolated hepatocytes from livers can be propagated ex vivo under appropriate condition. However, it has not been investigated so far whether ammonia-treated hepatocyte-like cells are able to proliferate in vitro. In this study, we employed the ammonia selection strategy to purify hepatocyte-like cells that were differentiated from human pluripotent stem cells (PSCs) that are embryonic stem cells (ESCs) and induced pluripotent stem cells. Hepatocyte-like cells after exposure to ammonia highly expressed the CPS1 gene that metabolizes ammonia to carbamoyl phosphate. The resistance to cytotoxicity or cell death by ammonia is probably attributed to the metabolic activity of ammonia in the cells. In addition to the ammonia metabolism-related genes, ammonia-selected PSC-derived hepatocytes increased expression of the CYP3A4 gene, one of the cytochrome P450 genes, that is mainly expressed in zone III hepatocytes. Ammonia-selected hepatocyte-like cells derived from both ESCs and iPSCs can be propagated in vitro up to 30 population doublings for more than 190 days without affecting expression of the liver-associated genes, implying that the ammonia-selected cells have immortality or equivalent life span on the appropriate feeder cells like ESCs and iPSCs. The long-term cultivation of ammonia-selected hepatocyte-like cells resulted in the increased expression of hepatocyte-associated genes such as the CPS1 and CYP3A4 genes. The ammonia selection method to enrich a hepatocyte population was also applicable to immortalized cells from the liver. Ammonia treatment in combination with in vitro propagation will be used to obtain large amounts of hepatocytes or hepatocyte-like cells for pharmacology, toxicology and regenerative medicine.

cell biology↗