Biology subjects

Kawano, H.

Publications and source records attributed to Kawano, H..

11 recordsLinked to original sources

bioRxiv2024

Targeting IL-1/IRAK1/4 signaling in Acute Myeloid Leukemia Stem Cells Following Treatment and Relapse

Therapies for acute myeloid leukemia (AML) face formidable challenges due to relapse, often driven by leukemia stem cells (LSCs). Strategies targeting LSCs hold promise for enhancing outcomes, yet paired comparisons of functionally defined LSCs at diagnosis and relapse remain underexplored. We present transcriptome analyses of functionally defined LSC populations at diagnosis and relapse, revealing significant alterations in IL-1 signaling. Interleukin-1 receptor type I (IL1R1) and interleukin-1 receptor accessory protein (IL1RAP) were notably upregulated in leukemia stem and progenitor cells at both diagnosis and relapse. Knockdown of IL1R1 and IL1RAP reduced the clonogenicity and/or engraftment of primary human AML cells. In leukemic MLL-AF9 mice, Il1r1 knockout reduced LSC frequency and extended survival. To target IL-1 signaling at both diagnosis and relapse, we developed UR241-2, a novel interleukin-1 receptor-associated kinase 1 and 4 (IRAK1/4) inhibitor. UR241-2 robustly suppressed IL-1/IRAK1/4 signaling, including NF-{kappa}B activation and phosphorylation of p65 and p38, following IL-1 stimulation. UR241-2 selectively inhibited LSC clonogenicity in primary human AML cells at both diagnosis and relapse, while sparing normal hematopoietic stem and progenitor cells. It also reduced AML engraftment in leukemic mice. Our findings highlight the therapeutic potential of UR241-2 in targeting IL-1/IRAK1/4 signaling to eradicate LSCs and improve AML outcomes.

cancer biology↗

bioRxiv2023

Myelodysplastic syndromes disable human CD271+VCAM1+CD146+ niches supporting normal hematopoietic stem/progenitor cells

Mesenchymal stem/stromal cells (MSCs) within the bone marrow microenvironment (BMME) support normal hematopoietic stem and progenitor cells (HSPCs). However, the heterogeneity of human MSCs has limited the understanding of their contribution to clonal dynamics and evolution to myelodysplastic syndromes (MDS). We combined three MSC cell surface markers, CD271, VCAM-1 (Vascular Cell Adhesion Molecule-1) and CD146, to isolate distinct subsets of human MSCs from bone marrow aspirates of healthy controls (Control BM). Based on transcriptional and functional analysis, CD271+CD106+CD146+ (NGFR+/VCAM1+/MCAM+/Lin-; NVML) cells display stem cell characteristics, are compatible with murine BM- derived Leptin receptor positive MSCs and provide superior support for normal HSPCs. MSC subsets from 17 patients with MDS demonstrated shared transcriptional changes in spite of mutational heterogeneity in the MDS clones, with loss of preferential support of normal HSPCs by MDS-derived NVML cells. Our data provide a new approach to dissect microenvironment-dependent mechanisms regulating clonal dynamics and progression to MDS.

cancer biology↗

bioRxiv2021

Rhomboid protease RHBDL4/RHBDD1 cleaves SREBP-1c at ER monitoring and regulating fatty acids

The ER-embedded transcription factors, sterol-regulatory element-binding proteins (SREBPs), master regulators of lipid biosynthesis, are transported to Golgi for proteolytic activation to tune cellular cholesterol levels and regulate lipogenesis. However, mechanisms by which the cell responds to the levels of saturated or unsaturated fatty acids remain underexplored. Here we show that RHBDL4/RHBDD1, a rhomboid family protease, directly cleaves SREBP-1c at ER. The p97/VCP, AAA-ATPase complex then acts as an auxiliary segregase to extract the remaining ER-embedded fragment of SREBP-1c. Importantly, the enzymatic activity of RHBDL4 is enhanced by saturated fatty acids (SFAs), but inhibited by polyunsaturated fatty acids (PUFAs). Genetic deletion of RHBDL4 in mice fed on a Western diet enriched in SFAs and cholesterol prevented SREBP-1c from inducing genes for lipogenesis, particularly for synthesis and incorporation of PUFAs, and secretion of lipoproteins. The RHBDL4-SREBP-1c pathway reveals a regulatory system for monitoring fatty acid composition and maintaining cellular lipid homeostasis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC="FIGDIR/small/457590v2_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@591eb2org.highwire.dtl.DTLVardef@ee6361org.highwire.dtl.DTLVardef@be893eorg.highwire.dtl.DTLVardef@1247d38_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2021

The excitatory/inhibitory balance of synaptic transmission is impaired at higher extracellular Ca2+ concentrations in Scn1a+/- mouse model of Dravet syndrome

Dravet syndrome (DS) is an intractable form of childhood epilepsy that occurs in infancy. More than 80% of all patients have a heterozygous abnormality in the SCN1A gene, which encodes a subunit of Na+ channels in the brain. However, the detailed pathogenesis of DS remains unclear. This study investigated the synaptic pathogenesis of this disease in terms of excitatory/inhibitory balance using a mouse model of DS. We show that excitatory postsynaptic currents were similar between Scn1a knock-in neurons (Scn1a+/- neurons) and wild-type neurons, but inhibitory postsynaptic currents were significantly lower in Scn1a+/- neurons. Moreover, both the vesicular release probability and the number of inhibitory synapses were significantly lower in Scn1a+/- neurons compared with wild-type neurons. There was no proportional increase in inhibitory postsynaptic current amplitude in response to increased extracellular Ca2+ concentrations. Our study revealed that the number of inhibitory synapses is significantly reduced in Scn1a+/- neurons, while the sensitivity of inhibitory synapses to extracellular Ca2+ concentrations is markedly increased. These data suggest that Ca2+ tethering in inhibitory nerve terminals may be disturbed following the synaptic burst, likely leading to epileptic symptoms.

neuroscience↗

bioRxiv2020

Correlating cell function and morphology by performing fluorescent immunocytochemical staining on the light-microscope stage

BackgroundCorrelation of fluorescence signals from functional changes in live cells with those from immunocytochemical indicators of their morphology following chemical fixation can be highly informative with regard to function-structure relationship. Such analyses can be technically challenging because they need consistently aligning the images between imaging sessions. Existing solutions include introducing artificial spatial landmarks and modifying the microscopes. However, these methods can require extensive changes to the experimental systems. New methodHere we introduce a simple approach for aligning images. It is based on two procedures: performing immunocytochemistry while a specimen stays on a microscope stage (on-stage), and aligning images using biological structures as landmarks after they are observed with transmitted-light optics in combination with fluorescence-filter sets. ResultsWe imaged a transient functional signal from a fluorescent Ca2+ indicator, and mapped it to neurites based on immunocytochemical staining of a structural marker. In the same preparation, we could identify presynaptically silent synapses, based on a lack of labeling with an indicator for synaptic vesicle recycling and on positive immunocytochemical staining for a structural marker of nerve terminals. On-stage immunocytochemistry minimized lateral translations and eliminated rotations, and transmitted-light images of neurites were sufficiently clear to enable spatial registration, effective at a single-pixel level. Comparison with existing methodsThis method aligned images with minimal change or investment in the experimental systems. ConclusionsThis method facilitates information retrieval across multiple imaging sessions, even when functional signals are transient or local, and when fluorescent signals in multiple imaging sessions do not match spatially.

neuroscience↗

bioRxiv2020

HSV Forms an HCMV-like Viral Assembly Center in Neuronal Cells

Herpes simplex virus (HSV) is a neuroinvasive virus that has been used as a model organism for studying common properties of all herpesviruses. HSV induces host organelle rearrangement and forms dispersed assembly compartments in epithelial cells, which complicates the study of HSV assembly. In this study, we show that HSV forms a visually distinct unitary cytoplasmic viral assembly center (cVAC) in both cancerous and primary neuronal cells that concentrates viral structural proteins and is the site of capsid envelopment. The HSV cVAC also concentrates host membranes that are important for viral assembly, such as Golgi- and recycling endosome-derived membranes. Lastly, we show that HSV cVAC formation and/or maintenance depends on an intact microtubule network and a viral tegument protein, pUL51. Our observations suggest that the neuronal cVAC is a uniquely useful model to study common herpesvirus assembly pathways, and cell-specific pathways for membrane reorganization. SummaryThis study shows that HSV forms a viral assembly center in neuronal cells by reorganization of host membranes. This system is a novel and powerful tool to study herpesvirus assembly pathways and host cell membrane dynamics.

cell biology↗

bioRxiv2020

Presynaptically silent synapses are modulated by the density of surrounding astrocytes

The astrocyte, a major glial cell type, is involved in formation and maturation of synapses, and thus contributes to sustainable synaptic transmission between neurons. Given that the animals in the higher phylogenetic tree have brains with higher density of glial cells with respect to neurons, there is a possibility that the relative astrocytic density directly influences synaptic transmission. However, the notion has not been tested thoroughly. Here we addressed it, by using a primary culture preparation where single hippocampal neurons are surrounded by a variable but countable number of cortical astrocytes in dot-patterned microislands, and recording synaptic transmission by patch-clamp electrophysiology. Neurons with a higher astrocytic density showed a higher amplitude of evoked excitatory postsynaptic current (EPSC) than that of neurons with a lower astrocytic density. The size of readily releasable pool of synaptic vesicles per neuron was significantly higher. The frequency of spontaneous synaptic transmission (miniature EPSC) was higher, but the amplitude was unchanged. The number of morphologically identified glutamatergic synapses was unchanged, but the number of functional ones was increased, indicating a lower ratio of presynaptically silent synapses. Taken together, the higher astrocytic density enhanced excitatory synaptic transmission by increasing the number of functional synapses through presynaptic un-silencing.

neuroscience↗

bioRxiv2019

Localization of the signal of dystonia-associated protein torsinA near the Golgi apparatus in cultured central neurons

A single in-frame deletion of a codon for a glutamic acid residue within the TOR1A gene is linked to the autosomal-dominant movement disorder DYT1 dystonia, a condition characterized by involuntary muscle contractions that cause abnormal posture. This gene encodes the protein torsinA, and the functions of both wild-type and mutant ({Delta}E-torsinA) forms remain poorly understood. Previous studies based on overexpression systems indicated that wild-type torsinA resides mainly in the endoplasmic reticulum but that {Delta}E-torsinA is localized to the nuclear envelope or intracellular inclusions. This mutation-associated mis-localization has been proposed to underlie at least a part of the pathophysiology of DYT1 dystonia. However, the subcellular localization of torsinA has not been extensively studied when expressed at the endogenous level. Here we report an immunocytochemical analysis of torsinA proteins in cultured mouse neurons from a {Delta}E-torsinA knock-in model of DYT1 dystonia, where torsinA proteins are not upregulated. In all examined neurons of wild-type, heterozygous and homozygous mice, torsinA signal was found mainly near the Golgi apparatus, and only weakly in the endoplasmic reticulum and nuclear envelope. These results suggest that, in the absence of overexpression, torsinA proteins are localized near the Golgi apparatus and may influence cellular function involving the organelle.

neuroscience↗

bioRxiv2019

Localization of immunoreactive, dystonia-associated protein torsinA near the Golgi apparatus of cultured rodent astrocytes

An in-frame deletion of a single glutamic acid codon in the TOR1A gene causes the neurological disorder DYT1 dystonia, but the cellular pathophysiology of this disorder remains elusive. A current model postulates that the wild-type (WT) torsinA protein is mainly localized to the endoplasmic reticulum (ER), but that the mutant form ({Delta}E-torsinA) is diverted to the nuclear envelope and cytoplasmic inclusion bodies. This mis-localization has been observed by overexpressing the proteins in neuronal and non-neuronal cells. However, it is not clear whether this model is valid for the astrocytic glial cells that support and modify neuronal functions. Here we report, using rodent astrocytes in primary culture, that the overexpressed torsinA proteins were distributed as predicted by the mis-localization model. We also found by immunocytochemistry that the cultured astrocytes express torsinA endogenously. Most of the signals from endogenous protein, whether the WT or {Delta}E form, were localized near a cis-Golgi marker GM130. Such localization of endogenous proteins was found in glial cells from several sources: the hippocampus of WT rats, the hippocampus and striatum of WT mice, and the hippocampus and striatum of {Delta}E-torsinA knock-in mice, a model of DYT1 dystonia. These results show that the mis-localization model is applicable to overexpressed torsinA proteins, but not applicable to those expressed at endogenous levels, at least in cultured rodent astrocytes. These discrepancies in the distribution of overexpressed versus endogenous torsinA proteins highlight the potential need for caution in interpreting the results of overexpression studies.

neuroscience↗

bioRxiv2019

Calcium-induced calcium release in noradrenergic neurons of the locus coeruleus

The locus coeruleus (LC) is a nucleus within the brainstem that consists of norepinephrine-releasing neurons. It is involved in broad processes including autonomic regulation, and cognitive and emotional functions such as arousal, attention and anxiety. Understanding the mechanisms that control the excitability of LC neurons is important because they innervate widespread regions of the central nervous system. One of the key regulators is the cytosolic calcium concentration ([Ca2+]c), the increases in which can be amplified by calcium-induced calcium release (CICR) from the intracellular calcium stores. Although the electrical activities of LC neurons are regulated by changes in [Ca2+]c, the extent of CICR involvement in this regulation has remained unclear. Here we show that CICR hyperpolarizes acutely dissociated LC neurons of the rat brain and demonstrate the pathway whereby it does this. When CICR was activated by extracellular application of 10 mM caffeine, LC neurons were hyperpolarized in the current-clamp mode of the patch-clamp recording, and the majority of neurons showed an outward current in the voltage-clamp mode. This outward current was accompanied by an increase in membrane conductance, and its reversal potential was close to the K+ equilibrium potential, indicating that it is mediated by the opening of K+ channels. The outward current was generated in the absence of extracellular calcium and was blocked when the calcium stores were inhibited by applying ryanodine. Pharmacological experiments indicated that the outward current was mediated by Ca2+-activated K+ channels of the non-small conductance type. Finally, the application of caffeine led to an increase in the [Ca2+]c in these neurons, as visualized by fluorescence microscopy. These findings delineate a mechanism whereby CICR suppresses the electrical activity of LC neurons, and indicate that it could play a dynamic role in modulating the LC-mediated noradrenergic tone in the brain.

neuroscience↗

bioRxiv2019

Transcriptome profiles in brains of mice heterozygous for a DYT1 dystonia-associated mutation in the endogenous Tor1a gene

In patients with the brain disorder dystonia, body movement is severely affected - with involuntary muscle contractions and abnormal postures, causing extensive deterioration of the patients quality of life. The most common inherited form of this disorder is DYT1 dystonia, which is caused by a mutation in TOR1A gene and autosomal dominant. The molecular mechanisms that underlie the effects of the TOR1A mutation on brain function remain unclear. To understand these, we examined the gene expression profiles (transcriptome) in four brain regions (cerebral cortex, hippocampus, striatum and cerebellum) in a mouse model, the heterozygous {Delta}E-torsinA knock-in mice which genetically reproduce the mutation in DYT1 dystonia. The samples were obtained at 2 to 3 weeks of age, a period during which synaptic abnormalities have been reported. Pairwise comparisons of brain regions revealed differential gene expression irrespective of genotype. A comparison of heterozygous to wild-type mice failed to reveal genotype-dependent differences in gene expression in any of the four brain regions when examined individually. However, genotype-dependent differences became apparent when the information for all brain regions was combined. These results suggest that any changes in the transcriptome within a brain region were subtle at this developmental stage, but that statistically significant changes occur across all brain regions. Such changes in the transcriptome, although subtle in degree, could underlie the processes that give rise to DYT1 dystonia.

neuroscience↗