Publications by authors named "Hiroki R Ueda"

119 Publications

Activation of Sympathetic Signaling in Macrophages Blocks Systemic Inflammation and Protects against Renal Ischemia-Reperfusion Injury.

J Am Soc Nephrol 2021 Apr 19. Epub 2021 Apr 19.

Division of CKD Pathophysiology, University of Tokyo Graduate School of Medicine, Tokyo, Japan.

Background: The sympathetic nervous system regulates immune cell dynamics. However, the detailed role of sympathetic signaling in inflammatory diseases is still unclear because it varies according to the disease situation and responsible cell types. This study focused on identifying the functions of sympathetic signaling in macrophages in LPS-induced sepsis and renal ischemia-reperfusion injury (IRI).

Methods: We performed RNA sequencing of mouse macrophage cell lines to identify the critical gene that mediates the anti-inflammatory effect of 2-adrenergic receptor (Adrb2) signaling. We also examined the effects of salbutamol (a selective Adrb2 agonist) in LPS-induced systemic inflammation and renal IRI. Macrophage-specific conditional knockout (cKO) mice and the adoptive transfer of salbutamol-treated macrophages were used to assess the involvement of macrophage Adrb2 signaling.

Results: , activation of Adrb2 signaling in macrophages induced the expression of T cell Ig and mucin domain 3 (), which contributes to anti-inflammatory phenotypic alterations. , salbutamol administration blocked LPS-induced systemic inflammation and protected against renal IRI; this protection was mitigated in macrophage-specific cKO mice. The adoptive transfer of salbutamol-treated macrophages also protected against renal IRI. Single-cell RNA sequencing revealed that this protection was associated with the accumulation of -expressing macrophages in the renal tissue.

Conclusions: The activation of Adrb2 signaling in macrophages induces anti-inflammatory phenotypic alterations partially via the induction of expression, which blocks LPS-induced systemic inflammation and protects against renal IRI.
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http://dx.doi.org/10.1681/ASN.2020121723DOI Listing
April 2021

Whole-organ analysis of TGF-β-mediated remodelling of the tumour microenvironment by tissue clearing.

Commun Biol 2021 Mar 5;4(1):294. Epub 2021 Mar 5.

Department of Molecular Pathology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Tissue clearing is one of the most powerful strategies for a comprehensive analysis of disease progression. Here, we established an integrated pipeline that combines tissue clearing, 3D imaging, and machine learning and applied to a mouse tumour model of experimental lung metastasis using human lung adenocarcinoma A549 cells. This pipeline provided the spatial information of the tumour microenvironment. We further explored the role of transforming growth factor-β (TGF-β) in cancer metastasis. TGF-β-stimulated cancer cells enhanced metastatic colonization of unstimulated-cancer cells in vivo when both cells were mixed. RNA-sequencing analysis showed that expression of the genes related to coagulation and inflammation were up-regulated in TGF-β-stimulated cancer cells. Further, whole-organ analysis revealed accumulation of platelets or macrophages with TGF-β-stimulated cancer cells, suggesting that TGF-β might promote remodelling of the tumour microenvironment, enhancing the colonization of cancer cells. Hence, our integrated pipeline for 3D profiling will help the understanding of the tumour microenvironment.
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http://dx.doi.org/10.1038/s42003-021-01786-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7935961PMC
March 2021

Malignant subclone drives metastasis of genetically and phenotypically heterogenous cell clusters through fibrotic niche generation.

Nat Commun 2021 02 8;12(1):863. Epub 2021 Feb 8.

Division of Genetics, Cancer Research Institute, Kanazawa University, Kanazawa, Japan.

A concept of polyclonal metastasis has recently been proposed, wherein tumor cell clusters break off from the primary site and are disseminated. However, the involvement of driver mutations in such polyclonal mechanism is not fully understood. Here, we show that non-metastatic AP cells metastasize to the liver with metastatic AKTP cells after co-transplantation to the spleen. Furthermore, AKTP cell depletion after the development of metastases results in the continuous proliferation of the remaining AP cells, indicating a role of AKTP cells in the early step of polyclonal metastasis. Importantly, AKTP cells, but not AP cells, induce fibrotic niche generation when arrested in the sinusoid, and such fibrotic microenvironment promotes the colonization of AP cells. These results indicate that non-metastatic cells can metastasize via the polyclonal metastasis mechanism using the fibrotic niche induced by malignant cells. Thus, targeting the fibrotic niche is an effective strategy for halting polyclonal metastasis.
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http://dx.doi.org/10.1038/s41467-021-21160-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7870854PMC
February 2021

A design principle for posttranslational chaotic oscillators.

iScience 2021 Jan 15;24(1):101946. Epub 2020 Dec 15.

Department of Systems Pharmacology, Graduate School of Medicine, the University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113-0033, Japan.

Chaos behavior has been observed in various cellular and molecular processes. Here, we modeled reversible phosphorylation dynamics to elucidate a design principle for autonomous chaos generation that may arise from generic enzymatic reactions. A comprehensive parameter search demonstrated that the reaction system composed of a set of kinases and phosphatases and two substrates with two modification sites exhibits chaos behavior. All reactions are described according to the Michaelis-Menten reaction scheme without exotic functions being applied to enzymes and substrates. Clustering analysis of parameter sets that can generate chaos behavior revealed the existence of motif structures. These chaos motifs allow the two-substrate species to interact via enzyme availability and constrain the two substrates' dynamic changes in phosphorylation status so that they occur at different timescales. This chaos motif structure is found in several enzymatic reactions, suggesting that chaos behavior may underlie cellular autonomy in a variety of biochemical systems.
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http://dx.doi.org/10.1016/j.isci.2020.101946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7786127PMC
January 2021

Protocol for Imaging and Analysis of Mouse Tumor Models with CUBIC Tissue Clearing.

STAR Protoc 2020 Dec 26;1(3):100191. Epub 2020 Nov 26.

Department of Molecular Pathology, Graduate School of Medicine, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

Tissue-clearing technologies have developed rapidly in the past decade, especially for use in neuroscience research. We previously reported that CUBIC, which is one tissue-clearing method, is useful for applications in cancer research. CUBIC cancer analysis can be used to detect cancer metastasis with single-cell resolution at whole mouse body/organ level. This approach can also analyze the tumor characteristics with high-quality 3D images. Here, we describe a detailed CUBIC cancer protocol from tissue clearing, capturing 3D images and post-immunohistochemistry. For complete details on the use and execution of this protocol, please refer to Kubota et al. (2017).
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http://dx.doi.org/10.1016/j.xpro.2020.100191DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7757565PMC
December 2020

Generation of gene-corrected iPSCs line (KEIUi001-A) from a PARK8 patient iPSCs with familial Parkinson's disease carrying the I2020T mutation in LRRK2.

Stem Cell Res 2020 12 3;49:102073. Epub 2020 Nov 3.

Department of Physiology, Keio University School of Medicine, Japan. Electronic address:

Leucine-rich repeat kinase 2 (LRRK2) is the causal gene of the autosomal dominant hereditary form of Parkinson's disease (PD), PARK8. We have previously reported that induced pluripotent stem cells (iPSCs) from a PARK8 patient with I2020T LRRK2 mutation replicated to some extent the pathologic phenotype evident in the brain of PD patients. In the present study, we generated gene-corrected iPSCs line, KEIUi001-A, using TALEN-mediated genome editing. KEIUi001-A retained a normal karyotype and pluripotency, i.e. the capacity to differentiate into cell types of the three germ layers. This iPSCs will be valuable for clarifying various aspects of LRRK2-related pathology.
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http://dx.doi.org/10.1016/j.scr.2020.102073DOI Listing
December 2020

Phosphorylation Hypothesis of Sleep.

Front Psychol 2020 2;11:575328. Epub 2020 Oct 2.

Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Sleep is a fundamental property conserved across species. The homeostatic induction of sleep indicates the presence of a mechanism that is progressively activated by the awake state and that induces sleep. Several lines of evidence support that such function, namely, sleep need, lies in the neuronal assemblies rather than specific brain regions and circuits. However, the molecular mechanism underlying the dynamics of sleep need is still unclear. This review aims to summarize recent studies mainly in rodents indicating that protein phosphorylation, especially at the synapses, could be the molecular entity associated with sleep need. Genetic studies in rodents have identified a set of kinases that promote sleep. The activity of sleep-promoting kinases appears to be elevated during the awake phase and in sleep deprivation. Furthermore, the proteomic analysis demonstrated that the phosphorylation status of synaptic protein is controlled by the sleep-wake cycle. Therefore, a plausible scenario may be that the awake-dependent activation of kinases modifies the phosphorylation status of synaptic proteins to promote sleep. We also discuss the possible importance of multisite phosphorylation on macromolecular protein complexes to achieve the slow dynamics and physiological functions of sleep in mammals.
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http://dx.doi.org/10.3389/fpsyg.2020.575328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566165PMC
October 2020

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

BMC Res Notes 2020 Oct 5;13(1):453. Epub 2020 Oct 5.

Laboratory for Integrated Biodevice, Center for Biosystems Dynamics Research, RIKEN, 1-3 Yamadaoka, Suita, Osaka, 565-0871, Japan.

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.
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http://dx.doi.org/10.1186/s13104-020-05294-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7534166PMC
October 2020

Generation of a p16 Reporter Mouse and Its Use to Characterize and Target p16 Cells In Vivo.

Cell Metab 2020 11 18;32(5):814-828.e6. Epub 2020 Sep 18.

Division of Molecular Regulation of Inflammatory and Immune Diseases, Research Institute of Biomedical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-0022, Japan.

Cell senescence plays a key role in age-associated organ dysfunction, but the in vivo pathogenesis is largely unclear. Here, we generated a p16-Cre-tdTomato mouse model to analyze the in vivo characteristics of p16 cells at a single-cell level. We found tdTomato-positive p16 cells detectable in all organs, which were enriched with age. We also found that these cells failed to proliferate and had half-lives ranging from 2.6 to 4.2 months, depending on the tissue examined. Single-cell transcriptomics in the liver and kidneys revealed that p16 cells were present in various cell types, though most dominant in hepatic endothelium and in renal proximal and distal tubule epithelia, and that these cells exhibited heterogeneous senescence-associated phenotypes. Further, elimination of p16 cells ameliorated nonalcoholic steatohepatitis-related hepatic lipidosis and immune cell infiltration. Our new mouse model and single-cell analysis provide a powerful resource to enable the discovery of previously unidentified senescence functions in vivo.
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http://dx.doi.org/10.1016/j.cmet.2020.09.006DOI Listing
November 2020

Reflections on the past two decades of neuroscience.

Nat Rev Neurosci 2020 10 2;21(10):524-534. Epub 2020 Sep 2.

Department of Systems Pharmacology, The University of Tokyo, Tokyo, Japan.

The first issue of Nature Reviews Neuroscience was published 20 years ago, in 2000. To mark this anniversary, in this Viewpoint article we asked a selection of researchers from across the field who have authored pieces published in the journal in recent years for their thoughts on notable and interesting developments in neuroscience, and particularly in their areas of the field, over the past two decades. They also provide some thoughts on current lines of research and questions that excite them.
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http://dx.doi.org/10.1038/s41583-020-0363-6DOI Listing
October 2020

Mass spectrometry-based absolute quantification of amyloid proteins in pathology tissue specimens: Merits and limitations.

PLoS One 2020 1;15(7):e0235143. Epub 2020 Jul 1.

Department of Pathology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

To clarify the significance of quantitative analyses of amyloid proteins in clinical practice and in research relating to systemic amyloidoses, we applied mass spectrometry-based quantification by isotope-labeled cell-free products (MS-QBIC) to formalin-fixed, paraffin-embedded (FFPE) tissues. The technique was applied to amyloid tissues collected by laser microdissection of Congo red-stained lesions of FFPE specimens. Twelve of 13 amyloid precursor proteins were successfully quantified, including serum amyloid A (SAA), transthyretin (TTR), immunoglobulin kappa light chain (IGK), immunoglobulin lambda light chain (IGL), beta-2-microglobulin (B2M), apolipoprotein (Apo) A1, Apo A4, Apo E, lysozyme, Apo A2, gelsolin, and fibrinogen alpha chain; leukocyte cell-derived chemotaxin-2 was not detected. The quantification of SAA, TTR, IGK, IGL, and B2M confirmed the responsible proteins, even when the immunohistochemical results were not decisive. Considerable amounts of Apo A1, Apo A4, and Apo E were deposited in parallel amounts with the responsible proteins. Quantification of amyloid protein by MS-QBIC is feasible and useful for the classification of and research on systemic amyloidoses.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0235143PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7329117PMC
September 2020

A cellular model of albumin endocytosis uncovers a link between membrane and nuclear proteins.

J Cell Sci 2020 07 6;133(13). Epub 2020 Jul 6.

Department of Pediatrics, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-8655, Japan.

Cubilin (CUBN) and amnionless (AMN), expressed in kidney and intestine, form a multiligand receptor complex called CUBAM that plays a crucial role in albumin absorption. To date, the mechanism of albumin endocytosis mediated by CUBAM remains to be elucidated. Here, we describe a quantitative assay to evaluate albumin uptake by CUBAM using cells expressing full-length CUBN and elucidate the crucial roles of the C-terminal part of CUBN and the endocytosis signal motifs of AMN in albumin endocytosis. We also demonstrate that nuclear valosin-containing protein-like 2 (NVL2), an interacting protein of AMN, is involved in this process. Although NVL2 was mainly localized in the nucleolus in cells without AMN expression, it was translocated to the extranuclear compartment when coexpressed with AMN. NVL2 knockdown significantly impaired internalization of the CUBN-albumin complex in cultured cells, demonstrating an involvement of NVL2 in endocytic regulation. These findings uncover a link between membrane and nucleolar proteins that is involved in endocytic processes.
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http://dx.doi.org/10.1242/jcs.242859DOI Listing
July 2020

Whole-Brain Profiling of Cells and Circuits in Mammals by Tissue Clearing and Light-Sheet Microscopy.

Neuron 2020 05;106(3):369-387

Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA.

Tissue clearing and light-sheet microscopy have a 100-year-plus history, yet these fields have been combined only recently to facilitate novel experiments and measurements in neuroscience. Since tissue-clearing methods were first combined with modernized light-sheet microscopy a decade ago, the performance of both technologies has rapidly improved, broadening their applications. Here, we review the state of the art of tissue-clearing methods and light-sheet microscopy and discuss applications of these techniques in profiling cells and circuits in mice. We examine outstanding challenges and future opportunities for expanding these techniques to achieve brain-wide profiling of cells and circuits in primates and humans. Such integration will help provide a systems-level understanding of the physiology and pathology of our central nervous system.
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http://dx.doi.org/10.1016/j.neuron.2020.03.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7213014PMC
May 2020

Versatile whole-organ/body staining and imaging based on electrolyte-gel properties of biological tissues.

Nat Commun 2020 04 27;11(1):1982. Epub 2020 Apr 27.

Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.

Whole-organ/body three-dimensional (3D) staining and imaging have been enduring challenges in histology. By dissecting the complex physicochemical environment of the staining system, we developed a highly optimized 3D staining imaging pipeline based on CUBIC. Based on our precise characterization of biological tissues as an electrolyte gel, we experimentally evaluated broad 3D staining conditions by using an artificial tissue-mimicking material. The combination of optimized conditions allows a bottom-up design of a superior 3D staining protocol that can uniformly label whole adult mouse brains, an adult marmoset brain hemisphere, an ~1 cm tissue block of a postmortem adult human cerebellum, and an entire infant marmoset body with dozens of antibodies and cell-impermeant nuclear stains. The whole-organ 3D images collected by light-sheet microscopy are used for computational analyses and whole-organ comparison analysis between species. This pipeline, named CUBIC-HistoVIsion, thus offers advanced opportunities for organ- and organism-scale histological analysis of multicellular systems.
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http://dx.doi.org/10.1038/s41467-020-15906-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7184626PMC
April 2020

The oral hypoxia-inducible factor prolyl hydroxylase inhibitor enarodustat counteracts alterations in renal energy metabolism in the early stages of diabetic kidney disease.

Kidney Int 2020 05 25;97(5):934-950. Epub 2019 Dec 25.

Division of Nephrology and Endocrinology, The University of Tokyo Graduate School of Medicine, Tokyo, Japan. Electronic address:

Hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors, also known as HIF stabilizers, increase endogenous erythropoietin production and serve as novel therapeutic agents against anemia in chronic kidney disease. HIF induces the expression of various genes related to energy metabolism as an adaptive response to hypoxia. However, it remains obscure how the metabolic reprogramming in renal tissue by HIF stabilization affects the pathophysiology of kidney diseases. Previous studies suggest that systemic metabolic disorders such as hyperglycemia and dyslipidemia cause alterations of renal metabolism, leading to renal dysfunction including diabetic kidney disease. Here, we analyze the effects of enarodustat (JTZ-951), an oral HIF stabilizer, on renal energy metabolism in the early stages of diabetic kidney disease, using streptozotocin-induced diabetic rats and alloxan-induced diabetic mice. Transcriptome analysis revealed that enarodustat counteracts the alterations in diabetic renal metabolism. Transcriptome analysis showed that fatty acid and amino acid metabolisms were upregulated in diabetic renal tissue and downregulated by enarodustat, whereas glucose metabolism was upregulated. These symmetric changes were confirmed by metabolome analysis. Whereas glycolysis and tricarboxylic acid cycle metabolites were accumulated and amino acids reduced in renal tissue of diabetic animals, these metabolic disturbances were mitigated by enarodustat. Furthermore, enarodustat increased the glutathione to glutathione disulfide ratio and relieved oxidative stress in renal tissue of diabetic animals. Thus, HIF stabilization counteracts alterations in renal energy metabolism occurring in incipient diabetic kidney disease.
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http://dx.doi.org/10.1016/j.kint.2019.12.007DOI Listing
May 2020

Publisher Correction: Tissue clearing and its applications in neuroscience.

Nat Rev Neurosci 2020 05;21(5):298

Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41583-020-0291-5DOI Listing
May 2020

Visualization and molecular characterization of whole-brain vascular networks with capillary resolution.

Nat Commun 2020 02 27;11(1):1104. Epub 2020 Feb 27.

Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan.

Structural elucidation and molecular scrutiny of cerebral vasculature is crucial for understanding the functions and diseases of the brain. Here, we introduce SeeNet, a method for near-complete three-dimensional visualization of cerebral vascular networks with high signal-to-noise ratios compatible with molecular phenotyping. SeeNet employs perfusion of a multifunctional crosslinker, vascular casting by temperature-controlled polymerization of hybrid hydrogels, and a bile salt-based tissue-clearing technique optimized for observation of vascular connectivity. SeeNet is capable of whole-brain visualization of molecularly characterized cerebral vasculatures at the single-microvessel level. Moreover, SeeNet reveals a hitherto unidentified vascular pathway bridging cerebral and hippocampal vessels, thus serving as a potential tool to evaluate the connectivity of cerebral vasculature.
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http://dx.doi.org/10.1038/s41467-020-14786-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7046771PMC
February 2020

Molecular Mechanisms of REM Sleep.

Front Neurosci 2019 14;13:1402. Epub 2020 Jan 14.

Laboratory for Synthetic Biology, RIKEN Center for Biosystems Dynamics Research, Osaka, Japan.

Rapid-eye movement (REM) sleep is a paradoxical sleep state characterized by brain activity similar to wakefulness, rapid-eye-movement, and lack of muscle tone. REM sleep is a fundamental brain function, evolutionary conserved across species, including human, mouse, bird, and even reptiles. The physiological importance of REM sleep is highlighted by severe sleep disorders incurred by a failure in REM sleep regulation. Despite the intense interest in the mechanism of REM sleep regulation, the molecular machinery is largely left to be investigated. In models of REM sleep regulation, acetylcholine has been a pivotal component. However, even newly emerged techniques such as pharmacogenetics and optogenetics have not fully clarified the function of acetylcholine either at the cellular level or neural-circuit level. Recently, we discovered that the G type muscarinic acetylcholine receptor genes, and , are essential for REM sleep. In this review, we develop the perspective of current knowledge on REM sleep from a molecular viewpoint. This should be a starting point to clarify the molecular and cellular machinery underlying REM sleep regulation and will provide insights to explore physiological functions of REM sleep and its pathological roles in REM-sleep-related disorders such as depression, PTSD, and neurodegenerative diseases.
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http://dx.doi.org/10.3389/fnins.2019.01402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6972504PMC
January 2020

Tissue clearing and its applications in neuroscience.

Nat Rev Neurosci 2020 02;21(2):61-79

Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA.

State-of-the-art tissue-clearing methods provide subcellular-level optical access to intact tissues from individual organs and even to some entire mammals. When combined with light-sheet microscopy and automated approaches to image analysis, existing tissue-clearing methods can speed up and may reduce the cost of conventional histology by several orders of magnitude. In addition, tissue-clearing chemistry allows whole-organ antibody labelling, which can be applied even to thick human tissues. By combining the most powerful labelling, clearing, imaging and data-analysis tools, scientists are extracting structural and functional cellular and subcellular information on complex mammalian bodies and large human specimens at an accelerated pace. The rapid generation of terabyte-scale imaging data furthermore creates a high demand for efficient computational approaches that tackle challenges in large-scale data analysis and management. In this Review, we discuss how tissue-clearing methods could provide an unbiased, system-level view of mammalian bodies and human specimens and discuss future opportunities for the use of these methods in human neuroscience.
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http://dx.doi.org/10.1038/s41583-019-0250-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8121164PMC
February 2020

Advanced CUBIC tissue clearing for whole-organ cell profiling.

Nat Protoc 2019 12 20;14(12):3506-3537. Epub 2019 Nov 20.

Laboratory for Synthetic Biology, RIKEN Center for Biosystems Dynamics Research, Osaka, Japan.

Tissue-clearing techniques are powerful tools for biological research and pathological diagnosis. Here, we describe advanced clear, unobstructed brain imaging cocktails and computational analysis (CUBIC) procedures that can be applied to biomedical research. This protocol enables preparation of high-transparency organs that retain fluorescent protein signals within 7-21 d by immersion in CUBIC reagents. A transparent mouse organ can then be imaged by a high-speed imaging system (>0.5 TB/h/color). In addition, to improve the understanding and simplify handling of the data, the positions of all detected cells in an organ (3-12 GB) can be extracted from a large image dataset (2.5-14 TB) within 3-12 h. As an example of how the protocol can be used, we counted the number of cells in an adult whole mouse brain and other distinct anatomical regions and determined the number of cells transduced with mCherry following whole-brain infection with adeno-associated virus (AAV)-PHP.eB. The improved throughput offered by this protocol allows analysis of numerous samples (e.g., >100 mouse brains per study), providing a platform for next-generation biomedical research.
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http://dx.doi.org/10.1038/s41596-019-0240-9DOI Listing
December 2019

A Microfluidic Platform Based on Robust Gas and Liquid Exchange for Long-term Culturing of Explanted Tissues.

Anal Sci 2019 ;35(10):1141-1147

Center for Biosystems Dynamics Research, RIKEN.

Microfluidic devices are important platforms to culture and observe biological tissues. Compared with conventional setups, microfluidic devices have advantages in perfusion, including an enhanced delivery of nutrients and gases to tissues. However, explanted tissues can maintain their functions for only hours to days in microfluidic devices, although their observations are desired for weeks. The suprachiasmatic nucleus (SCN) is a brain region composed of heterogeneous cells to control the biological clock system through synchronizing individual cells in this region. The synchronized and complicated cell-cell interactions of SCN cells are difficult to reproduce from seeded cells. Thus, the viability of explanted SCN contributes to the study of SCN functions. In this paper, we propose a new perfusion platform combining a PDMS microfluidic device with a porous membrane to culture an explanted SCN for 25 days. We expect that this platform will provide a universal interface for microfluidic manipulation of tissue explants.
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http://dx.doi.org/10.2116/analsci.19P099DOI Listing
February 2020

Novel 3D analysis using optical tissue clearing documents the evolution of murine rapidly progressive glomerulonephritis.

Kidney Int 2019 08 15;96(2):505-516. Epub 2019 Mar 15.

Department of Nephrology and Clinical Immunology, RWTH Aachen University Clinic, Aachen, Germany; Interdisciplinary Centre for Clinical Research (IZKF Aachen), RWTH Aachen University Hospital, Aachen, Germany; Heisenberg Chair for Preventive and Translational Nephrology, Division of Nephrology, RWTH Aachen University, Aachen, Germany. Electronic address:

Recent developments in optical tissue clearing have been difficult to apply for the morphometric analysis of organs with high cellular content and small functional structures, such as the kidney. Here, we establish combinations of genetic and immuno-labelling for single cell identification, tissue clearing and subsequent de-clarification for histoimmunopathology and transmission electron microscopy. Using advanced light microscopy and computational analyses, we investigated a murine model of crescentic nephritis, an inflammatory kidney disease typified by immune-mediated damage to glomeruli leading to the formation of hypercellular lesions and the rapid loss of kidney function induced by nephrotoxic serum. Results show a graded susceptibility of the glomeruli, significant podocyte loss and capillary injury. These effects are associated with activation of parietal epithelial cells and formation of glomerular lesions that may evolve and obstruct the kidney tubule, thereby explaining the loss of kidney function. Thus, our work provides new high-throughput endpoints for the analysis of complex tissues with single-cell resolution.
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http://dx.doi.org/10.1016/j.kint.2019.02.034DOI Listing
August 2019

A period without PER: understanding 24-hour rhythms without classic transcription and translation feedback loops.

F1000Res 2019 16;8. Epub 2019 Apr 16.

Laboratory for Synthetic Biology, RIKEN Center for Biosystems Dynamics Research, 1-3 Yamadaoka, Suita, Osaka, 565-0871, Japan.

Since Ronald Konopka and Seymour Benzer's discovery of the gene in the 1970s, the circadian rhythm field has diligently investigated regulatory mechanisms and intracellular transcriptional and translation feedback loops involving , and these investigations culminated in a 2017 Nobel Prize in Physiology or Medicine for Michael W. Young, Michael Rosbash, and Jeffrey C. Hall. Although research on 24-hour behavior rhythms started with , a series of discoveries in the past decade have shown us that post-transcriptional regulation and protein modification, such as phosphorylation and oxidation, are alternatives ways to building a ticking clock.
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http://dx.doi.org/10.12688/f1000research.18158.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468715PMC
June 2020

Comprehensive three-dimensional analysis (CUBIC-kidney) visualizes abnormal renal sympathetic nerves after ischemia/reperfusion injury.

Kidney Int 2019 07 9;96(1):129-138. Epub 2019 Apr 9.

Division of Nephrology and Endocrinology, The University of Tokyo Graduate School of Medicine, Tokyo, Japan.

The sympathetic nervous system is critical in maintaining the homeostasis of renal functions. However, its three-dimensional (3D) structures in the kidney have not been elucidated due to limitation of conventional imaging methods. CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis) is a newly developed tissue-clearing technique, which enables whole-organ 3D imaging without thin-sectioning. Comprehensive 3D imaging by CUBIC found that sympathetic nerves are primarily distributed around arteries in the mouse kidney. Notably, the sympathetic innervation density was significantly decreased 10 days after ischemia-reperfusion injury (voluminal ratio of innervation area to kidney) by about 70%. Moreover, norepinephrine levels in kidney tissue (output of sympathetic nerves) were significantly reduced in injured kidneys by 77%, confirming sympathetic denervation after ischemia-reperfusion injury. Time-course imaging indicated that innervation partially recovered although overall denervation persisted 28 days after injury, indicating a continuous sympathetic nervous abnormality during the progression of chronic kidney disease. Thus, CUBIC-kidney, the 3D imaging analysis, can be a strong imaging tool, providing comprehensive, macroscopic perspectives for kidney research.
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http://dx.doi.org/10.1016/j.kint.2019.02.011DOI Listing
July 2019

Next-generation human genetics for organism-level systems biology.

Curr Opin Biotechnol 2019 08 5;58:137-145. Epub 2019 Apr 5.

Laboratory for Synthetic Biology, RIKEN Center for Biosystems Dynamics Research, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan; Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; International Research Center for Neurointelligence (WPI-IRCN), UTIAS, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Electronic address:

Systems-biological approaches, such as comprehensive identification and analysis of system components and networks, are necessary to understand design principles of human physiology and pathology. Although reverse genetics using mouse models have been used previously, it is a low throughput method because of the need for repetitive crossing to produce mice having all cells of the body with knock-out or knock-in mutations. Moreover, there are often issues from the interspecific gap between humans and mice. To overcome these problems, high-throughput methods for producing knock-out or knock-in mice are necessary. In this review, we describe 'next-generation' human genetics, which can be defined as high-throughput mammalian genetics without crossing to knock out human-mouse ortholog genes or to knock in genetically humanized mutations.
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http://dx.doi.org/10.1016/j.copbio.2019.03.003DOI Listing
August 2019

Whole-Brain Analysis of Cells and Circuits by Tissue Clearing and Light-Sheet Microscopy.

J Neurosci 2018 10;38(44):9330-9337

International Research Center for Neurointelligence, UTIAS,

In this photo essay, we present a sampling of technologies from laboratories at the forefront of whole-brain clearing and imaging for high-resolution analysis of cell populations and neuronal circuits. The data presented here were provided for the eponymous Mini-Symposium presented at the Society for Neuroscience's 2018 annual meeting.
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http://dx.doi.org/10.1523/JNEUROSCI.1677-18.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6706004PMC
October 2018

Lost in clocks: non-canonical circadian oscillation discovered in cells.

Mol Syst Biol 2018 09 24;14(9):e8567. Epub 2018 Sep 24.

Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6151625PMC
http://dx.doi.org/10.15252/msb.20188567DOI Listing
September 2018

Easy and efficient production of completely embryonic-stem-cell-derived mice using a micro-aggregation device.

PLoS One 2018 19;13(9):e0203056. Epub 2018 Sep 19.

Laboratory for Integrated Biodevice, RIKEN Center for Biosystems Dynamics Research, 1-3 Yamadaoka, Suita, Osaka, Japan.

There is an increasing demand for genetically modified mice produced without crossing, for rapid phenotypic screening studies at the organismal level. For this purpose, generation of completely embryonic-stem-cell (ESC)-derived chimeric mice without crossing is now possible using a microinjection or aggregation method with 3i culture medium. However, the microinjection of ESCs into blastocyst, morula, or 8-cell-stage embryos requires a highly skilled operator. The aggregation method is an easier alternative, but the conventional aggregation protocol still requires special skills. To make the aggregation method easier and more precise, here we developed a micro-aggregation device. Unlike conventional 3-dimensional culture, which uses hanging-drop devices for aggregation, we fabricated a polystyrene funnel-like structure to smoothly drop ESCs into a small area (300-μm in diameter) at the bottom of the device. The bottom area was designed so that the surface tension of the liquid-air interface prevents the cells from falling. After aggregation, the cells can be recovered by simply exerting pressure on the liquid from the top. The microdevice can be set upon a regular 96-well plate, so it is compatible with multichannel pipette use or machine operation. Using the microdevice, we successfully obtained chimeric blastocysts, which when transplanted resulted in completely ESC-derived chimeric mice with high efficiency. By changing the number of ESCs in the aggregate, we found that the optimum number of co-cultured ESCs was around 90~120 per embryo. Under this condition, the efficiency of generating completely ESC-derived mice was the same or better than that of the injection method. These results indicated that our microdevice can be used to produce completely ESC-derived chimeric mice easily and with a high success rate, and thus represents a promising alternative to the conventional microinjection or aggregation method, especially for high-throughput, parallel experimental applications.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0203056PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6145547PMC
February 2019

Leak potassium channels regulate sleep duration.

Proc Natl Acad Sci U S A 2018 10 17;115(40):E9459-E9468. Epub 2018 Sep 17.

Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, 113-0033 Tokyo, Japan;

A primary goal of sleep research is to understand the molecular basis of sleep. Although some sleep/wake-promoting circuits and secreted substances have been identified, the detailed molecular mechanisms underlying the regulation of sleep duration have been elusive. Here, to address these mechanisms, we developed a simple computational model of a cortical neuron with five channels and a pump, which recapitulates the cortical electrophysiological characteristics of slow-wave sleep (SWS) and wakefulness. Comprehensive bifurcation and detailed mathematical analyses predicted that leak K channels play a role in generating the electrophysiological characteristics of SWS, leading to a hypothesis that leak K channels play a role in the regulation of sleep duration. To test this hypothesis experimentally, we comprehensively generated and analyzed 14 KO mice, and found that impairment of the leak K channel () decreased sleep duration. Based on these results, we hypothesize that leak K channels regulate sleep duration in mammals.
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http://dx.doi.org/10.1073/pnas.1806486115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6176580PMC
October 2018

Muscarinic Acetylcholine Receptors Chrm1 and Chrm3 Are Essential for REM Sleep.

Cell Rep 2018 08;24(9):2231-2247.e7

Laboratory for Synthetic Biology, RIKEN Center for Biosystems Dynamics Research, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan; Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; International Research Center for Neurointelligence (WPI-IRCN), The University of Tokyo Institutes for Advanced Study (UTIAS), The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Electronic address:

Sleep regulation involves interdependent signaling among specialized neurons in distributed brain regions. Although acetylcholine promotes wakefulness and rapid eye movement (REM) sleep, it is unclear whether the cholinergic pathway is essential (i.e., absolutely required) for REM sleep because of redundancy from neural circuits to molecules. First, we demonstrate that synaptic inhibition of TrkA+ cholinergic neurons causes a severe short-sleep phenotype and that sleep reduction is mostly attributable to a shortened sleep duration in the dark phase. Subsequent comprehensive knockout of acetylcholine receptor genes by the triple-target CRISPR method reveals that a similar short-sleep phenotype appears in the knockout of two Gq-type acetylcholine receptors Chrm1 and Chrm3. Strikingly, Chrm1 and Chrm3 double knockout chronically diminishes REM sleep to an almost undetectable level. These results suggest that muscarinic acetylcholine receptors, Chrm1 and Chrm3, are essential for REM sleep.
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http://dx.doi.org/10.1016/j.celrep.2018.07.082DOI Listing
August 2018