Biology subjects

Shimizu, T.

Publications and source records attributed to Shimizu, T..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Structural insight into the nuclear transportation mechanism of PPARg by Transportin-1

The spatial and temporal control of protein is essential for normal cellular function. Proteins working in the nucleus have nuclear localization signal (NLS) sequences and are escorted into the nucleus by cognate nuclear transport receptors. A wealth of experimental data about NLS has been accumulated, and nuclear transportation mechanisms are established at the biochemical and structural levels. The peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors that control various biological responses. We recently reported that the transportation of PPAR{gamma} is mediated by Transportin-1, but PPAR{gamma} lacks a typical NLS sequence recognized by Transportin-1. Moreover, the recognition mechanism remains largely unknown. In this study, we determined the Cryo-EM structure of PPAR{gamma} in complex with Transportin-1 and revealed that Transportin-1 gripped the folded DNA binding domain and the Hinge region of PPAR{gamma}, indicating that PPAR{gamma} recognizes a folded domain with an extended region as a nuclear localization signal, not a canonical unstructured signal sequence, confirmed by the mutation analyses in vitro and in cultured cells. Our study is the first snapshot structure working in nuclear transportation, not in transcription, of PPAR{gamma}.

molecular biology↗

bioRxiv2024

Evolutionary conserved RLF, a plant cytochrome b5-like heme-binding protein, is essential for organ development in Marchantia polymorpha

In Arabidopsis thaliana, REDUCED LATERAL ROOT FORMATION (RLF), a cytochrome b5-like heme-binding domain (Cytb5-HBD) protein, is necessary for proper lateral root formation. Whereas the other Cytb5-HBD proteins in A. thaliana regulate different metabolic reactions, RLF is unique as it specifically regulates organ development. However, it remains unknown whether heme binding to RLF is necessary for its function, and whether RLF orthologs in different plant species also regulate organ development. We demonstrate that RLF binds to heme in vitro and that the two histidine residues, which are conserved among Cytb5-HBD, are crucial for both heme binding and its biological function in A. thaliana. In addition, MpRLF, a RLF ortholog in Marchantia polymorpha, rescues the lateral root formation phenotype of the A. thaliana rlf mutant. Mprlfge, the loss-of-function mutation in the MpRLF, resulted in delayed thallus growth and inhibited both gemma cup and antheridiophore formation. Transcriptome analysis using Mprlfge revealed that MpRLF affects several metabolic pathways. Our findings indicate that MpRLF is essential for vegetative and reproductive development in M. polymorpha, suggesting that the RLF-dependent redox reaction systems are evolutionarily conserved as crucial mechanisms for organ development across diverse plant species.

plant biology↗

bioRxiv2024

2'-O-Methyl-guanosine 3-base RNA fragments mediate essential natural TLR7/8 antagonism

Recognition of RNA fragments by Toll-like receptors (TLR) 7 and 8 is a key contributor to the initiation of a protective innate immune response against pathogens. A long-standing enigma is how degradation products of host RNAs, generated by the daily phagocytic clearance of billions of apoptotic cells, fail to activate TLR7 and TLR8 signalling1. Here, we report that select 2-O-methyl (2-Ome) guanosine RNA fragments as short as 3 bases, including those derived from host-RNAs, are potent TLR7 and TLR8 antagonists that reduce TLR7 sensing in vivo. Mechanistically, antagonistic fragments are directed towards a distinct binding site on these proteins by 5-end 2-Ome guanosine. Our results indicate that host-RNAs evade detection by TLR7/8 due to a pool of abundant host ribosomal 2-Ome-modified RNA fragments that naturally antagonize TLR7 and TLR8 sensing to avoid auto-immunity. Crucially, rare TLR7 and TLR8 mutations located at this antagonistic site decrease the inhibitory activity of 2-Ome guanosine RNA fragments and lead to auto-immunity in patients. Our findings also establish that select chemically synthesised 3-base oligonucleotides can harness the protective anti-inflammatory activity of this natural immune checkpoint for therapeutic targeting of TLR7-driven diseases. One Sentence SummaryShort 2-O-Methyl RNA fragments are natural TLR7/8 antagonists

immunology↗

bioRxiv2024

TRPC6 is a mechanosensitive channel essential for ultrasound neuromodulation in mammalian brain

Ultrasound neuromodulation has become an innovative technology that enables non-invasive intervention in mammalian brain circuits with high spatiotemporal precision. Despite the expanding utility of ultrasound neuromodulation in the neuroscience research field and clinical applications, the molecular and cellular mechanisms by which ultrasound impacts neural activity in the brain are still largely unknown. Here, we report that transient receptor potential canonical 6 (TRPC6), a mechanosensitive non-selective cation channel, is essential for ultrasound neuromodulation of mammalian neurons in vitro and in vivo. We first demonstrated that ultrasound irradiation elicited rapid and robust Ca2+ transients mediated via extracellular Ca2+ influx in cultured mouse cortical and hippocampal neurons. Ultrasound-induced neuronal responses were massively diminished by blocking either the generation of action potential or synaptic transmission. Importantly, both pharmacological inhibition and genetic deficiency of TRPC6 almost completely abolished neuronal responses to ultrasound. Furthermore, we found that intracerebroventricular administration of a TRPC6 blocker significantly attenuated the population of neuronal firings in the cerebral cortex evoked by transcranial ultrasound irradiation in mice. Our findings indicate that TRPC6 is an indispensable molecule of ultrasound neuromodulation in the intact mammalian brains, providing fundamental understanding of biophysical molecular mechanisms of ultrasound neuromodulation as well as insight into its future feasibility in neuroscience and translational researches in humans.

neuroscience↗

bioRxiv2024

Impact of perinatal maternal docosahexaenoic acid-containing phospholipid synthesis on offspring growth and neurological symptoms

Mothers provide essential nutrients, including docosahexaenoic acid (DHA), an omega-3 fatty acid, during the perinatal period. DHA deficiency in perinatal mothers is linked to developmental abnormalities, especially in the central nervous system of the offspring; however, its specific impact on distinct events in fetal and neonatal brain development and prospective brain functions remains incompletely understood. We demonstrated using mice lacking Agpat3, a gene encoding the enzyme that synthesizes DHA-containing phospholipids (DHA-PLs), that maternal DHA-PL synthesis significantly contributes to the maternal- offspring DHA supply during the fetal period but not in infancy. Selective modulation of DHA-PL levels during fetal and postnatal periods in Agpat3-knockout mice showed that fetal stage-specific insufficiency in maternal DHA-PL supply potentially influences the neuropsychiatric phenotype in adult mice without affecting postnatal tissue DHA-PL levels, weight gain, and brain expansion. Collectively, enhancing maternal DHA-PL synthesis during pregnancy may help prevent prospective neuropsychiatric abnormalities in the offspring.

biochemistry↗

bioRxiv2023

Complete suspension culture of human induced pluripotent stem cells supplemented with suppressors of spontaneous differentiation

Human induced pluripotent stem cells (hiPSCs) are promising resources for producing various types of tissues in regenerative medicine; however, the improvement in a scalable culture system that can precisely control the cellular status of hiPSCs is needed. Utilizing suspension culture without microcarriers or special materials allows for massive production, automation, cost-effectiveness, and safety assurance in industrialized regenerative medicine. Here, we found that hiPSCs cultured in suspension conditions with continuous agitation without any microcarriers or extracellular matrix components were more prone to spontaneous differentiation than those cultured in conventional adherent conditions. Adding PKC{beta} and Wnt signaling pathway inhibitors in the suspension conditions suppressed the spontaneous differentiation of hiPSCs into ectoderm and mesendoderm, respectively. In these conditions, we successfully completed the culture processes of hiPSCs including the generation of hiPSCs from peripheral blood mononuclear cells with the expansion of bulk population and single-cell sorted clones, long-term culture with robust self-renewal characteristics, single-cell cloning, direct cryopreservation from suspension culture and their successful recovery, and efficient mass production of a clinical-grade hiPSC line. Our results demonstrate that precise control of the cellular status in suspension culture conditions paves the way for their stable and automated clinical application.

bioengineering↗

bioRxiv2023

Hyphal exploration strategies and habitat modification of an arbuscular mycorrhizal fungus in microengineered soil chips

Arbuscular mycorrhizal fungi (AMF) are considered ecosystem engineers, however, the exact mechanisms by which they modify and influence their immediate surroundings are largely unknown and difficult to study in soil. In this study, we used microfluidic chips, simulating artificial soil structures, to study foraging strategies and habitat modification of Rhizophagus irregularis in symbiotic state associated to carrot roots. Our results suggest that AMF hyphae forage over long distances in void spaces, prefer straight over tortuous passages, anastomose and show strong inducement of branching when encountering obstacles. We observed bi-directional vesicle transport inside active hyphae and documented strategic allocation of biomass within the mycelium e.g., truncated hyphal growth and cytoplasm retraction from inefficient paths. We found R. irregularis able to modify pore-spaces in the chips by producing irregularly shaped spores that filled up pores. We suggest that studying AMF hyphal behaviour in spatial settings can explain phenomena reported at bulk scale such as AMF modification of water retention in soils. The use of microfluidic soil chips in AMF research opens up novel opportunities to under very controlled conditions study ecophysiology and interactions of the mycelium with both biotic and abiotic factors.

microbiology↗

bioRxiv2023

Ablation of oligodendrogenesis in adult mice alters brain microstructure and activity independently of behavioural deficits

Oligodendrocytes continue to differentiate from their precursor cells even in adulthood, a process that can be modulated by neuronal activity and experience. Yet, our understanding of the functional role of adult oligodendrogenesis remains limited. Previous work has indicated that conditional ablation of oligodendrogenesis in adult mice can lead to learning and memory deficits in a range of behavioural tasks. Our results, reported here, have replicated a key finding that learning to run on a complex wheel with unevenly spaced rungs is disrupted by ablation of oligodendrogenesis. However, using ex vivo MRI (MTR and DTI), we also found that ablating oligodendrogenesis by itself alters brain microstructure, independent of behavioural experience. Furthermore, in vivo EEG recording in behaviourally naive mice with ablated oligodendrogenesis revealed altered brain activity in the form of increased EEG power density across a broad frequency range. Together, our data indicate that disrupting the formation of new oligodendrocytes directly alters brain microstructure and activity. This suggests a role for adult oligodendrogenesis in the maintenance of brain function and indicates that task-independent changes to brain structure and function might contribute to the learning and memory deficits associated with oligodendrogenesis ablation.

neuroscience↗

bioRxiv2023

Opioidergic activation of descending pain inhibitory system underlies placebo analgesia

Placebo analgesia is caused by inactive treatment, implicating endogenous brain function involvement. However, the underlying neurobiological mechanisms remain unclear. We found that -opioid signals in the medial prefrontal cortex (mPFC) activate the descending pain inhibitory system to initiate placebo analgesia in neuropathic pain rats. Chemogenetic manipulation demonstrated that specific activation of -opioid receptor-positive (MOR+) neurons in the mPFC or suppression of the mPFC-ventrolateral periaqueductal gray (vlPAG) circuit inhibited placebo analgesia in rats. MOR+ neurons in the mPFC are monosynaptically connected and directly inhibit L5 pyramidal neurons that project to the vlPAG via GABAA receptors. Thus, intrinsic opioid signaling in the mPFC disinhibits excitatory outflow to the vlPAG by suppressing MOR+ neurons, leading to descending pain inhibitory system activation that initiates placebo analgesia. One Sentence SummarySugar pills relieve pain by activating the intrinsic pain inhibitory system via opioidergic signals in the prefrontal cortex.

neuroscience↗

bioRxiv2023

Pax3 and Pax7 function in combination with Mitf to generate melanophores and xanthophores in medaka and zebrafish

Neural crest cells generate numerous derivatives, including pigment cells, and are a model for studying how fate specification from multipotent progenitors is controlled. In mammals, the core gene regulatory network (GRN) for melanocytes (their only pigment cell-type) contains three transcription factors Sox10, Pax3 and Mitf, with the latter considered a master regulator of melanocyte development. In teleosts, which have three to four pigment cell types (melanophores, iridophores and xanthophores, plus leucophores e.g. in medaka), GRNs governing fate specification are poorly understood, although Mitf function is considered conserved. Here, we show that the regulatory relationships between Sox10, Pax3 and Mitf are conserved in zebrafish, but the role for Mitf is more complex than previously emphasised, affecting xanthophore development too. Similarly, medaka Mitf is necessary for melanophore, xanthophore and leucophore formation. Furthermore, expression patterns and mutant phenotypes of pax3 and pax7 suggest that Pax3 and Pax7 act sequentially, activating mitf expression. Pax7 modulates Mitf function, driving co-expressing cells to differentiate as xanthophores and leucophores rather than melanophores. We propose that pigment cell fate specification is better considered as resulting from the combinatorial activity of Mitf with other transcription factors.

developmental biology↗

bioRxiv2023

Foxp- and Skor-family proteins control differentiation of Purkinje cells from Ptf1a and Neurogenin1-expressing progenitors in zebrafish

Cerebellar neurons such as Purkinje cells (PCs) and granule cells (GCs) are differentiated from neural progenitors expressing proneural genes. Zebrafish mutants of proneural genes ptf1a and neurogenin1 showed a reduction or loss of PCs, GABAergic interneurons (INs), and reduced expression of GC progenitor genes atoh1a/b/c. Lineage tracing revealed that the ptf1a-expressing progenitors gave rise to PCs, INs, and a part of GCs in zebrafish. These data indicate that the ptf1a/neurognin1-expressing neural progenitors can generate a variety of cerebellar neurons. In this study, we found that genes encoding transcriptional regulators Foxp1b and Foxp4, as well as Skor1b and Skor2, which are reportedly expressed in PCs, were not expressed in ptf1a;neurogenin1 mutants. foxp1b;foxp4 mutants showed a strong reduction in PCs, while skor1b;skor2 mutants completely lacked PCs but instead displayed an increase in immature GCs. Misexpression of skor2 in GC progenitors expressing atoh1c suppressed GC fate. These data indicate that Foxp1b/4 and Skor1b/2 function as key transcriptional regulators in the initial step of PC differentiation from ptf1a/neurogenin1-expressing neural progenitors, while Skor1b and Skor2 control PC differentiation by suppressing their differentiation into GCs.

developmental biology↗

bioRxiv2023

Comprehensive analysis of autophagic functions of WIPI family proteins and their implications for the pathogenesis of β-propeller associated neurodegeneration

{beta}-propellers that bind polyphosphoinositides (PROPPINs) are an autophagy-related protein family conserved throughout eukaryotes. The PROPPIN family includes Atg18, Atg21, and Hsv2 in yeast and WD-repeat protein interacting with phosphoinositides (WIPI)1-4 in mammals. Mutations in the WIPI genes are associated with human neuronal diseases, including {beta}-propeller associated neurodegeneration (BPAN) caused by mutations in WDR45 (encoding WIPI4). In contrast to yeast PROPPINs, the functions of mammalian WIPI1-WIPI4 have not been systematically investigated. Although the involvement of WIPI2 in autophagy has been clearly shown, the functions of WIPI1, WIPI3, and WIPI4 in autophagy remain poorly understood. In this study, we comprehensively analyzed the roles of WIPI proteins by using WIPI-knockout (single, double, and quadruple knockout) HEK293T cells and recently developed HaloTag-based reporters, which enable us to monitor autophagic flux sensitively and quantitatively. We found that WIPI2 was nearly essential for autophagy and partially redundant with WIPI1. Autophagic flux was unaffected or only slightly reduced by single deletion of WIPI3 (encoded by WDR45B) or WIPI4 but was profoundly reduced by double deletion of WIPI3 and WIPI4. Furthermore, we revealed variable effects of BPAN-related missense mutations on the autophagic activity of WIPI4. BPAN is characterized by neurodevelopmental and neurodegenerative abnormalities, and we found a possible association between the magnitude of the defect of the autophagic activity of WIPI4 mutants and the severity of neurodevelopmental symptoms. However, some of the BPAN-related missense mutations, which produce neurodegenerative signs, showed almost normal autophagic activity, suggesting that non-autophagic functions of WIPI4 may be related to neurodegeneration in BPAN.

cell biology↗

bioRxiv2023

Oligodendrocyte dynamics dictate individual performance outcomes of working memory training in mice

Motor skill learning stimulates and requires generation of myelinating oligodendrocytes (OLs) from their precursors (OLPs). We asked whether OL production is also required for non-motor learning and cognition, using T-maze and radial arm maze tasks that tax spatial working memory. Maze training stimulated OL production in the medial prefrontal cortex (mPFC), anterior corpus callosum (genu), dorsal thalamus and hippocampal formation; myelin sheath formation was also stimulated in the genu. Genetic blockade of OL differentiation and neo-myelination in Myrf conditional-knockout mice strongly impaired training-induced improvements in maze performance. Remarkably, there was a strong positive correlation between working memory performance of individual mice and the scale of OLP proliferation and OL generation during training, but not with the number or intensity of c-Fos+ neurons in the mPFC, underscoring the key role of OL lineage cells in cognitive performance.

neuroscience↗

bioRxiv2022

Median eminence myelin continuously turns over in adult mice

ObjectiveOligodendrocyte progenitor cell differentiation is regulated by nutritional signals in the adult median eminence (ME), but the consequences on local myelination are unknown. The aim of this study was to characterise myelin plasticity in the ME of adult mice in health or in response to chronic nutritional challenge. MethodsWe assessed new oligodendrocyte and myelin generation and stability in the ME of healthy adult male mice using bromodeoxyuridine labelling and genetic fate mapping tools. We assessed the contribution of microglia to ME myelin plasticity in PLX5622-treated C57BL6/J mice and in Pdgfra-Cre/ERT2;R26R-eYFP;Myrffl/fl mice, where adult oligodendrogenesis is genetically blunted. Finally, we investigated how 45% high fat diet or 70% caloric restriction feeding paradigms impact ME oligodendrocyte lineage progression and myelination in C57BL6/J mice. ResultsWe show that myelinating oligodendrocytes (OLs) are continuously and rapidly generated in the adult ME. Paradoxically, OL number and myelin amounts remain remarkably stable in the adult ME. In fact, the high rate of new OL and myelin generation in the ME is offset by continuous turnover of both. We show that microglia are required for continuous OL and myelin production, and that ME myelin plasticity regulates the recruitment of local immune cells. Finally, we provide evidence that ME myelination is regulated by the bodys energetic status, decreased in calorie-restricted animals, and increased in mice fed a high fat diet. ConclusionsThis study uncovers a previously unappreciated form of ME structural plasticity and mechanism of myelin remodeling in the adult brain.

neuroscience↗

bioRxiv2022

Optogenetic manipulation of second messengers in neurons and cardiomyocytes with microbial rhodopsins and adenylyl cyclase

Even though microbial photosensitive proteins have been used for optogenetics, their use should be optimized to precisely control second messengers in vivo. We exploited GtCCR4 and KnChR, cation channelrhodopsins from algae, BeGC1, a guanylyl cyclase rhodopsin from a fungus, and photoactivated adenylyl cyclases (PACs) from cyanobacteria (OPAC) or bacteria (bPAC), to control cell functions in zebrafish. Optical activation of GtCCR4 and KnChR in the hindbrain reticulospinal V2a neurons, which are involved in locomotion, immediately induced swimming behavior, whereas activation of BeGC1 or PACs was achieved at a short latency. KnChR had the highest locomotioninducing activity of all the channelrhodopsins examined. Activation of GtCCR4 and KnChR in cardiomyocytes induced cardiac arrest, whereas activation of bPAC gradually induced bradycardia. KnChR activation led to an increase in intracellular Ca2+ in the heart, suggesting that depolarization caused cardiac arrest. These data suggest that these optogenetic tools can be used to reveal the roles of second messengers in various cell types in vertebrates. Impact statementWe identified efficient and useful microbial channelrhodopsin, guanylyl cyclase rhodopsin, and photoactivated adenylyl cyclase that regulate neural activity and cardiac function in zebrafish. Major subject areasNeuroscience, Cell biology Research organismZebrafish (Danio rerio)

neuroscience↗

bioRxiv2022

Optogenetic manipulation of Gq-and Gi/o-coupled receptor signaling in neurons and heart muscle cells

G protein-coupled receptors (GPCRs) transmit extracellular signals into the cell depending on the type of G protein. To analyze the functions of GPCR signaling, we developed optogenetic tools using animal G protein-coupled bistable rhodopsins that can be controlled into active and inactive states by light irradiation. We expressed Gq- and Gi/o-coupled bistable rhodopsins in hindbrain reticulospinal V2a neurons, which are involved in locomotion, or in cardiomyocytes of zebrafish. Light stimulation of the reticulospinal V2a neurons expressing Gq-coupled spider Rh1 resulted in an increase in the level of cytoplasmic Ca2+ and evoked swimming behavior. Light stimulation of cardiomyocytes expressing the Gi/o-coupled mosquito Opn3, pufferfish TMT opsin, or lamprey parapinopsin induced cardiac arrest, and the effect was suppressed by treatment with pertussis toxin or barium, suggesting that Gi/o-dependent regulation of inward-rectifier K+ channels controls cardiac function. These data indicate that these rhodopsins are useful for optogenetic control of GPCR-mediated signaling in neurons and cardiomyocytes in vivo. Impact statementAnimal G protein-coupled bistable rhodopsins can regulate Gq and Gi-mediated signaling in a light-dependent manner in neurons and cardiomyocytes, making them useful for analyzing roles of GPCR signaling in vivo.

neuroscience↗

bioRxiv2022

TLR7/8 stress response drives histiocytosis in SLC29A3 disorders

SLC29A3, also known as ENT3, is a lysosomal transmembrane protein that transports nucleosides from the lysosomes to the cytoplasm1. Loss-of-function mutations in SLC29A3 cause lysosomal nucleoside storage and histiocytosis: phagocyte accumulation in multiple organs2,3. However, little is known about the mechanism through which lysosomal nucleoside storage drives histiocytosis. Herein, histiocytosis in Slc29a3-/- mice was demonstrated to depend on TLR7, which senses a combination of nucleosides and oligoribonucleotides4,5. TLR7 responded to lysosomal nucleoside storage and enhanced proliferation of Ly6Chi CX3CR1low immature monocytes and their maturation into Ly6Clow phagocytes in Slc29a3-/- mice. Because accumulated nucleosides primarily originated from cell corpse phagocytosis, TLR7 in immature monocytes recognized nucleoside storage as lysosomal stress and increased phagocyte numbers. This non-inflammatory compensatory response is referred to as the TLR7 stress response where Syk, GSK3{beta}, {beta}-catenin, and mTORC1 serve as downstream signalling molecules. In SLC29A3 disorders, histiocytosis accompanies inflammation6,7. Nucleoside storage failed to induce pro-inflammatory cytokine production in Slc29a3-/- mice, but enhanced ssRNA-dependent pro-inflammatory cytokine production in Ly6Chi classical monocytes and peripheral macrophages, not proliferating immature monocytes. Patient-derived monocytes harbouring G208R SLC29A3 mutation showed higher survival and proliferation in the presence of M-CSF and produced larger amounts of IL-6 upon ssRNA stimulation than did those derived from healthy subjects. A TLR8 antagonist inhibited the survival/proliferation of patient-derived macrophages. These results demonstrated that TLR7/8 responses to lysosomal nucleoside stress drive SLC29A3 disorders.

immunology↗

bioRxiv2022

Human Dectin-1 is O-glycosylated and serves as a ligand for C-type lectin receptor CLEC-2

C-type lectin receptors (CLRs) elicit immune responses upon recognition of glycoconjugates present on pathogens and self-components. While Dectin-1 is the best-characterized CLR recognizing {beta}-glucan on pathogens, the endogenous targets of Dectin-1 are not fully understood. Herein, we report that human Dectin-1 is a ligand for CLEC-2, another CLR expressed on platelets. Biochemical analyses revealed that Dectin-1 is a mucin-like protein as its stalk region is highly O-glycosylated. A sialylated core 1 glycan attached to the EDxxT motif of human Dectin-1, which is absent in mouse Dectin-1, provides a ligand moiety for CLEC-2. Strikingly, the expression of human Dectin-1 in mice rescued the lethality and lymphatic defect resulting from a deficiency of Podoplanin, a known CLEC-2 ligand. This finding is the first example of an innate immune receptor also functioning as a physiological ligand to regulate ontogeny upon glycosylation.

immunology↗