Biology subjects

Ishii, H.

Publications and source records attributed to Ishii, H..

35 records · Page 2Linked to original sources

bioRxiv2021

Astroglial CD38 regulates social memory and synapse formation through SPARCL1 in the medial prefrontal cortex

Social behavior is essential for the health, survival and reproduction of animals, yet the role of astrocytes in social behavior is largely unknown. CD38 is critical for social behaviors by regulating oxytocin release from hypothalamic neurons. On the other hand, CD38 is most abundantly expressed in astrocytes especially in the postnatal cortex, and is important for astroglial development. Here, we demonstrate that astroglial CD38 plays a pivotal role in the social behavior. Selective deletion of CD38 in postnatal astrocytes, but not in adult astrocytes, specifically impaired social memory without any other behavioral abnormalities. Morphological analysis revealed reductions in spine numbers, mature spines and excitatory synapse numbers in the pyramidal neurons of the medial prefrontal cortex (mPFC) due to deletion of astroglial CD38 in the postnatal brain. Astrocyte-conditioned medium (ACM) of CD38 KO astrocytes reduced synaptogenesis of cortical neurons by reducing extracellular SPARCL1, a synaptogenic protein. Finally, the release of SPARCL1 from astrocytes is regulated by CD38/cADPR/calcium signaling. Our data indicate that astroglial CD38 developmentally regulates social memory and neural circuit formation in the developing brain by promoting synaptogenesis through SPARCL1.

neuroscience↗

bioRxiv2021

Subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells in cynomolgus macaques

SARS-CoV-2 infection presents clinical manifestations ranging from asymptomatic to fatal respiratory failure. Despite the induction of functional SARS-CoV-2-specific CD8+ T-cell responses in convalescent individuals, the role of virus-specific CD8+ T-cell responses in the control of SARS-CoV-2 replication remains unknown. In the present study, we show that subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells in cynomolgus macaques. Eight macaques were intranasally inoculated with 105 or 106 TCID50 of SARS-CoV-2, and three of the eight macaques were treated with a monoclonal anti-CD8 antibody on days 5 and 7 post-infection. In these three macaques, CD8+ T cells were undetectable on day 7 and thereafter, while virus-specific CD8+ T-cell responses were induced in the remaining five untreated animals. Viral RNA was detected in nasopharyngeal swabs for 10-17 days post-infection in all macaques, and the kinetics of viral RNA levels in pharyngeal swabs and plasma neutralizing antibody titers were comparable between the anti-CD8 antibody treated and untreated animals. SARS-CoV-2 RNA was detected in the pharyngeal mucosa and/or retropharyngeal lymph node obtained at necropsy on day 21 in two of the untreated group but undetectable in all macaques treated with anti-CD8 antibody. CD8+ T-cell responses may contribute to viral control in SARS-CoV-2 infection, but our results indicate possible containment of subacute viral replication in the absence of CD8+ T cells, implying that CD8+ T-cell dysfunction may not solely lead to viral control failure. Author SummarySARS-CoV-2 infection presents a wide spectrum of clinical manifestations ranging from asymptomatic to fatal respiratory failure. The determinants for failure in viral control and/or fatal disease progression have not been elucidated fully. Both acquired immune effectors, antibodies and CD8+ T cells, are considered to contribute to viral control. However, it remains unknown whether a deficiency in either of these two arms is directly linked to failure in the control of SARS-CoV-2 replication. In the present study, to know the requirement of CD8+ T cells for viral control after the establishment of infection, we examined the effect of CD8+ cell depletion by monoclonal anti-CD8 antibody administration in the subacute phase on SARS-CoV-2 replication in cynomolgus macaques. Unexpectedly, our analysis revealed no significant impact of CD8+ cell depletion on viral replication, indicating that subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells. CD8+ T-cell responses may contribute to viral control in SARS-CoV-2 infection, but this study suggests that CD8+ T-cell dysfunction may not solely lead to viral control failure or fatal disease progression.

microbiology↗

bioRxiv2021

The ATF6β-calreticulin axis protects hippocampal neurons against endoplasmic reticulum stress and excitotoxicity

While ATF6 plays a central role in the endoplasmic reticulum (ER) stress response, the function of ATF6{beta} is largely unknown. Here, we demonstrate that ATF6{beta} is highly expressed in the hippocampus of the brain, and specifically regulates the expression of calreticulin, a molecular chaperone in the ER with a high Ca2+-binding capacity. Calreticulin expression was reduced to ~50% in the central nervous system of Atf6b-/- mice, and restored by ATF6{beta}. Analysis using cultured hippocampal neurons revealed that ATF6{beta} deficiency reduced Ca2+ stores in the ER and enhanced ER stress-induced death, which was rescued by ATF6{beta}, calreticulin, Ca2+-modulating reagents such as BAPTA-AM and 2-APB, and ER stress inhibitor salubrinal. In vivo, kainate-induced neuronal death was enhanced in hippocampi of Atf6b-/- and Calr+/- mice, and restored by 2-APB and salubrinal. These results suggest that the ATF6{beta}-calreticulin axis plays a critical role in the neuronal survival by improving Ca2+ homeostasis under ER stress.

cell biology↗

bioRxiv2020

Synthesis and preclinical evaluation of MTP38 as a novel PET ligand for phophodiesterase 7 in the brain

PurposePhosphodiesterase (PDE) 7 is a potential therapeutic target for neurological and inflammatory diseases, although in-vivo visualization of PDE7 has not been successful. In this study, we aimed to develop [11C]MTP38 as a novel positron emission tomography (PET) ligand for PDE7. Methods[11C]MTP38 was radiosynthesized by 11C-cyanation of a bromo precursor with [11C]HCN. PET scans of rat and rhesus monkey brains and in-vitro autoradiography of brain sections derived from these species were conducted with [11C]MTP38. In monkeys, dynamic PET data were analyzed with an arterial input function to calculate the total distribution volume (VT). The non-displaceable binding potential (BPND) in the striatum was also determined by a reference tissue model with cerebellar reference. Finally, striatal occupancy of PDE7 by an inhibitor was calculated in monkeys according to changes in BPND. Results[11C]MTP38 was synthesized with radiochemical purity [≥] 99.4% and molar activity of 38.6 {+/-} 12.6 GBq/mol. Autoradiography revealed high radioactivity in the striatum and its reduction by non-radiolabeled ligands, in contrast with unaltered autoradiographic signals in other regions. In-vivo PET after radioligand injection to rats and monkeys demonstrated that radioactivity was rapidly distributed to the brain and intensely accumulated in the striatum relative to the cerebellum. Correspondingly, estimated VT values in the monkey striatum and cerebellum were 3.59 and 2.69 mL/cm3, respectively. The cerebellar VT value was unchanged by pretreatment with unlabeled MTP38. Striatal BPND was reduced in a dose-dependent manner after pretreatment with MTP-X, a PDE7 inhibitor. Relationships between PDE7 occupancy by MTP-X and plasma MTP-X concentration could be described by Hills sigmoidal function. ConclusionWe have provided the first successful preclinical demonstration of in-vivo PDE7 imaging with a specific PET radioligand. [11C]MTP38 is a feasible radioligand for evaluating PDE7 in the brain and is currently being applied to a first-in-human PET study.

neuroscience↗

bioRxiv2020

Noise-induced scaling in skull suture interdigitation

Sutures, the thin, soft tissue between skull bones, serve as the major craniofacial growth centers during postnatal development. In a newborn skull, the sutures are straight; however, as the skull develops, the sutures wind dynamically to form an interdigitation pattern. Moreover, the final winding pattern had been shown to have fractal characteristics. Although various molecules involved in suture development have been identified, the mechanism underlying the pattern formation remains unknown. In a previous study, we reproduced the formation of the interdigitation pattern in a mathematical model combining an interface equation and a convolution kernel. However, the generated pattern had a specific characteristic length, and the model was unable to produce a fractal structure with the model.In the present study, we focused on the anterior part of the sagittal suture and formulated a new mathematical model with time–space-dependent noise that was able to generate the fractal structure. We reduced our previous model to represent the linear dynamics of the centerline of the suture tissue and included a time–space-dependent noise term. We showed theoretically that the final pattern from the model follows a scaling law due to the scaling of the dispersion relation in the full model, which we confirmed numerically. Furthermore, we observed experimentally that stochastic fluctuation of the osteogenic signal exists in the developing skull, and found that actual suture patterns followed a scaling law similar to that of the theoretical prediction.Author summary Skull sutures (thin, undifferentiated tissue between bones) act as the growth centers for the skull. Sutures are straight at birth but later develop an interdigitated pattern that ultimately becomes a fractal structure. While our previous mathematical model of sutures generated a periodic pattern, the mechanism underlying the fractal structure formation remained to be elucidated. Here, we focused only on the anterior part of the sagittal suture and formulated a reduced model representing the initial linear phase of pattern formation with the addition of a time–space-dependent noise term. We showed analytically that the model generates patterns with a scaling law. This result was confirmed numerically and experimentally.View Full Text

developmental biology↗

bioRxiv2020

A Genetically Encoded and Versatile Probe for Fluorescence Polarization Detection

Biomolecular assemblies govern the physiology of cells. Their function often depends on the changes in molecular arrangements of constituents, both in the positions and orientations. While recent advancements of fluorescence microscopy including super-resolution microscopy have enabled us to determine the positions of fluorophores with unprecedented accuracy, monitoring orientation of fluorescently labeled molecules within living cells in real-time is challenging. Fluorescence polarization microscopy (FPM) reports the orientation of emission dipoles and is therefore a promising solution. For imaging with FPM, target proteins need labeling with fluorescent probes in a sterically constrained manner, but due to difficulties in the rational three-dimensional design of protein connection, universal method for constrained tagging with fluorophore was not available. Here we report POLArIS, a genetically encoded and versatile probe for molecular orientation imaging. Instead of using a direct tagging approach, we used a recombinant binder connected to a fluorescent protein in a sterically constrained manner and can target arbitrary biomolecules by combining with phage-display screening. As an initial test case of POLArIS, we developed POLArISact, which specifically binds to F-actin in living cells. We confirmed that the orientation of F-actin can be monitored by observing cells expressing POLArISact with FPM. In living starfish early embryos expressing POLArISact, we found actin filaments radially extending from centrosomes in association with microtubule asters during mitosis. By taking advantage of the genetically encoded nature, POLArIS can be used in a variety of living specimens including whole bodies of developing embryos and animals, and also expressed in a cell-type/tissue specific manner.

cell biology↗

bioRxiv2020

Chloroplast acquisition without the gene transfer in kleptoplastic sea slugs

Some sea slugs sequester chloroplasts from algal food in their intestinal cells and photosynthesize for months. This phenomenon, kleptoplasty, poses a question of how the chloroplast retains its activity without the algal nucleus. There have been debates on the horizontal transfer of algal genes to the animal nucleus. To settle the arguments, this study reported the genome of a kleptoplastic sea slug Plakobranchus ocellatus and found no evidence of photosynthetic genes encoded on the nucleus. Nevertheless, it was confirmed that light illumination prolongs the life of mollusk under starvation. These data presented a paradigm that a complex adaptive trait, as typified by photosynthesis, can be transferred between eukaryotic kingdoms by a unique organelle transmission without nuclear gene transfer. Our phylogenomic analysis showed that genes for proteolysis and immunity undergo gene expansion and are up-regulated in chloroplast-enriched tissue, suggesting that these molluskan genes are involved in the DNA-independent phenotype acquisition.

evolutionary biology↗

bioRxiv2020

A comprehensive framework for handling location error in animal tracking data

Animal tracking data are being collected more frequently, in greater detail, and on smaller taxa than ever before. These data hold the promise to increase the relevance of animal movement for understanding ecological processes, but this potential will only be fully realized if their accompanying location error is properly addressed. Historically, coarsely-sampled movement data have proved invaluable for understanding large scale processes (e.g., home range, habitat selection, etc.), but modern fine-scale data promise to unlock far more ecological information. While GPS location error can often be ignored in coarsely sampled data, fine-scale data require more care, and tools to do this have not kept pace. Current approaches to dealing with location error largely fall into two categories--either discarding the least accurate location estimates prior to analysis or simultaneously fitting movement and error parameters in a hidden-state model. In some cases these approaches can provide a level of correction, but they have known limitations, and in some cases they can be worse than doing nothing. Here, we provide a general framework to account for location error in the analysis of triangulated and trilatcralizcd animal tracking data, which includes GPS, Argos Doppler-shift, triangulated VHF, trilatcralized acoustic and cellular location data. We apply our error-modelselection framework to 190 GPS, cellular, and acoustic devices representing 27 models from 14 manufacturers. Collectively, these devices were used to track a wide range of taxa comprising birds, fish, reptiles, and mammals of different sizes and with different behaviors, in urban, suburban, and wild settings. In almost half of the tested device models, error-model selection was necessary to obtain the best performing error model, and in almost a quarter of tested device models, the reported DOP values were actually misinformative. Then, using empirical tracking data from multiple species, we provide an overview of modern, error-informed movement analyses, including continuous-time path reconstruction, home-range distribution, home-range overlap, speed, and distance estimation. Adding to these techniques, we introduce new error-informed estimators for outlier detection and autocorrelation visualization. Because error-induced biases depend on many factors--sampling schedule, movement characteristics, tracking device, habitat, etc.--differential bias can easily confound biological inference and lead researchers to draw false conclusions. We demonstrate how error-informed analyses on calibrated tracking data can provide more accurate estimates are that are insensitive to location error, and allow researchers to use all of their data.

ecology↗

bioRxiv2020

CTCF Promotes Long-range Enhancer-promoter Interactions and Lineage-specific Gene Expression in Mammalian Cells

Topologically associating domains (TAD) and insulated neighborhoods (INs) have been proposed to constrain enhancer-promoter communications to enable cell-type specific transcription programs, but recent studies show that disruption of TADs and INs resulted in relatively mild changes in gene expression profiles. To better understand the role of chromatin architecture in dynamic enhancer-promoter contacts and lineage-specific gene expression, we have utilized the auxin-inducible degron system to acutely deplete CTCF, a key factor involved in TADs and IN formation, in mouse embryonic stem cells (mESCs) and examined chromatin architecture and gene regulation during neural differentiation. We find that while CTCF depletion leads to global weakening of TAD boundaries and loss of INs, only a minor fraction of enhancer-promoter contacts are lost, affecting a small subset of genes. The CTCF-dependent enhancer-promoter contacts tend to be long-range, spanning hundreds of kilobases, and are established directly by CTCF binding to promoters. Disruption of CTCF binding at the promoter reduces enhancer-promoter contacts and transcription, while artificial tethering of CTCF to the promoter restores the enhancer-promoter contacts and gene activation. Genome-wide analysis of CTCF binding and gene expression across multiple mouse tissues suggests that CTCF-dependent promoter-enhancer contacts may regulate expression of additional mouse genes, particularly those expressed in the brain. Our results uncover both CTCF-dependent and independent enhancer-promoter contacts, and highlight a distinct role for CTCF in promoting enhancer-promoter contacts and gene activation in addition to its insulator function.

genomics↗

bioRxiv2019

Spatial organization of cortical actin alignments for the ooplasmic segregation of ascidian Ciona eggs

Spatial reorganization of cytoplasm in zygotic cells is critically important for establishing the body plans of many animal species. In ascidian zygotes, maternal determinants (mRNAs) are first transported to the vegetal pole a few minutes after the fertilization, and then to the future posterior side of the zygotes in later phase of the cytoplasmic reorganization, before the first cell division. Here, by using a novel fluorescence polarization microscope that reports the position and the orientation of fluorescently labeled proteins in living cells, we mapped the local alignments and the time-dependent changes of cortical actin networks in Ciona eggs. The initial cytoplasmic reorganization started with the contraction of vegetal hemisphere [~]20s after the fertilization induced [Ca2+] increase. Timing of the vegetal contraction was consistent with the emergence of highly aligned actin filaments at the cell cortex of vegetal hemisphere which ran perpendicular to the animal-vegetal axis. We propose that the first ooplasmic segregation is initiated by the local contraction of laterally aligned cortical actomyosin in the vegetal hemisphere, which in turn generates the convectional flow of cytoplasm within whole eggs. SUMMARY STATEMENTLocally distinct, transient emergence of cortical F-actin alignments were observed in live ascidian Ciona eggs during the first ooplasmic segregation by using fluorescence polarization microscopy.

cell biology↗

bioRxiv2019

A Theoretical Model of Telomere Length Regulation

Telomere length is maintained by a negative feedback mechanism that inhibits the telomerase activity when the telomere is sufficiently long. A prevailing explanation is that the negative feedback is caused by a mechanism that \"counts\" the number of regulatory protein molecules that are bound to the telomere. However, how such protein counting is accomplished is not clear. In this paper, I introduce a simple theoretical model to consider how the telomerase inhibition can depend on the telomere length. I show that this model is able to explain some key features of regulation of telomere length. While the real telomeres are more complex, this simple model may capture the essence of the mechanism.

molecular biology↗

bioRxiv2018

Genome-wide association meta-analysis identifies novel GP2 gene risk variants for pancreatic cancer in the Japanese population

The etiology of pancreatic cancer remains largely unknown. Here, we report the results of a meta-analysis of three genome-wide association studies (GWASs) comprising 2,039 pancreatic cancer cases and 32,592 controls, the largest sample size in the Japanese population. We identified 3 (13q12.2, 13q22.1, and 16p12.3) genome-wide significant loci (P<5.0x10-8) and 4 suggestive loci (P<1.0x10-6) for pancreatic cancer. Of these risk loci, 16p12.3 is novel; the lead SNP maps to rs78193826 (odds ratio (OR)=1.46, 95% CI=1.29-1.66, P=4.28x10-9), an Asian-specific, nonsynonymous glycoprotein 2 (GP2) gene variant predicted to be highly deleterious. Additionally, the gene-based GWAS identified a novel gene, KRT8, which is linked to exocrine pancreatic and liver diseases. The identified GP2 gene variants were pleiotropic for multiple traits, including type 2 diabetes, hemoglobin A1c (HbA1c) levels, and pancreatic cancer. Mendelian randomization analyses corroborated causality between HbA1c and pancreatic cancer. These findings suggest that GP2 gene variants are associated with pancreatic cancer susceptibility in the Japanese population, prompting further functional characterization of this locus.

genetics↗

bioRxiv2018

Hereditary Pancreatitis Model by Blastocyst Complementation in Mouse

Summary StatementThe present study is the first report for reproducing human disease by utilizing blastocyst complementation method and would lead to the development of novel therapy for human disease.\n\nThe application of pluripotent stem cell is expected to contribute to the elucidation of the unknown mechanism of human diseases. However, in vitro induction of cells in several organs, such as the pancreas and liver, remains difficult; therefore, reproduction of those diseases in a model has not been feasible. To reproduce human hereditary pancreatitis (HP), which is most frequently caused by the mutations in the PRSS1 gene, we performed the blastocyst complementation (BC) method. In the BC method, mouse embryonic stem (ES) cells harboring CRISPR/CAS9-mediated mutations in the Prss1 were injected into the blastocysts of deficient Pdx1 gene mice, which is a critical transcription factor in the pancreas. The results showed that the blastocysts injected into the Prss1-mutant ES cells induced trypsin activation. This implied that the mouse phenotype mimics that of human HP and that the BC method was useful for the reproduction and study of pancreatic disorders. The present study opens the possibility of investigating uncharacterized human diseases by utilizing the BC method.

developmental biology↗

bioRxiv2018

Re-Evaluating One-step Generation of Mice Carrying Conditional Alleles by CRISPR-Cas9-Mediated Genome Editing Technology

CRISPR-Cas9 gene editing technology has considerably facilitated the generation of mouse knockout alleles, relieving many of the cumbersome and time-consuming steps of traditional mouse embryonic stem cell technology. However, the generation of conditional knockout alleles remains an important challenge. An earlier study reported up to 16% efficiency in generating conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN) (2sgRNA-2ssODN). We re-evaluated this method from a large data set generated from a consortium consisting of 17 transgenic core facilities or laboratories or programs across the world. The dataset constituted 17,887 microinjected or electroporated zygotes and 1,718 live born mice, of which only 15 (0.87%) mice harbored 2 correct LoxP insertions in cis configuration indicating a very low efficiency of the method. To determine the factors required to successfully generate conditional alleles using the 2sgRNA-2ssODN approach, we performed a generalized linear regression model. We show that factors such as the concentration of the sgRNA, Cas9 protein or the distance between the placement of LoxP insertions were not predictive for the success of this technique. The major predictor affecting the methods success was the probability of simultaneously inserting intact proximal and distal LoxP sequences, without the loss of the DNA segment between the two sgRNA cleavage sites. Our analysis of a large data set indicates that the 2sgRNA-2ssODN method generates a large number of undesired alleles (>99%), and a very small number of desired alleles (<1%) requiring, on average 1,192 zygotes.

genetics↗

bioRxiv2018

Draft Genome Sequence of the Asian Pear Scab Pathogen, Venturia nashicola

Venturia nashicola, which causes scab disease of Asian pear, is a host-specific, biotrophic fungus, with a sexual stage that occurs during saprobic growth. V. nashicola is endemic to Asia and is regarded as a quarantine threat to Asian pear production outside of this continent. Currently, fungicide applications are routinely used to control scab disease. However, fungicide resistance in V. nashicola, as in other fungal pathogens, is an ongoing challenge and alternative control or prevention measures that include, for example, the deployment of durable host resistance, are required. A close relative of V. nashicola, V. pirina, causes scab disease of European pear. European pear displays non-host resistance (NHR) to V. nashicola and Asian pears are non-hosts of V. pirina. It is anticipated that the host specificity of these two fungi is governed by differences in their effector arsenals, with a subset responsible for activating NHR. The Pyrus-Venturia pathosystems provide a unique opportunity to dissect the underlying genetics of non-host interactions and to understand coevolution in relation to this potentially more durable form of resistance. Here, we present the first V. nashicola draft whole genome sequence (WGS), which is made up of 40,800 scaffolds (totalling 45 Mb) and 11,094 predicted genes. Of these genes, 1,232 are predicted to encode a secreted protein by SignalP, with 273 of these predicted to be effectors by EffectorP. The V. nashicola WGS will enable comparison to the WGSs of other Venturia spp. to identify effectors that potentially activate NHR in the pear scab pathosystems.

genomics↗

bioRxiv2017

Preservation of Chromatin Organization after Acute Loss of CTCF in Mouse Embryonic Stem Cells

The CCCTC-binding factor (CTCF) is widely regarded as a key player in chromosome organization in mammalian cells, yet direct assessment of the impact of loss of CTCF on genome architecture has been difficult due to its essential role in cell proliferation and early embryogenesis. Here, using auxin-inducible degron techniques to acutely deplete CTCF in mouse embryonic stem cells, we show that cell growth is severely slowed yet chromatin organization remains largely intact after loss of CTCF. Depletion of CTCF reduces interactions between chromatin loop anchors, diminishes occupancy of cohesin complex genome-wide, and slightly weakens topologically associating domain (TAD) structure, but the active and inactive chromatin compartments are maintained and the vast majority of TAD boundaries persist. Furthermore, transcriptional regulation and histone marks associated with enhancers are broadly unchanged upon CTCF depletion. Our results suggest CTCF-independent mechanisms in maintenance of chromatin organization.

genomics↗

bioRxiv2017

Histone H3 Lysine 4 methyltransferases MLL3 and MLL4 Modulate Long-range Chromatin Interactions at Enhancers

Regulation of gene expression in mammalian cells depends on long-range chromatin interactions between enhancers and promoters. Currently, the exact mechanisms that connect distal enhancers to their specific target promoters remain to be fully elucidated. Here we show that the histone H3 Lysine 4 monomethylation (H3K4me1) writer proteins MLL3 and MLL4 (MLL3/4) play an active role in this process. We demonstrate that in differentiating mouse embryonic stem cells, MLL3/4-dependent deposition of H3K4me1 at enhancers correlates with increased levels of chromatin interactions, whereas loss of MLL3/4 leads to greatly reduced frequencies of chromatin interactions and failure of lineage-specific gene expression programs. We further show that H3K4me1 facilitates recruitment of the Cohesin complex to chromatin in vitro and in vivo, providing a potential mechanism for MLL3/4 to promote chromatin looping. Taken together, our results support an active role for MLL3/4 in modulating chromatin organization at enhancers in mammalian cells.

genomics↗