Biology subjects

Ohtsuka, M.

Publications and source records attributed to Ohtsuka, M..

16 recordsLinked to original sources

bioRxiv2026

Tumor-associated tissue-resident macrophages drive pancreatic cancer progression through IGF1-IGF1R signaling

The specific contribution of tissue-resident macrophages (TRMs) to pancreatic ductal adenocarcinoma (PDAC) progression remains unclear. Here, we found that a high abundance of TRM-derived tumor-associated macrophages (TRM-TAMs) is an independent indicator of poor prognosis in patients with PDAC. To elucidate the underlying mechanism, we established an advanced organoid platform (iMac-FPCO), which incorporates macrophages derived from human induced pluripotent stem cells to reflect the differentiation process of TRMs. Single-cell transcriptomic analysis revealed this model recapitulates the transcriptional identity of TRM-TAMs in patient tissue. We demonstrated that TRM-TAMs drive cancer cell proliferation, while maintaining chemoresistance, and identified TRM-derived insulin-like growth factor 1 (IGF1) as the critical mediator. This result provides a rationale for why previous trials targeting IGF1 receptor (IGF1R) failed to improve survival in unselected patient populations. We hypothesize that stratifying patients by TRM-TAM abundance could help identify a responsive subgroup, thereby reviving IGF1R-targeted therapy as a viable treatment for PDAC.

cancer biology↗

bioRxiv2026

uniSynK: a ratio-optimized bicistronic chemically induced dimerization tool for robust induction of dendritic spine enlargement

Synaptic plasticity is widely implicated in learning and brain-state regulation. Although correlations between synaptic modifications and diverse brain functions have been extensively reported, establishing causality requires tools that can directly and selectively perturb synapses in vivo. Recently, the chemogenetic actuator SYNCit-K (SynK) enabled causal induction of dendritic spine enlargement and synaptic potentiation. However, SynK relies on the co-expression of two independent components delivered by separate viral vectors, making it difficult to reliably achieve appropriate expression levels of both components within the same neuron. Here, using mouse neocortical dissociated culture, we sought to develop a unified, single-vector version of SynK (uniSynK) that enables robust and safe induction of dendritic spine enlargement. We first found that each SynK component induces distinct, concentration-dependent abnormalities in neuronal morphology when overexpressed, underscoring the need for precise control of their expression ratio. To address this challenge, we engineered a single construct in which the two components are linked by internal ribosome entry site (IRES) variants with graded translational efficiencies. Systematic screening of IRES variants identified an optimized configuration that preferentially drives each component within its respective concentration range that preserves normal spine morphology. The optimized uniSynK efficiently induces dendritic spine enlargement, while exhibiting fewer off-target morphological abnormalities than observed in the multi-vector system when expression levels were not properly controlled. By simplifying experimental design, this single-vector, ratio-optimized synaptic chemogenetic tool provides a practical and broadly applicable platform that should facilitate causal interrogation of synaptic plasticity across neural circuits and behavior. Significance StatementSynaptic plasticity is thought to shape learning, brain states, and neuropsychiatric disease, but testing its causal roles requires tools that can directly manipulate synapses with minimal side effects. Here, we developed uniSynK, a single-vector chemogenetic system that induces dendritic spine enlargement by coordinating the expression of two functional components. By defining safe expression ranges and using IRES variants to optimize their ratio, uniSynK achieves robust spine enlargement while reducing unintended morphological abnormalities associated with imbalanced expression. This work provides both a practical tool for synaptic manipulation and a general strategy for optimizing multi-component biological systems.

neuroscience↗

bioRxiv2025

Revisiting the in vivo Functions of Coilin in Mice

Cajal bodies (CBs), first identified in the early 20th century, are classic nuclear structures organized primarily by the protein coilin. Although CBs have long been implicated in snRNA modification and maturation, their precise roles in mice remain unclear. Here, we revisit the functions of CBs by using genome editing to generate mice that lack nearly the entire coilin protein, retaining only the first seven amino acids. Mass-spectrometry analyses showed reduced 2'-O-methylation of U2 snRNA in coilin mutant liver; however, these reductions had minimal impact on overall gene expression and pre-mRNA splicing patterns. Additionally, detailed analysis recapitulated previously reported testicular size reductions and newly revealed decreased proportions of high-quality sperm. We also identified novel coilin-containing condensates distinct from genuine CBs in the nuclear pocket of elongating spermatids. Our findings implicate that coilin orchestrates membraneless organelles in various compartments, playing multifunctional roles beyond classical CB formation.

molecular biology↗

bioRxiv2025

Blockade of associative spine enlargement impairs awake cortical function

Awake cognition is usually attributed to arousal systems and ongoing electrical activity, yet these do not explain how cortical processing is coupled to prior context and memory formation. Associative synaptic plasticity provides a cellular mechanism for such coupling, but is viewed as memory-related rather than an online requirement for cognition. Here we developed SynC, an A/C-activated chemogenetic perturbation that selectively and reversibly blocks associative spine enlargement while sparing non-associative spine dynamics, excitability, synaptic transmission and NMDA receptor-mediated responses. In mice with broad frontoparietal SynC expression, acute A/C reversibly impaired open-field centre exploration, abolished laser-dot chasing, and delayed feeding initiation. During quiet-wake immobile epochs, cortical firing rates and {gamma}-band power were preserved, but pairwise spike correlations were reduced. Mice also intermittently showed sudden behavioural arrest with reduced {gamma} activity but without the robust {delta} elevation of slow-wave sleep. Critically, in vivo two-photon glutamate uncaging induced seconds-scale associative spine enlargement in [~]24% of neocortical spines, acutely blocked by SynC-A/C. These A/C-induced behavioural, circuit and spine-enlargement effects recovered within 1 h. Thus, wake-like firing and {gamma} activity are not sufficient to sustain awake cognitive function. Rapid associative spine enlargement is required to maintain functional cortical coupling and stable wakefulness.

neuroscience↗

bioRxiv2024

Inhibition of p38-MK2 pathway enhances the efficacy of microtubule inhibitors in breast cancer cells

Microtubule-targeting agents (MTAs) have been successfully translated from basic research into clinical therapies and have been widely used as first- and second-line chemotherapy drugs for various cancers. However, current MTAs exhibit positive responses only in subsets of patients and are often accompanied by side effects due to their impact on normal cells. This underscores an urgent need to develop novel therapeutic strategies that enhance MTA efficacy while minimizing toxicity to normal tissues. In this study, we demonstrate that inhibition of the p38-MK2 (MAP kinase-activated protein kinase 2) pathway sensitizes cancer cells to MTA treatment. We utilize CMPD1, a dual-target inhibitor, to concurrently suppress the p38-MK2 pathway and microtubule dynamicity. In addition to its established role as an MK2 inhibitor, we find that CMPD1 rapidly induces microtubule depolymerization, preferentially at the microtubule plus-end, leading to the inhibition of tumor growth and cancer cell invasion in both in vitro and in vivo models. Notably, 10 nM CMPD1 is sufficient to induce irreversible mitotic defects in cancer cells, but not in non-transformed normal cells, highlighting its high specificity to cancer cells. We further validate that a specific p38-MK2 inhibitor significantly potentiates the efficacy of sub-clinical concentrations of MTA. In summary, our findings suggest that the p38-MK2 pathway presents a promising therapeutic target in combination with MTAs in cancer treatment.

cancer biology↗

bioRxiv2024

Development of bright fluorescent auxin

Polar transport of the phytohormone auxin plays a crucial role in plant growth and response to environmental stimuli. Small-molecule tools that visualize auxin distribution in intact plants enable us to understand how plants dynamically regulate auxin transport to modulate growth. In this study, we developed a new fluorescent auxin probe, BODIPY-IAA2, which effectively visualizes auxin distribution in various plant tissues. We designed this probe to be transported by auxin transporters while lacking the ability to elicit auxin signaling. Using BODIPY as the fluorophore provides bright and stable fluorescence signals, making it suitable for live-imaging under standard fluorescent microscopy. We tested the probe with auxin reporter lines in Arabidopsis and performed yeast two-hybrid assays. The results showed that BODIPY-IAA2 did not activate auxin signaling through the auxin receptor TIR1. However, BODIPY-IAA2 did mildly compete with both exogenous and endogenous auxins for transport, indicating that the probe is transported by auxin transporters in vivo. The probe not only enables visualization of its tissue distribution but also allows sub-cellular staining, including the endoplasmic reticulum and tip regions in elongating cells in moss. We also observed unusual staining patterns in the main root of non-model parasitic plants where genetic transformation is not feasible. Our new fluorescent auxin probe demonstrates significant potential for detailed studies on auxin transport and distribution across diverse plant species.

plant biology↗

bioRxiv2024

A single microRNA miR-195 rescues the arrested B cell development induced by EBF1 deficiency

Accumulated studies have reported that hematopoietic differentiation was primarily regulated by transcription factors. Early B cell factor 1 (EBF1) is an essential transcription factor for B lymphopoiesis. Contrary to the canonical notion, we found that a single miRNA, miRNA-195 (miR-195) transduction let EBF1 deficient hematopoietic progenitor cells (HPCs) express CD19, carry out V(D)J recombination and class switch recombination, which implied that B cell matured without EBF1. A part of the mechanism was caused by FOXO1 accumulation via inhibition of FOXO1 phosphorylation pathways in which targets of miR-195 are enriched. These results suggested that some miRNA transductions could function as alternatives to transcription factors.

immunology↗

bioRxiv2023

Epileptic responses in marmosets induced by knockdown of CREB Regulated Transcription Coactivator 1 (CRTC1)

Animal models have contributed greatly to the development of anticonvulsant drugs. However, 20-30% of epilepsy patients still do not achieve adequate seizure control. It is important to understand the biological mechanisms of epilepsy in order to develop treatments and novel anticonvulsant drugs. Here we report CRTC1 knockdown (KD) induced abnormal cFOS expression beyond the injection sites followed by epileptic response by three local injections of short hairpin RNA (shRNA) in marmoset V1. Longitudinal monitoring of cortical-wide neural activity revealed transient changes in high frequency oscillations by cortical electrocorticography throughout the cortex, and a lesion in the temporal lobe was observed by MRI and postmortem histology. shCRTC1 KD marmosets showed severe lesions surrounding the injection site several months after injection. Glial cell activation may occur simultaneously or prior to IEG induction after shCRTC1 injection. Thus, the epileptic process from seizure onset to remission can be studied in this animal model.

neuroscience↗

bioRxiv2023

Akr1b7 functions as a master regulator in ovarian aging

At the beginning of ovarian aging, the ovulation of immature oocytes is accelerated, leading to the arrest of ovulation despite the remaining oocytes. Here, RNA expression in the ovarian aging of mice is comprehensively analyzed during the estrous cycle after ovulation stimulation. The aldo-keto reductase Akr1b7 pathway transiently activated in the ovaries of young mice disappears in those of old mice. Akr1b7--/-- mice attenuate oocyte Akt activation essential for the follicular development in primordial follicles, and enhanced ovulation in immature oocytes. The estrous cycle is extended because of the prolonged diestrous stage by a sustained progesterone level in Akr1b7--/--mice ovaries, which is caused by the decline of Cyp17a1, a major metabolic enzyme of progesterone in Akr1b7-expressed theca cell layers. In summary, the decreased Akr1b7 pathway causes ovulation of immature oocytes and a prolonged estrous cycle, typical symptoms of ovarian aging.

molecular biology↗

bioRxiv2023

Delivering mRNAs to mouse tissues using the SEND system

mRNAs produced in a cell are almost always translated within the same cell. Some mRNAs are transported to other cells of the organism through processes involving membrane nanotubes or extracellular vesicles. A recent report describes a surprising new phenomenon of encapsulating mRNAs inside virus-like particles (VLPs) to deliver them to other cells in a process that was named SEND (Selective Endogenous eNcapsidation for cellular Delivery). Although the seminal work demonstrates the SEND process in cultured cells, it is unknown whether this phenomenon occurs in vivo. Here, we demonstrate the SEND process in living organisms using specially designed genetically engineered mouse models. Our proof of principle study lays a foundation for the SEND-VLP system to potentially be used as a gene therapy tool to deliver therapeutically important mRNAs to tissues.

genomics↗

bioRxiv2021

Novel System to Monitor In Vivo Neural Graft Activity After Spinal Cord Injury

Expectations for neural stem/progenitor cell (NS/PC) transplantation as a treatment for spinal cord injury (SCI) are increasing. However, whether and how grafted cells are incorporated into the host neural circuit and contribute to motor function recovery remain unknown. The aim of this project was to establish a novel non-invasive in vivo imaging system to visualize the activity of neural grafts by which we can simultaneously demonstrate the circuit-level integration between the graft and host, and the contribution of graft neuronal activity to host behaviour. We introduced Akaluc, a newly engineered luciferase, under control of a potent neuronal activity-dependent synthetic promoter, E-SARE, into NS/PCs and engrafted the cells into SCI model mice. Through the use of this system, we reveal that the activity of grafted cells was integrated with host behaviour and driven by host neural circuit inputs. This non-invasive system is expected to help elucidate the therapeutic mechanism of cell transplantation treatment for SCI and determine better therapy techniques that maximize the function of cells in the host circuit.

neuroscience↗

bioRxiv2020

Interleukin-11 is a Marker for Both Cancer- and Inflammation-Associated Fibroblasts that Contribute to Colorectal Cancer Progression

Interleukin (IL)-11 is a member of the IL-6 family of cytokines and involved in multiple cellular responses, including tumor development. However, the origin and functions of IL-11-producing (IL-11+) cells are not fully understood. To characterize IL-11+ cells in vivo, we generated Il11 reporter mice. IL-11+ cells appeared in the colon of three murine tumor models, and a murine acute colitis model. Il11ra1 or Il11 deletion attenuated the development of colitis-associated colorectal cancer. IL-11+ cells expressed fibroblast markers, and genes associated with cell proliferation and tissue repair. IL-11 induced STAT3 phosphorylation in colonic fibroblasts, suggesting the activation of IL-11+ fibroblasts. Analysis using the human cancer database revealed that genes enriched in IL-11+ fibroblasts were elevated in human colorectal cancer, and correlated with reduced disease-free survival. Together, our results suggested that tumor cells induced IL-11+ fibroblasts, and that a feed-forward loop between IL-11 and IL-11+ fibroblasts might contribute to tumor development.

cancer biology↗

bioRxiv2018

Alopecia areata susceptibility variant identified by MHC risk haplotype sequencing reproduces symptomatic patched hair loss in mice

BackgroundAlopecia areata (AA) is a highly heritable multifactorial and complex disease. However, no convincing susceptibility gene has yet been pinpointed in the major histocompatibility complex (MHC), a region in the human genome known to be associated with AA as compared to other regions.\n\nResultsBy sequencing MHC risk haplotypes, we identified a variant (rs142986308, p.Arg587Trp) in the coiled-coil alpha-helical rod protein 1 (CCHCR1) gene as the only non-synonymous variant in the AA risk haplotype. Using CRISPR/Cas9 for allele-specific genome editing, we then phenocopied AA symptomatic patched hair loss in mice engineered to carry the Cchcr1 risk allele. Skin biopsies of these alopecic mice showed strong up-regulation of hair-related genes, including hair keratin and keratin-associated proteins (KRTAPs). Using transcriptomics findings, we further identified CCHCR1 as a novel component of hair shafts and cuticles in areas where the engineered alopecic mice displayed fragile and impaired hair.\n\nConclusionsThese results suggest an alternative mechanism for the aetiology of AA based on aberrant keratinization, in addition to generally well-known autoimmune events.

genomics↗

bioRxiv2018

Identification of PTBP1 responsible for caspase dependent YRNA cleavage

Some RNAs such as 28S rRNA, U1 snRNA, and Y RNAs are known to be cleaved during apoptosis. As the underlying mechanism is yet unclear, the functions and biological significance of RNA degradation in apoptosis remain elusive. We previously identified novel, functional small RNAs named AGO-taxis small RNA (ASR) that are specifically bound to AGO1. Here, we investigated ASR biogenesis, which appears to be non-canonical. Y RNAs, non-coding RNAs degraded during apoptosis, were identified as the precursors of several ASRs. Cell-free analysis combined with fractionation methods revealed that the apoptosis-specific biogenesis of ASRs or Y RNA degradation was induced by PTBP1--an endoribonuclease inhibitor of Y RNAs. PTBP1, a splicing factor, was truncated by caspase 3, which subsequently activated endoribonuclease to induce biogenesis of ASRs and Y RNA cleavage.

molecular biology↗

bioRxiv2017

In situ genome editing method suitable for routine generation of germline modified animal models

Animal genome engineering experimental procedures involve three major steps: isolation of zygotes from pregnant females; microinjection of zygotes, and; transfer of injected zygotes into recipient females, that have been practiced for over three decades. The laboratory set ups intending to performing these procedures require to have sophisticated equipment as well as highly skilled technical personnel. Because of these reasons, animal transgenesis experiments are typically performed at centralized core facilities in most research organizations. We recently showed that all three steps, of animal transgensis, can be bypassed using a method termed GONAD (Genome-editing via Oviductal Nucleic Acids Delivery), by directly electroporating genome editing components into zygotes in situ. Although our first report demonstrated the genome-editing capability, its efficiency was lower than the standard methods using microinjection. Here we investigated critical parameters of GONAD to make it suitable for creating animal models of large genomic deletions, single nucleotide corrections and long sequence insertions. The efficiency of genome editing in the improved GONAD (i-GONAD) method reached to the levels comparable to traditional microinjection methods. The streamlined parameters, and the simplified experimental steps, in the i-GONAD method makes it suitable for routine genome editing applications performed both at centralized facilities as well as at the laboratories that lack highly skilled personnel and the sophisticated equipment.

genomics↗

bioRxiv2017

An Efficient CRISPR Protocol For Generating Conditional And Knock-In Mice Using Long Single-Stranded DNA Donors

The CRISPR/Cas9 tool can easily generate knockout mouse models by disrupting the gene sequence, but its efficiency for creating models that require either insertion of exogenous DNA (knock-in) or replacement of genomic segments is very poor. The majority of mouse models used in research are knock-in (reporters or recombinases) or gene-replacement (for example, conditional knockout alleles containing LoxP sites flanked exons). A few methods for creating such models are reported using double-stranded DNA as donors, but their efficiency is typically 1-10% and therefore not suitable for routine use. We recently demonstrated that long single-stranded DNAs serve as very efficient donors, both for insertion and for gene replacement. We call this method Easi-CRISPR (efficient additions with ssDNA inserts-CRISPR), a highly efficient technology (typically 25%-50%, and up to 100% in some cases), one that has worked at over a dozen loci thus far. Here, we provide detailed protocols for Easi-CRISPR.

genomics↗