Biology subjects

Ishii, H.

Publications and source records attributed to Ishii, H..

At least 19 recordsLinked to original sources

bioRxiv2026

Fully Two-Photon-Driven RESOLFT Microscopy for Super-Resolution Imaging inside Tissue

Two-photon excitation (2PE) microscopy reaches deep into tissue but remains diffraction-limited, and existing 2PE-STED and 2PE-RESOLFT schemes still rely on single-photon depletion or photoswitching. Here we present 2P-RESOLFT microscopy, in which excitation and reversible photoswitching are both two-photon-driven. Using the positive photoswitchable protein Padron2, we achieved 205-nm lateral resolution and resolved previously unreported membrane protrusions of {beta} cells inside pancreatic pseudoislets.

biophysics↗

bioRxiv2026

IL-10 Overexpression Improves Cerebral Microcirculation and Attenuates Cerebral Vasospasm After Experimental SAH

BACKGROUND: Subarachnoid hemorrhage (SAH) induces inflammatory responses and subsequent immune cell activation, which may contribute in cerebral vasospasm, microcirculatory impairment and poor neurological outcomes. Although cerebral vasospasm has traditionally been considered a major cause of delayed cerebral ischemia after SAH, therapies targeting angiographic vasospasm have not consistently improved functional outcomes. Early inflammatory responses may contribute to microcirculatory impairment, cerebral vasospasm, and subsequent neurological injury. Herein, we investigated whether interleukin-10 (IL-10), an anti-inflammatory cytokine, improves these outcomes in an experimental SAH model. METHODS: Mice received intramuscular injections of either an adeno-associated virus encoding IL-10 (AAV/IL-10) vector or an AAV expressing green fluorescent protein (AAV/GFP) vector (control). India ink angiography was performed to assess the diameter of the sphenoidal segment of the middle cerebral artery (MCA), the total length of the visible cortical arteries, and cortical staining intensity, as indices of cerebral vasospasm, microcirculatory impairment, and cerebral perfusion, respectively. Perivascular inflammatory cell infiltration and cytokine levels were assessed using immunohistochemistry and ELISA. We also evaluated the therapeutic efficacy of the AAV/IL-10 vector when administered immediately after SAH induction. RESULTS: IL-10 overexpression significantly improved neurological outcomes after SAH and was associated with attenuated cerebral vasospasm and microcirculatory impairment, as well as preservation of cerebral perfusion. It also significantly reduced neutrophil and macrophage infiltration around the internal carotid artery and attenuated SAH-induced elevations in IL-6 and matrix metalloproteinase-3 levels. Mice treated with the AAV/IL-10 vector immediately after SAH induction showed significant improvements in neurological scores and cerebral perfusion. CONCLUSIONS: AAV-mediated IL-10 overexpression improves neurological outcomes after SAH, likely by attenuating inflammatory responses, cerebral vasospasm, and microcirculatory impairment. These findings suggest that IL-10-based anti-inflammatory therapy is a promising therapeutic strategy for SAH.

pathology↗

bioRxiv2026

Age-associated erosion of organ-specific endothelial programs compromises tissue function and resilience

Endothelial cells (ECs) express organ-specific gene programs supporting tissue homeostasis and resilience. However, the mechanisms by which aging reshapes these organ-specific endothelial programs and how the resulting changes affect tissue homeostasis, resilience, and disease susceptibility remain largely unknown. Herein, we performed single-cell RNA sequencing of ECs harvested from five organs across the lifespan and found that aging progressively erodes organ-specific endothelial programs while inducing shared interferon-responsive and antigen-presentation programs across organs. Although vascular subtype identity and conserved capillary subset identity were mostly preserved, these organ-specific transcriptional programs were broadly attenuated with aging, indicating erosion of organ-specific endothelial identity to be a fundamental feature of endothelial aging. Importantly, these alterations were associated with declines in specialized EC functions, including alveolar barrier maintenance in the lung, scavenging activity in the liver, angiogenic capacity in the heart, and homeostatic programs in the kidneys and the brain, suggesting that age-related EC alterations compromise tissue homeostasis and resilience in multiple organs. Furthermore, we established a single-cell aging index for alveolar capillary ECs in mice and humans, revealing stress-associated endothelial activation to potentially be an intermediate state linking functional deterioration to cellular senescence, and also demonstrating marked heterogeneity in aging states even among ECs from animals of the same chronological age. Notably, alveolar capillary ECs exhibited progressive functional decline before reaching a senescent-like state, suggesting endothelial dysfunction to precede overt cellular senescence as the organism ages. Collectively, our findings establish progressive erosion of organ-specific endothelial programs as a central feature of vascular aging and provide a conceptual framework for elucidating how endothelial aging contributes to tissue dysfunction and reduced resilience across organs.

physiology↗

bioRxiv2026

In vivo brain temperature measurement using quantum dot imaging temperature sensors

Temperature regulation in the brain is essential for maintaining neuronal function and preventing thermally induced damage. Here, we report the development and in vivo application of quantum dots (QDs) to high-resolution thermometry in the mouse brain using two-photon excitation microscopy. These QDs, via the red-to-green photoluminescence (PL) intensity ratios, enabled stable temperature measurements in both normal and chronically hypo-perfused cerebral tissue. Our findings show that localized neuronal activity leads to transient heat generation, which is rapidly dissipated by cerebrovascular responses. In a chronic hypoperfusion model, impaired vascular function resulted in exaggerated and prolonged brain temperature elevations. This thermometry system provides unprecedented insight into the mechanisms of cerebral thermoregulation and highlights the importance of vascular cooling in protecting the brain from heat-induced stress, particularly in pathological conditions such as stroke.

bioengineering↗

bioRxiv2026

A chromatin relay from AIRE to ETS transcription factors sustains peripheral antigen expression in the thymic mimetic cells to ensure central tolerance.

Medullary thymic epithelial cells (mTECs) establish central tolerance by expressing a diverse repertoire of peripheral tissue-specific antigens (TSAs). This diversity is regulated not only by the transcriptional regulator AIRE, but also by lineage-defining factors in tissue-mimetic, AIRE-negative descendants of Aire mTECs (post-Aire mimetic TECs). However, whether and how prior AIRE activity contributes to TSA expression in post-AIRE mimetic TECs remains unclear. Here, we identify the ETS transcription factors, EHF and ELF3, as key regulators that sustain AIRE-primed gene expression in these cells. EHF and ELF3 preferentially bind distal genomic regions which are rendered accessible in advance by AIRE. Combined loss of EHF and ELF3 disrupts expression of AIRE-regulated genes expressed in mimetic TECs, leading to tissue-selective autoimmunity. Our findings reveal a relay mechanism in which AIRE primes chromatin for ETS factors to maintain TSA expression in post-AIRE mimetic TECs, thereby safeguarding self-tolerance.

immunology↗

bioRxiv2025

Age-related decline of synaptic plasticity is regulated by neuro-androgen and neuro-estrogen in normal aging of hippocampus

We revealed a good relationship between age-dependent decrease in the hippocampal dendritic spine density and age-dependent decrease in hippocampal androgen and estrogen levels with normal aging of male rats. Approximately 25% decrease in the spine density was observed in hippocampal CA1 region by going from 3 month-old (3m; young adult) to 24 month-old (24m; aged). We found a significant age-induced decrease in hippocampal neuro-androgen levels by going from 3m to 24 m using mass-spectrometric analysis. The hippocampal levels of testosterone (T) and dihydrotestosterone (DHT) dramatically decreased from 17 nM T and 7 nM DHT at 3 m to 17/100 nM T and 7/15 nM DHT at 24 m. On the other hand, hippocampal estradiol (E2) was moderately decreased with aging, from 8 nM at 3 m to 2 nM at 24m. Comprehensive analysis of mRNAs of hippocampal steroidogenic enzymes and receptors showed an age-dependent decrease in their expression levels by approximately 50% (P450(17)), 25% (17-hydroxysteroid dehydrogenase) and 0% (5-reductase and P450arom). Androgen receptor AR was moderately decreased but estrogen receptor ER was not decreased with aging. The 25% decrease in the spine density with aging may be due to a balance between considerably decreased T and DHT levels (spine decrease factor) and remained moderately high E2 level (spine increase factor) in the 24m hippocampus. Aged hippocampus still has moderate capacity of sex-steroid synthesis and their functions. Interestingly, DHT-supplementation and T-supplementation recovered the spine density at 24m.

neuroscience↗

bioRxiv2025

Chromatin organizer ASCL1 governs gene programs in thymic epithelial cells, defining immunological self

Immunological self-tolerance depends on medullary thymic epithelial cells (mTECs), which express a broad repertoire of self-antigens to support negative selection of autoreactive T cells and the development of regulatory T cells. Although the autoimmune regulator AIRE is essential for this process, additional factors are required to establish the full mTEC gene expression program. Because thymoma is frequently associated with autoimmunity, implicating defective thymic tolerance, we performed single-cell RNA sequencing of TECs from thymoma patients and identified the transcription factor ASCL1 as selectively downregulated in tumor mTECs. Deletion of Ascl1 in mouse TECs resulted in spontaneous autoimmunity without inducing thymic tumorigenesis. Transcriptomic and chromatin accessibility analyses revealed that ASCL1 influences mTEC gene expression programs and is associated with corresponding changes in chromatin accessibility. Genetic interaction analyses further suggested that ASCL1 activity in immature mTECs modulates the AIRE dependency of gene expression in mature mTECs. Together, these findings identify ASCL1 as a key regulator of mTEC function and central tolerance, providing insight into mechanisms that safeguard immune homeostasis and whose disruption may contribute to autoimmune disease.

immunology↗

bioRxiv2025

In vivo deep-brain microscopy at submicrometer resolution with refractive index-matched prism interfaces

The mammalian brain is a thick and densely layered structure comprising a huge number of neurons that work together to process information and regulate brain functions. Although various optical methods have been developed to investigate deep brain dynamics, they are limited by technical constraints, invasiveness, suboptimal spatial resolution, and/or a restricted field of view. To overcome these limitations, we developed an implantable, optically optimized microprism interface with a refractive index matched to that of brain tissue and water, enabling minimally-invasive, wide-field two-photon imaging method with enhanced brightness and sub-micron resolution in deep prefrontal areas.

neuroscience↗

bioRxiv2024

Inhibition of PDE5 induces rapid non-genomic increase in hippocampal dendritic spines via specific kinases

Cyclic guanosine monophosphate (cGMP) is a typical neuromodulator that is used in neuronal synapses. Inhibitor of phosphodiesterase 5 (PDE5) could regulates signaling pathways by elevating cGMP levels. In the current study, we found that the treatments with tadalafil, a PDE5 inhibitor, for 2 h rapidly and non-genomically increased the total density of dendritic spines, using confocal imaging of Lucifer Yellow-injected hippocampal CA1 pyramidal neurons. Since inhibition of PDE5 elevates cGMP levels at synapses, we analyzed downstream nongenomic signaling. We analyzed the involvement of kinase signaling, because rapid postsynaptic modulation often involves kinase networks as observed from many investigations of synaptic functions. Upon co-treatments of PKG inhibitor (KT5823) with tadalafil, the spine increase was considerably blocked. In addition, co-treatments of GSK-3 inhibitor (I8) also blocked the spine increase induced by tadalafil. However, inhibitors of other representative synaptic kinases, including LIMK, Erk/MAPK, PKA, PKC and PI3K, did not suppress the tadalafil-induced spine increases. PDE5 inhibitors have attracted attention on their recovery effects from pathological damages, including memory improvement from cognitive impairment by Stroke, Amyloid beta accumulation and tau phosphorylation. The current finding of tadalafil function about rapid modulation of neural plasticity may add new elemental steps of anti-aging capacity against cognitive decline.

neuroscience↗

bioRxiv2024

Direct and indirect RANK ligand and CD40 ligand signaling regulate the maintenance of thymic epithelial cell frequency and properties in the adult thymus

Medullary thymic epithelial cells (mTECs) play a crucial role in suppressing the onset of autoimmunity by eliminating autoreactive T cells and promoting the development of regulatory T cells in the thymus. Although mTECs undergo turnover in adults, the molecular mechanisms behind this process remain unclear. This study describes the direct and indirect roles of receptor activator of NF-{kappa}B ligand (RANKL) and CD40 ligand (CD40L) signaling in TECs in the adult thymus. Flow cytometric and single-cell RNA-seq (scRNA-seq) analyses suggest that the depletion of both RANKL and CD40L signaling inhibits mTEC differentiation from CCL21+ mTEC progenitors to transit-amplifying TECs in the adult thymus. Unexpectedly, this depletion also indirectly affects the gene expression of TEC progenitors and cortical TECs. Notably, AP-1 gene expression, which allows further subdivision of TEC progenitors, is upregulated following the depletion of RANKL and CD40L signaling. Overall, our data propose that RANKL and CD40L signaling cooperatively maintain mature mTEC frequency in the adult thymus and sustain the characteristics of TEC progenitors through an indirect mechanism.

immunology↗

bioRxiv2024

Amyloid-β-induced Alteration of Fast and Localized Calcium Elevations in Cultured Astrocytes

Alzheimers disease (AD) is a progressive neurodegenerative disorder that causes cognitive decline. Uncovering the mechanisms of neurodegeneration in the early stages is essential to establish a treatment for AD. Recent research has proposed the hypothesis that amyloid-{beta} (A{beta}) oligomers elicit an excessive glutamate release from astrocytes toward synapses through intracellular free Ca2+ ([Ca2+]i) elevations in astrocytes, finally resulting in neuronal dendritic spine loss. Under physiological conditions, astrocytic [Ca2+]i elevations range spatially from microdomains to network-wide propagation and temporally from milliseconds to tens of seconds. Astrocytic localized and fast [Ca2+]i elevations might correlate with glutamate release; however, the A{beta}-induced alteration of localized, fast astrocytic [Ca2+]i elevations remains unexplored. In this study, we quantitatively investigated the A{beta} dimers-induced changes in the spatial and temporal patterns of [Ca2+]i in a primary culture of astrocytes by two-photon excitation spinning-disk confocal microscopy. The frequency of fast [Ca2+]i elevations occurring locally in astrocytes ([≤]0.5 s, [≤]35 {micro}m2) and [Ca2+]i event occupancy relative to cell area significantly increased after exposure to A{beta} dimers. The effect of A{beta} dimers appeared dose-dependently above 500 nM, and these A{beta} dimers-induced [Ca2+]i elevations were primarily mediated by a metabotropic purinergic receptor (P2Y1 receptor) and Ca2+ release from the endoplasmic reticulum. Our findings suggest that the A{beta} dimers-induced alterations and hyperactivation of astrocytic [Ca2+]i is a candidate cellular mechanism in the early stages of AD.

neuroscience↗

bioRxiv2024

Second impact of an acute total body irradiation on thymic epithelial cells

The thymus, a crucial organ for T cell development, undergoes transient involution following exposure to sublethal total body irradiation. The impact of sublethal irradiation on the thymus is reportedly bimodal: thymic involution recurs after the recovery of the thymus from the initial impact of acute sublethal irradiation. While the second impact of acute irradiation has been acknowledged for thymocytes, its influence on thymic epithelial cells (TECs), which are crucial for thymic T cell differentiation and selection, remains to be elucidated. In this study, we aim to elucidate this influence. Mice were subjected to acute sublethal total body irradiation, and TECs were evaluated at three distinct time points: during the initial impact, during the recovery phase post-initial impact, and during the second impact phase. Flow cytometry analysis revealed that during the second impact phase, mTECs were reduced, whereas cTECs remained unaffected. Among mTECs, the subset expressing high levels of the co-stimulatory molecule CD80 (mTEChi) experienced the most pronounced reduction. RNA sequencing analysis of mTEChi cells at early differentiation stages revealed significant alterations in gene expression profiles during the second impact phase. Notably, gene signatures of tuft-like TECs and Aire-expressing TECs were preferentially influenced in these mTEChi subpopulations. These findings suggest that acute total body irradiation disrupts mTEC frequencies and gene expression in a bimodal manner, possibly compromising thymic functions over extended periods.

immunology↗

bioRxiv2024

Establishing a method for the cryopreservation of viable peripheral blood mononuclear cells in the International Space Station

The analysis of cells frozen within the International Space Station (ISS) will provide crucial insights into the impact of the space environment on cellular functions and properties. The objective of this study was to develop a method for cryopreserving blood cells under the specific constraints of the ISS. In a ground experiment, mouse blood was directly mixed with a cryoprotectant and gradually frozen at -80 {degrees}C. Thawing the frozen blood sample resulted in the successful recovery of viable mononuclear cells when using a mixed solution of dimethylsulfoxide and hydroxyethyl starch as a cryoprotectant. Additionally, we developed new freezing cases to minimize storage space utilization within the ISS freezer. Finally, we confirmed the recovery of major mononuclear immune cell subsets from the cryopreserved blood cells through a high dimensional analysis of flow cytometric data using 13 cell surface markers. Consequently, this ground study lays the foundation for the cryopreservation of viable blood cells on the ISS, enabling their analysis upon return to Earth. The application of this method in ISS studies will contribute to understanding the impact of space environments on human cells. Moreover, this method may find application in the cryopreservation of blood cells in situations where research facilities are inadequate.

cell biology↗

bioRxiv2024

CD34-positive monocytes are highly susceptible to HIV-1

HIV-1 persists in cellular reservoirs despite effective combined antiretroviral therapy (cART). CD4+ T cells are a well-known reservoir, but there is evidence suggesting that myeloid cells, including circulating monocytes, are also a clinically relevant reservoir. However, it is not fully understood which subsets of monocytes are preferentially infected in vivo. Here, we show that a monocyte fraction expressing a stem cell marker CD34 is more susceptible to HIV-1 infection than the CD34-negative major subset. In cART-untreated viremic individuals, the CD34+ fraction increased in the percentage in total monocytes, and harbored higher copies of proviral DNA than the major subset. Consistent with this, the CD34+ fraction expressed HIV-1 receptors CD4 and CCR5 at higher levels and HIV-1 restriction factors MX2 and SAMHD1 at lower levels. Interestingly, proviral DNA was still detectable in the CD34+ fraction of cART-treated virologically suppressed individuals. CD34+ monocytes were also present in lymph nodes, and expressed CD4 and CCR5 at higher levels than the major subset, as observed in peripheral blood. Moreover, CD34+ monocytes present in peripheral blood and lymph nodes highly expressed CCR7 and sphingosine-1-phosphate receptor 1 (S1PR1), critical regulators of in vivo cellular trafficking. Collectively, our findings raise the new possibility that lymph node CD34+ monocytes, which originate from the circulation, are infected with HIV-1 owing to their high susceptibility to HIV-1, and return to circulation, which explains the detection of proviral DNA in peripheral CD34+ monocytes even after long-term cART.

immunology↗

bioRxiv2023

Mitochondrial protein C15ORF48 is a stress-independent inducer of autophagy that regulates oxidative stress and autoimmunity

Autophagy is primarily activated by cellular stress, such as starvation or mitochondrial damage. However, stress-independent autophagy is activated by unknown mechanisms in several cell types, such as thymic epithelial cells (TECs). Here we report that the mitochondrial protein, C15ORF48, is a critical inducer of stress-independent autophagy. Mechanistically, C15ORF48 reduces the mitochondrial membrane potential and lowers intracellular ATP levels, thereby activating AMP-activated protein kinase and its downstream Unc-51-like kinase 1. Interestingly, C15ORF48 induction of autophagy upregulates intracellular glutathione levels, promoting cell survival by reducing oxidative stress. Mice deficient in C15orf48 showed a reduction in stress-independent autophagy in TECs, but not in typical starvation-induced autophagy in skeletal muscles. Moreover, C15orf48-/- mice developed autoimmunity, which is consistent with the fact that the stress-independent autophagy in TECs is crucial for the thymic self-tolerance. These results suggest that C15ORF48 induces stress-independent autophagy, thereby regulating oxidative stress and self-tolerance.

cell biology↗

bioRxiv2022

Robust capability of renal tubule fatty acid uptake from apical and basolateral membranes in physiology and disease

Excess lipid accumulation is associated with obesity-related chronic kidney disease, but the mechanisms of fatty acid (FA) uptake have been poorly understood. To this end, we investigated how FAs are taken up by tubular epithelial cells (TECs) in mice by using in vivo FA tracing and histological methods. Immunohistochemistry showed that CD36, which is a well-known FA transporter, was abundantly expressed on the basolateral side of proximal TECs (PTECs). The uptake of 125I-BMIPP (a radiolabeled FA tracer) was significantly reduced in CD36-knockout kidneys at 1 min after injection. In vivo imaging with multiphoton microscopy revealed that BODIPY-C12 (a fluorescence-labeled FA tracer) accumulated on both the basolateral and apical sides of PTECs. Numerous lipid droplets accumulated in PTECs after accelerated lipolysis. Furthermore, PTEC-specific injury via diphtheria toxin (DT) injection in transgenic mice expressing the DT receptor resulted in a compensatory increase in lipid accumulation in downstream TECs. Importantly, urinary FAs were undetectable, even in mice and humans with remarkable albuminuria. Our data demonstrate that renal TECs take up FAs from blood (CD36-dependent) and primary urine (CD36-independent) and can store excess FAs as neutral lipids. The results further show that renal tubules have hitherto largely unappreciated mechanisms by which the excretion of FAs into the urine is avoided.

biochemistry↗

bioRxiv2022

Environmental pH signals the release of monosaccharides from cell wall in coral symbiotic alga

Reef-building corals thrive in oligotrophic environments due to their possession of endosymbiotic algae. Confined to the low pH interior of the symbiosome within the cell, the algal symbiont provides the coral host with photosynthetically fixed carbon. However, it remains unknown how carbon is released from the algal symbiont for uptake by the host. Here we show, using cultured symbiotic dinoflagellate, Breviolum sp., that decreases in pH directly accelerates the release of monosaccharides, i.e., glucose and galactose, into the ambient environment. Under low pH conditions, the cell surface structures were deformed and genes related to cellulase were significantly upregulated in Breviolum. Importantly, the release of monosaccharides was suppressed by the cellulase inhibitor, glucopyranoside, linking the release of carbon to degradation of the agal cell wall. Our results suggest that the low pH signals the cellulase-mediated release of monosaccharides from the algal cell wall as an environmental response in coral reef ecosystems.

evolutionary biology↗

bioRxiv2022

Differential activation of NLRP3 inflammasome by Acinetobacter baumannii strains

Acinetobacter baumannii is an emerging nosocomial, opportunistic pathogen with growing clinical significance globally. A. baumannii has an exceptional ability to rapidly develop drug resistance. It is frequently responsible for ventilator-associated pneumonia in clinical settings and inflammation resulting in severe sepsis. The inflammatory response is mediated by host pattern-recognition receptors and the inflammasomes. Inflammasome activation triggers inflammatory responses, including the secretion of the pro-inflammatory cytokines IL-1{beta} and IL-18, the recruitment of innate immune effectors against A. baumannii infection, and the induction programmed cell death by pyroptosis. An important knowledge gap is how variation among clinical isolates affects the hosts innate response and activation of the inflammasome during A. baumannii infection. In this study, we compared nine A. baumannii strains, including clinical locally-acquired isolates, in their ability to induce activation of the inflammasome and programmed cell death pathway in primary macrophages and mice. We found a striking variability in survival outcomes of mice and bacterial dissemination in organs among three ATCC A. baumannii strains, likely due to the differences in virulence between strains. Interestingly, we found a stark contrast in activation of the NLRP3 inflammasome pathway, the non-canonical caspase-11 pathway, plasmatic secretion of the pro-inflammatory cytokines IL-1{beta} and IL-18 between A. baumannii strains. Our study highlights the importance of utilising multiple bacterial strains and clinical isolates with differential virulence to investigate the innate immune response to A. baumannii infection.

microbiology↗