Biology subjects

Nagai, T.

Publications and source records attributed to Nagai, T..

At least 19 recordsLinked to original sources

bioRxiv2026

Developmental prefrontal myelination shapes inhibitory interneuron function and adult social behavior

Early-life adversity and abnormal myelination are implicated in psychiatric disorders, but how developmental myelination influences adult social cognition remains unclear. Here, we tested whether disrupting developmental myelination in the medial prefrontal cortex (mPFC) impairs adult social behavior. Using complementary genetic strategies in male mice, we suppressed Myelin Regulatory Factor (Myrf)-dependent myelination locally in the juvenile mPFC or broadly in oligodendrocyte-lineage cells. Both approaches selectively impaired adult social novelty preference while sparing sociability, locomotion, and affective behaviors. Focal prefrontal dysmyelination also reduced social dominance. Spatial transcriptomics revealed coupling between myelin-related genes and parvalbumin (PV) interneuron markers in the healthy mPFC, which was disrupted by developmental hypomyelination. Consistent with this transcriptional alteration, PV interneurons showed reduced high-frequency firing despite preserved intrinsic excitability. In the human prefrontal cortex, MBP-PV expression was correlated in controls but not in individuals with schizophrenia. These findings indicate that developmental prefrontal myelination contributes to PV interneuron maturation and suggest that disrupted oligodendrocyte-interneuron coordination may contribute to impaired social recognition.

neuroscience↗

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

ATP-Free Fatty Aldehyde Biosynthesis Enables an Autonomous Lux-Based Bioluminescence System

Autonomous bioluminescence systems enable continuous light emission in engineered organisms by genetically encoding both luciferase enzymes and their substrate biosynthetic pathways, offering a powerful platform for non-invasive and long-term monitoring of biological processes. However, bioluminescence output is highly sensitive to substrate availability and host metabolic state, often leading to signal instability under energy-limited conditions. Here, we report an alternative luciferin biosynthetic strategy for the bacterial Lux bioluminescence system in which the adenosine triphosphate (ATP)-dependent LuxEC complex is replaced by -dioxygenase (DOX), an enzyme that directly converts fatty acids into fatty aldehydes without consuming ATP. Using machine learning-guided directed evolution, we engineered DOX variants that markedly enhanced bioluminescence intensity when coupled with bacterial luciferase. The resulting ATP-free Lux bioluminescence system enabled single-cell-level bioluminescence imaging and maintained stable light emission under diverse antibiotic treatments, demonstrating enhanced robustness against metabolic perturbations. SIGNIFICANCEAutonomous bioluminescence imaging has become an attractive method for long-term observation of biological phenomena without the need for exogenous substrate addition. However, the light output of existing autonomous bioluminescence systems, including bacterial and fungal pathways, remains ATP-dependent and often declines when cellular metabolism is perturbed, limiting their reliability for quantitative analysis. Here, we report the development of an ATP-independent substrate biosynthesis pathway for the bacterial luciferase system using DOX. To improve system performance, we applied machine learning- guided directed evolution, which significantly enhanced signal intensity and enabled single-cell bioluminescence imaging. Furthermore, the DOX-based bacterial luciferase system maintained stable luminescence under antibiotic treatments, in contrast to the conventional ATP-dependent bacterial luciferase system. In summary, our findings establish a robust ATP-independent autonomous bioluminescence imaging platform that enables monitoring of cellular events under metabolic perturbations.

synthetic biology↗

bioRxiv2026

KCNQ2/3 Regulates the Synaptic Remodeling Associated with Drug Seeking in Addiction

Drug-seeking behavior during withdrawal represents a critical obstacle to addiction treatment. In the nucleus accumbens, hyperactive dopamine D1 receptor-expressing medium spiny neurons (D1R-MSNs) promote cocaine-seeking through aberrant synaptic remodeling, including synapse formation and calcium-permeable AMPA receptor (CP-AMPAR) insertion. However, the intermediate molecular control mechanism remains unclear. We identified KCNQ2/3 potassium channels as key regulators of synaptic pathology during withdrawal. Cocaine-conditioned mice showed increased spine density, enhanced surface CP-AMPAR, and elevated neuronal activity 14 days after withdrawal. These phenotypes were reversed by repeated administration of KCNQ2/3 openers or D1R-MSN-specific expression of constitutively active KCNQ2. Functional restoration of KCNQ2/3 suppressed cocaine-seeking behavior and normalized D1R-MSN excitability. These findings suggest that sustained KCNQ2/3 deactivation drives synaptic remodeling during withdrawal, while channel activation offers a potential therapeutic strategy. Furthermore, the results position KCNQ2/3 as a master regulator of drug-seeking behavior and channel activation in D1R-MSNs as a logical target for relapse prevention in addiction.

neuroscience↗

bioRxiv2026

Fusion of a non-specific DNA-binding domain enhances Cas12a trans-cleavage for robust nucleic-acid diagnostics

CRISPR-Cas12a underlies powerful genome-editing and nucleic-acid detection technologies, yet its performance is limited by inefficient target engagement and low catalytic turnover, particularly at low target abundance and elevated temperatures. Here, we report a modular protein-engineering strategy to enhance Cas12a trans-activity by fusing the hyperthermophilic DNA-binding protein Sso7d to the N terminus of Lachnospiraceae bacterium ND2006 Cas12a (LbaCas12a). The resulting fusion enzyme shows a twofold improvement in detection sensitivity, a 4.6-fold increase in kcat, and substantially accelerated trans-cleavage kinetics compared with wild-type Cas12a. These enhancements are observed across multiple guide RNAs, diverse DNA substrates, and a broad temperature range from 37 to 60 {degrees}C. The engineered Cas12a enables robust detection of the intrinsic blaOXA-51 and acquired blaOXA-24 antibiotic resistance genes from multiple Acinetobacter baumannii strains with independently validated resistance profiles, whereas wild-type Cas12a produces little or no detectable signal. Signal output is increased by up to [~]30-fold, expanding the effective detection window and shortening time-to-positive. Together, these results establish fusion of an accessory DNA-binding domain as a proof-of-concept for Sso7d fusion to LbCas12a and that generalization to other CRISPR effectors or non-specific DNA-binding domains represents an exciting avenue for future investigation.

biochemistry↗

bioRxiv2026

The perception of realism is correlated with the concept of physical gamut

Realism and naturalness remain unresolved questions in vision science. This study investigates whether the physical gamut correlates with realism judgements. We conducted psychophysical experiments where observers judged the realism of natural scenes with target regions manipulated across the CIE 1931 color space. Results initially showed a moderate-to-strong correlation between judgements and a theoretical physical gamut derived from optimal colors. Further analysis revealed that the most detrimental points were in the saturated green region of the CIE 1931 xy chromaticity diagram; removing them yielded a very strong correlation. To explain this discrepancy, we modeled a real-world physical gamut based on USGS and ECOSTRESS spectral libraries. The analysis revealed that the detrimental green chromaticities might be non-existent in the real-world. Since physical gamut theory posits that the visual system constructs internal references through empirical observation of the world, the absence of these colors in nature might be a plausible explanation to the theoretical models failure. Ultimately, the real-world gamut exhibited an even stronger correlation with judgements, supporting our hypothesis while suggesting that the theoretical model may not be the optimal approximation of the actual physical gamut. These findings contribute to discussions on perceptual realism and offer a framework for enhancing rendering technologies.

neuroscience↗

bioRxiv2025

Retinal Origins of Circadian Photoregulation's Specialized Dynamic Range and Temporal Integration

The day-night cycle drives profound environmental changes, which animals anticipate using the circadian clock. The clock synchronizes with this cycle by extracting features of light that indicate solar elevation. It uses a specialized dynamic range to sense relevant photon rates and precise, extended temporal integration to encode total photon counts. This feature selectivity is distinct from that of visual perception and is thought to arise in the brain. We have found it within the retina's M1 melanopsin photoreceptors, which signal light to the clock. M1s have heterogeneous intrinsic and synaptic drives, and collectively account for the dynamic range of circadian photoregulation. Both individually and as a population, M1s approach the level of temporal integration observed behaviorally. M1s and behavior match closely when the pupillary light reflex, another M1-driven function, dynamically shapes retinal illumination. Thus, the earliest steps of photoreception are tuned to signifiers of Earth's rotation.

neuroscience↗

bioRxiv2025

Temporal dynamics of mRNA translation dysregulation and codon decoding during murine stroke evolution.

Ischemic stroke imposes acute oxidative and metabolic stress, yet how this reshapes mRNA translation remains poorly defined. We generated a time-resolved RNA-seq and ribosome-profiling atlas of mouse ischemic cortex at 1, 6 and 24 hours. Stroke produced early discordance between mRNA abundance and ribosome occupancy, revealing translational efficiency changes not captured by transcriptomics. The hyperacute phase was marked by a transient shift toward G/C-ending codon usage and a burst of 3' untranslated region ribosome footprints consistent with stop-codon readthrough-like termination stress. Machine-learning analyses linked this response to stop-proximal ribosome queuing, 3' untranslated region frame usage, local RNA structure and transcript architecture rather. From 6 to 24 hours, ischemic cortex developed progressive A-site pausing and altered coding-frame occupancy. Hidden Markov modeling localized a subset of frame-disruption events to discrete coding-sequence windows and machine learning revealed reproducible codon, amino-acid, nucleotide-composition and RNA-structure features associated with frameshifting. ORF-level analyses showed that many frame changes coincided with differential open reading frame usage. Late injury was characterized by increased upstream open reading frame engagement and 5' untranslated region ribosome occupancy, inversely correlating with coding-sequence translation. These data identify staged translational remodeling after stroke that could not be captured via traditional mRNA or protein expression analysis.

molecular biology↗

bioRxiv2025

Optimal colors can predict luminosity thresholds in natural scenes

Luminosity thresholds define the luminance boundary at which a surface color shifts in appearance from being perceived as an illuminated surface to appearing self-luminous. Previous research suggests that the human visual system infers these thresholds based on internal references of physically realizable surface colors under a given illumination, referred to as the physical gamut. A surface is perceived as self-luminous when its luminance exceeds the upper limit of this empirically internalized gamut. However, the precise structure and boundaries of these gamuts remain uncertain. Optimal colors, which represent theoretical surface reflectances under specific illuminants, have been shown to provide an effective model for visualizing and computing the physical gamuts. In prior studies, optimal colors have successfully predicted luminosity thresholds; however, these findings were limited to highly simplified, abstract stimuli. Whether this framework generalizes to more naturalistic viewing conditions has remained an open question. In the present study, we demonstrate that the theory of an internal reference in the form of an empirically constructed physical gamut, visualized through optimal colors, remains valid under more natural conditions. Our results confirm that optimal colors can still accurately predict luminosity thresholds in such settings. Moreover, our findings suggest that the luminosity thresholds encompass both self-luminosity and naturalness concepts. Subsequently, this may imply that the notion of physical gamut could envelope both concepts as well and could be defined as "all physically possible colors in a scene for an object that does not emit light." These insights can have profound potential implications for both applied fields (i.e., XR or projection mapping) and fundamental science (e.g., understanding human visual processing mechanisms).

neuroscience↗

bioRxiv2025

Distinct effects of nonselective Rho-kinase inhibitor fasudil and selective Rho-kinase 2 inhibitor KD025 on serotonin and dopamine release in the nucleus accumbens of mice

Recent studies have indicated that the Rho GTPase family and Rho-kinases are associated with psychiatric diseases, such as schizophrenia. Rho-kinases have two subtypes, Rho-kinases 1 and 2 that regulate actin dynamics and mediate neurite outgrowth, spine morphology in neurons, and neurotransmitter release in vitro and ex vivo. However, the precise role of Rho-kinases in neurotransmitter release in vivo remains unclear. To clarify the role of Rho-kinases 1 and 2 in serotonin and dopamine release in the nucleus accumbens (NAc) of mice in vivo, we investigated the effect of a nonselective Rho-kinase inhibitor, fasudil, and a selective Rho-kinase 2 inhibitor, KD025, using an in vivo microdialysis technique. Fasudil perfusion (1-20 M) into the NAc increased the basal extracellular serotonin level but did not affect dopamine levels, whereas KD025 (10-20 M) had little effect on basal serotonin and dopamine levels. Notably, fasudil perfusion into the NAc suppressed depolarization-induced serotonin and dopamine release in a dose-dependent manner, whereas KD025 selectively suppressed depolarization-induced serotonin release. Our results suggested that Rho-kinases 1 and 2 are associated with dopamine and serotonin release, respectively, and that both may have significant but distinct roles in the regulation of serotonin and dopamine release in the NAc.

neuroscience↗

bioRxiv2025

Cortical excitatory and inhibitory neuron deficits may underlie the cognitive and social impairments in a mouse model of schizophrenia with exonic Reln deletion

Reelin is an essential extracellular matrix glycoprotein involved in the formation of cortical layers and has been associated with several neuropsychiatric conditions, such as schizophrenia (SCZ). To explore its role in brain function and its potential involvement in SCZ, we developed a Reln heterozygous deletion (Relndel/+) mouse model that mimics a genetic deletion observed in a Japanese patient with SCZ. In previous studies, we demonstrated that Relndel/+ mice exhibit cognitive impairments in a visual discrimination test. Here, we found that Relndel/+ mice displayed impairments in social novelty recognition, while social preference remained intact. Immunohistochemical analyses revealed a significant decrease in the numbers of calcium/calmodulin-dependent protein kinase II (CaMKII)-positive glutamatergic pyramidal neurons, gamma-aminobutyric acid (GABA)-ergic interneurons, and parvalbumin (PV)-positive interneurons in the medial prefrontal cortex (mPFC) of Relndel/+ mice. Furthermore, Relndel/+ mice exhibited significant deficits in excitatory spine density and morphology, as well as a decrease number of PV boutons in the mPFC compared to wild-type (WT) mice. Finally, we demonstrated that injection of AAV-R36-Myc virus into the mPFC can improve social novelty impairments in Relndel/+ mice, but no effects on WT control. These findings indicate that Relndel/+ mice could be a valuable model for exploring the neurobiological mechanisms underlying cognitive and social impairments in SCZ. Futhermore, our results with AAV-R36-Myc also suggest the therapeutic potential of Reelin replacement, warranting further investigation as a possible treatment strategy for SCZ. HighlightsO_LIReelin deficiency disrupts social novelty, but not social preference, in Relndel/+ mice C_LIO_LIRelndel/+ mice serve as a novel model to investigate impairments of neuronal mechanisms on schizophrenia C_LIO_LIAAV-R36-myc injection rescues social novelty deficits in Relndel/+ mice C_LIO_LIReelin replacement therapy is a potential treatment for schizophrenia C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=143 HEIGHT=200 SRC="FIGDIR/small/666057v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1f4921aorg.highwire.dtl.DTLVardef@1a41b33org.highwire.dtl.DTLVardef@f5f2bcorg.highwire.dtl.DTLVardef@1134d8d_HPS_FORMAT_FIGEXP M_FIG C_FIG

animal behavior and cognition↗

bioRxiv2025

Twinfilin-1 phosphorylation in reelin signaling regulates actin dynamics and spine development

Reelin is an extracellular glycoprotein essential for neuronal migration, spine development, and synaptic plasticity. Impaired reelin signaling is linked to neurological disorders, including schizophrenia and autism. While reelin mutant (reeler) mice exhibit behavioral deficits associated with impaired spine formation, the underlying molecular mechanisms remain unclear. We identified Twinfilin-1 (Twf1) as a downstream effector of reelin signaling via phosphoproteomic analysis, based on its reduced tyrosine phosphorylation in reeler mice. We found that Src regulated Twf1 phosphorylation at tyrosine 309, and reelin stimulation increased Twf1 phosphorylation in neurons, an effect blocked by the Src inhibitor PP2. A phospho-resistant Twf1 mutant (Twf1 Y309F) showed reduced capping protein binding and a lower F/G-actin ratio. Twf1Y309F mice exhibited cognitive deficits, reduced spine density, smaller spine head size, and a decreased F/G-actin ratio in synaptosomes. These findings highlight Twf1 phosphorylation as a key component of reelin signaling involved in actin remodeling and spine development.

neuroscience↗

bioRxiv2025

Theoretical physical color gamuts define luminosity and naturalness perceptual limits in natural scenes

Realism in augmented reality (AR) hinges on the seamless blending of virtual elements into real-world environments. One possible factor influencing this realism may be the physical gamut: an internal representation of all perceivable colors within a natural scene. Previous studies on luminosity thresholds suggest that this gamut, rooted in optimal colors theory, constrains perceptual judgments. While promising, such findings were based on abstract and two-dimensional stimuli only. Before extending this framework to more realistic AR scenarios, an essential next step is to assess whether the physical gamut theory also applies to naturalistic stimuli. This study addresses that gap. Our results reveal that the physical gamut remains a valid construct for natural objects viewed in realistic scenes. Moreover, observers judgments of luminosity thresholds appear guided not only by a criterion of self-luminosity, but also by an implicit sense of naturalness. These insights pave the way for exploring AR realism through the lens of physical gamut theory.

neuroscience↗

bioRxiv2025

Elucidating Chiral Myosin-Induced Actin Dynamics: From Single Filament Behavior to Collective Structures

The myosin superfamily comprises over 70 classes, each with multiple subclasses, and shows substantial diversity in properties such as velocity, ATPase activity, duty ratio, and directionality. This functional diversity enables the specialized roles of each myosin in various organisms, organs, and cell types. Recent studies have revealed that certain myosins induce chiral curved motions of actin filaments. However, this newly identified property remains largely unexplored. Here, we investigated this chiral motion in vitro using Chara corallina myosin XI (CcXI), which drives fast counterclockwise (CCW) movement of actin filaments. This chiral motion arises from asymmetric displacement at the filaments leading tip, and its curvature depends on the surface density of myosin. Surprisingly, at near-physiological actin concentrations, actin filaments exhibiting chiral curved motion undergo collective motion, spontaneously forming a novel structure--termed the actin chiral ring (ACR)--that exhibits persistent CCW rotation. ACRs display remarkable stability, continuing to rotate at their formation site until ATP is depleted, while maintaining their structure even after rotation ceases. This stability is unprecedented among reported collective motions of cytoskeletal proteins driven by various motors. Our findings demonstrate that myosins with chiral activity can autonomously organize actin filaments into stable, chiral structures through collective motion, providing new insights into actin self-organization by unconventional myosins. This study advances our understanding of the diverse functional roles of unconventional myosins and introduces a new paradigm for cytoskeletal organization. Significance StatementMyosins are motor proteins that move along actin filaments and support various intracellular functions. Recently, some myosins have been found to drive actin filaments along chiral curved paths, but the mechanisms and significance of this behavior remain poorly understood. Here, we analyzed this activity and discovered that it not only drives chiral curved motion of single actin filaments, but also organizes them into stable, unidirectionally rotating ring structures through collective motion. These rings, termed actin chiral rings (ACRs), spontaneously emerge at near-physiological actin concentrations. Our findings uncover a previously unrecognized organizing principle of actin self-assembly driven by myosin with chiral activity and provide a new framework for understanding how cytoskeletal chirality is made.

cell biology↗

bioRxiv2024

The crucial role of intercellular calcium wave propagation triggered by influenza A virus in promoting infection

Influenza A viruses (IAVs) initially infect a few host cells, and the infection subsequently spreads to neighboring cells. However, the molecular mechanisms underlying this expansion remain unclear. Here, we show that IAV infection upregulates the frequency of intercellular calcium wave propagations (iCWPs) that mediate the spread of IAVs. ADP released from initially infected cells mediated iCWPs via the P2Y1 receptor. The generation of iCWPs and spread of viral infection were inhibited by a P2Y1 antagonist. Enhanced endocytosis in the surrounding cells that received ADP signaling upregulated viral entry. Expression of IAV matrix protein 2 (M2) in initially infected cells triggered iCWPs through ADP diffusion, thereby increasing infection, whereas an ion permeability-deficient mutation of M2 or inhibition of its ion channel activity suppressed iCWPs. Therefore, intercellular calcium signaling is essential for early expansion and potential establishment of IAV infection, which offers a promising target for IAV prophylaxis.

cell biology↗

bioRxiv2024

Top-Down Effects on Translucency Perception in Relation to Shape Cues

0.It is well established that object shape perception significantly influences the perception of translucency. However, how object shape cues such as motion and binocular disparity affect the perception of translucency in rich environments, like virtual reality or real visual environments, remains unclear. This study aims to psychophysically measure the extent to which multiple object shape cues influence the perception of translucency. Additionally, we examined whether top-down factors, such as changes in cognitive attitude caused by the sequence of experiments, affect translucency perception. The results revealed that while motion and binocular disparity enhance translucency perception, this effect is confined to situations where shape cues are poor. Moreover, the effect became particularly pronounced when the experiments began with weak specular reflection stimuli, followed by the experiments using stimuli with specular reflection. In the case of translucent objects without specular reflection, strong shape information cannot be derived solely from shading patterns. These findings thus suggest that top-down factors related to shape modulate the influence of shape cues on translucency perception.

neuroscience↗

bioRxiv2024

Antipsychotic-like effects of the selective Rho-kinase 2 inhibitor KD025 in genetic and pharmacological mouse models of schizophrenia

1Copy number variations in the ARHGAP10 gene encoding Rho GTPase-activating protein 10 are significantly associated with schizophrenia. ARHGAP10 negatively regulates RhoA/Rho-kinase (ROCK) signaling. We previously demonstrated that fasudil, a non-selective ROCK inhibitor, exhibited antipsychotic-like effects in several mouse models of schizophrenia. ROCK has two subtypes, ROCK1 and ROCK2. ROCK1 is mainly expressed in the thymus and blood, while ROCK2 is predominantly expressed in the brain. Therefore, it is expected that like fasudil, selective ROCK2 inhibitors will exhibit antipsychotic-like effects, accompanied by a lower incidence of adverse effects due to ROCK1 inhibition. Here, we used genetic and pharmacological models of schizophrenia to investigate whether the selective ROCK2 inhibitor KD025 would show antipsychotic-like effects with a favorable adverse effect profile. Oral administration of KD025 suppressed the abnormal increase in the phosphorylation level of myosin phosphatase-targeting subunit 1, a substrate of ROCK, and ameliorated the decreased spine density of layer 2/3 pyramidal neurons in the medial prefrontal cortex of Arhgap10 S490P/NHEJ mice. Furthermore, KD025 mitigated the methamphetamine-induced impairment of visual discrimination (VD) in Arhgap10 S490P/NHEJ and wild-type mice. KD025 also reduced MK-801-induced impairments of VD, novel object recognition, and hyperlocomotion. Regarding side effects that are commonly seen with typical antipsychotics, KD025 did not affect systolic blood pressure and did not induce extrapyramidal symptoms, hyperprolactinemia, or hyperglycemia at the effective dosage in naive wild-type mice. Taken together, KD025 shows antipsychotic-like effects with a favorable adverse effect profile in genetic and pharmacological mouse models of schizophrenia.

pharmacology and toxicology↗

bioRxiv2024

VR HMD color calibration and accurate control of emitted light using Three.js

Virtual Reality (VR) can be used to design and create new types of psychophysical experiments. Its main advantage is that it frees us from the physical limitations of real-life experiments and the hardware and software limitations of experiments running on 2D displays and computer graphics. However, color calibration of the displays is often required in vision science studies. Recent studies have shown that a standard color calibration of a Head-Mounted Display (HMD) can be very challenging and comes with significant drawbacks. In this paper, we introduce a new approach that allows for successful color calibration of an HMD and overcomes the disadvantages associated with other solutions. We utilize a new VR engine, Three.js, which offers several advantages. This paper details our setup and methodology, and provides all the elements required to reproduce the method, including the source code. We also apply our method to evaluate and compare three different HMDs: HTC Vive Pro Eye, Meta Quest Pro, and Meta Quest 3. The results show that the HTC Vive Pro Eye performs excellently, the Meta Quest Pro performs well, and the Meta Quest 3 performs poorly.

neuroscience↗