Biology subjects

Hirai, H.

Publications and source records attributed to Hirai, H..

At least 19 recordsLinked to original sources

bioRxiv2026

Megalin-mediated deterministic pseudotransduction in proximal tubules fundamentally challenges current understanding of AAV kidney transduction

Genetic kidney diseases are promising targets for gene therapy. Emerging AAV capsids that reportedly enable effective gene delivery to proximal tubules (PTs) following intravenous injection have raised hopes for treating diseases affecting renal tubules. Despite these promising developments, the scientific literature contains contradictory and non-reproducible findings, which prompted us to conduct a detailed and rigorous investigation of the mechanisms of AAV vector-mediated PT transduction. Here we provide compelling evidence that previously reported effective PT transduction following intravenous AAV-GFP or AAV-mCherry administration largely reflects megalin-mediated deterministic pseudotransduction rather than stochastic vector-mediated gene delivery. We demonstrate that this process is mediated by megalin-dependent uptake of circulating reporter proteins released from transduced organs including the liver and filtered through the glomeruli. These observations highlight the ongoing challenge of achieving true proximal tubule transduction via systemic AAV delivery and caution against relying on small reporters like GFP or mCherry, which can produce misleading signals.

bioengineering↗

bioRxiv2026

The vagus nerve drives cognitive function through the default mode network

The afferent vagus nerve transmits interoceptive signals to the brain but a systematic understanding of how vagal signals affect brain-wide neuronal dynamics remains elusive. Here, comprehensive spatiotemporal analyses across the entire mouse brain revealed that vagus nerve stimulation activated neuronal ensembles within the default mode network (DMN). In contrast, vagal-independent mechanisms predominantly affected the salience network. The vagal-DMN association was mediated by locus coeruleus neuronal subpopulations. Selective activation or silencing of vagal-responsive DMN neuronal ensembles replicated or abolished VNS-induced enhancement of cognitive behaviors and progressive problem-solving behaviors. These findings establish a unifying framework for how the vagus nerve drives cognitive functions by engaging the DMN.

neuroscience↗

bioRxiv2026

BRCA2 loss triggers a downward spiral of genomic instability via ROS-dependent metabolic collapse

BRCA2 plays a central role in maintaining genome integrity through homologous recombination and replication-fork protection, yet the compensatory networks sustaining BRCA2-deficient cells remain unclear. Here we show BRCA2 enforces a homeostatic mechanism aligning mitochondrial respiration with DNA repair capacity in both cancer and non-cancer contexts. Genome-wide CRISPR screening identified glutathione metabolism and base-excision repair as the key compensatory networks sustaining BRCA2-deficient cells by detoxifying mitochondria-derived reactive oxygen species. BRCA2 loss provokes an acute mitochondrial ROS surge, causing 8-oxoguanine accumulation and a systemic metabolic crisis marked by NAD+ and glutathione depletion. PARP inhibitor targets DNA replication vulnerabilities, increasing the cellular requirement for BRCA2. The resulting oxidative burden primes cells for TP53-dependent apoptosis in G1 during olaparib treatment, which extends cytotoxicity beyond canonical S-phase stress. These findings indicate BRCA2 prevents metabolic flux from outpacing repair capacity, providing a rationale for combining PARP inhibition with redox modulation to enhance efficacy and overcome resistance. HighlightsO_LIAcute BRCA2 loss induces ROS and mitochondrial dysfunction creating a metabolic scar C_LIO_LIOxidative lesions drive PARP hyperactivation and precipitate a cellular NAD crisis C_LIO_LIPARP inhibitors provoke TP53-dependent apoptosis in G1 beyond replication stress in S phase C_LIO_LIGlutathione deficiency exacerbates bone marrow failure under BRCA2 depletion C_LIO_LIBRCA2 tightly couples mitochondrial redox homeostasis to genomic maintenance C_LI

cell biology↗

bioRxiv2025

A compact glutamic acid decarboxylase 67 promoter enables inhibitory neuron-targeted AAV gene therapy for treatment-resistant epilepsy

Epilepsy often becomes treatment-resistant, partly due to impaired inhibitory neurotransmission and reduced {gamma}-aminobutyric acid (GABA) function. Enhancing inhibitory neuron activity via gene therapy may restore excitation-inhibition (E/I) balance. We developed a compact 410-bp glutamic acid decarboxylase 67 promoter (cmGAD67) that enables strong, selective transgene expression in inhibitory neurons while preserving AAV packaging capacity. When delivered systemically, AAV vectors carrying cmGAD67 preferentially targeted parvalbumin interneurons and supported effective circuit manipulation. To evaluate therapeutic potential, we expressed glutamic acid decarboxylase 65 (GAD65) under cmGAD67 (AAV-GAD65) in pentylenetetrazole (PTZ) epilepsy models. Systemic AAV-GAD65 suppressed abnormal delta oscillations, reduced seizure-like events, normalized anxiety-like behavior, and improved survival in a severe PTZ paradigm. Biochemical analyses confirmed increased cortical and hippocampal GABA levels, linking behavioral and electrophysiological improvements to enhanced inhibitory neurotransmitter synthesis. Prior clinical evidence indicates that AAV-GAD65 delivery to the subthalamic nucleus is safe and effective in Parkinsons disease. Building on this foundation, our findings establish the cmGAD67 promoter as a powerful platform for inhibitory neuron-targeted AAV gene therapy and highlight AAV-cmGAD67-GAD65 as a promising approach for treatment-resistant epilepsy and other disorders involving disrupted E/I balance.

neuroscience↗

bioRxiv2025

VCAP-102 Achieves Exceptional BBB Penetration in Marmosets After Ten Years of AAV Capsid Evaluation

Efficient passage of adenoassociated virus (AAV) vectors across the blood-brain barrier (BBB) is essential for achieving broad central nervous system transduction following systemic delivery. Since PHP.B was reported in 2016 to exhibit high BBB permeability in C57BL/6J mice, many BBB-penetrant capsids have been developed, yet their performance often varies substantially across species. From 2016 to 2025, we produced each newly reported BBB-penetrant capsid as its sequence became available and evaluated it under standardized intravenous administration conditions in marmosets. This included PHP.B, PHP.eB, AAV-F, CAP-B22, CAP-B10, CPP.16, CAP-Mac, and BR1N. Although several of these capsids have been reported to penetrate the marmoset BBB, none reproduced these effects under our unified evaluation conditions. We also pursued several directed-evolution approaches to engineer AAV9-based variants with improved BBB permeability; however, none surpassed AAV9. In 2025, VCAP-102 was reported as an AAV9-derived capsid with BBB permeability in mice and non-human primates. When evaluated using our standardized framework, VCAP-102 consistently mediated markedly stronger and more widespread whole-brain transduction than AAV9 across all three animals tested. These findings provide a decade-long, systematic assessment of BBB-penetrant AAV capsids in marmosets and identify VCAP-102 as a highly promising candidate for future systemically delivered AAV-based gene therapies.

neuroscience↗

bioRxiv2025

Axis-specific neuro-musculoskeletal contributions to postural stability and vibration avoidance in mice

Animals must appropriately regulate their responses to external mechanical oscillations such as whole-body (WB) vibration to ensure postural stability. However, knowledge of mouse responses to WB vibration remains limited. Here, we used high-speed video-based quantitative analyses to characterize body-part movements in awake and anesthetized mice under vertical, longitudinal, and lateral WB vibration. Awake mice exhibited lower resonance frequencies and smaller displacement amplitudes than anesthetized mice during vertical and lateral WB vibration, but not longitudinal vibration, indicating that the neuro-musculoskeletal (NMS) system contributes to postural stability in a vibration axis-dependent manner. Vibration modeling suggests that the NMS system acts as an active vibration absorber by reducing effective stiffness and dynamically adjusting effective damping, analogous to active vehicle suspensions. Axis-specific vibration control may reflect an evolutionary balance between postural stability and locomotor efficiency in quadrupeds, paralleling the design principles of rear-wheel-drive vehicles. Behavioral preference tests revealed that mice selectively avoid vertical WB vibration at specific frequencies, but neither lateral nor longitudinal WB vibration. These findings reveal an axis-specific NMS control principle analogous to vehicle design, linking postural regulation, locomotor efficiency and avoidance behavior, and advancing our understanding of the biomechanics and behavior of quadrupeds exposed to WB vibration.

biophysics↗

bioRxiv2025

Establishment of spinocerebellar ataxia type 34 model mice accompanied by early glial activation and degeneration of cerebellar neurons

Spinocerebellar ataxia type 34 (SCA34) is an autosomal dominant neurodegenerative disease primarily characterized by progressive cerebellar atrophy and ataxia, frequently accompanied by cognitive dysfunction and erythrokeratodermia variabilis. In 2014, missense mutations in the gene encoding elongation of very long chain fatty acids protein 4 (ELOVL4) were identified as the causative gene for SCA34. ELOVL4, which is involved in the synthesis of very long chain fatty acids, is highly expressed in the cerebellum compared to other brain regions, with predominant expression in neurons. We attempted to establish a mouse model of SCA34 by expressing mutant ELOVL4 in cerebellar neurons using adeno-associated virus (AAV) vectors and to elucidate the underlying pathogenic mechanisms. Expression of W246G mutant ELOVL4 successfully induced progressive motor dysfunction beginning at two weeks post-AAV vector injection. Immunohistochemical analyses revealed that the degeneration of cerebellar Purkinje cells and neurons in the deep cerebellar nuclei (DCN) paralleled the observed motor decline. Importantly, microglial activation was detected in the molecular layer of the cerebellar cortices and the DCN prior to the onset of both neurodegeneration and motor dysfunction. Furthermore, after the onset of motor symptoms, the SCA34 model mice exhibited decreased synaptic inputs from climbing fibers to Purkinje cells, as well as reduced inputs from Purkinje cells to DCN neurons. These findings suggest that early microglial activation and the resulting synaptic disturbance are critical preceding events that lead to the progressive cerebellar neurodegeneration and motor dysfunction observed in this SCA34 mouse model.

neuroscience↗

bioRxiv2025

Functional and structural profiling of circulation via genetically encoded modular fluorescent probes.

Sustained labeling of fluids is crucial for their investigation in animal models. Here, we introduce a mouse line (Alb-mSc-ST), where blood and interstitial fluid are labeled with the red fluorescent protein mScarlet and SpyTag. The SpyTag-SpyCatcher technology is exploited to monitor circulating fluid properties by biosensors or detect blood-brain barrier disruption. This approach represents a valuable tool for studying vascular structure, permeability and microenvironment in body organs in vivo.

neuroscience↗

bioRxiv2024

Optimization of microRNA Target Sequence Arrangement in AAV Vectors Dramatically Enhances Specificity and Efficiency of Transgene Expression in Cortical Microglia

Microglia play a critical role in diseases such as Alzheimers and stroke, making them a significant target for therapeutic intervention. However, due to their immune functions in detecting and combating viral invasion, efficient gene delivery to microglia remains challenging. We achieved specific and efficient gene delivery to microglia using an adeno- associated virus (AAV) vector designed for this purpose. This microglia-targeting AAV vector includes the mouse microglia/macrophage-specific ionized calcium-binding adaptor molecule 1 (mIba1) promoter, green fluorescent protein (GFP), microRNA target sequences (miR.Ts), woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), and polyadenylation (polyA) signal, positioned between inverted terminal repeats. When miR.Ts were placed downstream of WPRE (between WPRE and polyA), gene expression occurred not only in microglia but also in a substantial number of neurons. However, when miR.Ts were positioned upstream of WPRE (between GFP and WPRE) or on both sides of WPRE, neuronal expression was significantly suppressed, resulting in selective GFP expression in microglia. Notably, positioning miR.Ts on both sides of WPRE achieved over 90% specificity and more than 60% efficiency in transgene expression in microglia three weeks after viral administration. This vector also enabled GCaMP expression in microglia, facilitating real-time monitoring of calcium dynamics and microglial process activity in the cortex. Additionally, intravenous administration of this vector with the blood-brain barrier-penetrant AAV-9P31 capsid variant resulted in extensive GFP expression selectively in microglia throughout the brain. These findings establish this AAV vector system as a robust tool for long-term, specific, and efficient gene expression in microglia.

neuroscience↗

bioRxiv2024

Distinct release properties of glutamate/GABA co-transmission serve as a frequency-dependent filtering of supramammillary inputs

Glutamate and GABA co-transmitting neurons exist in several brain regions; however, the mechanism by which these two neurotransmitters are co-released from the same synaptic terminals remains unclear. Here, we show that the supramammillary nucleus (SuM) to dentate granule cell synapses, which co-release glutamate and GABA, exhibit differences between glutamate and GABA release properties in paired-pulse ratio, Ca2+-sensitivity, presynaptic receptor modulation, and Ca2+ channel-vesicle coupling configuration. Moreover, uniquantal synaptic responses show independent glutamatergic and GABAergic responses. Morphological analysis reveals that most SuM terminals form distinct glutamatergic and GABAergic synapses in proximity, each characterized by GluN1 and GABAA1 labeling, respectively. Notably, glutamate/GABA co-transmission exhibits distinct short-term plasticities, with frequency-dependent depression of glutamate and frequency-independent stable depression of GABA. Our findings suggest that glutamate and GABA are co-released from different synaptic vesicles within the SuM terminals, and reveal that distinct transmission modes of glutamate/GABA co-release serve as frequency-dependent filters of SuM inputs.

neuroscience↗

bioRxiv2024

Optimal AAV capsid/promoter combinations to target specific cell types in the common marmoset cerebral cortex

To achieve cell type-specific gene expression, using target cell-tropic AAV capsids is advantageous. However, their tropism across brain cell types remains unexplored in non-human primates. We assessed the tropism of nine AAV serotype capsids (AAV1, 2, 5, 6, 7, 8, 9, rh.10 (rh10), and DJ) on marmoset cerebral cortical cell types. Marmoset cerebral cortex was injected with different serotype AAVs expressing enhanced GFP (EGFP) by the ubiquitous chicken {beta}-actin hybrid (CBh) promoter. After 4 weeks, all nine AAV capsid vectors, especially AAV9 and AAVrh10, caused highly neuron-selective EGFP expression. Some AAV capsids, including AAV5, caused EGFP expression in oligodendrocytes to a lesser extent, with minimal or no expression in astrocytes and microglia. Different ubiquitous CMV and CAG promoters showed similar neuron-predominant transduction. Conversely, all nine AAV capsid vectors with the astrocyte-specific hGFA(ABC1D) promoter selectively transduced astrocytes, except AAV5, which transduced oligodendrocytes modestly. Oligodendrocyte-specific mouse myeline basic protein (mMBP) promoter in AAV5 vectors transduced oligodendrocytes specifically and efficiently. Our results suggest optimal combinations of capsids and promoters for cell type-specific expression: AAV9 or AAVrh10 and ubiquitous CBh, CMV, or CAG promoter for neuron-specific transduction; AAV2 or 7 and hGFA(ABC1D) promoter for astrocyte-specific transduction; and AAV5 and mMBP promoter for oligodendrocyte-specific transduction.

neuroscience↗

bioRxiv2023

Virally induced CRISPR/Cas9-based knock-in of fluorescent albumin allows long-term visualization of cerebral circulation in infant and adult mice

Albumin, a protein produced by liver hepatocytes, represents the most abundant protein in blood plasma. We have previously engineered a liver-targeting adeno-associated viral vector (AAV) that expresses fluorescent protein-tagged albumin to visualize blood plasma in mice. While this approach is versatile for imaging in adult mice, transgene expression vanishes when AAV is administered in neonates due to dilution of the episomal AAV genome in the rapidly growing liver. Here, we use CRISPR/Cas9 genome editing to insert the fluorescent protein mNeonGreen (mNG) gene into the albumin (Alb) locus of hepatocytes to produce fluorescently labeled albumin (Alb-mNG). We constructed a CRISPR AAV that includes [~]1 kb homologous arms around Alb exon 14 to express Alb-mNG. Subcutaneous injection of this AAV with AAV-CMV-Cas9 in postnatal day 3 mice resulted in two-photon visualization of the cerebral cortex vasculature within ten days. The expression levels of Alb-mNG were persistent for at least three months and were so robust that vasomotion and capillary blood flow could be assessed transcranially in early postnatal mice. This knock-in approach provides powerful means for micro- and macroscopic imaging of cerebral vascular dynamics in postnatal and adult mice.

neuroscience↗

bioRxiv2022

Dynamic Modulation of Pulsatile Activities of Oxytocin Neurons in Lactating Wild-type Mice

Breastfeeding, which is essential for the survival of mammalian infants, is critically mediated by pulsatile secretion of the pituitary hormone oxytocin from the central oxytocin neurons located in the paraventricular and supraoptic hypothalamic nuclei of mothers. Despite its importance, the molecular and neural circuit mechanisms of the milk ejection reflex remain poorly understood, in part because a mouse model to study lactation was only recently established. In our previous study, we successfully introduced fiber photometry-based chronic imaging of the pulsatile activities of oxytocin neurons during lactation. However, the necessity of Cre recombinase-based double knock-in mice substantially compromised the use of various Cre-dependent neuroscience toolkits. To overcome this obstacle, we developed a simple Cre-free method for monitoring oxytocin neurons by an adeno-associated virus vector driving GCaMP6s under a 2.6 kb mouse oxytocin mini-promoter. Using this method, we monitored calcium ion transients of oxytocin neurons in the paraventricular nucleus in wild-type C57BL/6N and ICR mothers without genetic crossing. By combining this method with video recordings of mothers and pups, we found that the pulsatile activities of oxytocin neurons require physical mother-pup contact for the milk ejection reflex. Notably, the frequencies of photometric signals were dynamically modulated by mother-pup reunions after isolation and during natural weaning stages. Collectively, the present study illuminates the temporal dynamics of pulsatile activities of oxytocin neurons in wild-type mice and provides a tool to characterize maternal oxytocin functions.

neuroscience↗

bioRxiv2022

Gene mapping methodology powered by induced genome rearrangements

Phenotypic variation occurs through genome rearrangements and mutations in certain responsible genes; however, systematic gene identification methodologies based on genome rearrangements have not been fully established. Here, we explored the loci responsible for the given phenotype using the TAQing system and compared it with a conventional mutagenesis-based method. Two yeast strains with different genetic backgrounds and flocculation phenotypes were fused and genomic rearrangements were induced by transient DNA breaks. Then, selection pressure was applied and multiple mutants were generated, showing different flocculation abilities. We also raised mutants with altered cohesiveness due to spontaneous mutations during long-term recursive passages of haploid strains without TAQing treatment. Comparative genomic analysis of the TAQed mutants revealed three chromosomal regions harboring pivotal flocculation genes, whereas conventional mutagenesis generated a more diverse list of candidate loci after prolonged selection. The combined use of these approaches will accelerate the identification of genes involved in complex phenotypes.

genetics↗

bioRxiv2022

Liver-secreted fluorescent blood plasma markers enable chronic imaging of microcirculation

Studying blood microcirculation is vital for gaining insights into vascular diseases. Acute administration of fluorescent tracers is currently used for deep tissue blood flow imaging. This is invasive, and the plasma fluorescence decreases within an hour of administration. We report a novel approach for the longitudinal study of vasculature. Using a single systemic administration of viral vectors, we express fluorescent secretory albumin-fusion proteins in the liver to label the blood in mice. All segments of the vasculature in brain and peripheral tissue are observable by two-photon microscopy within two weeks of vector administration. This approach allows for observation of circulation without the need for repeated administration for several months. We demonstrate the chronic assessment of vascular functions at micro-and mesoscopic scales. This genetic plasma labeling approach represents a versatile and cost-effective method for the chronic investigation of vasculature functions across the body in health and disease.

physiology↗

bioRxiv2021

Mis6/CENP-I maintains CENP-A nucleosomes against centromeric non-coding transcription during mitosis

Centromeres are established by nucleosomes containing the histone H3 variant CENP-A. CENP-A is recruited to centromeres by the Mis18-HJURP machinery. During mitosis, CENP-A recruitment ceases, implying the necessity of CENP-A maintenance at centromeres, although the exact underlying mechanism remains elusive. Herein, we show that the kinetochore protein Mis6 (CENP-I) retains CENP-A during mitosis in fission yeast. Eliminating Mis6 during mitosis caused immediate loss of pre-existing CENP-A at centromeres. CENP-A loss occurred due to the transcriptional upregulation of non-coding RNAs at the central core region of centromeres, as confirmed by the observation RNA polymerase II inhibition preventing CENP-A loss from centromeres in the mis6 mutant. Thus, we concluded that Mis6 blocks the indiscriminate transcription of non-coding RNAs at the core centromere, thereby retaining the epigenetic inheritance of CENP-A during mitosis.

cell biology↗

bioRxiv2021

Plasticity of Neural Connections Underlying Oxytocin-mediated Parental Behaviors of Male Mice

The adult brain can flexibly adapt behaviors to specific life-stage demands. For example, while sexually naive male mice are aggressive to the conspecific young, they start to provide caregiving to infants around the time when their own young are expected. How such behavioral plasticity is implemented at the level of neural connections remains poorly understood. Using viral-genetic approaches, here we establish hypothalamic oxytocin neurons as key regulators of parental caregiving behaviors of male mice. We then used rabies virus-mediated unbiased screen to identify excitatory neural connections originating from the lateral hypothalamus to the oxytocin neurons to be drastically strengthened when male mice become fathers. These connections are functionally relevant, as their activation suppresses pup-directed aggression in virgin males. These results demonstrate the life-stage associated, long-distance, and cell-type-specific plasticity of neural connections in the hypothalamus, the brain region classically assumed to be hard-wired. Highlight- OT is indispensable for parental caregiving behavior of male mice - Activation of OT neurons triggers paternal caregiving behavior in otherwise infanticidal sexually-naive male mice partly via OT ligand - Unbiased rabies virus-mediated screening reveals enhanced connectivity originated from excitatory LHA neurons to OT neurons in fathers. - This structural plasticity can support behavioral plasticity

neuroscience↗

bioRxiv2021

Development of novel potent ligands for GPR85, an orphan G protein-coupled receptor expressed in the brain

GPR85 is a member of the G protein-coupled receptor and is a super-conserved receptor expressed in the brain sub-family (SREB) with GPR27 and GPR173. These three receptors are "orphan receptors"; however, their endogenous ligands have not been identified. SREB has garnered the interest of many scientists because it is expressed in the central nervous system and is evolutionarily conserved. In particular, brain mass is reported to be increased and learning and memory are improved in GPR85 knockout mice (Matsumoto et al., 2008). In this study, we characterized newly synthesized compounds using a GPR85-Gs fusion protein and the [35S]GTP{gamma}S binding assay and identified novel GPR85 inverse-agonists with IC50 values of approximately 1 {micro}M. To analyze the neurochemical character of the compounds and investigate the physiological significance of GPR85, we used cerebellar Purkinje cells expressing GPR85 and an electrophysiological technique. Based on the results, the inverse-agonist compound for GPR85 modulated potassium channel opening. Together with the results of previous gene analysis of GPR85, we expect that the development of the GPR85 ligand will provide new insights into a few types of neurological disorders.

pharmacology and toxicology↗