Biology subjects

Tsunekawa, Y.

Publications and source records attributed to Tsunekawa, Y..

8 recordsLinked to original sources

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↗

bioRxiv2025

A Novel MiRNA Detection System Combining TWJ-SDA,Multistep L-TEAM, and CRISPR-Cas3

MicroRNAs (miRNAs) serve critical regulatory roles in gene expression and are valuable biomarkers for early disease detection. However, their inherent low concentration in biological fluids poses significant detection challenges. Although traditional methods like real-time quantitative PCR (RT-qPCR) are highly sensitive, they require thermal cycling, limiting their application in point-of-care testing (POCT). Here, we present an isothermal amplification-based miRNA detection system integrating Three-Way Junction (TWJ) formation, Multistep Low-Temperature Amplification (L-TEAM), and CRISPR-Cas3-mediated signal amplification. The integration of the Multistep L-TEAM with the TWJ method achieves high sensitivity, detecting miRNA at concentrations as low as 10 femtomolar within 50 minutes, and effectively distinguishes single-nucleotide mismatches. When CRISPR-Cas3-mediated reaction was integrated, it still proved effective for confirming the presence of the target, but its quantitative reliability requires further optimization. We developed a predictive model using machine learning to facilitate rational optimization of experimental conditions through contribution analysis and to establish a methodology for designing more favorable sequences. The modular nature of our method permits adaptation to diverse miRNA targets without modifications to the fundamental amplification mechanism.

molecular biology↗

bioRxiv2024

PURIFICATION METHOD FOR ADENO-ASSOCIATED VIRAL VECTOR SEROTYPE 9 USING CERAMIC HYDROXYAPATITE CHROMATOGRAPHY AND ITS QUALIFICATION

Adeno-associated virus (AAV) vectors can efficiently transduce exogenous genes into various tissues in vivo. Owing to their convenience, high efficiency, long-term stable gene expression, and minimal side effects, AAV vectors have become one of the gold standards for investigating gene functions in vivo, especially in non-clinical studies. However, challenges persist in efficiently preparing a substantial quantity of high-quality AAV vectors that comprise only genome-packaged full particles. Commercial AAV vectors are typically associated with high costs. Further, in-laboratory production is hindered by the lack of specific laboratory equipment, such as ultracentrifuges. Therefore, a simple, quick, and scalable preparation method for AAV vectors is needed for proof-of-concept experiments. Herein, we present an optimized method for producing and purifying high-quality AAV serotype 9 (AAV9) vectors using standard laboratory equipment and chromatography. Using ceramic hydroxyapatite as a mixed-mode chromatography medium can markedly increase the quality of purified AAV vectors. Basic protocols and optional methods for evaluating purified AAV vectors are also described.

molecular biology↗

bioRxiv2023

Efficient workflow for validating homology-independent targeted integration-mediated gene insertion in rod photoreceptor cells to treat dominant-negative mutations causing retinitis pigmentosa

Among the genome-editing methods for repairing disease-causing mutations resulting in dominant inhibition, homology-independent targeted integration (HITI)-mediated gene insertion of the normal form of the causative gene is useful because it allows the development of mutation-agnostic therapeutic products. For the rapid optimization and validation of highly effective HITI-treatment gene constructs against dominant-negative inheritance of inherited retinal dystrophy, we improved the gene constructs available in both plasmid and adeno-associated virus (AAV) vectors, and established a workflow that uses in vivo electroporation to verify the in vivo efficacy. By targeting the mouse Rhodopsin gene, we derived a construct in which HITI-mediated gene insertion occurs in 80%-90% of transduced mouse rod photoreceptor cells. This construct suppressed degeneration and induced visual restoration in the mutant mice. The HITI-treatment constructs for the rhodopsin gene were shown to be effective in AAV vectors, and this construction is available for the mouse Peripherin 2 gene. These findings suggest that the workflow reported here may be useful for the generation of HITI-treatment constructs for various target genes and for the development of gene therapy products.

molecular biology↗

bioRxiv2023

Evasion of APOBEC1-mediated Intrinsic Immunity by a Herpesvirus Uracil DNA Glycosylase Is a Determinant of Viral Encephalitis

Herpes simplex virus 1 (HSV-1) is the most common cause of viral encephalitis, which can be lethal or result in severe neurological defects, even when treated with antiviral therapy. We demonstrated that activation of HSV-1 uracil-DNA glycosylase (vUNG) by phosphorylation, essential for its enzymatic activity, counteracted APOBEC1 to promote viral replication and encephalitis in the central nervous system (CNS) of mice. The activation of vUNG protected HSV-1 genomes from APOBEC1-mediated DNA editing, allowing efficient viral replication to occur. The presence of APOBEC1 markedly improved lethal encephalitis in mice infected with an HSV-1 mutant carrying a mutation in the phosphorylation site and an UNG inhibitor protected wild-type HSV-1-infected mice from lethal encephalitis. These findings re-define vUNG as an important factor that allows evasion from intrinsic anti-viral immunity mediated by APOBEC1 in the CNS, and suggest a new therapeutic approach for the treatment of fetal and critical HSV-1 encephalitis.

microbiology↗

bioRxiv2023

Engineering a highly durable adeno-associated virus receptor for analytical applications

Adeno-associated virus (AAV) is a major viral vector used in gene therapy. There are multiple AAV serotypes, and many engineered AAV serotypes with modified capsids altering tissue tropism are enthusiastically being developed. The universal AAV receptor (AAVR) is an essential receptor for multiple AAV infections. Since most AAV serotypes used in gene therapy infect cells via interaction with AAVR, the quantification of the vector-binding ability of AAV to AAVR could be an important quality check for therapeutic AAV vectors. To enable a steady evaluation of the AAV-AAVR interaction, we created an engineered AAVR through mutagenesis. Engineered AAVR showed high durability against acid while retaining its AAV-binding activity and an affinity chromatography column with engineered AAVR was also developed. This column enabled repeated binding and acid dissociation measurements of AAVR with various AAV serotypes. Our data showed that the binding affinities of AAV to AAVR were diverse among serotypes, providing insight into the relationship with the infection efficiency of AAV vectors. Thus, this affinity column can be used in process development for quality checks, quantitating capsid titers, and affinity purification of AAV vectors. Furthermore, this column may work for a useful tool of novel AAV vector capsid engineering.

bioengineering↗

bioRxiv2022

Single cell transcriptomics of ferrets reveal a common temporal pattern of progenitors in brain development of gyrencephalic mammals

The diversity of neural stem cells is a hallmark of the cerebral cortex development in gyrencephalic mammals, such as Primates and Carnivora. Among them, ferrets are a good model for mechanistic studies. However, information on their neural progenitor cells (NPC), termed radial glia (RG), is limited. Here, we surveyed the temporal series of single-cell transcriptomes of progenitors regarding ferret corticogenesis and, found a conserved diversity and temporal trajectory between human and ferret NPC, despite the large timescale difference. We found truncated RG (tRG) in ferret cortical development, a progenitor subtype previously described in humans. The combination of in silico and in vivo analyses identified that tRG differentiate into both ependymal and astrogenic cells. Via transcriptomic comparison, we predict that this is also the case in humans. Our findings suggest that tRG plays a role in the formation of adult ventricles, thereby providing the architectural bases for brain expansion.

developmental biology↗

bioRxiv2020

Subcellular mRNA localization and local translation of Arhgap11a in radial glial cells regulates cortical development

mRNA localization and local translation enable exquisite spatial and temporal control of gene expression, particularly in highly polarized and elongated cells. These features are especially prominent in radial glial cells (RGCs), which serve as neural and glial precursors of the developing cerebral cortex, and scaffolds for migrating neurons. Yet the mechanisms by which distinct sub-cellular compartments of RGCs accomplish their diverse functions are poorly understood. Here, we demonstrate that subcellular RNA localization and translation of the RhoGAP Arhgap11a controls RGC morphology and mediates cortical cytoarchitecture. Arhgap11a mRNA and protein exhibit conserved localization to RGC basal structures in mice and humans, conferred by a 5'UTR cis-element. Proper RGC morphology relies upon active Arhgap11a mRNA transport and localization to basal structures, where ARHGAP11A is locally synthesized. Thus, RhoA activity is spatially and acutely activated via local translation in RGCs to promote neuron positioning and cortical cytoarchitecture. Altogether, our study demonstrates that mRNA localization and local translation mediate compartmentalization of neural progenitor functions to control brain development. HighlightsO_LIArhgap11a in radial glia non-cell autonomously promotes neuronal migration and lamination C_LIO_LIArhgap11a mRNA localizes to radial glial endfeet via a 5 UTR cis element C_LIO_LIARHGAP11A expression in basal process and endfeet depends upon its localized mRNA C_LIO_LILocalized mRNA and RhoA-GAP activity in endfeet control radial glial morphology C_LI

neuroscience↗