Biology subjects

Saga, Y.

Publications and source records attributed to Saga, Y..

14 recordsLinked to original sources

bioRxiv2026

Activation of the NAD⁺-Sirtuin Axis Protects Against Chronic Doxorubicin-Induced Subclinical Renal Tubular Injury Through Restoration of Mitochondrial Homeostasis and Suppression of Inflammation

Background and purposeAnthracyclines, such as doxorubicin (DOX), are associated with late-onset kidney dysfunction; however, the mechanisms underlying chronic tubular injury remain poorly understood. We investigated whether chronic low-dose DOX exposure induces persistent mitochondrial dysfunction in renal tubules and evaluated the therapeutic potential of activating the NAD-Sirtuin axis. Experimental ApproachC57BL/6 mice were repeatedly administered low-dose DOX with or without resveratrol (RSV) or nicotinamide mononucleotide (NMN), a sirtuin activator. Renal injury was assessed using neutrophil gelatinase-associated lipocalin (NGAL) staining. Integrated proteomic and RNA sequencing analyses were performed to identify molecular alterations. Mitochondrial morphology and function were evaluated using structured illumination microscopy (SIM) of Massons trichrome-stained paraffin sections and ex vivo Seahorse analysis of freshly isolated renal tubules. Key ResultsChronic DOX administration induced tubular injury, despite preserving serum creatinine levels. Multi-omics analyses consistently demonstrated the suppression of mitochondrial pathways, including oxidative phosphorylation, fatty acid oxidation, and mitochondrial gene expression. SIM revealed mitochondrial fragmentation in tubular epithelial cells, whereas the Seahorse assay showed impaired mitochondrial respiratory capacity in isolated renal tubules. DOX also increased tubular acetylated superoxide dismutase 2 (SOD2) levels and activated inflammatory pathways. Importantly, both RSV and NMN attenuated tubular injury, restored mitochondrial metabolic pathways, reduced SOD2 acetylation, improved mitochondrial morphology, and suppressed inflammatory responses. Conclusions and ImplicationsChronic low-dose DOX exposure induces subclinical renal tubular injury characterized by mitochondrial dysfunction and inflammation. The pharmacological activation of sirtuins confers reno-protective effects by preserving mitochondrial homeostasis. These findings identify mitochondrial dysfunction as a central therapeutic target in DOX-induced nephrotoxicity and support sirtuin modulation as a potential strategy for preventing chemotherapy-related chronic kidney injury. Bullet point summaryO_ST_ABSWhat is already knownC_ST_ABSO_LIDoxorubicin causes cardiotoxicity through mitochondrial dysfunction and oxidative stress. C_LIO_LIDoxorubicin-induced tubular injury and the associated late-onset kidney dysfunction are clinically proven. C_LI What does this study addO_LIChronic low-dose doxorubicin induces tubular mitochondrial dysfunction, as identified by integrated multi-omics analyses. C_LIO_LIResveratrol and NMN preserve mitochondrial integrity and suppress inflammatory responses in renal tubules. C_LI Clinical significanceO_LIMitochondrial dysfunction may represent an early therapeutic target in doxorubicin-associated nephrotoxicity. C_LIO_LIActivation of the NAD-Sirtuin axis could prevent chronic kidney injury in cancer survivors. C_LI

pharmacology and toxicology↗

bioRxiv2025

AID-mediated protein knockdown reveals the requirement of NANOS2 in prenatal gonocytes for establishing functional spermatogonial stem cells

The RNA-binding protein NANOS2 plays a crucial role in male gonocyte development and the maintenance of spermatogonial stem cells. In the absence of the Nanos2 gene (Nanos2-KO), germ cells fail to enter G0 arrest and initiate the male differentiation program (including DNA methylation and the piRNA pathway), ultimately undergoing apoptosis before birth. Nanos2 transcription begins at embryonic day 12.5 (E12.5) and terminates at E15.5. However, as the NANOS2 protein continues to be stably expressed beyond E15.5, it is important to elucidate the function of NANOS2 during this post-E15.5 period in germ cell fate determination. To address the functional significance of sustained NANOS2 protein expression, we employed an auxin-inducible degron (AID2) system to achieve rapid degradation of NANOS2 after E15.5. Within 24 hours of 5-Ph-IAA administration, NANOS2 protein was efficiently depleted. As a result, germ cells resumed the cell cycle, exhibited aberrant gene expression patterns similar to Nanos2- KO gonocytes, and underwent apoptosis if NANOS2 depletion occurred at E15.5 or E16.5. Although some surviving cells initiated spermatogenesis and expressed PLZF and GFRA1 after birth, further spermatogenesis was not observed. These findings reveal that sustained NANOS2 protein expression during the embryonic stage is essential for establishing functional spermatogonial stem cells, highlighting a previously unrecognized regulatory mechanism in male germ cell development.

developmental biology↗

bioRxiv2025

Establishment of a second-generation transgenic marmoset model of polyglutamine disease recapitulating neurological symptoms and pathology

Neurodegenerative diseases, including polyglutamine diseases, remain a major clinical challenge, partly because of limited animal models that recapitulate human disease. Here, we describe a second-generation transgenic marmoset model of spinocerebellar ataxia 3 (SCA3), a polyglutamine disease, which stably expresses expanded CAG repeats in ATXN3. All five offspring of the founder marmoset harbored the transgene with reduced transgene integration sites and without repeat instability or genetic mosaicism, offering improved construct validity. Three of the five marmosets developed progressive motor impairments that segregated into two distinct phenotypes: early onset with rapid progression and late onset with mild progression, accompanied by corresponding patterns in body weight gain and grip strength. Pathological analysis revealed cerebellar Purkinje cell loss, spinal cord neurodegeneration, and widespread intranuclear inclusions. The severity of motor phenotypes correlated with transgene expression levels in disease-relevant brain regions, including the cerebellum, spinal cord, and striatum. By overcoming the common translational limitations of rodent systems, our second-generation model offers a powerful platform for investigating disease mechanisms and testing potential therapeutic interventions. Our results advance the utility of transgenic marmosets as clinically relevant models of neurodegenerative diseases. Summary StatementSecond-generation transgenic marmoset models of spinocerebellar ataxia 3 replicated the progressive motor deficits and neuropathology of the founder marmoset, providing a powerful platform for studying disease mechanisms and developing therapies.

neuroscience↗

bioRxiv2023

Comprehensive analysis of nasal IgA antibodies induced by intranasal administration of the SARS-CoV-2 spike protein

Intranasal vaccination is an attractive strategy for preventing COVID-19 disease as it stimulates the production of multimeric secretory immunoglobulin A (IgA), the predominant antibody isotype in the mucosal immune system, at the target site of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry. Currently, intranasal vaccine efficacy is evaluated based on the measurement of polyclonal antibody titers in nasal lavage fluid. However, how individual multimeric secretory IgA protects the mucosa from SARS-CoV-2 infection remains to be elucidated. To understand the precise contribution and molecular nature of multimeric secretory IgA induced by intranasal vaccines, we developed 99 monoclonal IgA clones from nasal mucosa and 114 monoclonal IgA or IgG clones from nonmucosal tissues of mice that were intranasally immunized with the SARS-CoV-2 spike protein. The nonmucosal IgA clones exhibited shared origins and common and unique somatic mutations with the related nasal IgA clones, indicating that the antigen-specific plasma cells in the nonmucosal tissues originated from B cells stimulated at the nasal mucosa. Comparing the spike protein binding reactivity, angiotensin-converting enzyme-2-blocking and in vitro SARS-CoV-2 virus neutralization of monomeric and multimeric secretory IgA pairs recognizing different epitopes showed that even non-neutralizing monomeric IgAs, which represents 70% of the nasal IgA repertoire, can protect against SARS-CoV-2 infection when expressed as multimeric secretory IgAs. We also demonstrated that the intranasal administration of multimeric secretory IgA delivered as prophylaxis in the hamster model reduced infection-induced weight loss. Our investigation is the first to demonstrate the function of nasal IgA at the monoclonal level, showing that nasal immunization can provide effective immunity against SARS-CoV-2 by inducing multimeric secretory IgAs at the target site of the virus infection.

immunology↗

bioRxiv2023

Neutralizing antibody levels and epidemiological information of patients with breakthrough COVID-19 infection in Toyama, Japan

Breakthrough infection (BI) after coronavirus disease 2019 (COVID-19) vaccination has exploded owing to the emergence of various SARS-CoV-2 variants and has become a major problem at present. In this study, we analyzed the epidemiological information and possession status of neutralizing antibodies in patients with BI using SARS-CoV-2 pseudotyped viruses (SARS-CoV-2pv). Analysis of 44 specimens diagnosed with COVID-19 after two or more vaccinations showed high inhibition of infection by 90% or more against the Wuhan strain and the Alpha and Delta variants of pseudotyped viruses in 40 specimens. In contrast, almost no neutralizing activity was observed against the Omicron BA.1 variant. Many cases without neutralizing activity or BI were immunosuppressed individuals. The results of this study show that BI occurs even when there are sufficient neutralizing antibodies in the blood due to exposure to close contacts at the time of infection. Thus, even after vaccination, sufficient precautions must be taken to prevent infection.

microbiology↗

bioRxiv2023

Evaluation of SARS-CoV-2 isolation in cell culture from nasal/nasopharyngeal swabs or saliva specimens of patients with COVID-19

It has been revealed that SARS-CoV-2 can be efficiently isolated from clinical specimens such as nasal/nasopharyngeal swabs or saliva in cultured cells. In this study, we examined the efficiency of viral isolation including SARS-CoV-2 mutant strains between nasal/nasopharyngeal swab or saliva specimens. Furthermore, we also examined the comparison of viral isolation rates by sample species using simulated specimens for COVID- 19. As a result, it was found that the isolation efficiency of SARS-CoV-2 in the saliva specimens was significantly lower than that in the nasal/nasopharyngeal swab specimens. In order to determine which component of saliva is responsible for the lower isolation rate of saliva specimens, we tested the abilities of lactoferrin, amylase, cathelicidin, and mucin, which are considered to be abundant in saliva, to inhibit the infection of SARS-CoV-2 pseudotyped viruses (SARS-CoV-2pv). Lactoferrin and amylase were found to inhibit SARS-CoV-2pv infection. In conclusion, even if the same number of viral genome copies was detected by the real-time RT-PCR test, infection of SARS-CoV-2 present in saliva is thought to be inhibited by inhibitory factors such as lactoferrin and amylase, compared to nasal/nasopharyngeal swab specimens.

microbiology↗

bioRxiv2022

Activation of SARS-CoV-2 by trypsin-like proteases in the clinical specimens of patients with COVID-19

SARS-CoV-2 enters host cells through the angiotensin converting enzyme 2 (ACE2) receptor and/or transmembrane protease, serine 2 (TMPRSS2). Serine proteases, such as TMPRSS2 and trypsin, promote viral entry. In this study, we investigated whether proteases increased SARS-CoV-2 infectivity using pseudotyped viruses and clinical specimens from patients with COVID-19. First, we investigated how trypsin increased infectivity using the pseudotyped virus. Our findings revealed that trypsin increased infectivity after the virus was adsorbed on the cells, but no increase in infectivity was observed when the virus was treated with trypsin. We examined the effect of trypsin on SARS-CoV-2 infection in clinical specimens and found that the infectivity of the SARS-CoV-2 delta variant increased 36,000-fold after trypsin treatment. By contrast, the infectivity of SARS-CoV-2 omicron variant increased to less than 20-fold in the clinical specimens. Finally, infectivity of clinical specimens containing culture supernatants of Fusobacterium necrophorum was increased from several- to 10-fold. Because SARS-CoV-2 infectivity increases in the oral cavity, which may contain anaerobic bacteria, keeping the oral cavities clean may help prevent SARS-CoV-2 infection. ImportanceIn this study, we examined whether trypsin-like proteases increased the infectivity of SARS-CoV-2. We found that trypsin-like proteases increased the infectivity of both the pseudotyped viruses and the live virus in the clinical specimens. The increase in infectivity was significantly higher for the delta than the omicron variant. A large amount of protease in the oral cavity during SARS-CoV-2 infection is expected to increase infectivity. Therefore, keeping the oral cavity clean is important for preventing infection.

microbiology↗

bioRxiv2022

Nhlh1 and Nhlh2, a global transcriptional mechanism regulating commissural axon projection via activating Robo3

SUMMARYCommissural neurons are highly heterogeneous in their developmental origins, neurotransmitter type and function, but all share the common feature of projecting axons across the midline. The floor plate-crossing commissural axons in mammals, from the spinal cord to the midbrain, are guided by a conserved molecular mechanism relying primarily on Netrin-1/DCC/Robo3 signaling. Up to date, we know very little about the upstream transcriptional program that specify commissural axon laterality, neither do we know if a common mechanism operates in all commissural neurons. Here, we identified a pair of highly related helix-loop-helix transcription factors, Nhlh1 and Nhlh2, as a global transcriptional mechanism that controls the laterality of all floor plate-crossing commissural axons. Forced expression of Nhlh1/2 induce ectopic Robo3 expression and contralateral axon projections. And mutant mice deficient in both genes show a remarkable reduction in Robo3 expression and a total lack of ventral commissures from the spinal cord to the midbrain. This global mechanism may interact with neuron type specific mechanism to achieve specific generation of commissural circuits.

neuroscience↗

bioRxiv2021

Myosin phosphatase target subunit 1 governs integrity of the embryonic gut epithelium to circumvent atresia development in medaka, Oryzias latipes

Congenital intestinal atresia (IA) is a birth defect characterised by the absence or closure of part of the intestine. Although genetic factors are implicated, mechanistic understanding has been hindered by the lack of suitable animal models. Here, we describe a medaka (Oryzias latipes) mutant, generated by N-ethyl-N-nitrosourea (ENU) mutagenesis, that develops IA during embryogenesis. Positional cloning identified a nonsense mutation in mypt1, encoding myosin phosphatase target subunit 1. Mutant embryos exhibited ectopic accumulation of F-actin and phosphorylated myosin regulatory light chain (Mrlc) in the intestinal epithelium, consistent with disrupted actomyosin regulation. These cytoskeletal abnormalities were accompanied by epithelial disorganisation without notable alterations in cell proliferation, motility, or apoptosis. Inhibition of myh11a, encoding smooth muscle (SM) myosin heavy chain, ameliorated the IA phenotype but Blebbistatin treatment completely rescued the defect, suggesting a non-contractile role prior to SM maturation. Together, these findings demonstrate that mypt1 loss disrupts intestinal morphogenesis through actomyosin dysregulation. Given the recent clinical identification of IA associated with MYPT1 mutations, this medaka model offers a valuable platform to investigate the developmental and molecular basis of MYPT1-associated IA in human.

developmental biology↗

bioRxiv2021

The RNA helicase DDX6 controls early mouse embryogenesis by repressing aberrant inhibition of BMP signaling through miRNA-mediated gene silencing

The evolutionarily conserved RNA helicase DDX6 is a central player of post-transcriptional regulation, but its role during embryogenesis remains elusive. We here demonstrated that DDX6 enables proper cell lineage specification from pluripotent cells by analyzing Ddx6 KO mouse embryos and in vitro epiblast-like cell (EpiLC) induction system. Our study unveiled a great impact of DDX6-mediated RNA regulation on signaling pathways. Deletion of Ddx6 caused the aberrant transcriptional upregulation of the negative regulators of BMP signaling, which accompanied with enhanced Nodal signaling. Ddx6{bigtriangleup}/{bigtriangleup} pluripotent cells acquired higher pluripotency with a strong inclination toward neural lineage commitment. During gastrulation, abnormally expanded Nodal expression in the primitive streak likely promoted endoderm cell fate specification while inhibiting mesoderm development. We further clarified the mechanism how DDX6 regulates cell fate determination of pluripotent cells by genetically dissecting major DDX6 pathways: processing body (P-body) formation, translational repression, mRNA decay, and miRNA-mediated silencing. P-body-related functions were dispensable, but the miRNA pathway was essential for the DDX6 function. DDX6 may prevent aberrant transcriptional upregulation of the negative regulators of BMP signaling by repressing translation of certain transcription factors through the interaction with miRNA-induced silencing complexes (miRISCs). Overall, this delineates how DDX6 affects development of the three primary germ layers during early mouse embryogenesis and the underlying mechanism of DDX6 function. Author summaryGene expression occurs through the two steps: transcription (DNA to RNA) and translation (RNA to protein). Cells have very sophisticated regulatory processes working on various levels for the accurate gene expression. Post-transcriptional regulation, which includes all RNA-related controls, is crucial because it enables fine-tuning and rapid alteration of gene expression. RNA- binding proteins and non-coding RNAs are the two main players of post-transcriptional regulation. DDX6, the subject of our study, is an RNA-binding protein, more specifically an RNA helicase, which can unwind or rearrange RNA secondary structures. Its diverse molecular and cellular functions have been reported, but its embryogenic role is unknown. Here, we describe DDX6 function during early mouse embryogenesis and the underlying mechanism using genetic methodology. DDX6 enables proper cell lineage specification of pluripotent stem cells by mainly regulating BMP signaling through miRNA-mediated gene silencing. As DDX6- mediated RNA regulation affected signaling pathways, the loss of Ddx6 had a wide impact on developmental processes from pluripotency to embryo patterning. In addition, we identified which DDX6 molecular function is essential during early embryogenesis by genetically dissecting its main pathways.

genetics↗

bioRxiv2021

A novel hamster model of SARS-CoV-2 respiratory infection using a pseudotyped virus

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a biosafety level (BSL)-3 pathogen; therefore, its research environment is strictly limited. Pseudotyped viruses that mimic SARS-CoV-2 have been widely used for in vitro evaluation because they are available in BSL-2 containment laboratories; however, in vivo application is inadequate. Therefore, animal models that can be instigated with animal BSL-2 will increase opportunities for in vivo evaluations. MethodsHamsters (6-to 10-week-old males) were intratracheally inoculated with luciferase-expressing vesicular stomatitis virus (VSV)-based SARS-CoV-2 pseudotyped virus. The lungs were harvested 24 h after inoculation, and luminescence was measured using an in vivo imaging system. ResultsLung luminescence after inoculation with the SARS-CoV-2 pseudotyped virus increased in a dose-dependent manner. VSV-G (envelope [G]) pseudotyped virus also induced luminescence; however, a 100-fold concentration was required to reach a level similar to that of the SARS-CoV-2 pseudotyped virus. ConclusionsThe SARS-CoV-2 pseudotyped virus is applicable to SARS-CoV-2 respiratory infections in a hamster model. Because of the single-round infectious virus, the model can be used to study the steps from viral binding to entry, which will be useful for future research regarding SARS-CoV-2 entry without using live SARS-CoV-2 or transgenic animals.

microbiology↗

bioRxiv2021

Repurposing of the enhancer-promoter communication underlies the compensation of Mesp2 by Mesp1

Organisms are inherently equipped with buffering systems against genetic perturbations. Upregulation of homologous genes responding to gene loss, termed genetic compensation, is one such buffering mechanism. Recently, a well-conserved compensatory mechanism was proposed: transcriptional adaptation of homologs under the nonsense-mediated mRNA decay pathways. However, this model cannot explain the onset of all compensatory events. We report a novel genetic compensation mechanism operating over the Mesp gene locus. Mesp1 and Mesp2 are homologs located adjacently in the genome. Mesp2 loss is partially rescued by Mesp1 upregulation in the presomitic mesoderm (PSM). Using a cultured PSM induction system, we reproduced the compensatory response in vitro and found that the Mesp2-enhancer is required to promote Mesp1. We revealed that the Mesp2-enhancer directly interacts with the Mesp1 promoter, thereby upregulating Mesp1 expression upon the loss of Mesp2. Of note, this interaction is established by genomic arrangement upon PSM development independently of Mesp2 disruption. We propose that the repurposing of this established enhancer-promoter communication is the mechanism underlying this compensatory response for the upregulation of the adjacent homolog.

molecular biology↗

bioRxiv2020

NANOS2 suppresses the cell cycle by repressing mTORC1 activators in embryonic male germ cells

During murine germ cell development, male germ cells enter the mitotically arrested G0 stage, which is an initial step of sexually dimorphic differentiation. The male specific RNA-binding protein NANOS2 has a key role in suppressing the cell cycle in germ cells. However, the detailed mechanism of how NANOS2 regulates the cell cycle remains unclear. Using single-cell RNA sequencing (scRNA-seq), we extracted the cell cycle state of each germ cell in wild-type and Nanos2-KO testes, and revealed that Nanos2 expression starts in mitotic cells and induces mitotic arrest. We also found that NANOS2 and p38 MAPK work in parallel to regulate the cell cycle, suggesting that several different cascades are involved in the induction of cell cycle arrest. Furthermore, we identified Rheb, a regulator of mTORC1, and Ptma as possible targets of NANOS2. We propose that the repression of the cell cycle is a primary function of NANOS2 and that it is mediated via the suppression of mTORC1 activity by repressing Rheb in a post-transcriptional manner.

developmental biology↗

bioRxiv2019

Sox17 expression in endocardium precursor cells regulates heart development in mice

The endocardium is the endothelial component of the vertebrate heart and plays a key role in heart development. Cardiac progenitor cells (CPCs) that express the homeobox gene Nkx2-5 give rise to the endocardium. Where, when, and how the endocardium segregates during embryogenesis have remained largely unknown, however. We now show that Nkx2-5+ CPCs that express the Sry-type HMG box gene Sox17 specifically differentiate into the endocardium in mouse embryos. Approximately 20% to 30% of Nkx2-5+CPCs transiently express Sox17 from embryonic day (E) 7.5 to E8.5.Although Sox17 is not essential or sufficient for endocardium fate, it can bias the fate of CPCs toward the endocardium. On the other hand, Sox17 expression in the endocardium is required for heart development. Deletion of Sox17 specifically in the mesoderm markedly impaired endocardium development with regard to cell proliferation and behavior. The proliferation of cardiomyocytes, ventricular trabeculation, and myocardium thickening were also impaired in a non-cell-autonomous manner in the Sox17 mutant, resulting in anomalous morphology of the heart, likely as a consequence of down-regulation of NOTCH signaling. Changes in gene expression profile in both the endocardium and myocardium preceded the reduction in NOTCH-related gene expression in the mutant embryos, suggesting that Sox17 expression in the endocardium regulates an unknown signal required for nurturing of the myocardium. Our results thus provide insight into differentiation of the endocardium and its role in heart development. SignificanceThe endocardium is vital for vertebrate heart development; however, the molecular mechanisms regulating fate determination and differentiation remain largely unknown. Here, we show that a part of the earliest cardiac progenitor cells (CPCs) transiently and exclusively express Sry-type HMG box gene Sox17 in the mouse embryo. Sox17-expressing CPCs specifically differentiate to the endocardium. Sox17 biases the fate of CPCs toward the endocardium, and regulates proliferation and cellular behavior cell autonomously. Conversely, Sox17 in the endocardium regulates the myocardium non-cell autonomously. Notably, Sox17 is required for the ventricular trabeculation via the NOTCH signal that is not directly induced but maintained by Sox17. This study, thus, sheds light on endocardium development.

developmental biology↗