Shinya Ohata

Ependymomas are glial tumors that exhibit high resistance to chemotherapy. ZFTA-fusion-positive supratentorial ependymoma (ST-EPN-ZFTA) represents a subclass with a poor prognosis. ST-EPN-ZFTA expresses fusion proteins, which consist of zinc finger translocation-associated (ZFTA) combined with a transcriptional regulator, predominantly RELA. Unlike wild-type RELA, which translocates to the nucleus in a stimulus-dependent manner, ZFTA-RELA constitutively localizes to the nucleus and induces oncogenic gene expression. To identify inhibitors of this aberrant gene expression, 9,600 structurally diverse compounds were screened using a ZFTA-RELA-responsive luciferase reporter system. The screen identified a colchicine derivative, and subsequent evaluation revealed colchicine, a microtubule polymerization inhibitor, as the most potent inhibitor (IC50 of 90 nM). Colchicine attenuated ZFTA-RELA-upregulated endogenous genes, including L1CAM. Colchicine and another microtubule polymerization inhibitor, vinblastine, partially inhibited ZFTA-RELA nuclear localization, similar to the dynein motor protein inhibitors, ciliobrevin D and dynarrestin. This inhibition was mediated through the RELA region of ZFTA-RELA, indicating that RELA retains its microtubule-dependent nuclear import machinery, even in a fusion scenario. Among other ZFTA-fusion proteins, colchicine reduced nuclear localization of ZFTA-NCOA2 and ZFTA-MKL2, but not ZFTA-MAML2 and ZFTA-MAML3. In these fusion proteins, we identified three candidate nuclear localization signals (NLS) exhibiting colchicine-sensitive nuclear localization activity, two within the ZFTA regions absent in ZFTA-RELA and one within MKL2. The results indicate that the microtubule dependence of nuclear localization varies among different ZFTA-fusion partners, which may be attributable to unique NLS sequences in each fusion protein. Our results suggest that microtubule-dependent nuclear transport represents a therapeutic target in ZFTA-fusion-positive tumors.

Abstract Terbinafine (TBF) is a first-line allylamine antifungal used to treat dermatophytosis caused by Trichophyton species, targeting squalene epoxidase (SQLE). The recent emergence of TBF-resistant Trichophyton rubrum (T. rubrum) strains represents a significant global clinical challenge. Although the SQLE Leu393Phe substitution is a commonly reported resistance mechanism in Japan, direct genetic evidence demonstrating its effect when introduced into a T. rubrum recipient strain has been lacking. In this study, we established the causal relationship between the SQLE Leu393Phe substitution and TBF resistance via targeted gene replacement in T. rubrum. A homologous recombination plasmid containing SQLE from the TBF-resistant T. rubrum strain BGUTR13, which harbours the 1177TTA→TTC mutation encoding Leu393Phe, together with the neomycin phosphotransferase II (nptII) selection marker, was constructed and introduced into the TBF-susceptible strain T. rubrum CBS118892 Δku80. The resulting mutant transformants exhibited a >8,000-fold increase in TBF minimum inhibitory concentration compared with the parental strain, whereas control transformants carrying nptII but lacking the SQLE Leu393Phe substitution remained susceptible. Structural modelling indicated that the Leu393Phe substitution causes substantial steric hindrance within the SQLE binding pocket, displacing TBF’s aliphatic chain and disrupting key hydrophobic interactions. Molecular docking simulations predicted reduced TBF binding affinity (ΔG changed from −6.3 to −0.8 kcal/mol) following the mutation. These findings provide the first robust genetic evidence that the single SQLE Leu393Phe substitution is sufficient to confer high-level TBF resistance in T. rubrum. These findings highlight the importance of monitoring SQLE mutations in clinical isolates and provide a foundation for developing strategies to address antifungal resistance in dermatophytes.

ABSTRACT Lysin motif (LysM) domain-containing receptors are evolutionarily conserved pattern recognition receptors (PRRs) that serve as key mediators of glycan sensing and innate immune activation in plants and mammals. In invertebrates, however, their role in activating innate immunity remains poorly understood, although some evidence for immunosuppressive functions exists. In this study, we performed in silico structural analyses and identified a putative Bombyx mori LYSMD3 homolog ( XP_004933441.1 ). This protein exhibits high structural similarity in the LysM domain to human LYSMD3, with a root-mean-square deviation (RMSD) of 0.559 Å, indicating close structural alignment. RNA-seq analysis of hemocytes isolated from silkworm larvae injected with N -acetylchitohexaose (GN6), a chitin-derived oligosaccharide and known ligand of human LYSMD3, revealed transcriptional activation of innate immune effectors, including antimicrobial peptide (AMP) genes such as cecropins . GN6 also induced cecropin transcription in isolated hemocytes in vitro , and Western blotting of hemolymph confirmed elevated cecropin B protein levels. Furthermore, GN6 and chitin significantly improved survival outcomes against P. aeruginosa infection, with median effective doses (ED₅₀) values of 0.62 and 0.48  µg/larva, respectively. In contrast, N -acetylglucosamine (GlcNAc) and shorter oligosaccharides (GN2–GN5) were ineffective. These findings provide the first molecular-level evidence of a putative glycan receptor in silkworms based on the structural similarity to known LysM domains. Moreover, GN6-induced antimicrobial peptide expression and enhanced infection resistance demonstrate immune activation in this model, supporting an evolutionarily conserved glycan-sensing pathway in invertebrates.

ABSTRACT Pathogenic fungi pose significant societal challenges due to limited therapeutic targets resulting from the eukaryotic nature of fungi. This limitation emphasizes the importance of enhancing susceptibility to inhibitors of Cyp51, a crucial enzyme in ergosterol biosynthesis targeted by azole antifungals. In Cyp51 isozyme deletion strains (Δ cyp51A and Δ cyp51B ) of Trichophyton rubrum , the predominant dermatophyte species, we found that Cyp51B is essential for basal mycelial growth, while Cyp51A functions as an inducible isozyme associated with azole tolerance. Based on these differential functions, we hypothesized that each isozyme would show distinct susceptibility to azole antifungals. Our study demonstrated that most azoles exhibited increased antifungal activity against Δ cyp51A , while select agents demonstrated increased antifungal activity against Δ cyp51B . Remarkably, fluconazole, sulconazole, and imazalil exhibited relatively increased activity against Δ cyp51A , whereas prochloraz demonstrated increased activity against Δ cyp51B . Combining these isozyme-selective agents exerted synergistic effects against the wild-type strain and the parent ku80 -knockout strain but not against individual Cyp51 knockout mutants. Our data revealed that the two Cyp51 isozymes can be selectively inhibited by different azole antifungals, resulting in a synergistic effect when combined. This synergistic effect was also observed on another fungal species, Aspergillus welwitschiae , which also has two Cyp51 isozymes. These data demonstrate that combining azole antifungals with different Cyp51 isozyme selectivities exerts synergistic effects against fungi possessing multiple Cyp51 isozymes. These findings advance antifungal therapeutic strategies by demonstrating that the combination of antifungals with different Cyp51 isozyme selectivities offers a promising approach for treating fungal infections, opening new avenues for isozyme-specific drug development.

ABSTRACT The increasing prevalence of dermatophyte resistance to terbinafine, a key drug in the treatment of dermatophytosis, represents a significant obstacle to treatment. Trichophyton rubrum is the most commonly isolated fungus in dermatophytosis. In T. rubrum , we identified TERG_07844, a gene encoding a previously uncharacterized putative protein kinase, as an ortholog of budding yeast Saccharomyces cerevisiae polyamine transport kinase 2 (Ptk2), and found that T. rubrum Ptk2 (TrPtk2) is involved in terbinafine tolerance. In both T. rubrum and S. cerevisiae , Ptk2 knockout strains were more sensitive to terbinafine compared with the wild types, suggesting that promotion of terbinafine tolerance is a conserved function of fungal Ptk2. Pma1 is activated through phosphorylation by Ptk2 in S. cerevisiae . Overexpression of T. rubrum Pma1 (TrPma1) in T. rubrum Ptk2 knockout strain (ΔTrPtk2) suppressed terbinafine sensitivity, suggesting that the induction of terbinafine tolerance by TrPtk2 is mediated by TrPma1. Furthermore, omeprazole, an inhibitor of plasma membrane proton pump Pma1, increased the terbinafine sensitivity of clinically isolated terbinafine-resistant strains. These findings suggest that, in dermatophytes, the TrPtk2-TrPma1 pathway plays a key role in promoting intrinsic terbinafine tolerance and may serve as a potential target for combinational antifungal therapy against terbinafine-resistant dermatophytes.

Dermatophytes invade and colonize host superficial tissues via hyphal growth. Although cytoskeletal reorganization and its regulation are essential for hyphal growth, the molecular mechanisms in dermatophytes and their applicability as antifungal drug targets remain poorly understood. The p21-activated kinase (PAK) is a downstream effector of the small GTPases Rac and CDC42, also known as p21, and is involved in various molecular and cellular functions, including actin polymerization and cell morphogenesis. In this study, we investigated the contribution of the PAK protein TrCla4 to morphogenesis and mycelial growth in Trichophyton rubrum, the most frequently isolated fungus in dermatophytosis (athlete’s foot). The actin polymerization inhibitor, cytochalasin A inhibited actin accumulation at the hyphal tip and mycelial growth of T. rubrum, suggesting the involvement of the actin cytoskeleton in mycelial growth. In the Trcla4 knock-out strain (ΔTrcla4), we observed defects in mycelial growth, hyphal branching, and the accumulation of polymerized actin at the hyphal tip. Chemical inhibitors of TrRac-dependent TrCla4 kinase activity, FRAX486 and IPA-3, also inhibited spore germination and mycelial growth. Interestingly, ΔTrcla4 showed no additional inhibition of mycelial growth when treated with these inhibitors, indicating that their inhibitory effects are primarily mediated through TrCla4. In an invertebrate dermatophyte infection model, animals infected with ΔTrcla4 had higher survival rates than those infected with the wild-type, and IPA-3 and FRAX486 treatments both slightly but significantly improved animal survival rates. These results suggest that the dermatophyte PAK promotes mycelial growth by facilitating actin polymerization at the hyphal tip, making it a potential therapeutic target for dermatophytosis.

Nuclear factor-kappa B (NF-κB) signaling is an intracellular signaling pathway involved in inflammatory responses and the pathogenesis of various cancers, including ependymoma, which is a rare and chemotherapy-resistant glioma. Several isoforms of fusion proteins that consist of a nuclear protein, zinc finger translocation associated (ZFTA), and RELA (ZFTA-RELA), an NF-κB-signaling effector transcription factor, cause excessive activation of the NF-κB signaling pathway and result in supratentorial ependymomas (ST-EPN-RELA). As inhibitors of NF-κB activity induced by ZFTA-RELA are expected to be therapeutic agents for ST-EPN-RELA, we established an NF-κB responsive luciferase reporter cell line that expresses the most common isoform of ZFTA-RELA in a doxycycline-dependent manner. Using this reporter cell line, we screened fungus extracts for compounds that inhibit the NF-κB activity induced by ZFTA-RELA expression and identified aszonalenin, an alkaloid from Aspergillus novofumigatus. We also purified analogs of aszonalenin, namely acetylaszonalenin and epi-aszonalenin B and C. In a luciferase assay using cells constitutively expressing luciferase (counter assay), acetylaszonalenin and epi-aszonalenin C showed non-specific inhibition of the luciferase activity. Aszonalenin and epi-aszonalenin B inhibited the NF-κB responsive luciferase activity by expressing ZFTA-RELA more strongly than the luciferase activity in the counter assay. The upregulation of endogenous NF-κB responsive genes, such as CCND1, ICAM1, and L1CAM, by ZFTA-RELA expression was inhibited by epi-aszonalenin B, but not by aszonalenin. This study suggests that epi-aszonalenin B may be a lead compound for the therapeutic development of ST-EPN-RELA.

<title>Abstract</title>The fusion protein of uncharacterised zinc finger translocation associated (ZFTA) and effector transcription factor of tumorigenic NF-κB signalling, RELA (ZFTA-RELA), is expressed in more than two-thirds of supratentorial ependymoma (ST-EPN-RELA), but ZFTA’s expression profile and functional analysis in multiciliated ependymal (E1) cells have not been examined. Here, we showed the mRNA expression of mouse <italic>Zfta</italic> peaks on embryonic day (E) 17.5 in the wholemount of the lateral walls of the lateral ventricle. Zfta was expressed in the nuclei of FoxJ1-positive immature E1 (pre-E1) cells in E18.5 mouse embryonic brain. Interestingly, the transcription factors promoting ciliogenesis (ciliary TFs) (e.g., multicilin) and ZFTA-RELA upregulated luciferase activity using a 5′ upstream sequence of <italic>ZFTA</italic> in cultured cells. <italic>Zfta</italic>^{<italic>tm1/tm1</italic>} knock-in mice did not show developmental defects or abnormal fertility. In the <italic>Zfta</italic>^{<italic>tm1/tm1</italic>} E1 cells, morphology, gene expression, ciliary beating frequency and ependymal flow were unaffected. These results suggest that Zfta is expressed in pre-E1 cells, possibly under the control of ciliary TFs, but is not essential for ependymal development or flow. This study sheds light on the mechanism of the ZFTA-RELA expression in the pathogenesis of ST-EPN-RELA: Ciliary TFs initiate ZFTA-RELA expression in pre-E1 cells, and ZFTA-RELA enhances its own expression using positive feedback.

Lysosomes are acidic organelles responsible for degrading both exogenous and endogenous materials. The small GTPase Arl8 localizes primarily to lysosomes and is involved in lysosomal function. In the present study, using Arl8b gene-trapped mutant (Arl8b-/- ) mice, we show that Arl8b is required for the development of dorsal structures of the neural tube, including the thalamus and hippocampus. In embryonic day (E) 10.5 Arl8b-/- embryos, Sox1 (a neuroepithelium marker) was ectopically expressed in the roof plate, whereas the expression of Gdf7 and Msx1 (roof plate markers) was reduced in the dorsal midline of the midbrain. Ectopic expression of Sox1 in Arl8b-/- embryos was detected also at E9.0 in the neural fold, which gives rise to the roof plate. In addition, the levels of Bmp receptor IA and phosphorylated Smad 1/5/8 (downstream of BMP signaling) were increased in the neural fold of E9.0 Arl8b-/- embryos. These results suggest that Arl8b is involved in the development of the neural fold and the subsequently formed roof plate, possibly via control of BMP signaling.

In many animal species, the production of new neurons (neurogenesis) occurs throughout life, in a specialized germinal region called the ventricular-subventricular zone (V-SVZ). In this region, neural stem cells undergo self-renewal and generate neural progenitor cells and new neurons. In the olfactory system, the new neurons migrate rostrally toward the olfactory bulb, where they differentiate into mature interneurons. V-SVZ-derived new neurons can also migrate toward sites of brain injury, where they contribute to neural regeneration. Recent studies indicate that two major branches of the Wnt signaling pathway, the Wnt/β-catenin and Wnt/planar cell polarity pathways, play essential roles in various facets of adult neurogenesis. Here, we review the Wnt signaling-mediated regulation of adult neurogenesis in the V-SVZ under physiological and pathological conditions.