Biology subjects

Koike, A.

Publications and source records attributed to Koike, A..

6 recordsLinked to original sources

bioRxiv2025

Canonical WNT signalling governs Echinococcus metacestode development

Alveolar echinococcosis (AE) is a lethal zoonosis caused by infiltrative growth of the metacestode larva of the tapeworm Echinococcus multilocularis in host organs. We previously showed that the Echinococcus metacestode is an evolutionarily unique, broadly posteriorized tissue, leading us to hypothesize that canonical WNT (cWNT) signalling--which patterns the body axis across metazoans--might be critical for metacestode formation. Here, we report effective RNAi-mediated knockdown of the E. multilocularis {beta}-catenin gene (bcat-1), the central effector of cWNT signalling, in a primary parasite cell culture system that produces metacestode vesicles. bcat-1(RNAi) cultures were markedly impaired in vesicle formation, exhibited stem-cell hyperproliferation, and displayed disrupted muscle-fibre organisation. Genome-wide transcriptomics revealed a general anteriorization of gene expression, and in situ hybridization showed an overproduction of cells expressing head-inducing factors such as sfrp upon bcat-1 knockdown. Conversely, metacestode-specific genes--including the tegumental factors muc-1, TNFR, and antigen B--as well as the posterior marker post2b were significantly downregulated, consistent with the observed vesicle-formation defects. In situ analyses further identified anterior markers--frizzled-10, nou-darake, notum, and follistatin--that were overexpressed in bcat-1(RNAi) cultures and localized to the future anterior pole at the earliest stages of protoscolex formation. Finally, pharmacological inhibition of WNT signalling with pyrvinium pamoate caused complete loss of posterior tissue in Echinococcus protoscoleces, killed metacestode vesicles, and reduced stem-cell proliferation at nanomolar concentrations. Together, these findings establish a central role for cWNT signalling in directing Echinococcus body-axis formation and the posteriorization events driving metacestode growth within the host, providing insight into asexual parasite proliferation mediated by this biologically unique larval stage and pointing to potential targets for chemotherapy against AE. Author SummaryAlveolar echinococcosis (AE) is a lethal disease caused by the cancer-like growth of the metacestode larva of the tapeworm Echinococcus multilocularis. From a developmental perspective, the Echinococcus metacestode is an unusual biological structure and even atypical among tapeworms. Previous work indicated that metacestode formation involves re-patterning of the body axis, eliminating head structures and producing broadly posteriorized tissue. How this is controlled at the molecular and cellular levels, however, was unknown. In this study, we perturbed expression of the {beta}-catenin gene (bcat-1), a central regulator of canonical WNT signalling, using RNA interference (RNAi). bcat-1(RNAi) parasite cultures failed to generate metacestode vesicles and instead showed stem-cell hyperproliferation and muscle-cell distortion. Genes required for posteriorized metacestode tissue were downregulated, whereas genes directing head formation in adult worms (follistatin, sfrp, fz10, ndk) were upregulated, indicating a general anteriorization of the culture system. Pharmacological inhibition of WNT signalling with pyrvinium pamoate caused complete loss of posterior structures in protoscoleces, reduced stem-cell proliferation, and killed metacestode tissue. These findings identify {beta}-catenin and the canonical WNT pathway as crucial regulators of the posteriorization that underlies metacestode formation. Given that WNT signalling is deregulated in many human cancers and that small-molecule inhibitors are available, our results suggest new avenues for anti-AE drug development.

developmental biology↗

bioRxiv2024

Putative EGF ligand and receptor of Echinococcus multilocularis that are critical for parasite development

The neglected zoonosis alveolar echinococcosis (AE) is caused by infiltrative growth of the metacestode larval stage of the cestode Echinococcus multilocularis within host organs. We previously demonstrated that metacestode growth depends on the mitotic activity of a population of parasite stem cells, called germinative cells, but it is not yet clear which molecular mechanisms govern Echinococcus stem cell dynamics such as cell-cycle progression, self-renewal and differentiation. Based on previous reports showing that epidermal growth factor (EGF) signalling contributes to Echinococcus stem cell regulation, we herein characterized three EGF receptors of the parasite and demonstrated by RNAi and inhibitor assays that one of these, EmER1, is crucial for the development of metacestode vesicles from parasite stem cells. We also showed that EmER1 serves as a target for afatinib, an EGF receptor inhibitor with profound anti-parasitic activities in vitro and in vivo. By bioinformatic analyses and membrane-bound yeast two-hybrid assays, we identified a parasite-derived, neuregulin-like cognate ligand for EmER1, EmNRG, the expression of which is strongly upregulated in metacestode vesicles during clonal expansion of germinative cells. Furthermore, we demonstrate that RNAi knockdown of the EmNRG encoding gene drastically affects the ability of germinative cells to produce metacestode vesicles. We propose that EmNRG and EmER1 form a cognate ligand-receptor system utilized by E. multilocularis to regulate asymmetric versus symmetric division decisions of stem cells. These data are relevant for further studies into Echinococcus stem cell dynamics and for the development of EGF signalling-based anti-infectives against echinococcosis.

microbiology↗

bioRxiv2022

Targeting Echinococcus multilocularis PIM kinase for improving anti-parasitic chemotherapy

BackgroundThe potentially lethal zoonosis alveolar echinococcosis (AE) is caused by the metacestode larval stage of the tapeworm Echinococcus multilocularis. Current AE treatment options are limited and rely on surgery as well as on chemotherapy involving benzimidazoles (BZ). BZ treatment, however, is parasitostatic only, must be given for prolonged time periods, and is associated with adverse side effects. Novel treatment options are thus urgently needed. Methodology/Principal findingsBy applying a broad range of kinase inhibitors to E. multilocularis stem cell cultures we identified the proto-oncogene PIM kinase as a promising target for anti-AE chemotherapy. The gene encoding the respective E. multilocularis ortholog, EmPIM, was characterized and in situ hybridization assays indicated its expression in parasite stem cells. By yeast two-hybrid assays we demonstrate interaction of EmPIM with E. multilocularis CDC25, indicating an involvement of EmPIM in parasite cell cycle regulation. Small molecule compounds SGI-1776 and CX-6258, originally found to effectively inhibit human PIM kinases, exhibited detrimental effects on in vitro cultured parasite metacestode vesicles and prevented the formation of mature vesicles from parasite stem cell cultures. To improve compound specificity for EmPIM, we applied a high throughput in silico modelling approach, leading to the identification of compound Z196138710. When applied to in vitro cultured metacestode vesicles and parasite cell cultures, Z196138710 proved equally detrimental as SGI-1776 and CX-6258, but displayed significantly reduced toxicity towards human HEK293T and HepG2 cells. Conclusions/SignificanceRepurposing of kinase inhibitors initially designed to affect mammalian kinases for helminth disease treatment is often hampered by adverse side effects of respective compounds on human cells. Here we demonstrate the utility of high throughput in silico approaches to design small molecule compounds of higher specificity for parasite cells. We propose EmPIM as a promising target for respective approaches towards AE treatment. Author summaryThe larva of the tapeworm E. multilocularis grows tumor-like within the host liver, thus causing the lethal disease alveolar echinococcosis (AE). Anti-parasitic treatment relies on chemotherapy with benzimidazoles, which do not kill the parasite and must be applied for years. As druggable enzymes with key functions in growth control, protein kinases are promising drug targets and many kinase inhibitors have been identified during cancer research. Optimized for binding to human kinases, however, repurposing of such drugs for parasitic disease treatment is associated with adverse side effects. Herein, the authors applied an in silico approach to identify small molecule compounds that show higher specificity for a parasite kinase, EmPIM, over its mammalian homologs. The authors demonstrate expression of EmPIM in Echinococcus stem cells, which are the drivers of parasite growth, and show that mammalian PIM kinase inhibitors SGI-1776 and CX-6258 also affect parasite development in vitro. Finally, they show that one of the in silico screened compounds is equally effective against the parasite as SGI-1776 and CX-6258, but significantly less toxic to human cells. These results demonstrate the utility of in silico approaches to identify parasite-specific kinase inhibitors.

microbiology↗

bioRxiv2020

Statins attenuate antiviral IFN- and ISG expression via inhibiting IRF3/JAK/STAT signaling in poly(I:C)-treated hyperlipidemic mice and macrophages

Viral infection is a significant burden to healthcare worldwide. Statins, 3-hydroxy-3-methyl glutaryl coenzyme A reductase inhibitors, are widely used as cholesterol-lowering drugs. Recently, long term statin therapy was shown to reduce the antiviral immune response; however, the underlying molecular mechanisms are unclear. Here, we found that simvastatin decreased polyinosinic-polycytidylic acid [poly(I:C)]-induced expression of antiviral interferon (IFN)-{beta} and IFN-stimulated genes (ISGs) in the bronchoalveolar lavage fluid and lungs of mice with high-fat diet-induced hyperlipidemia. As macrophages were the dominant cell type in the bronchoalveolar lavage fluid of poly(I:C)-treated mice, we examined the molecular mechanisms of statin-mediated inhibition of antiviral gene expression using murine J774.1/JA-4 macrophages. Simvastatin and pitavastatin decreased poly(I:C)-induced expression of IFN-{beta} and ISGs. Moreover, they repressed poly(I:C)-induced phosphorylation of IFN regulatory factor (IRF) 3 and signal transducers and activators of transcription (STAT) 1, which is involved in Janus kinase (JAK)/STAT signaling. Mevalonate and geranylgeranylpyrophosphate (GGPP), but not cholesterol, counteracted the negative effect of statins on IFN-{beta} and ISG expression and phosphorylation of IRF3 and STAT1. These results suggest that statins suppressed the expression of IFN-{beta} and ISGs in poly(I:C)-treated hyperlipidemic mice and murine macrophages, and that these effects occured through the inhibition of IRF3-mediated JAK/STAT signaling in macrophages. Furthermore, GGPP recovered the statin-suppressed IRF3/JAK/STAT signaling pathway in poly(I:C)-treated macrophages.

biochemistry↗

bioRxiv2020

TGF-β inhibitor accelerates BMP4-induced cochlear gap junction formation during in vitro differentiation of embryonic stem cells

Mutations in the connexin 26 (CX26)/gap junction beta-2 (GJB2) gene are the most frequent cause of hereditary deafness worldwide. Using mouse induced pluripotent stem cells (iPSCs) and a BMP4 signal-based floating and adherent culture system, we recently produced in vitro responsible for GJB2-related deafness (CX26-gap junction plaque-forming cells, CX26GJCs). However, to use these cells as a disease model platform for high-throughput drug screening or regenerative therapy, cell yields must be substantially increased. In addition to BMP4, presently uncharacterized factors may also induce CX26 gap junction (GJ) formation. A floating culture with embryonic stem cell (ESC) treatment and BMP4/TGF-{beta} inhibitor (SB431542:SB) has been shown to result in greater production of isolatable CX26-positive small vesicles (CX26+ vesicles) and higher Gjb2 mRNA levels than BMP4 treatment alone, suggesting that SB may promote BMP4-mediated production of CX26+ vesicles in a dose-dependent manner, thereby increasing the yield of highly purified CX26GJCs. In the present study, we first demonstrated that SB accelerates BMP4 induced GJ formation during stem cell differentiation. By controlling the concentration and timing of SB supplementation with CX26+ vesicle purification, large-scale production of highly purified CX26GJCs suitable for high-throughput drug screening or regenerative therapy for GJB2-related deafness may be possible.

cell biology↗

bioRxiv2018

Acetylation of nuclear localization signal controls importin-mediated nuclear transport of Ku70

Ku70 participates in various intra-and extra-nucleic processes. For multifunctional control, machinery that precisely regulates the intracellular localization of Ku70 is essential. Recently, it was reported that acetylation of Ku70 regulates its function. Here, we demonstrate that specific lysine residues in Ku70 that are targets of acetylation are critical for regulating nuclear transport in vivo. Ku70-GFP fusion proteins transiently expressed in cultured cells localized in the nucleus, whereas mimicking acetylation of K553 or K556 in the Ku70 nuclear localization signal (NLS) by substituting these lysine residues with glutamine markedly decreased the nuclear localization of Ku70. Moreover, the Ku70-importin interaction was suppressed in the K553Q and K556Q mutants. Theoretical estimations indicated that the binding energy between the Ku70 NLS and importin- decreases with acetylation of lysine residues in the Ku70 NLS, similar to the case when these lysine residues are substituted with glutamine. These results suggest that acetylation of specific lysine residues in the Ku70 NLS is a key switch that controls the localization of Ku70 by modulating interactions between Ku70 and nuclear transport factors.

cell biology↗