Rapamycin-sensitive mechanisms confine the growth of fission yeast below the temperatures detrimental to cell physiology.

Cells cease to proliferate above their growth-permissible temperatures, a ubiquitous phenomenon generally attributed to heat damage to cellular macromolecules. We here report that, in the presence of rapamycin, a potent inhibitor of Target of Rapamycin Complex 1 (TORC1), the fission yeast Schizosaccharomyces pombe can proliferate at high temperatures that usually arrest its growth. Consistently, mutations to the TORC1 subunit RAPTOR/Mip1 and the TORC1 substrate Sck1 significantly improve cellular heat resistance, suggesting that TORC1 restricts fission yeast growth at high temperatures. Aiming for a more comprehensive understanding of the negative regulation of high-temperature growth, we conducted genome-wide screens, which identified additional factors that suppress cell proliferation at high temperatures. Among them is Mks1, which is phosphorylated in a TORC1-dependent manner, forms a complex with the 14-3-3 protein Rad24, and suppresses the high-temperature growth independently of Sck1. Our study has uncovered unexpected mechanisms of growth restraint even below the temperatures deleterious to cell physiology.

Microcystin-LR-induced epithelial-mesenchymal transition-like cells acquire resistance to multi-toxins.

The protein phosphatase inhibitor microcystin-LR (MC-LR), a hepatocyte-selective cyanotoxin, induces phenotypic changes in HEK293 OATP1B3-expressing (HEK293-OATP1B3) cells, which include cytoskeletal reorganization (HEK293-OATP1B3-AD) and anoikis resistance (HEK293-OATP1B3-FL) transformed cells, respectively. These cells acquire resistance to MC-LR and partial epithelial-mesenchymal transition (EMT) characteristics. In cancer cells, EMT is generally involved in multi-drug resistance. Here, we focused on the multi-drug resistance of HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells. The MTT assay and immunoblotting were conducted to examine the responses of HEK293-OATP1B3, HEK293-OATP1B3-AD, and HEK293-OATP1B3-FL cells to multiple toxins and drugs that function as substrates for OATP1B3, including MC-LR, nodularin (Nod), okadaic acid (OA), and cisplatin (CDDP). HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells were more resistant to MC-LR, Nod, and OA than HEK293-OATP1B3 cells. Conversely, the three cell types were equivalently sensitive to CDDP. By using protein phosphatase assay, the reduction of the inhibitory effect of MC-LR and Nod on phosphatase activity might be one reason for the resistance to MC-LR and Nod in HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells. Furthermore, the parental HEK293-OATP1B3 cells showed enhanced p53 phosphorylation and stabilization after MC-LR exposure, while p53 phosphorylation was attenuated in HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells. Moreover, in HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells, AKT phosphorylation was higher than that of the parental HEK293-OATP1B3 cell line. These results suggest that the multi-toxin resistance observed in HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells is associated with AKT activation and p53 inactivation.

Fission Yeast TORC1 Promotes Cell Proliferation through Sfp1, a Transcription Factor Involved in Ribosome Biogenesis.

Target of rapamycin complex 1 (TORC1) is activated in response to nutrient availability and growth factors, promoting cellular anabolism and proliferation. To explore the mechanism of TORC1-mediated proliferation control, we performed a genetic screen in fission yeast and identified Sfp1, a zinc-finger transcription factor, as a multicopy suppressor of temperature-sensitive TORC1 mutants. Our observations suggest that TORC1 phosphorylates Sfp1 and protects Sfp1 from proteasomal degradation. Transcription analysis revealed that Sfp1 positively regulates genes involved in ribosome production together with two additional transcription factors, Ifh1/Crf1 and Fhl1. Ifh1 physically interacts with Fhl1, and the nuclear localization of Ifh1 is regulated in response to nutrient levels in a manner dependent on TORC1 and Sfp1. Taken together, our data suggest that the transcriptional regulation of the genes involved in ribosome biosynthesis by Sfp1, Ifh1, and Fhl1 is one of the key pathways through which nutrient-activated TORC1 promotes cell proliferation.

Glucosyl hesperidin exhibits more potent anxiolytic activity than hesperidin accompanied by the attenuation of noradrenaline induction in a zebrafish model.

Anxiety is a symptom of various mental disorders, including depression. Severe anxiety can significantly affect the quality of life. Hesperidin (Hes), a flavonoid found in the peel of citrus fruits, reportedly has various functional properties, one of which is its ability to relieve acute and chronic stress. However, Hes is insoluble in water, resulting in a low absorption rate in the body and low bioavailability. Glucosyl hesperidin (GHes) is produced by adding one glucose molecule to hesperidin. Its water solubility is significantly higher than that of Hes, which is expected to improve its absorption into the body and enhance its effects. However, its efficacy in alleviating anxiety has not yet been investigated. Therefore, in this study, the anxiolytic effects of GHes were examined in a zebrafish model of anxiety. Long-term administration of diets supplemented with GHes did not cause any toxicity in the zebrafish. In the novel tank test, zebrafish in the control condition exhibited an anxious behavior called freezing, which was significantly suppressed in GHes-fed zebrafish. In the black-white preference test, which also induces visual stress, GHes-fed zebrafish showed significantly increased swimming time in the white side area. Furthermore, in tactile (low water-level stress) and olfactory-mediated stress (alarm substance administration test) tests, GHes suppressed anxious behavior, and these effects were stronger than those of Hes. Increased noradrenaline levels in the brain generally cause freezing; however, in zebrafish treated with GHes, the amount of noradrenaline after stress was lower than that in the control group. Activation of c-fos/ERK/Th, which is upstream of the noradrenaline synthesis pathway, was also suppressed, while activation of the CREB/BDNF system, which is vital for neuroprotective effects, was significantly increased. These results indicate that GHes has a more potent anxiolytic effect than Hes in vivo, which may have potential applications in drug discovery and functional food development.

l-fucoside localization in the gills of the genus Takifugu and its possible implication in the parasitism of Heterobothrium okamotoi (Monogenea: Diclidophoridae).

BACKGROUND: The monogenean parasite Heterobothrium okamotoi only parasitizes the gills of Takifugu rubripes. In this study, we hypothesized that the carbohydrates contribute to high host specificity of H. okamotoi. METHODS: T. rubripes, T. niphobles, T. snyderi, and T. pardalis were used for UEA I staining of the gills and an in vivo challenge test against H. okamotoi. To examine the effect of l-fucose, an in vitro detachment test was conducted using the host's gills. Additionally, fucosylated proteins were isolated from the membrane proteins of T. niphobles gills. RESULTS: The location of l-fucoside and the infection dynamics in four species were correlated to some extent; H. okamotoi detached relatively quickly from T. niphobles possessing l-fucoside both on the surface of the gills and in certain types of cells, including mucus cells, but detached slowly from T. snyderi possessing l-fucoside in only certain types of cells, including mucus cells. Under the conditions examined, H. okamotoi exhibited minimal detachment from T. rubripes and T. pardalis, and l-fucoside was not detected. The significantly higher detachment rate of H. okamotoi from the host's gills incubated in l-fucose-containing medium compared with the controls suggests that l-fucose in the non-host gills induced detachment of H. okamotoi. Four fucosylated proteins, including mucin5AC-like, were identified as potential factors for the detachment of H. okamotoi. CONCLUSIONS: Fucosylated proteins covering the surface of non-host gills might contribute to H. okamotoi detachment. GENERAL SIGNIFICANCE: This research shows the possible involvement of oligosaccharides in the host specificity of monogenean parasites.

Comparative study of the effects of the three kinds of Kampo-hozai: Ninjinyoeito, Hochuekkito, and Juzentaihoto on anxious and low sociability behavior using NPY-knockout zebrafish.

Ninjinyoeito, Hochuekkito, and Juzentaihoto are the three types of Kampo-hozai used to support the treatment of various diseases by energizing patients through improved mental health. While Kampo-hozais are clinically used to improve mental energy decline, a comparison between their effects on neuropsychiatric symptoms like anxiety and sociability and the strength of their effects has not been conducted. Therefore, this study compared the effects of Ninjinyoeito, Hochuekkito, and Juzentaihoto on psychiatric symptoms using neuropeptide Y knockout (NPY-KO) zebrafish, a suitable animal model for anxiety and low sociability. Neuropeptide Y knockout zebrafish were fed a Ninjinyoeito, Hochuekkito, or Juzentaihoto-supplemented diet for 4 days. Then, sociability was analyzed using a three-Chambers test and anxiety-like behavior was evaluated using the cold stress and novel tank tests. The results showed that Ninjinyoeito treatment improved the low sociability of neuropeptide Y knockout, while Hochuekkito and Juzentaihoto did not. Neuropeptide Y knockout exhibited anxiety-like behaviors, such as freezing and swimming in the wall area under cold stress, but Ninjinyoeito treatment improved these behaviors. However, these anxiety-like behaviors were not improved by Hochuekkito and Juzentaihoto. Ninjinyoeito treatment also improved anxiety-like behaviors of neuropeptide Y knockout in the novel tank test. However, no improvement was shown in the Hochuekkito and Juzentaihoto groups. This trend was also confirmed in the low water stress test using wild-type zebrafish. This study exhibits that among the three types of Kampo-hozai, Ninjinyoeito is the most effective in psychiatric disorders associated with anxiety and low sociability.

Elucidation of the mechanism of nuclear localization of Mexican tetra Neu4 via bipartite nuclear localization signal and less conserved regions.

Nuclear sialoglycans are minor components in the nucleus, and their biological significance was not well understood. Recently, Nile tilapia Neu4 sialidase (OnNeu4) was identified and reported as the first nuclear sialidase in vertebrates. Although OnNeu4 possesses the nuclear localization signal (NLS) required for nuclear localization, other fish Neu4 sialidases, such as zebrafish and Japanese medaka, also possess NLS, but their subcellular localizations are not nucleus. To understand the nuclear localization mechanism of fish Neu4, we focused on Mexican tetra Neu4 (AmNeu4), which, unlike Neu4 in other fishes, has a bipartite NLS. AmNeu4 exhibited a wide range of optimal pH and substrate specificity, and its gene expression was specifically detected in the liver, spleen, and gut in adult fish. AmNeu4, like OnNeu4, exhibited nuclear localization, which was attenuated by importin inhibitor, and deletion of the bipartite NLS completely reduced the nuclear localization. In addition, the conjugation of the bipartite NLS of AmNeu4 made GFP show nuclear localization. To understand the mechanism of nuclear localization of AmNeu4 and OnNeu4, we compared fish Neu4 amino acid sequences and focused on the less conserved region of Neu4 sialidase (LCR). LCR-deletion mutants of AmNeu4 and OnNeu4 showed significantly reduced the nuclear localization. The LCR region in AmNeu4 and OnNeu4 possessed consecutive Ser/Thr. The Neu4 mutants in which consecutive Ser/Thr in LCR were changed to Ala or deleted significantly suppressed the nuclear localization. These results suggest that the nuclear localization of Neu4 in Nile tilapia and Mexican tetra may be regulated by NLS and LCR.

Acteoside from Conandron ramondioides Reduces Microcystin-LR Cytotoxicity by Inhibiting Intracellular Uptake Mediated by OATP1B3.

The hepatotoxin microcystin-LR is a strong inhibitor of serine/threonine protein phosphatase (PP) 1 and PP2A. The onset of its cytotoxicity depends on its selective uptake via the hepatocyte uptake transporters, organic anion transporting polypeptide (OATP) 1B1 and OATP1B3. Understanding and preventing the cytotoxicity of microcystin-LR is crucial to maintain human health. This chemoprevention study demonstrates that the herbal plant extract of iwajisha (20 µg/mL) reduced microcystin-LR cytotoxicity in OATP1B3-expressing cells by approximately six times. In addition, 20 µM acteoside, which is one of the major compounds in iwajisha, reduced microcystin-LR cytotoxicity by approximately 7.4 times. Acteoside could also reduce the cytotoxicity of other compounds, such as okadaic acid and nodularin, which are both substrates of OATP1B3 and inhibitors of PP1/PP2A. To investigate the mechanism by which the cytotoxicity of microcystin-LR is attenuated by acteosides, microcystin-LR and microcystin-LR-binding proteins in cells were examined after microcystin-LR and acteosides were co-exposed. Thus, acteoside noncompetitively inhibited microcystin-LR uptake by OATP1B3-expressing cells. Furthermore, acteoside inhibited the intracellular interaction of microcystin-LR with its binding protein(s), including the 22 kDa protein. Furthermore, using immunoblot analysis, acteoside induced the phosphorylation of extracellular signal-regulated kinase (ERK), which is one of the survival signaling molecules. These results suggest that acteoside reduces microcystin-LR cytotoxicity through several mechanisms, including the inhibition of microcystin-LR uptake via OATP1B3, and decreased interaction between microcystin-LR and its binding protein(s), and that ERK signaling activation contributes to the attenuation effect of acteoside against microcystin-LR cytotoxicity.

Ninjinyoeito improves social behavior disorder in neuropeptide Y deficient zebrafish.

Sociability is an essential component of the linkage structure in human and other vertebrate communication. Low sociability is defined as a poor social approach, including social withdrawal and apathy, and is implicated in a variety of psychiatric disorders. Ninjinyoeito (NYT), a traditional Japanese herbal medicine, has been used in the medical field. This study aimed to determine the effect of NYT on low sociality in NPY-KO zebrafish. NPY-KO zebrafish were fed a 3% NYT-supplemented diet for 4 days and subjected to behavioral tests. In the mirror test, NPY-KO zebrafish fed a control diet showed avoidance behavior toward their mirror counterparts. In contrast, the treatment of NPY-KO zebrafish with NYT significantly increased their interaction with their counterparts in the mirror. In addition, a 3-chambers test was conducted to confirm the effect of NYT on the low sociality of NPY-KO zebrafish. NPY-KO zebrafish fed the control diet showed less interaction with fish chambers, while NYT treatment increased the interaction. Phosphorylation of ERK, a marker of neuronal activity, was significantly reduced in the whole brain of NYT-fed NPY-KO zebrafish, compared to the control diet. NYT treatment significantly suppressed hypothalamic-pituitary-adrenal-related genes (gr, pomc, and crh) and sympathetic-adrenal-medullary-related genes (th1, th2, and cck) in NPY-KO zebrafish. NYT administration significantly reduced mRNA levels of gad1b compared to the control diet, suggesting the involvement of GABAergic neurons in NYT-induced improvement of low sociability. Furthermore, the expression of CREB was suppressed when NPY-KO zebrafish were fed NYT. Next, we attempted to identify the effective herb responsible for the NYT-induced improvement of low sociability. NPY-KO zebrafish were fed an experimental diet containing the target herb for 4 days, and its effect on sociability was evaluated using the 3-chambers test. Results showed that Cinnamon Bark and Polygala Root treatments significantly increased time spent in the fish tank area compared to the control diet, while the other 10 herbs did not. We confirmed that these two herbs suppressed the activity of HPA-, SAM-, and GABAergic neurons, as well as NYT-treated zebrafish, accompanied by downregulation of CREB signaling. This study suggests the potential use of NYT as a drug for sociability disorders.

Alteration of the neuronal and glial cell profiles in Neu1-deficient zebrafish.

Neu1 is a glycosidase that releases sialic acids from the non-reducing ends of glycoconjugates, and its enzymatic properties are conserved among vertebrates. Recently, Neu1-KO zebrafish were generated using genome editing technology, and the KO fish showed abnormal emotional behavior, such as low schooling, low aggressiveness, and excess exploratory behavior, accompanied by the downregulation of anxiety-related genes. To examine the alteration of neuronal and glial cells in Neu1-KO zebrafish, we analyzed the molecular profiles in the zebrafish brain, focusing on the midbrain and telencephalon. Using immunohistochemistry, we found that signals of Maackia amurensis (MAM) lectin that recognizes Sia α2-3 linked glycoconjugates were highly increased in Neu1-KO zebrafish brains, accompanied by an increase in Lamp1a. Neu1-KO zebrafish suppressed the gene expression of AMPA-type glutamate receptors such as gria1a, gria2a, and gria3b, and vesicular glutamate transporter 1. Additionally, Neu1-KO zebrafish induced the hyperactivation of astrocytes accompanied by an increase in Gfap and phosphorylated ERK levels, while the mRNA levels of astrocyte glutamate transporters (eaat1a, eaat1c, and eaat2) were downregulated. The mRNA levels of sypb and ho1b, which are markers of synaptic plasticity, were also suppressed by Neu1 deficiency. Abnormal activity of microglia was also revealed by IHC, and the expressions of iNOS and IL-1ß, an inflammatory cytokine, were increased in Neu1-KO zebrafish. Furthermore, drastic neuronal degeneration was detected in Neu1-KO zebrafish using Fluoro-Jade B staining. Collectively, the neuronal and glial abnormalities in Neu1-KO zebrafish may be caused by changes in the excitatory neurotransmitter glutamate and involved in the emotional abnormalities.

Neu1 deficiency increases the susceptibility of zebrafish to Edwardsiella piscicida infection via lysosomal dysfunction.

Neu1 is a lysosomal glycosidase that catalyzes the removal of sialic acids from glycoconjugates. Although Neu1 sialidase is highly conserved among vertebrates, the role of fish Neu1 is not fully understood because of its unique aquatic living situation. Compared to land animals, fish have a higher chance of bacterial infection, and to understand the role of fish Neu1, the susceptibility of Neu1 knockout zebrafish (Neu1-KO) was evaluated using Edwardsiella piscicida, a fish pathogen. Neu1-KO larvae showed high susceptibility to E. piscicida, despite the activation of macrophages, and presented increased lysosomal signals induced by the accumulation of Sia α2-3 linked oligosaccharides. The accumulation coincided with the signal of the macrophage marker, suggesting that the dysfunction of lysosomes in macrophages would result in a high susceptibility of Neu1-KO to E. piscicida. Chloroquine, an inhibitor of lysosomal degradation, induced high mortality of wild type zebrafish with E. piscicida infection accompanied by increased lysosomal accumulation, similar to Neu1-KO zebrafish. This study revealed that Neu1 sialidase plays a crucial role in the lysosomal degradation of macrophages with a bacterial infection.

Involvement of N-acetylneuraminate cytidylyltransferase in Edwardsiella piscicida pathogenicity.

Edwardsiella piscicida is a gram-negative bacterium that causes Edwardsiellosis in cultured fish. Edwardsiellosis is accompanied by symptoms such as skin lesions, hemorrhage, and necrosis in fish organs, which leads to significant economic losses in the aquaculture industry. Recently, we found that bacterial sialoglycoconjugates may be involved in the infectivity of E. piscicida. The more infectious strains of E. piscicida contain more sialic acid in the bacterial body, and the mRNA level of putative CMP-Neu5Ac synthase (css) is upregulated compared to that in the non-pathogenic strain. However, this putative css gene is yet to be cloned, and the involvement of CSS in E. piscicida pathogenicity remains unclear. Here, we cloned and transferred the css gene from E. piscicida into the FPC498 strain. CSS promoted infection in cultured cells originating from different fish species, and enhanced the mortality of E. piscicida-infected zebrafish larvae. CSS enhanced cell attachment and motility in E. piscicida, which differs from the decreased bacterial growth observed with the sialic acid-supplemented M9 medium. Both fractions (chloroform-methanol)-soluble and -insoluble fraction) prepared from E. piscicida pellet exhibited the increment of sialo-conjugates induced by CSS. Further, lectin blotting revealed the increment of Sia α2-3- and α2-6-, but not α2-8-, -linked glycoprotein in CSS-overexpressing E. piscicida. Overall, these findings indicate the physiological significance of CSS and the role of sialylation in E. piscicida pathogenicity.

Enhancement of Edwardsiella piscicida infection, biofilm formation, and motility caused by N-acetylneuraminate lyase.

Sialic acid and its catabolism are involved in bacterial pathogenicity. N-acetylneuraminate lyase (NAL), which catalyzes the reversible aldol cleavage of sialic acid to form N-acetyl-D-mannosamine in the first step of sialic acid degradation, has been recently investigated to elucidate whether NAL enhances bacterial virulence; however, the role of NAL in bacterial pathogenicity remains unclear. In the present study, we demonstrated that the existence of two enzymes in Edwardsiella piscicida, referred to as dihydrodipicolinate synthase (DHDPS) and NAL, induced the cleavage/condensation activity toward sialic acids such as N-acetylneuraminic acid, N-glycolylneuraminic acid and 3-deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid. NAL enhanced cellular infection in vitro and suppressed the survival rate in zebrafish larvae in bath-infection in vivo, whereas DHDPS did not. Furthermore, NAL strongly activated the expression of E. piscicida phenotypes such as biofilm formation and motility, whereas DHDPS did not. Besides, the gene expression level of nanK, nanE, and glmU were up-regulated in the NAL-overexpressing strain, along with an increase in the total amount of N-acetylglucosamine.

Fission yeast TOR complex 1 phosphorylates Psk1 through an evolutionarily conserved interaction mediated by the TOS motif.

TOR complex 1 (TORC1) is a multi-subunit protein kinase complex that controls cellular growth in response to environmental cues. The regulatory subunits of mammalian TORC1 (mTORC1) include RAPTOR (also known as RPTOR), which recruits mTORC1 substrates, such as S6K1 (also known as RPS6KB1) and 4EBP1 (EIF4EBP1), by interacting with their TOR signaling (TOS) motif. Despite the evolutionary conservation of TORC1, no TOS motif has been described in lower eukaryotes. In the present study, we show that the fission yeast S6 kinase Psk1 contains a TOS motif that interacts with Mip1, a RAPTOR ortholog. The TOS motif in Psk1 resembles those in mammals, including the conserved phenylalanine and aspartic acid residues essential for the Mip1 interaction and TORC1-dependent phosphorylation of Psk1. The binding of the TOS motif to Mip1 is dependent on Mip1 Tyr-533, whose equivalent in RAPTOR is known to interact with the TOS motif in their co-crystals. Furthermore, we utilized the mip1-Y533A mutation to screen the known TORC1 substrates in fission yeast and successfully identified Atg13 as a novel TOS-motif-containing substrate. These results strongly suggest that the TOS motif represents an evolutionarily conserved mechanism of the substrate recognition by TORC1.

Neu1 deficiency induces abnormal emotional behavior in zebrafish.

NEU1 sialidase hydrolyzes sialic acids from glycoconjugates in lysosomes. Deficiency of NEU1 causes sialidosis with symptoms including facial dysmorphism, bone dysplasia, and neurodegeneration. However, the effects of NEU1 deficiency on emotional activity have not been explored. Here, we conducted the behavioral analysis using Neu1-knockout zebrafish (Neu1-KO). Neu1-KO zebrafish showed normal swimming similar to wild-type zebrafish (WT), whereas shoaling was decreased and accompanied by greater inter-fish distance than WT zebrafish. The aggression test showed a reduced aggressive behavior in Neu1-KO zebrafish than in WT zebrafish. In the mirror and 3-chambers test, Neu1-KO zebrafish showed more interest toward the opponent in the mirror and multiple unfamiliar zebrafish, respectively, than WT zebrafish. Furthermore, Neu1-KO zebrafish also showed increased interaction with different fish species, whereas WT zebrafish avoided them. In the black-white preference test, Neu1-KO zebrafish showed an abnormal preference for the white region, whereas WT zebrafish preferred the black region. Neu1-KO zebrafish were characterized by a downregulation of the anxiety-related genes of the hypothalamic-pituitary-adrenal axis and upregulation of lamp1a, an activator of lysosomal exocytosis, with their brains accumulating several sphingoglycolipids. This study revealed that Neu1 deficiency caused abnormal emotional behavior in zebrafish, possibly due to neuronal dysfunction induced by lysosomal exocytosis.

Multiplexed suppression of TOR complex 1 induces autophagy during starvation.

Target of rapamycin complex 1 (TORC1) promotes cellular anabolism and suppresses macroautophagy/autophagy. In mammalian cells starved of amino acid, the GATOR1 complex, a negative regulator of TORC1, is released from its inhibitor GATOR2 and inactivates TORC1. We have recently identified the evolutionarily conserved GATOR2 components in fission yeast including Sea3, an ortholog of mammalian WDR59, but, unexpectedly, Sea3 acts as a part of GATOR1 to suppress TORC1. Moreover, fission yeast GATOR1 is not required for the amino-acid starvation-induced TORC1 attenuation, which is instead mediated by the Gcn2 pathway. Conversely, absence of a nitrogen source suppresses TORC1 in a manner dependent on GATOR1 as well as the Tsc1-Tsc2 complex, whose mammalian equivalent functions as a growth-factor sensitive TORC1 inhibitor. Thus, the evolutionarily conserved signaling modules are utilized differently between fission yeast and mammals to control TORC1 activity and autophagy.

Host lactosylceramide enhances Edwardsiella tarda infection.

Edwardsiella tarda is a Gram-negative bacterium causing economic damage in aquaculture. The interaction of E. tarda with microdomains is an important step in the invasion, but the target molecules in microdomains remain undefined. Here, we found that intraperitoneal injection of E. tarda altered splenic glycosphingolipid patterns in the model host medaka (Oryzias latipes) accompanied by alteration of glycosphingolipid metabolism-related gene expressions, suggesting that glycosphingolipid levels are involved in E. tarda infection. To ascertain the significance of glycosphingolipids in the infection, fish cell lines, DIT29 cells with a high amount of lactosylceramide (LacCer) and glucosylceramide (GlcCer), and GAKS cells with a low amount of these lipids, were treated with methyl-ß-cyclodextrin to disrupt the microdomain. E. tarda infection was suppressed in DIT29 cells, but not in GAKS cells, suggesting the involvement of microdomain LacCer and GlcCer in the infection. DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, an inhibitor of glycosphingolipid-synthesis, attenuated the infection in DIT29 cells, while Neu3-overexpressing GAKS cells, which accumulated LacCer, enhanced the infection. E. tarda possessed binding ability towards LacCer, but not GlcCer, and LacCer preincubation declined the infection towards fish cells, possibly due to the masking of binding sites. The present study suggests that LacCer may be a positive regulator of E. tarda invasion.

Ninjinyoeito improves anxiety behavior in neuropeptide Y deficient zebrafish.

Anxiety induced by excess mental or physical stress is deeply involved in the onset of human psychiatric diseases such as depression, bipolar disorder, and panic disorder. Recently, Kampo medicines have received focus as antidepressant drugs for clinical use because of their synergistic and additive effects. Thus, we evaluated the anxiolytic activity of Ninjinyoeito (NYT) using neuropeptide Y-knockout (NPY-KO) zebrafish that exhibit severe anxiety responses to acute stress. Adult NPY-KO zebrafish were fed either a 3% NYT-supplemented or normal diet (i.e., the control diet) for four days and were then examined via behavioral tests. After short-term cold stress (10 °C, 2 s) was applied, control-fed NPY-KO zebrafish exhibited anxiety behaviors such as freezing, erratic movement, and increased swimming time along the tank wall. On the other hand, NYT-fed NPY-KO zebrafish significantly suppressed these anxiety behaviors, accompanied by a downregulation of tyrosine hydroxylase levels and phosphorylation of extracellular signal-regulated kinases in the brain. To understand the responsible component(s) in NYT, twelve kinds of herbal medicines that composed NYT were tested in behavioral trials with the zebrafish. Among them, nine significantly reduced freezing behavior in NPY-KO zebrafish. In particular, Schisandra fruit induced the most potent effect on abnormal zebrafish behavior, even in the lower amount (0.3% equivalent to NYT), followed by Atractylodes rhizome and Cinnamon bark. Subsequently, four lignans uniquely found in Schisandra fruit (i.e., gomisin A, gomisin N, schizandrin, and schizandrin B) were investigated for their anxiolytic activity in NPY-KO zebrafish. As a result, schizandrin was identified as a responsible compound in the anxiolytic effect of NYT. These results suggest that NYT has a positive effect on mental stress-induced anxiety and may be a promising therapeutic for psychiatric diseases.

The involvement of Nile tilapia (Oreochromis niloticus) Neu4 sialidase in neural differentiation during early ontogenesis.

Neurogenesis is an important process for the formation of the central nervous system during ontogenesis. Mammalian sialidases are involved in neurogenesis through desialylation of sialo-glycoconjugates. However, the significance of fish sialidases, unlike that of mammals, in neurogenesis has not been investigated. The present study focuses on Nile tilapia (Oreochromis niloticus) because of its unique profiles of sialidases related to enzymatic properties, subcellular localization, and tissue-specific gene expression. First, the fish were cultured under aphotic condition, which is known to cause the delayed development of the retina and brain in various fish. Next, we investigate the effect of aphotic condition on the levels of tilapia sialidases. Our results revealed that the tilapia showed a decrease in the number of ganglion cell in the retina. The expression level of neu4 mRNA is up-regulated in the eyes from tilapia reared in Dark accompanied by the increase of retinal differentiation markers. These results indicated that tilapia Neu4 is involved in retinal development in Nile tilapia. Furthermore, we tried to clarify the function of tilapia Neu4 in the neuronal cells using two neuroblast cell lines (SH-SY5Y and Neuro2a cell lines). Tilapia Neu4 decreased sialic acid level of both nuclear glycoproteins as well as glycolipids. Moreover, tilapia Neu4 accelerated neurite formation in both two neural cell lines and, increased the acetylcholinesterase activity, but it did not affect cell proliferation. Collectively, these results suggest that Neu4 accelerates neurite differentiation during ontogenesis in tilapia.

Tripartite suppression of fission yeast TORC1 signaling by the GATOR1-Sea3 complex, the TSC complex, and Gcn2 kinase.

Mammalian target of rapamycin complex 1 (TORC1) is controlled by the GATOR complex composed of the GATOR1 subcomplex and its inhibitor, the GATOR2 subcomplex, sensitive to amino acid starvation. Previously, we identified fission yeast GATOR1 that prevents deregulated activation of TORC1 (Chia et al., 2017). Here, we report identification and characterization of GATOR2 in fission yeast. Unexpectedly, the GATOR2 subunit Sea3, an ortholog of mammalian WDR59, is physically and functionally proximal to GATOR1, rather than GATOR2, attenuating TORC1 activity. The fission yeast GATOR complex is dispensable for TORC1 regulation in response to amino acid starvation, which instead activates the Gcn2 pathway to inhibit TORC1 and induce autophagy. On the other hand, nitrogen starvation suppresses TORC1 through the combined actions of the GATOR1-Sea3 complex, the Gcn2 pathway, and the TSC complex, another conserved TORC1 inhibitor. Thus, multiple, parallel signaling pathways implement negative regulation of TORC1 to ensure proper cellular starvation responses.