Maternal stress suppresses cell proliferation in the forebrain of zebrafish larvae.

Exposure to early life stress (ELS) can increase vulnerability to various psychiatric disorders. Although ELS has been shown to alter structure and functions of the hippocampus, amygdala and prefrontal cortex in the adult mammalian brain, it remains largely unclear whether ELS also affects embryonic or early-stage brain development. In this study, I investigated the effects of a maternal stress (maternal starvation for 4 days) of adult zebrafish on offspring's larval brain development. Although maternal starvation did not largely affect proliferation rate in the midbrain and hindbrain, it significantly decreased that in the forebrain of larvae at 5 days post-fertilization (dpf). I also found that embryos at 10 hr post-fertilization (hpf) born from a starved mother showed elevated cortisol levels compared to those born from a control mother. Furthermore, cortisol treatment was sufficient to decrease proliferating cells in the forebrain of 5 dpf larvae. Our findings thus demonstrate for the first time that maternal starvation induces neurodevelopmental changes in the forebrain of zebrafish larvae and points to a possible role of maternal cortisol in mediating this effect of maternal stress to offsprings.

Selective cross-linking behavior of oligodeoxyribonucleotides containing 2'-O-[N-(4,5',8-trimethylpsoralen-4'-ylmethylcarbamoyl)]adenosine to mutant H-ras DNA.

Point mutations are well characterized activators of oncogenes but are often indistinguishable using common gene technologies. In general, the precise sites of point-mutated oncogenes are difficult to distinguish under physiological conditions primarily because single nucleotide mismatch do not affect the annealing temperatures of DNA probes sufficiently. To address this limitation, we developed photo-responsive oligodeoxyribonucleotides containing 2'-O-[N-(4,5',8-trimethylpsoralen-4'-ylmethylcarbamoyl)]adenosine (Ps-amd-Oligo), which can be used to selectively manipulate and identify genes with point mutations. Here we present time course analyses of the photo-cross-linking efficiency of Ps-amd-Oligo with DNA and RNA and show promising selectivity for the oncogene H-ras.

PDK1-Akt pathway regulates radial neuronal migration and microtubules in the developing mouse neocortex.

Neurons migrate a long radial distance by a process known as locomotion in the developing mammalian neocortex. During locomotion, immature neurons undergo saltatory movement along radial glia fibers. The molecular mechanisms that regulate the speed of locomotion are largely unknown. We now show that the serine/threonine kinase Akt and its activator phosphoinositide-dependent protein kinase 1 (PDK1) regulate the speed of locomotion of mouse neocortical neurons through the cortical plate. Inactivation of the PDK1-Akt pathway impaired the coordinated movement of the nucleus and centrosome, a microtubule-dependent process, during neuronal migration. Moreover, the PDK1-Akt pathway was found to control microtubules, likely by regulating the binding of accessory proteins including the dynactin subunit p150(glued) Consistent with this notion, we found that PDK1 regulates the expression of cytoplasmic dynein intermediate chain and light intermediate chain at a posttranscriptional level in the developing neocortex. Our results thus reveal an essential role for the PDK1-Akt pathway in the regulation of a key step of neuronal migration.

The ASK family kinases differentially mediate induction of type I interferon and apoptosis during the antiviral response.

Viral infection activates host defense mechanisms, including the production of type I interferon (IFN) and the apoptosis of infected cells. We investigated whether these two antiviral responses were differentially regulated in infected cells. We showed that the mitogen-activated protein kinase (MAPK) kinase kinase (MAPKKK) apoptosis signal-regulating kinase 1 (ASK1) was activated in cells by the synthetic double-stranded RNA analog polyinosinic:polycytidylic acid [poly(I:C)] and by RNA viruses, and that ASK1 played an essential role in both the induction of the gene encoding IFN-ß (IFNB) and apoptotic cell death. In contrast, we found that the MAPKKK ASK2, a modulator of ASK1 signaling, was essential for ASK1-dependent apoptosis, but not for inducing IFNB expression. Furthermore, genetic deletion of either ASK1 or ASK2 in mice promoted the replication of influenza A virus in the lung. These results indicated that ASK1 and ASK2 are components of the antiviral defense mechanism and suggested that ASK2 acts as a key modulator that promotes apoptosis rather than the type I IFN response. Because ASK2 is selectively present in epithelium-rich tissues, such as the lung, ASK2-dependent apoptosis may contribute to an antiviral defense in tissues with a rapid repair rate in which cells could be readily replaced.

Mitochondrial localization of the antiviral signaling adaptor IPS-1 is important for its induction of caspase activation.

The RIG-I-like receptor (RLR) family of intracellular receptors detects viral nucleic acids and transmits an antiviral signal through the adaptor IPS-1. IPS-1 activation triggers host defense mechanisms, including rapid production of type I interferon (IFN), such as IFN-ß, and induction of apoptosis. IPS-1 is mainly localized to mitochondria, and this localization has been proposed to be essential for inducing production of type I IFN and IFN-stimulated genes (ISGs). However, the importance of this mitochondrial localization of IPS-1 in executing apoptosis has remained unclear. Here, using IPS-1 mutants that were directed to specific subcellular locations such as cytoplasm, plasma membrane and mitochondria, we found that IPS-1's localization to mitochondria is important to activate caspase, but not to signal for IFN-ß gene induction. We also found that IPS-1 possesses a BH3-like motif, which is commonly found among members of the Bcl-2 family. Mutations within this motif promoted IPS-1-induced caspase activation, suggesting that this domain acts as an intrinsic inhibitor domain of apoptosis induction. These results establish that the mitochondrial location of IPS-1 is essential to its ability to induce apoptosis.

Akt1 promotes focal adhesion disassembly and cell motility through phosphorylation of FAK in growth factor-stimulated cells.

The crosstalk between spatial adhesion signals and temporal soluble signals is key in regulating cellular responses such as cell migration. Here we show that soluble growth factors enhance integrin signaling through Akt phosphorylation of FAK at Ser695 and Thr700. PDGF treatment or overexpression of active Akt1 in fibroblasts increased autophosphorylation of FAK at Tyr397, an essential event for integrin turnover and cell migration. Phosphorylation-defective mutants of FAK (S695A and T700A) underwent autophosphorylation at Tyr397 and promoted cell migration in response to the integrin ligand fibronectin, but importantly, not in response to PDGF. This study has unveiled a novel function of Akt as an 'ignition kinase' of FAK in growth factor signaling and may shed light on the mechanism by which growth factors regulate integrin signaling.

Generation of metabolically functioning hepatocytes from human pluripotent stem cells by FOXA2 and HNF1α transduction.

BACKGROUND & AIMS: Hepatocyte-like cells differentiated from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) can be utilized as a tool for screening for hepatotoxicity in the early phase of pharmaceutical development. We have recently reported that hepatic differentiation is promoted by sequential transduction of SOX17, HEX, and HNF4α into hESC- or hiPSC-derived cells, but further maturation of hepatocyte-like cells is required for widespread use of drug screening. METHODS: To screen for hepatic differentiation-promoting factors, we tested the seven candidate genes related to liver development. RESULTS: The combination of two transcription factors, FOXA2 and HNF1α, promoted efficient hepatic differentiation from hESCs and hiPSCs. The expression profile of hepatocyte-related genes (such as genes encoding cytochrome P450 enzymes, conjugating enzymes, hepatic transporters, and hepatic nuclear receptors) achieved with FOXA2 and HNF1α transduction was comparable to that obtained in primary human hepatocytes. The hepatocyte-like cells generated by FOXA2 and HNF1α transduction exerted various hepatocyte functions including albumin and urea secretion, and the uptake of indocyanine green and low density lipoprotein. Moreover, these cells had the capacity to metabolize all nine tested drugs and were successfully employed to evaluate drug-induced cytotoxicity. CONCLUSIONS: Our method employing the transduction of FOXA2 and HNF1α represents a useful tool for the efficient generation of metabolically functional hepatocytes from hESCs and hiPSCs, and the screening of drug-induced cytotoxicity.

Suppression of hepatitis C virus replicon by adenovirus vector-mediated expression of tough decoy RNA against miR-122a.

Recent studies have demonstrated that the liver-specific microRNA (miRNA) miR-122a plays an important role in the replication of hepatitis C virus (HCV). Antisense nucleotides against miR-122a, including locked nucleic acid (LNA), have shown promising results for suppression of HCV replication; however, a liver-specific delivery system of antisense nucleotides has not been fully developed. In this study, an adenovirus (Ad) vector that expresses tough decoy (TuD)-RNA against miR-122a (TuD-122a) was developed to suppress the HCV replication in the liver hepatocytes. Ad vectors have been well established to exhibit a marked hepatotropism following systemic administration. An in vitro reporter gene expression assay demonstrated that Ad vector-mediated expression of TuD-122a efficiently blocked the miR-122a in Huh-7 cells. Furthermore, transduction with the Ad vector expressing TuD-122a in HCV replicon-expressing cells resulted in significant reduction in the HCV replicon levels. These results indicate that Ad vector-mediated expression of TuD-122a would be a promising tool for treatment of HCV infection.

Efficient generation of functional hepatocytes from human embryonic stem cells and induced pluripotent stem cells by HNF4α transduction.

Hepatocyte-like cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are expected to be a useful source of cells drug discovery. Although we recently reported that hepatic commitment is promoted by transduction of SOX17 and HEX into human ESC- and iPSC-derived cells, these hepatocyte-like cells were not sufficiently mature for drug screening. To promote hepatic maturation, we utilized transduction of the hepatocyte nuclear factor 4α (HNF4α) gene, which is known as a master regulator of liver-specific gene expression. Adenovirus vector-mediated overexpression of HNF4α in hepatoblasts induced by SOX17 and HEX transduction led to upregulation of epithelial and mature hepatic markers such as cytochrome P450 (CYP) enzymes, and promoted hepatic maturation by activating the mesenchymal-to-epithelial transition (MET). Thus HNF4α might play an important role in the hepatic differentiation from human ESC-derived hepatoblasts by activating the MET. Furthermore, the hepatocyte like-cells could catalyze the toxication of several compounds. Our method would be a valuable tool for the efficient generation of functional hepatocytes derived from human ESCs and iPSCs, and the hepatocyte-like cells could be used for predicting drug toxicity.

Specific regulation of point-mutated K-ras-immortalized cell proliferation by a photodynamic antisense strategy.

It has been reported that point mutations in genes are responsible for various cancers, and the selective regulation of gene expression is an important factor in developing new types of anticancer drugs. To develop effective drugs for the regulation of point-mutated genes, we focused on photoreactive antisense oligonucleotides. Previously, we reported that photoreactive oligonucleotides containing 2'-O-psoralenylmethoxyethyl adenosine (2'-Ps-eom) showed drastic photoreactivity in a strictly sequence-specific manner. Here, we demonstrated the specific gene regulatory effects of 2'-Ps-eom on [(12)Val]K-ras mutant (GGT --> GTT). Photo-cross-linking between target mRNAs and 2'-Ps-eom was sequence-specific, and the effect was UVA irradiation-dependent. Furthermore, 2'-Ps-eom was able to inhibit K-ras-immortalized cell proliferation (K12V) but not Vco cells that have the wild-type K-ras gene. These results suggest that the 2'-Ps-eom will be a powerful nucleic acid drug to inhibit the expression of disease-causing point mutation genes, and has great therapeutic potential in the treatment of cancer.

P-glycoprotein mediates efflux transport of darunavir in human intestinal Caco-2 and ABCB1 gene-transfected renal LLC-PK1 cell lines.

Darunavir (DRV) is a nonpeptidic protease inhibitor (PI) approved for the treatment of human immunodeficiency virus (HIV) infection. DRV displays potent activity against HIV strains resistant to other available PIs. Coadministration with ritonavir (RTV) improves the oral bioavailability of DRV. Inhibition of cytochrome P450 by RTV has been proposed as a mechanism for enhanced DRV bioavailability. However, interaction of these drugs with intestinal transporters has not been elucidated. This study was performed to explore the involvement of P-glycoprotein in transcellular DRV transport in monolayers of human intestinal Caco-2 and in ABCB1 multidrug resistance 1, (MDR1) gene-transfected renal LLC-PK1 (L-MDR1) cell lines. Transepithelial transport of DRV in Caco-2 cell monolayers was 2-fold greater in the basal-to-apical direction compared to that in the opposite direction. RTV had a significant inhibitory effect on the efflux transport of DRV in Caco-2 cells. The apical-to-basal DRV transport was enhanced by P-glycoprotein inhibitors, cyclosporin A and verapamil, as well as multidrug resistance-related protein (MRP/ABCC) inhibitors, probenecid and MK571. Using the L-MDR1 cell line, basal-to-apical DRV transport was much greater than in the opposite direction. Furthermore, cyclosporin A markedly inhibited the basal-to-apical DRV transport. RTV significantly increased the apical-to-basal transport of DRV in L-MDR1 cells, but reduced transport in the opposite direction. DRV inhibited P-glycoprotein-mediated efflux of calcein-acetoxymethyl ester in L-MDR1 cells with the inhibitory potency of 121 microM. These findings suggest that DRV is a substrate of P-glycoprotein and MRP, most likely MRP2. RTV appeared to inhibit P-glycoprotein, thereby enhancing the absorptive transport of DRV.

Synthesis of antisense oligonucleotides containing 2'-O-psoralenylmethoxyalkyl adenosine for photodynamic regulation of point mutations in RNA.

2'-O-psoralen-conjugated antisense oligonucleotide was able to recognize a point mutation of mRNA. It had outstanding ability to photo-cross-link only to oligoribonucleotides (ORN) having a point mutation. This type of antisense molecule is the only one of its kind so far. To give high photo-cross-linking efficiency and sequence selectivity to antisense molecules, we synthesized novel photo-reactive oligonucleotides (2'-Ps-xom) containing psoralen at the 2'-O-position adenosine via an ethoxymethylene (2'-Ps-eom), propoxymethylene (2'-Ps-pom) and butoxymethylene (2'-Ps-bom) linker, respectively. We evaluated the photo-cross-linking efficiency and sequence selectivity in photo-cross-linking of 2'-Ps-xom to the complementary ORN and to an ORN having a mismatch base. Among them, 2'-Ps-eom exhibited superior photo-cross-linking efficiency with high sequence selectivity.

Scaffolding function of PAK in the PDK1-Akt pathway.

Many extracellular signals stimulate phosphatidylinositol-3-kinase, which in turn activates the Rac1 GTPase, the protein kinase Akt and the Akt Thr 308 upstream kinase PDK1. Active Rac1 stimulates a number of events, including substrate phosphorylation by a subgroup of the PAK family of kinases. The combined effects of Rac1, PDK1 and Akt are crucial for cell migration, growth, survival, metabolism and tumorigenesis. Here we show that Rac1 stimulates a second, kinase-independent function of PAK1. The PAK1 kinase domain serves as a scaffold to facilitate Akt stimulation by PDK1 and to aid recruitment of Akt to the membrane. PAK differentially activates subpopulations of Akt. These findings reveal scaffolding functions of PAK that regulate the efficiency, localization and specificity of the PDK1-Akt pathway.

Photodynamic antisense regulation of mRNA having a point mutation with psoralen-conjugated oligonucleotide.

Nucleic acid-based drugs, such as antisense oligonucleotide, ribozyme, and small interfering RNA, are specific compounds that inhibit gene expression at the post-transcriptional level. To develop more effective nucleic acid-based drugs, we focused on photo-reactive antisense oligonucleotides. We have optimized the structure of psoralen-conjugated oligonucleotide to improve their sequence selectivity and photo-crosslinking efficiency. Previously, we reported that photo reactive oligonucleotides containing 2'-O-psoralenyl-methoxyethyl adenosine (2'-Ps-eom) showed drastic photo-reactivity with a strictly sequence specific manner in vitro. In this report, we evaluated the binding ability toward intracellular target mRNA. The 2'-Ps-eom selectively photo-cross-linked to the target mRNA extracted from cells. The 2'-Ps-eom also cross-linked to target mRNA in cells. Furthermore, 2'-Ps-eom did not cross-link to mRNA having a mismatch base. These results suggest that 2'-Ps-eom is a powerful antisense molecule to inhibit the expression of mRNA having a point mutation.

JNK phosphorylates synaptotagmin-4 and enhances Ca2+-evoked release.

Ca2+ influx induced by membrane depolarization triggers the exocytosis of secretory vesicles in various cell types such as endocrine cells and neurons. Peptidyl growth factors enhance Ca2+-evoked release, an effect that may underlie important adaptive responses such as the long-term potentiation of synaptic transmission induced by growth factors. Here, we show that activation of the c-Jun N-terminal kinase (JNK) plays an essential role in nerve growth factor (NGF) enhancement of Ca2+-evoked release in PC12 neuroendocrine cells. Moreover, JNK associated with phosphorylated synaptotagmin-4 (Syt 4), a key mediator of NGF enhancement of Ca2+-evoked release in this system. NGF treatment led to phosphorylation of endogenous Syt 4 at Ser135 and translocation of Syt 4 from immature to mature secretory vesicles in a JNK-dependent manner. Furthermore, mutation of Ser135 abrogated enhancement of Ca2+-evoked release by Syt 4. These results provide a molecular basis for the effect of growth factors on Ca2+-mediated secretion.

Homogeneous fluorescence assays for RNA diagnosis by pyrene-conjugated 2'-O-methyloligoribonucleotides.

We developed a bispyrene-conjugated 2'-O-methyloligoribonucleotide as an RNA-specific RNA-probe. The probe hybridized with the complementary RNA, greatly enhancing fluorescence and discriminating RNA from DNA. The assay was carried out in homogeneous aqueous media without removing the unbound probe from the detection solution. This homogeneous fluorescence assay also discriminated mismatch sequences in the target RNA. These pyrene probes could possess high potential to detect RNA in biological specimens simply.

Gene regulation by decoy approach (II): Development of photo-cross-linked oligonucleotides duplex as a decoy DNA for estrogen receptor.

We have developed a photo-cross-linked oligonucleotide (clip-ODN) for a novel type of gene regulator molecule. Here, we examined the ability of the clip-ODN as a decoy DNA on regulation of the transcriptional activity of estrogen receptor (ER). A photo-cross-linking reagent, 4,5',8-[4'-(aminoethyl-amino) methyl]-trimethylpsoralen (aeAMT) was conjugated with an ODN at the 5'-end, and the aeAMT was cross-linked with the thymine residue of the complementary oligonucleotide upon UVA irradiation (365nm). The clip-ODN drastically inhibited the proliferation of breast cancer cell line (MCF-7) than non-cliped one in a sequence specific manner. This finding revealed that photo-cross-linking of double stranded ODN improve the regulatory ability as a decoy DNA, and clip-ODN may be a valuable tool in gene therapy protocols for inhibiting breast cancer cells' proliferation.

Selective regulation of mutant K-ras mRNA expression by photo-cross-linking antisense oligonucleotide.

It has been reported that point mutations in genes are responsible for various cancers and the selective regulation of the gene expression is an important issue to develop new types of anticancer drugs. In this report, we report new types of antisense molecules that photo-cross-link to target mRNA having a point mutation site in a sequence specific manner. We designed and synthesized photo-reactive antisense oligonucleotides containing a 2'-O-psoralen-conjugated adenosine (2'-Ps-oligo). They contain a psoralen via an ethoxymethylene linker (2'-Ps-eom), a propoxymethylene linker (2'-Ps-pom) and a buthoxymethylene linker (2'-Ps-bom), respectively. We evaluated the photo-cross-linking efficiency and the sequence specificity toward complementary RNA. 2'-Ps-eom showed the highest photo-cross-linking efficiency among 2'-Ps-oligos.

Synaptic adhesion molecule OBCAM; synaptogenesis and dynamic internalization.

Opioid-binding cell adhesion molecule (OBCAM) is the member of the IgLON family, a subgroup of the immunoglobulin superfamily. In the present study, the functions and dynamics of OBCAM were investigated in hippocampal neurons in vitro. Western blotting revealed that OBCAM expression was low at early stages of culture but it was increased as culture development. Double labeling immunofluorescence microscopy showed that OBCAM immunoreactivity was localized mainly at postsynaptic spines labeled with phalloidin and anti-PSD-95. The inhibition of OBCAM function with the specific antibody resulted in a significant decrease in the number of synapses on dendrites compared with control mouse IgG. The suppression of OBCAM expression using the antisense oligodeoxynucleotide also impaired the formation of synapses compared with control universal ones. The overexpression of OBCAM mRNA using a plasmid vector augmented the formation of synapses. Moreover, the internalization of OBCAM was promoted with increased neuronal activity by 4-aminopyridine. This internalization was reduced with the treatment of filipin, a sterol agent, indicating that this process is a raft-dependent pathway. These results indicate that OBCAM is a synaptic cell adhesion molecule concerning synaptogenesis and its surface localization is dynamically regulated in response to neuronal activity.

Selective photo-cross-linking of 2'-O-psoralen-conjugated oligonucleotide with RNAs having point mutations.

It has been reported that point mutations in genes are responsible for various cancers and the selective regulation of the gene expression is an important issue to develop a new type of anticancer drugs. In this report, we present a new type of antisense molecule that photo-cross-links to an oligoribonucleotide having a point mutation site in a sequence specific manner. 2'-O-psoralen-conjugated adenosine was synthesized in four steps from adenosine and introduced in the middle of an oligodeoxyribonucleotide (2'-Ps-oligo). Compared with 5'-O-psoralen-conjugated oligodeoxyribonucleotide (5'-Ps-oligo), which has a psoralen at the 5'-end, 2'-Ps-oligo more selectively photo-cross-linked to a pyrimidine base of the site of alteration from purine to pyrimidine in the oligoribonucleotide.