Mitsuko Ishihara-Sugano

Over-expression of AhR (aryl hydrocarbon receptor) induces neural differentiation of Neuro2a cells: neurotoxicology study

BackgroundDioxins and related compounds are suspected of causing neurological disruption in human and experimental animal offspring following perinatal exposure during development and growth. The molecular mechanism(s) of the actions in the brain, however, have not been fully investigated. A major participant in the process of the dioxin-toxicity is the dioxin receptor, namely the aryl hydrocarbon receptor (AhR). AhR regulates the transcription of diverse genes through binding to the xenobiotic-responsive element (XRE). Since the AhR has also been detected in various regions of the brain, the AhR may play a key role in the developmental neurotoxicity of dioxins. This study focused on the effect of AhR activation in the developing neuron.MethodsThe influence of the AhR on the developing neuron was assessed using the Neuro2a-AhR transfectant. The undifferentiated murine neuroblastoma Neuro2a cell line (ATCC) was stably transfected with AhR cDNA and the established cell line was named N2a-Rα. The activation of exogenous AhR in N2a-Rα cells was confirmed using RNAi, with si-AhR suppressing the expression of exogenous AhR. The neurological properties of N2a-Rα based on AhR activation were evaluated by immunohistochemical analysis of cytoskeletal molecules and by RT-PCR analysis of mRNA expression of neurotransmitter-production related molecules, such as tyrosine hydroxylase (TH).ResultsN2a-Rα cells exhibited constant activation of the exogenous AhR. CYP1A1, a typical XRE-regulated gene, mRNA was induced without the application of ligand to the culture medium. N2a-Rα cells exhibited two significant functional features. Morphologically, N2a-Rα cells bore spontaneous neurites exhibiting axon-like properties with the localization of NF-H. In addition, cdc42 expression was increased in comparison to the control cell line. The other is the catecholaminergic neuron-like property. N2a-Rα cells expressed tyrosine hydroxylase (TH) mRNA as a functional marker of catecholaminergic neurotransmitter production. Thus, exogenous AhR induced catecholaminergic differentiation in N2a-Rα cells.ConclusionThe excessive activation of AhR resulted in neural differentiation of Neuro2a cells. This result revealed that dioxins may affect the nervous system through the AhR-signaling pathway. Activated AhR may disrupt the strictly regulated brain formation with irregular differentiation occurring rather than cell death.

Effect of dioxins on regulation of tyrosine hydroxylase gene expression by aryl hydrocarbon receptor: a neurotoxicology study

BackgroundDioxins and related compounds are suspected of causing neurological disruption. Epidemiological studies indicated that exposure to these compounds caused neurodevelopmental disturbances such as learning disability and attention deficit hyperactivity disorder, which are thought to be closely related to dopaminergic dysfunction. Although the molecular mechanism of their actions has not been fully investigated, a major participant in the process is aryl hydrocarbon receptor (AhR). This study focused on the effect of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) exposure on the regulation of TH, a rate-limiting enzyme of dopamine synthesis, gene expression by AhR.MethodsN2a-Rβ cells were established by transfecting murine neuroblastoma Neuro2a with the rat AhR cDNA. TH expression induced by TCDD was assessed by RT-PCR and Western blotting. Participation of AhR in TCDD-induced TH gene expression was confirmed by suppressing AhR expression using the siRNA method. Catecholamines including dopamine were measured by high-performance liquid chromatography. A reporter gene assay was used to identify regulatory motifs in the promoter region of TH gene. Binding of AhR with the regulatory motif was confirmed by an electrophoretic mobility shift assay (EMSA).ResultsInduction of TH by TCDD through AhR activation was detected at mRNA and protein levels. Induced TH protein was functional and its expression increased dopamine synthesis. The reporter gene assay and EMSA indicated that AhR directly regulated TH gene expression. Regulatory sequence called aryl hydrocarbon receptor responsive element III (AHRE-III) was identified upstream of the TH gene from -285 bp to -167 bp. Under TCDD exposure, an AhR complex was bound to AHRE-III as well as the xenobiotic response element (XRE), though AHRE-III was not identical to XRE, the conventional AhR-binding motif.ConclusionOur results suggest TCDD directly regulate the dopamine system by TH gene transactivation via an AhR-AHRE-III-mediated pathway. The AhR- mediated pathway could have a particular AhR-mediated genomic control pathway transmitting the effects of TCDD action to target cells in the development of dopaminergic disabilities.

Specific interactions between aryl hydrocarbon receptor and dioxin congeners: ab initio fragment molecular orbital calculations.

Aryl hydrocarbon receptor (AhR) is a transcription factor and its function is activated by the binding of halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 1,2,4-trichlorodibenzo-p-dioxin (TrCDD). TCDD is highly toxic to rat, whereas its congener TrCDD shows only a weak effect on gene expression. In order to elucidate the reason of this remarkable difference in the effect of TCDD and TrCDD, we here obtained stable structures of the complexes with rat AhR (rAhR) and TCDD/TrCDD and investigated their electronic properties by using the ab initio fragment molecular orbital (FMO) method. The results indicate that TCDD binds more strongly to rAhR than TrCDD, which is consistent with the experimentally observed toxicity of TCDD and TrCDD. Furthermore, ab initio FMO calculations elucidate that His324 and Gln381 of rAhR are important for binding TCDD, while His324 and Ser334 are important for TrCDD binding.

A Subclone of HuH-7 with Enhanced Intracellular Hepatitis C Virus Production and Evasion of Virus Related-Cell Cycle Arrest

Hepatitis C virus (HCV) cell culture system with JFH-1 strain and HuH-7 cells enabled us to produce infectious HCV particles in vitro, and such system is useful to explore the anti-HCV compounds and to develop the vaccine against HCV. In the present study, we describe the derivation of a cell line that permits improved production of HCV particles. Specifically, we characterized several subclones that were isolated from the original HuH-7 cell line by limiting dilution. These HuH-7 subclones displayed a notable range of HCV production levels following transfection by full-genome JFH-1 RNA. Among these subclones, HuH-7T1 produced HCV more efficiently than other subclones and Huh-7.5.1 that is known to be highly permissive for HCV replication. Upon transfection with full-genome RNA, HCV production was increased ten-fold in HuH-7T1 compared to Huh-7.5.1. This increase in viral production correlated with increased efficiency of intracellular infectious virus production. Furthermore, HCV replication did not induce cell cycle arrest in HuH-7T1, whereas it did in Huh-7.5.1. Consequently, the use of HuH-7T1 as host cells could provide increased population of HCV-positive cells and elevated viral titer. In conclusion, we isolated a HuH-7 subclone, HuH-7T1, that supports efficient HCV production. High efficiency of intracellular infectious virus production and evasion of cell cycle arrest were important for this phenotype. We expect that the use of this cell line will facilitate analysis of the underlying mechanisms for HCV particle assembly and the cell cycle arrest caused by HCV.

Fetal exposure to 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin transactivates aryl hydrocarbon receptor‐responsive element III in the tyrosine hydroxylase immunoreactive neurons of the mouse midbrain

Fetal exposure to dioxins and related compounds is known to disrupt normal development of the midbrain dopaminergic system, which regulates behavior, cognition and emotion. The toxicity of these chemicals is mediated mainly by aryl hydrocarbon receptor (AhR) signaling. Previously, we identified a novel binding motif of AhR, the AhR‐responsive element III (AHRE‐III), in vitro. This motif is located upstream from the gene encoding tyrosine hydroxylase (TH), the rate‐limiting enzyme of dopamine biosynthesis. To provide in vivo evidence, we investigated whether 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) could regulate AHRE‐III transcriptional activity in midbrain dopaminergic neurons. We produced transgenic mice with inserted constructs of the AHRE‐III enhancers, TH gene promoter and the c‐myc‐tagged luciferase gene. Single oral administrations of TCDD (0–2000 ng kg–1 body weight) to the transgenic dams markedly enhanced TH‐immunoreactive (ir) intensity in the A9, A10 and A8 areas of their offspring at 3 days and 8 weeks of age. The offspring of dams treated with 200 ng kg–1 TCDD exhibited significant increases in the numbers of TH‐ and double (TH and c‐myc)‐ir neurons in area A9 compared with controls at 8 weeks. These results show that fetal exposure to TCDD upregulates TH expression and increases TH‐ir neurons in the midbrain. Moreover, the results suggest that TCDD directly transactivates the TH promoter via the AhR‐AHRE‐III‐mediated pathway in area A9. Fetal exposure to TCDD caused stable upregulation of TH via the AhR‐AHRE‐III signaling pathway and overgrowth of TH‐ir neurons in the midbrain, implying possible involvement in the etiology of neurodevelopmental disorders such as attention‐deficit/hyperactivity disorder (ADHD). Copyright

Tyrosine hydroxylase assay: a bioassay for aryl hydrocarbon receptor-active compounds based on tyrosine hydroxylase promoter activation

Tyrosine hydroxylase (TH) assay is a novel bioassay for screening aryl hydrocarbon receptor (AhR)-active compounds with diverse toxicities. The TH assay employs reporter genes to evaluate TH promoter activity, which is linked to dopamine synthesis and regulated by the AhR-aryl hydrocarbon receptor responsive element-III (AHRE-III)-mediated pathway. The toxic equivalency factors for higher chlorinated dioxin congeners in the TH assay (TH-TEF) exhibit the same tendency as those for the WHO-TEF, indicating that the activity of the TH assay is consistent with that of existing methods. Furthermore, other chemicals, such as lower chlorinated dioxin congeners that were minimally investigated in the past could be detected preferentially by this assay.

Immunohistochemical analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity on the developmental dentate gyrus and hippocampal fimbria in fetal mice

Dioxins are widespread persistent environmental contaminants with adverse impacts on humans and experimental animals. Behavioral and cognitive functions are impaired by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure. TCDD exerts its toxicity via the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor. The hippocampus, which plays important roles in episodic memory and spatial function, is considered vulnerable to TCDD-induced neurotoxicity, because it contains the AhR. We herein investigated the effects of TCDD toxicity on hippocampal development in embryonic mice. TCDD was administered to dams at 8.5 days postcoitum with a single dose of 20, 200, 2,000 and 5,000 ng/kg body weight (groups T20, T200, T2000 and T5000, respectively), and the brains were dissected from their pups at embryonic day 18.5. Immunohistochemical analysis demonstrated that the Glial Fibrillary Acidic Protein (GFAP) immunoreactivities in the dentate gyrus (DG) were reduced in the T5000 group. Granular GFAP immunoreactivity was observed in the hippocampal fimbria, and the number of immunoreactive fimbria was significantly decreased in the T5000 group. The number of Proliferating Cell Nuclear Antigen (PCNA)-positive cells was decreased in all TCDD-exposed groups and significantly reduced in the T20, T200 and T5000 groups. Together, these results demonstrate that maternal TCDD exposure has adverse impacts on neural stem cells (NSCs), neural precursor cells (NPCs) and granular cells in the DG and disrupts the NSC maintenance and timing of differentiation in the hippocampal fimbria, which in turn interrupt neuronal development in future generations of mice.

Binding affinity between AhR and exogenous/endogenous ligands: molecular simulations and biological experiment

Aryl hydrocarbon receptor (AhR) plays critical roles in cell differentiation, and its mechanism is controlled by exogenous and endogenous ligands. However, structures of AhR and its complex with ligand have not been determined by experimental structural biology. We here obtain stable structures of the complexes with rat AhR (rAhR) and some ligands in water by molecular simulations based on homology modelling, protein–ligand docking, classical molecular mechanics optimisation and ab initio fragment molecular orbital (FMO) calculations. In addition, the binding affinities and the specific interactions between rAhR and the ligands are investigated by ab initio FMO calculations and biological experiments. The experiments reveal the dependence of the rAhR-mediated transcriptional activation on the ligand binding. On the other hand, the results of FMO calculations elucidate that the exogenous ligands interact with many residues of rAhR, while the endogenous ligands interact specifically with a few residues, and that the side chain of Gln381 of rAhR interacts strongly with the oxygen atom located at the centre of the ligand. Furthermore, we evaluate the binding energies between rAhR and the ligands by the FMO method and compare them with the transcriptional activation obtained by the experiment.

Developmentally regulated mRNA splicing of clathrin assembly protein 3 (AP-3)

Clathrin assembly protein 3 (AP-3) is a neuron-specific component of clathrin coated vesicles. Because it promotes the assembly of uniform clathrin cages, AP-3 may play a regulatory role in synaptic vesicle recycling. Previously, using the monoclonal antibody MabR-18, we demonstrated that AP-3 expression starts from the embryonic stage and is maintained at high levels from the early postnatal stages through adult. In order to study the expression of AP-3 during early postnatal development at the mRNA level, RT-PCR analysis was performed. We divided the coding region of AP-3 into 10 regions and designed primers to amplify each region. As a result, developmentally regulated splicing sites were found in two regions. In one region, a PCR product with a 108-bp deletion was detected from postnatal day 10 (P10). In the other region, a product with a 15-bp deletion was increased compensating for the decrease of the undeleted product. The expression of isoforms changed mainly from around P7 to P10, whereas the level of AP-3 protein remained relatively constant throughout postnatal development. These results suggest that the expression of AP-3 isoforms with mRNA splicing is developmentally regulated in the brain and may be involved in the maturation of synaptic vesicle recycling.

Neuro-biological modeling of the brain function (II) — Cloning of glutamate decarboxylase cDNA by PCR-based eficient cloning method —

Several quantitative methods for gene expression have been developed by using PCR. however. most of them require special apparatuses or extra DNA fragments such as a competitor In the present study, we tested a simple quantitative method based on PCR Total RNA was extracted from a part of the rat brain, then reverse-transcribed into cDNA using random hexamers Obtained cDNA was serially diluted and PCR-amplified using primers for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), GABA receptor subunit 0 I (GABA ii I ), or phospholipase C subunit B 4 (PLC B 4) After agarose-gel electrophoresis, PCR products were visualized and quantitated by ethidium staining In each sample. the linear ranges of amplification of each cDNA of interest were established From the relation between volume of cDNA and