| Schizophrenia, described as the "worst disease affecting mankind", is a severeand disabling mental disorder that affects1%of the population worldwide. Primarilyowning to its lack of pathological hallmarks, schizophrenia continues to be one of theleast understood. It is generally accepted that schizophrenia is result from interactionsof multiple genes under influence of environment and neurodevelopmental factorsinvolved. Genetic research has found a number of important candidate genesassociated with schizophrenia, in which both the encoding sequences of neuregulin1(NRG1) and its receptor ErbB4were identified as susceptibility genes. Brain samplesfrom patients with schizophrenia showed upregulated NRG1and ErbB4expressionand enhanced signalling. Mice with hypomorphic NRG1and ErbB4showedschizophrenia-like symptoms. Recent studies have provided compelling evidence thatNRG1/ErbB4signalling pathways is involved in neural development and synapticplasticity with influence to dopaminergic, glutamatergic and GABAergic and otherneurotransmitter systems. Gene expression in different brain regions and differenttime points may affect the nervous system with a variety of features and contribute todisease differently. In order to investigate the roles of NRG1/ErbB4expressionregulation in the pathogenesis of schizophrenia, in this study, lentivirus-mediatedErbB4specific miRNA interference was used to specific inhibit ErbB4geneexpression in animal brain spatially and temporally and behavior studies were done.The expression changes of NRG1and ErbB4were also detected in different animalmodels of schizophrenia. Results are as follows.1. ErbB4gene specific miRNA plasmids were constructed. In order to achievethe specific regulation of ErbB4gene expression, the specific pre-miRNA singlestranded DNA oligos targeting rat ErbB4were designed and synthesized. Therecombined interfering plasmids containing miRNA targeting ErbB4were constructed.After extracting RNA from rat brain, the ErbB4targeting sequence expression vectorwas cloned by RT-PCR. HEK293T cells were co-transfected with ErbB4targetingsequence expression vector and miRNA expression plasmids. Real time RT-PCRshowed that some miRNA recombined plasmids could effectively inhibit theexpression of their targeting sequence more than70%.2. A lentiviral vector containing miRNA targeting ErbB4was constructed andhigh titer lentivirus were packaged. In order to express ErbB4-specific miRNA stablyin neurons in vivo, interference sequence was transferred into the lentiviral expressionplasmid. After co-transfecting HEK293T producer cell line with the lentiviral expression construct and the packaging mix, lentivirus were harvest and concentrated.Counting GFP positive cells in10-fold serial dilutions of lentivirus stocks transductedHEK293T cells by flow cytometry showed a titer of1.0×109transducing units(TU)/ml.3. Gene was expressed stably after lentivirus stereotactic micro-infusion andneurons were the primary cell type transduced by lentivirus. Gene expression wasobserved on hippocampus dentate gyrus in mice brain sections even6months afterlentivirus injection, showing the stable expression mediated by lentivirus. Mostlentivirus transduced cells were labled for NeuN and no cell double-labled for GFPand GFAP, revealed that neurons were the primary cell type transduced by lentivirus.4. No significant effect of miRNA inhibiting of ErbB4expression inhippocampal dentate gyrus on the behavior of adult mice. Behavior test wereperformed after ErbB4-specific miRNA lentivirus stereotactic micro-infusion intohippocampus dentate gyrus in Balb/c mice. Mice in the lentiviral-mediated ErbB4inhibiting group showed no difference to control subjects in the spontaneous activity,latency in step down passive avoidance test and behavioral sensitivity to MK-801.These results suggest that interfering ErbB4expression with lentivirus-mediatedmiRNA in adult hippocampal dentate gyrus may have no obvious effect on behaviorsof mice. Perhaps, dysfunction of NRG1/ErbB4in single brain region in normallydeveloped adult brain is insufficient to cause significant behavioral change.5. Expression of NRG1and ErbB4were not changed in the rat model of socialisolation. Post-weaning male SD rats were randomized. Isolation reared animals werehoused in individual cage, while group reared ones were housed in four per cage.After six weeks of isolation, compare to controls, the isolation reared rats showedincreased bodyweight, hyper-reactivity to a novel environment, social interactionbehavior change and learning and memory behavior nuances in Morris water mazetest. Western blot analysis of NRG1and ErbB4protein expression showed nochanges between two groups, suggesting post-weaning social isolation procedure isnot sufficient to cause the Nrg1and ErbB4protein expression changes.6. Expressions of NRG1and ErbB4were increased in rat hippocampus ofschizophrenia model induced by chronic ketamine administration. Noncompetitiveantagonist of NMDA receptors as phencyclidine (PCP), ketamine and MK-801whichcan deteriorate the schizophrenic symptoms of patients and induce schizophrenia-likesymptoms in normal individuals, are widely used to modeling schizophrenia. SD ratswere intraperitoneal administrated with ketamine15mg/kg,30mg/kg,60mg/kg ornormal saline control for7days,2times a day. Western-blot analysis showed thatketamine increased NRG1and ErbB4expression in hippocampus. NRG1/ErbB4protein up-regulation induced by NMDA receptor antagonists may be one of thepathological mechanisms of schizophrenia. 7. Neonatal repeated treatment with ketamine damaged cognitive function ofadult rats. SD rats were administrated subcutaneously with ketamine15mg/kg,30mg/kg,60mg/kg or normal saline control since post-natal day7to day21andbehavior test were performed. A slower body weight gain was observed during thedrug administration session. In adulthood, ketamine had no effect on locomotoractivity and anxiety behavior in elevated zero maze test. In the object recognition test,rats repeatedly treated with ketamine during neonatal period showed smallerdiscrimination ratio of novel objects compared with controls. In the Morris watermaze, although ketamine-treated rats behavior normal during the spatial acquisitionand reference memory task, but performed poorly during the spatial working memorytasks. These results suggest long-term effects of neonatal ketamine on cognitivedeficits.8. Neonatal repeated ketamine treatment and maternal deprivation inducedneuropsychological behavior in adult rats, and NRG1were up-regulated. SD rats wererandomly divided into control group, maternal deprivation group, neonatal ketamine30mg/kg treatment group and the neonatal ketamine30mg/kg treatment combinedwith maternal deprivation group. SD rats were administrated subcutaneously withketamine30mg/kg or saline control twice daily since post-natal day7to day21..Maternal deprivation, separating pups from their mothers in a24h period wereperformed in post-natal day9. Behavior test were performed in adulthood. Bothneonatal repeated ketamine treatment and maternal deprivation didn't influencelocomotor activity and behavior in elevated zero maze test, but impaired cognitionfunction in object recognition test and induced hypersensitivity to methamphetaminechallenge. Only adult male rats suffered from both neonatal repeated ketaminetreatment and maternal deprivation showed deficient sensorimotor gating in PPI testof the startle reflex, showing a interaction. Western-blot detection showed that proteinlevels of NRG1were elevated while protein levels of ErbB4were not altered. Theresults showed that neonatal repeated ketamine treatment and maternal deprivationinduced schizophrenia-like features in adult animals, altered NRG1/ErbB4signalingpathway may be one of the pathological mechanisms of schizophrenia.9. ErbB specific tyrosine kinase inhibitor can partly counteract the hyperactivityinduced by MK-801in mice. MK-8010.25mg/kg intraperitoneal injection inducedhyperactivity in mice. Intraventricular injection of ErbB specific tyrosine kinaseinhibitor lowered the hyperactivity induced by MK-801, suggesting the ErbB receptoractivity are related to some symptoms of schizophrenia and inhibition of ErbBreceptor activity may ameliorate some symptoms of schizophrenia.In this study, gene were stably expressed mediated by lentivirus in adult animalbrain in space and time suggesting that lentivirus can be used as a tool for spatial andtemporal specific regulation of gene expression to promote the function study of susceptibility gene in different brain regions in different neurodevelopmental point.Above results suggest that the expression of NRG1and ErbB4were differentlychanged in different animal models of schizophrenia, suggesting that differentinducing factors may have different effects on the nervous system at differentdevelopmental stages through regulation of different genes. While abnormal ofsusceptibility genes in different developmental stages in specific brain region maycontribute to disease distinctly. Further suggest the importance of neurodevelopmentin schizophrenia. Whether ErbB4receptor activity can be used as the target ofantipsychotic treatment is worthy of further exploration. |
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