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Poster #10 EFFECTS OF MATERNAL IMMUNE ACTIVATION ON GENE EXPRESSION PATTERNS IN THE FETAL BRAIN
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Abstracts of the 3rd Biennial Schizophrenia International Research Conference / Schizophrenia Research 136, Supplement 1 (2012) S1–S375
Poster #9 ANTAGONISTS AT N-METHYL-D-ASPARTATE RECEPTOR (NMDA-R) WITH DIFFERENT CLINICAL PROFILE AFFECT DIFFERENTLY THE TRANSCRIPTS OF CONSTITUTIVE AND INDUCIBLE EARLY GENES AT POSTSYNAPTIC DENSITY Felice Iasevoli 1 , Chiara Sarappa 1 , Anna Eramo 2 , Elisabetta F. Bonaguro 1 , Federica Marmo 1 , Andrea de Bartolomeis 1 1 Laboratory of Molecular Psychiatry - Department of Neuroscience - University School of Medicine Federico II, Naples, Italy; 2 H Lundbeck A/S, Copenhagen, Denmark Background: The administration of non-competitive antagonists at NMDA receptor (NMDA-R), such as ketamine, may induce psychotic-like symptoms in healthy individuals and reproduce an animal model of schizophrenia in rats, consistent with the NMDA-R hypofunction hypothesis of psychosis. NMDA-R is the core of the postsynaptic density (PSD), a protein network involved in glutamate signalling and implicated in behavioural disorders, and systemic ketamine has been found to affect the expression of the PSD gene Homer. Memantine, another NMDA-R antagonist, has been recently introduced as a pro-cognitive agent in Alzheimer and in some experimental proof of concept studies has been used as an adjunct therapy to antipsychotics. The aim of the study was to investigate whether types of NMDA-R antagonists may be associated with differential transcripts expression of inducible early genes involved in glutamate signalling and/or in neuronal activity. Methods: Male Sprague-Dawley rats were injected intraperitoneally with vehicle (NaCl), memantine (5mg/kg), MK-801 (0.8mg/kg), or ketamine (at either non-neurotoxic 25mg/kg or neurotoxic (50mg/kg) sub-anaesthetic doses). Rats were sacrificed 90 minutes after the treatment transcript image analysis by in situ hybridization histochemistry was performed to investigate the transcripts of PSD-genes (Homer 1a, Homer 1b, Arc andPSD-95) and c-fosin forebrain regions of interest (i.e.: frontal cortex and striatum). Results: NMDA-R antagonists preferentially affected the expression of inducible genes demonstrated to modulate synaptic plasticity, while the expression of constitutive genes was no changed in this paradigm. Changes in inducible genes’ expression were significantly different among compounds used. Homer1aexpression was decreased by memantine in the motor and somatosensory cortices and increased by sub-convulsant dose of ketamine in the somatosensory and insular cortices. No significant changes in gene expression were detected in the striatum. ArcmRNA was induced significantly in multiple cortical areas by NMDA-R antagonists. In striatum, Arcexpression was induced by the neurotoxic dose of ketamine and MK-801 and in the ventral aspects only, while memantine did not affect gene expression. C-fosexpression was activated by MK-801, ketamine, and memantine in premotor and insular cortices, while gene expression was unaffected in the other cortical regions. In striatum, c-fosexpression was decreased by ketamine 50mg/kg in the lateral caudate-putamen and increased by MK-801 in the dorsolateral putamen. No other significant changes were detected in this area. Discussion: The results of this study demonstrate that NMDA-R antagonists with different clinical profile may impact differently and in a regionspecific manner the expression of IEGs relevant for glutamate signalling and/or neuronal plasticity.
Poster #10 EFFECTS OF MATERNAL IMMUNE ACTIVATION ON GENE EXPRESSION PATTERNS IN THE FETAL BRAIN Krassimira A. Garbett 1 , Elaine Y. Hsiao 2 , Sára Kálmán 1,3 , Paul H. Patterson 2 , Károly Mirnics 1 1 Vanderbilt University, Nashville, Tennessee, USA; 2 California Institute of Technology, Pasadena, California, USA; 3 University of Szeged, Szeged, Hungary Background: A wide variety of infections during pregnancy (viral, bacterial, parasitic) are associated with increased risk for schizophrenia and autism in the offspring. To mimic this environmental risk factor, several animal models for maternal immune activation (MIA) have been used successfully. These include infection with human influenza virus, exposure to the synthetic dsRNA, poly(I:C) and injection of the cytokine IL-6 in pregnant mice. Adult mice born to MIA-exposed mothers display behavioral ab-
normalities that are relevant to both schizophrenia and autism, such as social interaction, prepulse inhibition (PPI), and open field and novel object exploration. Molecular and cellular studies of adult MIA offspring also reveal abnormalities that are informative of schizophrenia pathophysiology, such as increased levels of GABAA receptor α2 immunoreactivity, dopamine hyperfunction, delay in hippocampal myelination, reduced NMDA receptor expression in hippocampus, reduced numbers of reelin- and parvalbuminpositive cellsand reduced dopamine D1 and D2 receptors in prefrontal cortex, and enhanced tyrosine hydroxlyase in striatal structures. However, the immediate effects of MIA on the fetal brain are not well understood. Methods: To explore how maternal infection increases the risk for schizophrenia in the offspring, we induced MIA in pregnant mice with human influenza virus, poly(I:C), and recombinant IL-6 (rIL-6) under conditions that were previously shown to induce behavioral abnormalities in the offspring. Pregnant females for the flu group were anesthetized intraperitoneally (i.p.) and inoculated intra-nasally with human influenza virus on E9.5. Other groups of pregnant mice were injected with saline, poly(I:C) or rIL-6 on E12.5, the stage at which IL-6 levels peak when flu virus is given at E9.5. All mice were sacrificed on E12.5 (3h after poly(I:C) or rIL-6 injection and 3 days after influenza inoculation), and embryonic brains collected. RNA was isolated from individual embryonic brains, but pooled within each litter, and then used for microarray hybridization. Individual embryonic brain RNA or pooled RNA samples were used to generate cDNA for qPCR verification of gene expression. Individual placental weight was also correlated with gene expression findings. Results: Our study of the acute effects of MIA on the embryonic brain transcriptome reveals that: 1) all three MIA treatments (Flu, poly(I:C) and IL-6) evoke strong gene expression changes in the embryonic brain; 2) the three MIA treatments yield diverse as well as overlapping transcriptome signatures; 3) all MIA treatments strongly up-regulate the crystallin gene family, represented by alpha, beta and gamma members; 4) up-regulation of crystallin genes is an acute reaction that does not persist into adulthood; 5) the level of crystallin gene expression is correlated with the degree of MIA as measured by decrements in placental weight. In addition, our data also reveal a remarkable variability in crystallin induction among the individual brains in the Flu MIA model, indicating that this form of MIA does not affect all of the embryos equally within each dam Discussion: The overall gene expression changes suggest that the response to MIA is a neuroprotective attempt by the developing brain to counteract environmental stress, but at a possible cost of disrupting typical neuronal differentiation and axonal growth. We propose that this cascade of events might parallel the mechanisms by which environmental insults contribute to the risk of neurodevelopmental disorders such as schizophrenia and autism.
Poster #11 THE SP4 HYPOMORPHIC MOUSE AS A MODEL RELEVANT TO SCHIZOPHRENIA Xianjin Zhou, Mark A. Geyer University of California at San Diego, La Jolla, CA, USA Background: Sp4 is a transcription factor in the SP1/SP4 family that regulates the expression of a variety of brain genes and plays an important role in brain embryogenesis. We generated a Sp4 hypomorphic mouse by knock-in of a LacZ-Lox construct that reduced SP4 expression by 95%. Sp4 hypomorphs exhibited vacuolization and astrogliosis in dentate gyrus of the hippocampus, diminished prepulse inhibition of startle (PPI) with normal startle reactivity, and decreased contextual fear conditioning. Rescue of the Sp4 gene by breeding with Cre mice reversed the behavioral deficits. Regional rescue of SP4 expression by AAV-Cre injection into the hippocampus reversed deficits in PPI. Reduced Sp4 expression impaired the generation of LTP in hippocampal CA1, led to deficient spatial learning in the Barnes Maze, and markedly decreased the expression of NMDAR1 (NR1), the core subunit for all NMDA receptors, throughout brain. Sp4 maps to human chromosome 7 near a region implicated in linkage studies of schizophrenia. Genetic studies revealed that Sp4 SNPs are associated with both bipolar disorder and schizophrenia in both Caucasian and Chinese populations. Therefore, we suggested that Sp4 functions as a susceptibility gene for psychosis by modulating the expression of its downstream effector gene NMDAR1 and perhaps other genes. Based on the glutamate
Abstracts of the 3rd Biennial Schizophrenia International Research Conference / Schizophrenia Research 136, Supplement 1 (2012) S1–S375
Poster #9 ANTAGONISTS AT N-METHYL-D-ASPARTATE RECEPTOR (NMDA-R) WITH DIFFERENT CLINICAL PROFILE AFFECT DIFFERENTLY THE TRANSCRIPTS OF CONSTITUTIVE AND INDUCIBLE EARLY GENES AT POSTSYNAPTIC DENSITY Felice Iasevoli 1 , Chiara Sarappa 1 , Anna Eramo 2 , Elisabetta F. Bonaguro 1 , Federica Marmo 1 , Andrea de Bartolomeis 1 1 Laboratory of Molecular Psychiatry - Department of Neuroscience - University School of Medicine Federico II, Naples, Italy; 2 H Lundbeck A/S, Copenhagen, Denmark Background: The administration of non-competitive antagonists at NMDA receptor (NMDA-R), such as ketamine, may induce psychotic-like symptoms in healthy individuals and reproduce an animal model of schizophrenia in rats, consistent with the NMDA-R hypofunction hypothesis of psychosis. NMDA-R is the core of the postsynaptic density (PSD), a protein network involved in glutamate signalling and implicated in behavioural disorders, and systemic ketamine has been found to affect the expression of the PSD gene Homer. Memantine, another NMDA-R antagonist, has been recently introduced as a pro-cognitive agent in Alzheimer and in some experimental proof of concept studies has been used as an adjunct therapy to antipsychotics. The aim of the study was to investigate whether types of NMDA-R antagonists may be associated with differential transcripts expression of inducible early genes involved in glutamate signalling and/or in neuronal activity. Methods: Male Sprague-Dawley rats were injected intraperitoneally with vehicle (NaCl), memantine (5mg/kg), MK-801 (0.8mg/kg), or ketamine (at either non-neurotoxic 25mg/kg or neurotoxic (50mg/kg) sub-anaesthetic doses). Rats were sacrificed 90 minutes after the treatment transcript image analysis by in situ hybridization histochemistry was performed to investigate the transcripts of PSD-genes (Homer 1a, Homer 1b, Arc andPSD-95) and c-fosin forebrain regions of interest (i.e.: frontal cortex and striatum). Results: NMDA-R antagonists preferentially affected the expression of inducible genes demonstrated to modulate synaptic plasticity, while the expression of constitutive genes was no changed in this paradigm. Changes in inducible genes’ expression were significantly different among compounds used. Homer1aexpression was decreased by memantine in the motor and somatosensory cortices and increased by sub-convulsant dose of ketamine in the somatosensory and insular cortices. No significant changes in gene expression were detected in the striatum. ArcmRNA was induced significantly in multiple cortical areas by NMDA-R antagonists. In striatum, Arcexpression was induced by the neurotoxic dose of ketamine and MK-801 and in the ventral aspects only, while memantine did not affect gene expression. C-fosexpression was activated by MK-801, ketamine, and memantine in premotor and insular cortices, while gene expression was unaffected in the other cortical regions. In striatum, c-fosexpression was decreased by ketamine 50mg/kg in the lateral caudate-putamen and increased by MK-801 in the dorsolateral putamen. No other significant changes were detected in this area. Discussion: The results of this study demonstrate that NMDA-R antagonists with different clinical profile may impact differently and in a regionspecific manner the expression of IEGs relevant for glutamate signalling and/or neuronal plasticity.
Poster #10 EFFECTS OF MATERNAL IMMUNE ACTIVATION ON GENE EXPRESSION PATTERNS IN THE FETAL BRAIN Krassimira A. Garbett 1 , Elaine Y. Hsiao 2 , Sára Kálmán 1,3 , Paul H. Patterson 2 , Károly Mirnics 1 1 Vanderbilt University, Nashville, Tennessee, USA; 2 California Institute of Technology, Pasadena, California, USA; 3 University of Szeged, Szeged, Hungary Background: A wide variety of infections during pregnancy (viral, bacterial, parasitic) are associated with increased risk for schizophrenia and autism in the offspring. To mimic this environmental risk factor, several animal models for maternal immune activation (MIA) have been used successfully. These include infection with human influenza virus, exposure to the synthetic dsRNA, poly(I:C) and injection of the cytokine IL-6 in pregnant mice. Adult mice born to MIA-exposed mothers display behavioral ab-
normalities that are relevant to both schizophrenia and autism, such as social interaction, prepulse inhibition (PPI), and open field and novel object exploration. Molecular and cellular studies of adult MIA offspring also reveal abnormalities that are informative of schizophrenia pathophysiology, such as increased levels of GABAA receptor α2 immunoreactivity, dopamine hyperfunction, delay in hippocampal myelination, reduced NMDA receptor expression in hippocampus, reduced numbers of reelin- and parvalbuminpositive cellsand reduced dopamine D1 and D2 receptors in prefrontal cortex, and enhanced tyrosine hydroxlyase in striatal structures. However, the immediate effects of MIA on the fetal brain are not well understood. Methods: To explore how maternal infection increases the risk for schizophrenia in the offspring, we induced MIA in pregnant mice with human influenza virus, poly(I:C), and recombinant IL-6 (rIL-6) under conditions that were previously shown to induce behavioral abnormalities in the offspring. Pregnant females for the flu group were anesthetized intraperitoneally (i.p.) and inoculated intra-nasally with human influenza virus on E9.5. Other groups of pregnant mice were injected with saline, poly(I:C) or rIL-6 on E12.5, the stage at which IL-6 levels peak when flu virus is given at E9.5. All mice were sacrificed on E12.5 (3h after poly(I:C) or rIL-6 injection and 3 days after influenza inoculation), and embryonic brains collected. RNA was isolated from individual embryonic brains, but pooled within each litter, and then used for microarray hybridization. Individual embryonic brain RNA or pooled RNA samples were used to generate cDNA for qPCR verification of gene expression. Individual placental weight was also correlated with gene expression findings. Results: Our study of the acute effects of MIA on the embryonic brain transcriptome reveals that: 1) all three MIA treatments (Flu, poly(I:C) and IL-6) evoke strong gene expression changes in the embryonic brain; 2) the three MIA treatments yield diverse as well as overlapping transcriptome signatures; 3) all MIA treatments strongly up-regulate the crystallin gene family, represented by alpha, beta and gamma members; 4) up-regulation of crystallin genes is an acute reaction that does not persist into adulthood; 5) the level of crystallin gene expression is correlated with the degree of MIA as measured by decrements in placental weight. In addition, our data also reveal a remarkable variability in crystallin induction among the individual brains in the Flu MIA model, indicating that this form of MIA does not affect all of the embryos equally within each dam Discussion: The overall gene expression changes suggest that the response to MIA is a neuroprotective attempt by the developing brain to counteract environmental stress, but at a possible cost of disrupting typical neuronal differentiation and axonal growth. We propose that this cascade of events might parallel the mechanisms by which environmental insults contribute to the risk of neurodevelopmental disorders such as schizophrenia and autism.
Poster #11 THE SP4 HYPOMORPHIC MOUSE AS A MODEL RELEVANT TO SCHIZOPHRENIA Xianjin Zhou, Mark A. Geyer University of California at San Diego, La Jolla, CA, USA Background: Sp4 is a transcription factor in the SP1/SP4 family that regulates the expression of a variety of brain genes and plays an important role in brain embryogenesis. We generated a Sp4 hypomorphic mouse by knock-in of a LacZ-Lox construct that reduced SP4 expression by 95%. Sp4 hypomorphs exhibited vacuolization and astrogliosis in dentate gyrus of the hippocampus, diminished prepulse inhibition of startle (PPI) with normal startle reactivity, and decreased contextual fear conditioning. Rescue of the Sp4 gene by breeding with Cre mice reversed the behavioral deficits. Regional rescue of SP4 expression by AAV-Cre injection into the hippocampus reversed deficits in PPI. Reduced Sp4 expression impaired the generation of LTP in hippocampal CA1, led to deficient spatial learning in the Barnes Maze, and markedly decreased the expression of NMDAR1 (NR1), the core subunit for all NMDA receptors, throughout brain. Sp4 maps to human chromosome 7 near a region implicated in linkage studies of schizophrenia. Genetic studies revealed that Sp4 SNPs are associated with both bipolar disorder and schizophrenia in both Caucasian and Chinese populations. Therefore, we suggested that Sp4 functions as a susceptibility gene for psychosis by modulating the expression of its downstream effector gene NMDAR1 and perhaps other genes. Based on the glutamate