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REGULATION OF PSYCHOSIS GENE NPAS3 BY MICRORNA DURING POSTNATAL DEVELOPMENT AND IN SCHIZOPHRENIA
490
Abstracts
Results: Expression of Dysbindin1 and NRG1 type2 genes were observed in immortalized lymphocytes. Expression of NRG1 type1, 3, 4 genes were below the detection limit of real-time quantitative RT-PCR. No difference was observed between patients with schizophrenia and controls in the expression of Dysbindin1 and NRG1 type2 genes. Discussion: We found no difference between patients with schizophrenia and controls in the expression of Dysbindin1 and NRG1 type2 genes. Previous study showed that Dysbindin1 isoform a and NRG1 type2 isoforms GGF, GGF2 expression in immortalized lymphocyte from patients with schizophrenia were decreased. This discrepancy might be attributed to the number of cases used and the isoforms observed. In this study, we used about four times larger number of cases than the previous study and observed the expression of all the isoforms. Our findings show that immortalized lymphocyte gene expression profile in schizophrenia is different from Post-mortem brain tissue at least in Dysbindin1 and NRG1 genes. Further studies are required to assess whether immortalized lymphocyte is an appropriate alternative to neuronal tissue.
doi:10.1016/j.schres.2010.02.928
Poster 168 PARANOID SCHIZOPHRENIA IS CHARACTERISED BY INCREASED CANNABINOID CB1 RECEPTOR BINDING IN THE DORSOLATERAL PREFRONTAL CORTEX Katerina Zavitsanou, Victoria S. Dalton Australian Nuclear Science and Technology Organisation Sydney, NSW, Australia Schizophrenia Research Institute Sydney, NSW, Australia Background: Cannabis consumption may induce psychotic states in normal individuals, worsen psychotic symptoms of schizophrenic patients, and may facilitate precipitation of schizophrenia in vulnerable individuals. Previous studies using post-mortem human brain tissue suggest that binding to the cannabinoid CB1 receptor is increased in the anterior1 and posterior2 cingulate cortices and Brodmann's area 93 in schizophrenia. In the present study we examined CB1 receptor binding in the dorsolateral prefrontal cortex (DLPC, Brodmann's area 46), a region associated with altered function in both cannabis use and schizophrenia. Methods: Receptor density was investigated in this area using in vitro autoradiography with the CB1 receptor ligand [3H] CP55,940 in a cohort of 13 patients with paranoid schizophrenia, 24 patients with non-paranoid schizophrenia and 37 controls matched for age, PMI and pH. The non-paranoid schizophrenia group included cases that met criteria for undifferentiated, residual, disorganised, bipolar and depressive type schizophrenia. Seven cases from the non-paranoid group also met the criteria for schizoaffective disorder. All cases were obtained from the University of Sydney Tissue Resource Centre. Results were analysed using ANOVA followed by post hoc Bonferroni tests. Results: There was a main effect of diagnosis on [3H] CP55,940 binding quantified across all layers of the DLPC (F= 3.532, df = 2, P = 0.034). Post hoc tests indicated that this main effect was due to patients with paranoid schizophrenia having 24% higher levels of CB1 binding compared to the control group (59.5 ± 3.9 versus 48.0 ± 2.07 fmoles/mg tissue equivalent, respectively, p < 0.05). Factors such as post-mortem interval time, gender and agonal state were not found to have an effect on CB1 binding. There were however significant correlations between age at death, pH, and CB1 binding. Within the schizophrenia group, CB1 binding was not affected by the final recorded antipsychotic drug dose. Discussion: These results confirm previous studies and suggest that increased CB1 receptor binding is a feature of paranoid
schizophrenia. The data add to a growing biological and genetic evidence for the cannabinoid hypothesis of schizophrenia and point to the involvement of the endogenous cannabinoid system in the pathophysiology and pharmacotherapy of the disorder. doi:10.1016/j.schres.2010.02.929
Poster 169 REGULATION OF PSYCHOSIS GENE NPAS3 BY MICRORNA DURING POSTNATAL DEVELOPMENT AND IN SCHIZOPHRENIA Cyndi S. Weickert1,2,3, Carlotta Duncan1,2, Natalie Beveridge4,1, Jenny Wong1,2,5, Maree J. Webster6, Murray Cairns1,5 1 Schizophrenia Research Institute Sydney, NSW, Australia; 2Prince of Wales Medical Research Institute Sydney, NSW, Australia; 3School of Psychiatry, Faculty of Medicine, University of New South Wales Sydney, NSW, Australia; 4School of Biomedical Sciences and Pharmacy, The University of Newcastle Newcastle, NSW, Australia; 5School of Medical Sciences, Faculty of Medicine, University of New South Wales Sydney, NSW, Australia; 6Stanley Medical Research Institute Bethesda, MD, USA Background: Neuronal Per-Arnt-Sim domain protein 3 (NPAS3) is a brain-specific transcription factor localized to the breakpoint of a chromosomal translocation in a mother and daughter with psychiatric illness. NPAS3 has also been genetically associated with bipolar disorder and schizophrenia in a large cohort. NPAS3 knockout mice have reduced body size, decreased hippocampal volume, an underdeveloped corpus callosum and enlarged ventricles. They display incoordination, hyperactivity and deficits in sensorimotor gating and learning. In the cortex, NPAS3 is localized to GABAergic interneurons where it regulates expression of synaptic markers and reelin – a protein involved in neuronal migration and decreased in the frontal cortex of patients with schizophrenia. This links NPAS3 to behavioural, morphological and neurodevelopmental abnormalities which have been implicated in schizophrenia. However, little is known about the expression of the NPAS3 gene in human cortical development or in schizophrenia. Methods: We have measured NPAS3 mRNA by microarray and qPCR and NPAS3 protein by Western blotting in tissue from the middle frontal gyrus of developing humans and in adult humans with schizophrenia compared to controls. Results: In the postnatal human prefrontal cortex, NPAS3 mRNA is most highly expressed in the neonatal brain, decreasing to half maximal expression in the first few years of life, after which it is constantly expressed into adulthood (p < 0.001 by both microarray and qPCR). In contrast, NPAS3 protein increases in expression in the first decade of life and peaks in school age children then decreases to half maximal expression in the adult brain (p < 0.02). This suggested that there is not a direct translation of increased NPAS3 mRNA into increased protein, so we determined if microRNAs that putatively regulate NPAS3 levels were also changed across development. We found by microRNA array that miR-17 expression is highest in neonates compared to all other age groups (p < 0.02) suggesting that NPAS3 mRNA levels may undergo greater miRNAmediated decay early in life yielding less protein. In support of this, we have preliminary evidence that the NPAS3 3′UTR is post transcriptionally regulated by miR-17, in the context of a luciferase reporter assays in SHSY5Y cells (p < 0.001). We also found gender dimorphic expression of NPAS3 mRNA and protein in the adult cortex with normal females having almost double the amount of NPAS3 as compared to normal males. Furthermore, females with schizophrenia had significantly less NPAS3 protein as compared to normal females (p < 0.01). In preliminary analysis, we detected a trend towards a diagnosis by gender interaction for miR-17 mRNA
Abstracts
levels; however the pattern of mir-17 changes were similar to changes in the primary NPAS3 transcript (p = 0.07). Discussion: Multiple lines of convergent evidence indicate that NPAS3 is an interesting new candidate gene for susceptibility to psychiatric illness. Our study indicates that NPAS3 mRNA and protein are divergently regulated during cortical neurodevelopment in humans. This mismatch may involve developmental regulation of microRNAs that target the NPAS primary transcript. Considering the gender bias of certain psychiatric illnesses, such as schizophrenia, alterations in gender dimorphic expression of NPAS3 may constitute an underlying factor in disease severity. doi:10.1016/j.schres.2010.02.930
491
severity of DOR and spurious information detected in the contour integration test (Spearman's rho = 0.45) and the babble task (Spearman's rho = 0.42), although the results did not reach statistical significance because of the sample size. Spurious information processing in the auditory and visual tasks also showed strong correlation (Spearman's rho = 0.65, p = 0.056). Data collection for both parts of the study is expected to be completed by March 2010. Discussion: This study tests the hypothesis that excessive top-down processing and aberrant DMN function seen in schizophrenia is specifically related to DOR, a group of important symptom central to psychotic disorders. DOR are found in up to 67% of schizophrenic patients, and their roles as prodromal and relapse signal and schizotypal trait highlight their potential as a state and trait marker for schizophrenia. Identification of the neurocognitive and neurophysiological mechanisms of DOR will provide important insight into understanding psychosis.
Poster 170 DELUSIONS OF REFERENCE, EXCESSIVE TOP-DOWN PROCESSING, AND DEFAULT MODE NETWORK IN FIRST-EPISODE SCHIZOPHRENIA Gloria H.Y. Wong1, Haojuan Tao2, Zhong He2, Haihong Liu2, Cindy P.Y. Chiu1, Sherry W.K. Chan1, May M.L. Lam1, Christy L.M. Hui1, Jennifer Y.M. Tang1, Yunhua Wang2, Zhimin Xue2, Zhening Liu2, Eric Y.H. Chen1 1 The University of Hong Kong Hong Kong China; 2Second Xiangya Hospital of Central South University Changsha China Background: Delusions of reference (DOR) refer to the detection of spurious self-information in otherwise neutral or ambiguous environmental stimuli. Empirical studies of DOR using an information processing framework are lacking. We hypothesize, at the neurocognitive level, that DOR may be related to an excessive use of an internally generated, top-down processing strategy; whereas at the neurophysiological level, this may be related to the hyperactivity and hyperconnectivity in the default mode network (DMN) of the brain. The DMN has been implicated in self-focused attention and ‘stimulusindependent thoughts’, as well as baseline monitoring and automatic attention to salient environmental stimuli, and a link to psychotic symptoms has been observed. The first part of this study explore whether a relationship exists between DOR and excessive top-down processing; the second part of this study test the hypothesis that patients with DOR as chief compliant specifically present with aberrant DMN function and increased top-down processing. Methods: In Study 1, a total of 30 schizophrenic patients are assessed for DOR using the Ideas of Reference Interview Schedule (IRIS), and are tested using a visual processing (“contour integration”) and a verbal processing (“babbling”) task. Excessive topdown processing is measured by the score of spurious information perceived in the two tasks. In Study 2, to test the hypothesis that DOR is specifically related to DMN and increased top-down processing, 45 first-episode schizophrenic patients are recruited into one of three groups according to their symptomatology: (A) patient with I/DOR as chief presentation (n = 15); (B) patients with positive symptoms other than I/DOR as chief presentation (n = 15); and (C) patients without clinically significant positive symptoms (n = 15). A group of normal controls (n = 15) is also included. Participants are matched by age, sex and education level. Functional MRI scanning is performed under a resting condition and a blockdesign 0- and 2-back working memory task condition. ANOVA F test is used for the analysis of between group differences in DMN activity. For functional connectivity analysis, Pearson's correlation is performed in seed regions of interest according to previously defined components of the DMN. Results: In the exploratory study, interim data analysis (n = 9; 5 men, mean age 23.8 years) showed a positive correlation between
doi:10.1016/j.schres.2010.02.931
Poster 171 INTERACTION BETWEEN ESTROGEN RECEPTOR ALPHA AND TRKB SUGGEST CONVERGENCE IN DEVELOPMENTAL PATHWAYS IMPLICATED IN SCHIZOPHRENIA Jenny Wong1,2,3, Cynthia Shannon Weickert1,2,4 1 Prince of Wales Medical Research Institute Sydney, New South Wales, Australia; 2Schizophrenia Research Institute Sydney, New South Wales, Australia; 3School of Medical Sciences, Faculty of Medicine, University of New South Wales Sydney, New South Wales, Australia; 4School of Psychiatry, Faculty of Medicine, University of New South Wales Sydney, New South Wales, Australia Background: Schizophrenia is a heterogeneous disease resulting from the alteration of genes and pathways required for normal brain development, function and cognition. Molecular research implicates lower brain derived neurotrophic factor (BDNF) and lower BDNF receptor (TrkB) levels in the cortical neuropathology of schizophrenia. Since we have evidence suggesting that the cortical pathology in schizophrenia may also include the failure of the brain to respond to sex steroids, we set out to determine how altered estrogen receptor alpha (ERa) signalling and altered TrkB signalling may mechanistically converge at the cellular level. Considering that ERa and TrkB are capable of mediating overlapping cellular signalling cascades, we tested whether ligands for ERa were able to activate TrkB and vice versa. Methods: Transfection of cloned wild-type ERa and full-length TrkB were performed in cultured cells. To assay for transcriptional activity, Chinese Hamster Ovary cells (CHOK1) and neuronal cells (SHSY5Y) were co-transfected with TrkB and wild-type ERa in combination with a 3x ERE luciferase reporter construct. Changes in ERE-mediated transcription were measured by luciferase reporter assay. To test if protein interactions were direct, protein extract from cells transfected with TrkB were immunoprecipitated with an antibody specific for ERa and western blotting for TrkB was determined. Confocal microscopy for tagged proteins was used to anatomically localize TrkB within cells. Results: In CHOK1 cells, we found that TrkB overexpression significantly increased transcriptional activity from an estrogen responsive promoter relative to the empty vector control (t = 1.92, df = 4, p = 0.0002). When we explored the mechanism by which TrkB elicited this effect, we found that BDNF treatment increased ERa phosphorylation at Ser118 and Ser167 and increased total ERa protein. In contrast, estrogen treatment was unable to stimulate phosphorylation of TrkB. Interestingly, co-transfection of TrkB and wild-type ERa showed that overexpression of TrkB can potentiate
Abstracts
Results: Expression of Dysbindin1 and NRG1 type2 genes were observed in immortalized lymphocytes. Expression of NRG1 type1, 3, 4 genes were below the detection limit of real-time quantitative RT-PCR. No difference was observed between patients with schizophrenia and controls in the expression of Dysbindin1 and NRG1 type2 genes. Discussion: We found no difference between patients with schizophrenia and controls in the expression of Dysbindin1 and NRG1 type2 genes. Previous study showed that Dysbindin1 isoform a and NRG1 type2 isoforms GGF, GGF2 expression in immortalized lymphocyte from patients with schizophrenia were decreased. This discrepancy might be attributed to the number of cases used and the isoforms observed. In this study, we used about four times larger number of cases than the previous study and observed the expression of all the isoforms. Our findings show that immortalized lymphocyte gene expression profile in schizophrenia is different from Post-mortem brain tissue at least in Dysbindin1 and NRG1 genes. Further studies are required to assess whether immortalized lymphocyte is an appropriate alternative to neuronal tissue.
doi:10.1016/j.schres.2010.02.928
Poster 168 PARANOID SCHIZOPHRENIA IS CHARACTERISED BY INCREASED CANNABINOID CB1 RECEPTOR BINDING IN THE DORSOLATERAL PREFRONTAL CORTEX Katerina Zavitsanou, Victoria S. Dalton Australian Nuclear Science and Technology Organisation Sydney, NSW, Australia Schizophrenia Research Institute Sydney, NSW, Australia Background: Cannabis consumption may induce psychotic states in normal individuals, worsen psychotic symptoms of schizophrenic patients, and may facilitate precipitation of schizophrenia in vulnerable individuals. Previous studies using post-mortem human brain tissue suggest that binding to the cannabinoid CB1 receptor is increased in the anterior1 and posterior2 cingulate cortices and Brodmann's area 93 in schizophrenia. In the present study we examined CB1 receptor binding in the dorsolateral prefrontal cortex (DLPC, Brodmann's area 46), a region associated with altered function in both cannabis use and schizophrenia. Methods: Receptor density was investigated in this area using in vitro autoradiography with the CB1 receptor ligand [3H] CP55,940 in a cohort of 13 patients with paranoid schizophrenia, 24 patients with non-paranoid schizophrenia and 37 controls matched for age, PMI and pH. The non-paranoid schizophrenia group included cases that met criteria for undifferentiated, residual, disorganised, bipolar and depressive type schizophrenia. Seven cases from the non-paranoid group also met the criteria for schizoaffective disorder. All cases were obtained from the University of Sydney Tissue Resource Centre. Results were analysed using ANOVA followed by post hoc Bonferroni tests. Results: There was a main effect of diagnosis on [3H] CP55,940 binding quantified across all layers of the DLPC (F= 3.532, df = 2, P = 0.034). Post hoc tests indicated that this main effect was due to patients with paranoid schizophrenia having 24% higher levels of CB1 binding compared to the control group (59.5 ± 3.9 versus 48.0 ± 2.07 fmoles/mg tissue equivalent, respectively, p < 0.05). Factors such as post-mortem interval time, gender and agonal state were not found to have an effect on CB1 binding. There were however significant correlations between age at death, pH, and CB1 binding. Within the schizophrenia group, CB1 binding was not affected by the final recorded antipsychotic drug dose. Discussion: These results confirm previous studies and suggest that increased CB1 receptor binding is a feature of paranoid
schizophrenia. The data add to a growing biological and genetic evidence for the cannabinoid hypothesis of schizophrenia and point to the involvement of the endogenous cannabinoid system in the pathophysiology and pharmacotherapy of the disorder. doi:10.1016/j.schres.2010.02.929
Poster 169 REGULATION OF PSYCHOSIS GENE NPAS3 BY MICRORNA DURING POSTNATAL DEVELOPMENT AND IN SCHIZOPHRENIA Cyndi S. Weickert1,2,3, Carlotta Duncan1,2, Natalie Beveridge4,1, Jenny Wong1,2,5, Maree J. Webster6, Murray Cairns1,5 1 Schizophrenia Research Institute Sydney, NSW, Australia; 2Prince of Wales Medical Research Institute Sydney, NSW, Australia; 3School of Psychiatry, Faculty of Medicine, University of New South Wales Sydney, NSW, Australia; 4School of Biomedical Sciences and Pharmacy, The University of Newcastle Newcastle, NSW, Australia; 5School of Medical Sciences, Faculty of Medicine, University of New South Wales Sydney, NSW, Australia; 6Stanley Medical Research Institute Bethesda, MD, USA Background: Neuronal Per-Arnt-Sim domain protein 3 (NPAS3) is a brain-specific transcription factor localized to the breakpoint of a chromosomal translocation in a mother and daughter with psychiatric illness. NPAS3 has also been genetically associated with bipolar disorder and schizophrenia in a large cohort. NPAS3 knockout mice have reduced body size, decreased hippocampal volume, an underdeveloped corpus callosum and enlarged ventricles. They display incoordination, hyperactivity and deficits in sensorimotor gating and learning. In the cortex, NPAS3 is localized to GABAergic interneurons where it regulates expression of synaptic markers and reelin – a protein involved in neuronal migration and decreased in the frontal cortex of patients with schizophrenia. This links NPAS3 to behavioural, morphological and neurodevelopmental abnormalities which have been implicated in schizophrenia. However, little is known about the expression of the NPAS3 gene in human cortical development or in schizophrenia. Methods: We have measured NPAS3 mRNA by microarray and qPCR and NPAS3 protein by Western blotting in tissue from the middle frontal gyrus of developing humans and in adult humans with schizophrenia compared to controls. Results: In the postnatal human prefrontal cortex, NPAS3 mRNA is most highly expressed in the neonatal brain, decreasing to half maximal expression in the first few years of life, after which it is constantly expressed into adulthood (p < 0.001 by both microarray and qPCR). In contrast, NPAS3 protein increases in expression in the first decade of life and peaks in school age children then decreases to half maximal expression in the adult brain (p < 0.02). This suggested that there is not a direct translation of increased NPAS3 mRNA into increased protein, so we determined if microRNAs that putatively regulate NPAS3 levels were also changed across development. We found by microRNA array that miR-17 expression is highest in neonates compared to all other age groups (p < 0.02) suggesting that NPAS3 mRNA levels may undergo greater miRNAmediated decay early in life yielding less protein. In support of this, we have preliminary evidence that the NPAS3 3′UTR is post transcriptionally regulated by miR-17, in the context of a luciferase reporter assays in SHSY5Y cells (p < 0.001). We also found gender dimorphic expression of NPAS3 mRNA and protein in the adult cortex with normal females having almost double the amount of NPAS3 as compared to normal males. Furthermore, females with schizophrenia had significantly less NPAS3 protein as compared to normal females (p < 0.01). In preliminary analysis, we detected a trend towards a diagnosis by gender interaction for miR-17 mRNA
Abstracts
levels; however the pattern of mir-17 changes were similar to changes in the primary NPAS3 transcript (p = 0.07). Discussion: Multiple lines of convergent evidence indicate that NPAS3 is an interesting new candidate gene for susceptibility to psychiatric illness. Our study indicates that NPAS3 mRNA and protein are divergently regulated during cortical neurodevelopment in humans. This mismatch may involve developmental regulation of microRNAs that target the NPAS primary transcript. Considering the gender bias of certain psychiatric illnesses, such as schizophrenia, alterations in gender dimorphic expression of NPAS3 may constitute an underlying factor in disease severity. doi:10.1016/j.schres.2010.02.930
491
severity of DOR and spurious information detected in the contour integration test (Spearman's rho = 0.45) and the babble task (Spearman's rho = 0.42), although the results did not reach statistical significance because of the sample size. Spurious information processing in the auditory and visual tasks also showed strong correlation (Spearman's rho = 0.65, p = 0.056). Data collection for both parts of the study is expected to be completed by March 2010. Discussion: This study tests the hypothesis that excessive top-down processing and aberrant DMN function seen in schizophrenia is specifically related to DOR, a group of important symptom central to psychotic disorders. DOR are found in up to 67% of schizophrenic patients, and their roles as prodromal and relapse signal and schizotypal trait highlight their potential as a state and trait marker for schizophrenia. Identification of the neurocognitive and neurophysiological mechanisms of DOR will provide important insight into understanding psychosis.
Poster 170 DELUSIONS OF REFERENCE, EXCESSIVE TOP-DOWN PROCESSING, AND DEFAULT MODE NETWORK IN FIRST-EPISODE SCHIZOPHRENIA Gloria H.Y. Wong1, Haojuan Tao2, Zhong He2, Haihong Liu2, Cindy P.Y. Chiu1, Sherry W.K. Chan1, May M.L. Lam1, Christy L.M. Hui1, Jennifer Y.M. Tang1, Yunhua Wang2, Zhimin Xue2, Zhening Liu2, Eric Y.H. Chen1 1 The University of Hong Kong Hong Kong China; 2Second Xiangya Hospital of Central South University Changsha China Background: Delusions of reference (DOR) refer to the detection of spurious self-information in otherwise neutral or ambiguous environmental stimuli. Empirical studies of DOR using an information processing framework are lacking. We hypothesize, at the neurocognitive level, that DOR may be related to an excessive use of an internally generated, top-down processing strategy; whereas at the neurophysiological level, this may be related to the hyperactivity and hyperconnectivity in the default mode network (DMN) of the brain. The DMN has been implicated in self-focused attention and ‘stimulusindependent thoughts’, as well as baseline monitoring and automatic attention to salient environmental stimuli, and a link to psychotic symptoms has been observed. The first part of this study explore whether a relationship exists between DOR and excessive top-down processing; the second part of this study test the hypothesis that patients with DOR as chief compliant specifically present with aberrant DMN function and increased top-down processing. Methods: In Study 1, a total of 30 schizophrenic patients are assessed for DOR using the Ideas of Reference Interview Schedule (IRIS), and are tested using a visual processing (“contour integration”) and a verbal processing (“babbling”) task. Excessive topdown processing is measured by the score of spurious information perceived in the two tasks. In Study 2, to test the hypothesis that DOR is specifically related to DMN and increased top-down processing, 45 first-episode schizophrenic patients are recruited into one of three groups according to their symptomatology: (A) patient with I/DOR as chief presentation (n = 15); (B) patients with positive symptoms other than I/DOR as chief presentation (n = 15); and (C) patients without clinically significant positive symptoms (n = 15). A group of normal controls (n = 15) is also included. Participants are matched by age, sex and education level. Functional MRI scanning is performed under a resting condition and a blockdesign 0- and 2-back working memory task condition. ANOVA F test is used for the analysis of between group differences in DMN activity. For functional connectivity analysis, Pearson's correlation is performed in seed regions of interest according to previously defined components of the DMN. Results: In the exploratory study, interim data analysis (n = 9; 5 men, mean age 23.8 years) showed a positive correlation between
doi:10.1016/j.schres.2010.02.931
Poster 171 INTERACTION BETWEEN ESTROGEN RECEPTOR ALPHA AND TRKB SUGGEST CONVERGENCE IN DEVELOPMENTAL PATHWAYS IMPLICATED IN SCHIZOPHRENIA Jenny Wong1,2,3, Cynthia Shannon Weickert1,2,4 1 Prince of Wales Medical Research Institute Sydney, New South Wales, Australia; 2Schizophrenia Research Institute Sydney, New South Wales, Australia; 3School of Medical Sciences, Faculty of Medicine, University of New South Wales Sydney, New South Wales, Australia; 4School of Psychiatry, Faculty of Medicine, University of New South Wales Sydney, New South Wales, Australia Background: Schizophrenia is a heterogeneous disease resulting from the alteration of genes and pathways required for normal brain development, function and cognition. Molecular research implicates lower brain derived neurotrophic factor (BDNF) and lower BDNF receptor (TrkB) levels in the cortical neuropathology of schizophrenia. Since we have evidence suggesting that the cortical pathology in schizophrenia may also include the failure of the brain to respond to sex steroids, we set out to determine how altered estrogen receptor alpha (ERa) signalling and altered TrkB signalling may mechanistically converge at the cellular level. Considering that ERa and TrkB are capable of mediating overlapping cellular signalling cascades, we tested whether ligands for ERa were able to activate TrkB and vice versa. Methods: Transfection of cloned wild-type ERa and full-length TrkB were performed in cultured cells. To assay for transcriptional activity, Chinese Hamster Ovary cells (CHOK1) and neuronal cells (SHSY5Y) were co-transfected with TrkB and wild-type ERa in combination with a 3x ERE luciferase reporter construct. Changes in ERE-mediated transcription were measured by luciferase reporter assay. To test if protein interactions were direct, protein extract from cells transfected with TrkB were immunoprecipitated with an antibody specific for ERa and western blotting for TrkB was determined. Confocal microscopy for tagged proteins was used to anatomically localize TrkB within cells. Results: In CHOK1 cells, we found that TrkB overexpression significantly increased transcriptional activity from an estrogen responsive promoter relative to the empty vector control (t = 1.92, df = 4, p = 0.0002). When we explored the mechanism by which TrkB elicited this effect, we found that BDNF treatment increased ERa phosphorylation at Ser118 and Ser167 and increased total ERa protein. In contrast, estrogen treatment was unable to stimulate phosphorylation of TrkB. Interestingly, co-transfection of TrkB and wild-type ERa showed that overexpression of TrkB can potentiate