- Email: [email protected]
Using zebrafish to understand the neurodevelopment role of susceptibility genes for autism spectrum disorder
686
Poster Abstracts / Int. J. Devl Neuroscience 28 (2010) 655–719
tions presenting with hydrocephalus. The data we have obtained from the limited number of affected babies suggests a similar folate imbalance to that found in the rat model indicating that the human condition may also be amenable to the same combination folate supplements.Keywords: Hydrocephalus; Fetus; Cerebral cortex; Folate Reference: Cains, S., A. Shepherd, et al. (2009). “Addressing a folate imbalance in fetal cerebrospinal fluid can decrease the incidence of congenital hydrocephalus.” J. Neuropathol. Exp. Neurol. 68(4): 404–16. doi:10.1016/j.ijdevneu.2010.07.131 [P2.02] Repeated carbenoxolone injection during late pregnancy decreased SPAR but increased Snk expression in the hippocampus of rat pups N. Jutapakdeegul ∗ , P. achatyanukul
Surakul, R.
Vanichviriyakit, W.
Weer-
Mahidol University, Thailand Homeostasis of circulating cortisol is maintained by two enzymes of 11-hydroxysteroid dehydrogenase, 11-HSD1 and 11-HSD2. 11-HSD2 is NAD-dependent dehydrogenase that inactivates cortisol into cortisone. In addition, 11-HSD2 is abundantly expressed in the placenta where it protects the fetus from active maternal glucocorticoids. Administration of carbenoxolone (CBX), a powerful 11-HSD2 inhibitor, leads to an increased of fetal cortisol level and decreased plasma cortisone levels. Previous data showed that intrauterine environment plays crucial roles in determining hippocampal structure and function in later life. Exposure to high level of corticosteroid during pregnancy lead to a low birth weight offspring, increased risk of aged related memory and cognitive deficits, however, mechanism still unclear. We proposed that repeated CBX injections during late pregnancy may alter scaffolding proteins of NMDA receptor in the hippocampus of rat pups. Here we show that pregnant rats receiving CBX injections (30 mg/kg) during GD14-21 lead to a significant decreased of SPAR (spine Associated Rap Guanylate kinase activating protein) (p < 0.01) but significant increased of Snk (Serum inducible kinase) (p < 0.01) in the pup’s hippocampus at P40 and P60, while the level of PSD-95 remain unchanged. NMDA receptor function has been shown to be influenced by turnover of the scaffolding proteins via ubiquitin proteasome system (UPS). Normally, Snk is induced by neuronal activity and plays an important role in phosphorylate SPAR. The phosphorylated SPAR was then recognized and degraded by ubiquitin-proteasome system, causing the depletion of SPAR and PSD-95 from the spines and leading to spine shrinkage and loss. The results suggest that fetal exposure with excessive corticosteroids may activate Snk/SPAR pathway and lead to the depletion of SPAR. Since, corticosteroid drugs are commonly used in various obstetric and paediatric conditions, it is important to consider the risk–benefit of prenatal GC exposure to prevent the neurodevelopment delay in the offspring.Keywords: Carbenoxolone; SPAR; Snk; Hippocampus doi:10.1016/j.ijdevneu.2010.07.132
[P2.03] Repeated maternal restraint stress down-regulated NMDA receptor subunits and scaffolding proteins in the hippocampus of postnatal rat P. Surakul ∗ , R. Vanichviriyakit, W.A. Weerachatyanukul, N. Jutapakdeegul Mahidol University, Thailand Early life stress has been linked with a substantially increased risk of aged related memory and cognitive deficits. Maternal stress hormone plays a crucial role in determining pup’s hippocampal structure and function, however mechanism is still unclear. We proposed that prenatal stress (PS) may alter developmental expression of NR2A, NR2B subunits of NMDA receptor and its scaffolding proteins in the hippocampus of rat pups. To investigate this hypothesis, pregnant rats were restrained during late gestation (GD14-21) and the expression of NR2A, NR2B subunits, PSD-95 (Postsynaptic density protein-95), SPAR (sSpine associated Rap guanylate kinase activating protein) and Snk (Serum inducible kinase) were measured in the hippocampus of rat pups from P7–P60. First, we found that, PS pups show a significant decreased of NR2A and NR2B subunit at P40 (p < 0.001 and p < 0.05, respectively) and P60 (p < 0.01). Then we found that PS caused a significant increased of PSD-95 in the pup’s hippocampus during P7–P14 (p < 0.05) and P28 (p < 0.01), but decreased significantly at P40 (p < 0.001) and P60 (p < 0.01) as compare to control. The decreased of PSD-95 and NMDAR subunits can be explained by alteration of Snk-SPAR pathway. We also found that PS caused a significant increased of SPAR during P7 and P14 (p < 0.05), but showed a significant decreased during P40 (P < 0.001) and P60 (p < 0.01). The most interested finding is that PS pups show significant increased of Snk (P < 0.001) in the hippocampus throughout their childhood period from P7-P40 as compared to control. These data suggested that early life stress exert long term effects on the development of NMDAR in the pup’s hippocampus. Prenatal stress alters development of scaffolding proteins and NMDAR subunits may underlies the mechanism link between early life stress and increased risk of aged related memory and cognitive deficits and heightened vulnerability to psychological disorders in later life. Keywords: Prenatal stress; NMDA receptor; Scaffolding proteins; Hippocampus doi:10.1016/j.ijdevneu.2010.07.133 [P2.04] Using zebrafish to understand the neurodevelopment role of susceptibility genes for autism spectrum disorder B. Key University of Queensland, Australia Several studies in the last three years have revealed that members of a synaptic cell adhesion network are candidate susceptibility genes for Autism spectrum disorder (ASD). These genes include neurexin-1 (NRXN1), neuroligin-1 (NLGN1), NLGN4, contactin associated protein 2 (CNTNAP2 or previously known as CASPR2) and Shank3. ASD is increasingly attributed to a disorder of brain function rather than brain anatomy. We have begun to address the role of gene-gene interactions within the synaptic cell adhesion pathway involved in neural circuits associated with simple behaviours using the zebrafish animal model. We are focusing on interactions between identified susceptibility genes NLGN-1, NLGN-4, NRXN-1, Shank3 and CNTNAP2 as well as on interactions of these genes with other known synaptic cell adhe-
Poster Abstracts / Int. J. Devl Neuroscience 28 (2010) 655–719
sion pathway genes (LRRTM2, PSD-95 and CASK) in order to begin to understand the function of gene networks underlying the emergence of early behaviours. We have generated morpholinos against NRXN-1a, NRXN1b and CNTNAP2 and injected them individually into one cell embryos and then assessed the touch and escape responses at 30 and 45 h, respectively. Knock down of either NRXN1a, NRXN1b or CNTAP2 significantly reduced the touch response at 30 hpf to a similar extent. The high penetrance of these phenotypes (71–84%) suggest that these genes are playing a major role in the development of the underlying neural circuitry responsible for this behaviour. In contrast, at 45 hpf knock down of NRXN-1a had no effect on the escape response, knock down of NRXN1b either extinguished or reduced the response, while knock down of CNTNAP2 produced an abnormal response. These very different phenotypes suggest very different roles of these synaptic adhesion network genes in the underlying neural circuitry. Our analyses are beginning to reveal the most critical genes involved in development of neural circuits underlying a simple behaviour. Keywords: Zebrafish; Synapse; Behavior; Adhesion doi:10.1016/j.ijdevneu.2010.07.134 [P2.05] The canonical Wnt pathway regulates Tbr2-positive intermediate progenitor maturation in the neocortex R.N. Munji ∗ , Y. Choe, S.J. Pleasure University of California-San Francisco, USA Keywords: Wnt; Cortex; Intermediate progenitors; Periventricular heterotopia In this study, we focus on the role of the canonical Wnt pathway in neocortical neurogenesis. At present, conclusions from previous studies have not converged on a parsimonious model for the role of the canonical Wnt pathway in neocortical neurogenesis, some suggesting a role in promoting neural progenitor differentiation while others in promoting primary progenitor (PP) self-renewal and, in consequence, inhibition of intermediate progenitor (IP) production. We hypothesized that the canonical Wnt pathway might function to regulate both differentiation and selfrenewal, possibly in different progenitor populations and/or at different developmental time-points. To address our hypothesis, we analyzed canonical Wnt function from E13.5, a time-point when horizontal expansion of the neocortical epithelium has slowed while neuronal production continues, and up to P2, when embryonic neurogenesis has primarily subsided, and utilized in utero electroporation (EP) of secreted effectors of the pathway, Wnt3a ligand and Dkk1 inhibitor, to perturb the pathway upstream of intracellular events. Overexpression of Wnt3a at E13.5 horizontally expanded the neocortical epithelium and induced premature differentiation of Tbr2-positive IPs into neurons. These prematurely born neurons predominantly fail to reach the cortical plate and accumulate deep in the cortical epithelium, leading to the formation of periventricular heterotopias. In contrast, overexpression of Dkk1 reduced neuronal production. Our results suggest that the canonical Wnt pathway has a dual role in neocortical neurogenesis, promoting self-renewal of PPs and differentiation of Tbr2-positive IPs. Moreover, these results contribute to our understanding of cortical organization and suggest a connection between the canonical Wnt pathway and periventricular heterotopia disorders. doi:10.1016/j.ijdevneu.2010.07.135
687
[P2.06] Wnts regulate the timing and efficiency of OPC generation in the telencephalon A.J. Langseth 1,∗ , R.N. Munji 1 , Y. 1,2 1 Pozniak , S.J. Pleasure
Choe 1 , T.
Huynh 1 , C.D.
1
University of California, San Francisco, USA Genentech, Inc., USA Keywords: Oligodendrocyte; Neural Precursor; Wnt; Cell Fate 2
Oligodendrocyte precursor cells (OPCs) are generated from multiple progenitor domains in the telencephalon in developmental succession from ventral to dorsal. Each domain also generates neurons at earlier developmental stages. Previous studies have demonstrated that Wnt signaling inhibits the differentiation of OPCs into mature oligodendrocytes, but we also wanted to explore the hypothesis that Wnt signaling limits the generation of OPCs from multipotential neural precursors during forebrain development. First, we manipulated both extracellular Wnt ligands and inhibitors and the transcriptional output of the Wnt pathway in neural progenitor cultures and found that Wnt signaling acts directly on progenitor cells to regulate the production of OPCs. Furthermore, we found that endogenous Wnt signaling in these cultures is a major limitation on the efficiency of generating OPCs from multipotent neural precursors. To examine similar events in vivo, we electroporated a soluble Wnt inhibitor or a dominant-negative transcriptional regulator into embryonic mouse neocortical ventricular zone several days before the usual onset of OPC production and showed that decreasing Wnt signaling in this progenitor domain results in enhanced early production of OPCs. Our studies indicate that Wnt signaling is a regulator of the timing and extent of OPC production in the developing mammalian telencephalon. It is hoped that understanding the mechanisms that regulate OPC specification will aid in the development of therapies for oligodendrocyte replacement in demyelinating diseases including perinatal hypoxic-ischemic brain injury, leukodystrophies, and multiple sclerosis. doi:10.1016/j.ijdevneu.2010.07.136 [P2.07] Doublecortin expression in the prefrontal cortex of developing human and in adults with schizophrenia S.J. Fung 1,2,∗ , S. Sivagnanasunduram 1 , M.J. Webster 3 , C. Shannon Weickert 1,2 1
Schizophrenia Research Institute, Australia University of New South Wales, Australia 3 Stanley Medical Research Institute, USA Keywords: Doublecortin; Schizophrenia; Prefrontal cortex 2
Postnatal neurogenesis occurs in the subventricular zone and hippocampus of the primate brain. There is also increasing evidence to suggest that new neurons may be present in other regions of the mature brain, including the prefrontal cortex, which is involved in higher executive functions and cognition and is impaired in schizophrenia. To determine if postnatal migration of new neurons may occur in the prefrontal cortex and to address whether migration of immature neurons might be altered in disease we measured the expression of doublecortin, a marker of immature migrating neurons, in human dorsolateral prefrontal cortex (DLPFC) during postnatal development and in schizophrenia. We used quantitative RT-PCR and western blot analysis to measure doublecortin expression in a developmental cohort with
Poster Abstracts / Int. J. Devl Neuroscience 28 (2010) 655–719
tions presenting with hydrocephalus. The data we have obtained from the limited number of affected babies suggests a similar folate imbalance to that found in the rat model indicating that the human condition may also be amenable to the same combination folate supplements.Keywords: Hydrocephalus; Fetus; Cerebral cortex; Folate Reference: Cains, S., A. Shepherd, et al. (2009). “Addressing a folate imbalance in fetal cerebrospinal fluid can decrease the incidence of congenital hydrocephalus.” J. Neuropathol. Exp. Neurol. 68(4): 404–16. doi:10.1016/j.ijdevneu.2010.07.131 [P2.02] Repeated carbenoxolone injection during late pregnancy decreased SPAR but increased Snk expression in the hippocampus of rat pups N. Jutapakdeegul ∗ , P. achatyanukul
Surakul, R.
Vanichviriyakit, W.
Weer-
Mahidol University, Thailand Homeostasis of circulating cortisol is maintained by two enzymes of 11-hydroxysteroid dehydrogenase, 11-HSD1 and 11-HSD2. 11-HSD2 is NAD-dependent dehydrogenase that inactivates cortisol into cortisone. In addition, 11-HSD2 is abundantly expressed in the placenta where it protects the fetus from active maternal glucocorticoids. Administration of carbenoxolone (CBX), a powerful 11-HSD2 inhibitor, leads to an increased of fetal cortisol level and decreased plasma cortisone levels. Previous data showed that intrauterine environment plays crucial roles in determining hippocampal structure and function in later life. Exposure to high level of corticosteroid during pregnancy lead to a low birth weight offspring, increased risk of aged related memory and cognitive deficits, however, mechanism still unclear. We proposed that repeated CBX injections during late pregnancy may alter scaffolding proteins of NMDA receptor in the hippocampus of rat pups. Here we show that pregnant rats receiving CBX injections (30 mg/kg) during GD14-21 lead to a significant decreased of SPAR (spine Associated Rap Guanylate kinase activating protein) (p < 0.01) but significant increased of Snk (Serum inducible kinase) (p < 0.01) in the pup’s hippocampus at P40 and P60, while the level of PSD-95 remain unchanged. NMDA receptor function has been shown to be influenced by turnover of the scaffolding proteins via ubiquitin proteasome system (UPS). Normally, Snk is induced by neuronal activity and plays an important role in phosphorylate SPAR. The phosphorylated SPAR was then recognized and degraded by ubiquitin-proteasome system, causing the depletion of SPAR and PSD-95 from the spines and leading to spine shrinkage and loss. The results suggest that fetal exposure with excessive corticosteroids may activate Snk/SPAR pathway and lead to the depletion of SPAR. Since, corticosteroid drugs are commonly used in various obstetric and paediatric conditions, it is important to consider the risk–benefit of prenatal GC exposure to prevent the neurodevelopment delay in the offspring.Keywords: Carbenoxolone; SPAR; Snk; Hippocampus doi:10.1016/j.ijdevneu.2010.07.132
[P2.03] Repeated maternal restraint stress down-regulated NMDA receptor subunits and scaffolding proteins in the hippocampus of postnatal rat P. Surakul ∗ , R. Vanichviriyakit, W.A. Weerachatyanukul, N. Jutapakdeegul Mahidol University, Thailand Early life stress has been linked with a substantially increased risk of aged related memory and cognitive deficits. Maternal stress hormone plays a crucial role in determining pup’s hippocampal structure and function, however mechanism is still unclear. We proposed that prenatal stress (PS) may alter developmental expression of NR2A, NR2B subunits of NMDA receptor and its scaffolding proteins in the hippocampus of rat pups. To investigate this hypothesis, pregnant rats were restrained during late gestation (GD14-21) and the expression of NR2A, NR2B subunits, PSD-95 (Postsynaptic density protein-95), SPAR (sSpine associated Rap guanylate kinase activating protein) and Snk (Serum inducible kinase) were measured in the hippocampus of rat pups from P7–P60. First, we found that, PS pups show a significant decreased of NR2A and NR2B subunit at P40 (p < 0.001 and p < 0.05, respectively) and P60 (p < 0.01). Then we found that PS caused a significant increased of PSD-95 in the pup’s hippocampus during P7–P14 (p < 0.05) and P28 (p < 0.01), but decreased significantly at P40 (p < 0.001) and P60 (p < 0.01) as compare to control. The decreased of PSD-95 and NMDAR subunits can be explained by alteration of Snk-SPAR pathway. We also found that PS caused a significant increased of SPAR during P7 and P14 (p < 0.05), but showed a significant decreased during P40 (P < 0.001) and P60 (p < 0.01). The most interested finding is that PS pups show significant increased of Snk (P < 0.001) in the hippocampus throughout their childhood period from P7-P40 as compared to control. These data suggested that early life stress exert long term effects on the development of NMDAR in the pup’s hippocampus. Prenatal stress alters development of scaffolding proteins and NMDAR subunits may underlies the mechanism link between early life stress and increased risk of aged related memory and cognitive deficits and heightened vulnerability to psychological disorders in later life. Keywords: Prenatal stress; NMDA receptor; Scaffolding proteins; Hippocampus doi:10.1016/j.ijdevneu.2010.07.133 [P2.04] Using zebrafish to understand the neurodevelopment role of susceptibility genes for autism spectrum disorder B. Key University of Queensland, Australia Several studies in the last three years have revealed that members of a synaptic cell adhesion network are candidate susceptibility genes for Autism spectrum disorder (ASD). These genes include neurexin-1 (NRXN1), neuroligin-1 (NLGN1), NLGN4, contactin associated protein 2 (CNTNAP2 or previously known as CASPR2) and Shank3. ASD is increasingly attributed to a disorder of brain function rather than brain anatomy. We have begun to address the role of gene-gene interactions within the synaptic cell adhesion pathway involved in neural circuits associated with simple behaviours using the zebrafish animal model. We are focusing on interactions between identified susceptibility genes NLGN-1, NLGN-4, NRXN-1, Shank3 and CNTNAP2 as well as on interactions of these genes with other known synaptic cell adhe-
Poster Abstracts / Int. J. Devl Neuroscience 28 (2010) 655–719
sion pathway genes (LRRTM2, PSD-95 and CASK) in order to begin to understand the function of gene networks underlying the emergence of early behaviours. We have generated morpholinos against NRXN-1a, NRXN1b and CNTNAP2 and injected them individually into one cell embryos and then assessed the touch and escape responses at 30 and 45 h, respectively. Knock down of either NRXN1a, NRXN1b or CNTAP2 significantly reduced the touch response at 30 hpf to a similar extent. The high penetrance of these phenotypes (71–84%) suggest that these genes are playing a major role in the development of the underlying neural circuitry responsible for this behaviour. In contrast, at 45 hpf knock down of NRXN-1a had no effect on the escape response, knock down of NRXN1b either extinguished or reduced the response, while knock down of CNTNAP2 produced an abnormal response. These very different phenotypes suggest very different roles of these synaptic adhesion network genes in the underlying neural circuitry. Our analyses are beginning to reveal the most critical genes involved in development of neural circuits underlying a simple behaviour. Keywords: Zebrafish; Synapse; Behavior; Adhesion doi:10.1016/j.ijdevneu.2010.07.134 [P2.05] The canonical Wnt pathway regulates Tbr2-positive intermediate progenitor maturation in the neocortex R.N. Munji ∗ , Y. Choe, S.J. Pleasure University of California-San Francisco, USA Keywords: Wnt; Cortex; Intermediate progenitors; Periventricular heterotopia In this study, we focus on the role of the canonical Wnt pathway in neocortical neurogenesis. At present, conclusions from previous studies have not converged on a parsimonious model for the role of the canonical Wnt pathway in neocortical neurogenesis, some suggesting a role in promoting neural progenitor differentiation while others in promoting primary progenitor (PP) self-renewal and, in consequence, inhibition of intermediate progenitor (IP) production. We hypothesized that the canonical Wnt pathway might function to regulate both differentiation and selfrenewal, possibly in different progenitor populations and/or at different developmental time-points. To address our hypothesis, we analyzed canonical Wnt function from E13.5, a time-point when horizontal expansion of the neocortical epithelium has slowed while neuronal production continues, and up to P2, when embryonic neurogenesis has primarily subsided, and utilized in utero electroporation (EP) of secreted effectors of the pathway, Wnt3a ligand and Dkk1 inhibitor, to perturb the pathway upstream of intracellular events. Overexpression of Wnt3a at E13.5 horizontally expanded the neocortical epithelium and induced premature differentiation of Tbr2-positive IPs into neurons. These prematurely born neurons predominantly fail to reach the cortical plate and accumulate deep in the cortical epithelium, leading to the formation of periventricular heterotopias. In contrast, overexpression of Dkk1 reduced neuronal production. Our results suggest that the canonical Wnt pathway has a dual role in neocortical neurogenesis, promoting self-renewal of PPs and differentiation of Tbr2-positive IPs. Moreover, these results contribute to our understanding of cortical organization and suggest a connection between the canonical Wnt pathway and periventricular heterotopia disorders. doi:10.1016/j.ijdevneu.2010.07.135
687
[P2.06] Wnts regulate the timing and efficiency of OPC generation in the telencephalon A.J. Langseth 1,∗ , R.N. Munji 1 , Y. 1,2 1 Pozniak , S.J. Pleasure
Choe 1 , T.
Huynh 1 , C.D.
1
University of California, San Francisco, USA Genentech, Inc., USA Keywords: Oligodendrocyte; Neural Precursor; Wnt; Cell Fate 2
Oligodendrocyte precursor cells (OPCs) are generated from multiple progenitor domains in the telencephalon in developmental succession from ventral to dorsal. Each domain also generates neurons at earlier developmental stages. Previous studies have demonstrated that Wnt signaling inhibits the differentiation of OPCs into mature oligodendrocytes, but we also wanted to explore the hypothesis that Wnt signaling limits the generation of OPCs from multipotential neural precursors during forebrain development. First, we manipulated both extracellular Wnt ligands and inhibitors and the transcriptional output of the Wnt pathway in neural progenitor cultures and found that Wnt signaling acts directly on progenitor cells to regulate the production of OPCs. Furthermore, we found that endogenous Wnt signaling in these cultures is a major limitation on the efficiency of generating OPCs from multipotent neural precursors. To examine similar events in vivo, we electroporated a soluble Wnt inhibitor or a dominant-negative transcriptional regulator into embryonic mouse neocortical ventricular zone several days before the usual onset of OPC production and showed that decreasing Wnt signaling in this progenitor domain results in enhanced early production of OPCs. Our studies indicate that Wnt signaling is a regulator of the timing and extent of OPC production in the developing mammalian telencephalon. It is hoped that understanding the mechanisms that regulate OPC specification will aid in the development of therapies for oligodendrocyte replacement in demyelinating diseases including perinatal hypoxic-ischemic brain injury, leukodystrophies, and multiple sclerosis. doi:10.1016/j.ijdevneu.2010.07.136 [P2.07] Doublecortin expression in the prefrontal cortex of developing human and in adults with schizophrenia S.J. Fung 1,2,∗ , S. Sivagnanasunduram 1 , M.J. Webster 3 , C. Shannon Weickert 1,2 1
Schizophrenia Research Institute, Australia University of New South Wales, Australia 3 Stanley Medical Research Institute, USA Keywords: Doublecortin; Schizophrenia; Prefrontal cortex 2
Postnatal neurogenesis occurs in the subventricular zone and hippocampus of the primate brain. There is also increasing evidence to suggest that new neurons may be present in other regions of the mature brain, including the prefrontal cortex, which is involved in higher executive functions and cognition and is impaired in schizophrenia. To determine if postnatal migration of new neurons may occur in the prefrontal cortex and to address whether migration of immature neurons might be altered in disease we measured the expression of doublecortin, a marker of immature migrating neurons, in human dorsolateral prefrontal cortex (DLPFC) during postnatal development and in schizophrenia. We used quantitative RT-PCR and western blot analysis to measure doublecortin expression in a developmental cohort with