- Email: [email protected]
Regulation of synaptic growth signaling at the Drosophila neuromuscular junction
Symposium Abstracts / Int. J. Devl Neuroscience 28 (2010) 643–653
and MKS3/TMEM67, encoding the proteins MKS1 and meckelin, a novel receptor. Remarkably, MKS is allelic and overlaps in phenotype with the neurodevelopmental disorder Joubert syndrome (JS), with some of the causative genes implicated in regulation of the Hedgehog signalling pathway. However, our recent work has suggested a role for meckelin and some other MKS proteins in modulating non-canonical Wnt signalling and remodelling the actin cytoskeleton. Meckelin is localized at the apical cell surface, basal bodies and ciliary axoneme of ciliated cell lines and tissues, but also interacts with other MKS proteins and the actin-binding proteins nesprin-2 and filamin A. Loss of expression of MKS genes following RNAi-mediated knockdown or in MKS patient fibroblasts: (1) prevents the movement of the basal body to the apical cell surface prior to ciliogenesis; (2) causes hyperactivation of the small GTPase RhoA and Dishevelled, both implicated in the control of apical docking of basal bodies and planar polarization of epithelial cells; and (3) remodels the actin cytoskeleton. These findings are reiterated in the Mks3/Tmem67 knock-out mouse model of MKS/JS. In contrast, MKS1 is implicated in constraining canonical Wnt signalling. These findings therefore underline the critical role of MKS proteins in ciliogenesis and regulation of Wnt signalling, through interactions with apical cell surface proteins associated with the actin cytoskeleton and implicated in basal body docking. doi:10.1016/j.ijdevneu.2010.07.015 [S2.4] Cilia and mammalian hedgehog signaling C.Y. Su, C.E. Larkins, M.J. Hillman, T. Caspary ∗ Emory University School of Medicine, USA Keywords: Sonic Hedgehog; Neural tube patterning; Cilia; Cilia membrane We identified the ciliary protein Arl13b as a novel GTPase of the ARF family through our work with the ENU-induced mouse mutant, hennin (hnn). Arl13b is a 48 kD protein composed of a 20 kD ARF domain and an additional 28 kD C-terminus with no identifiable motifs. Arl13bhnn embryos have short cilia with a structural defect in the microtubule outer doublet. The precise role of Arl13b in cilia is unclear but most of the 30 ARF family proteins have been linked to processes such as vesicle trafficking and microtubule stability. We showed through immunofluorescence that Arl13b is membrane associated. Consistent with this, we used fluorescence recovery after photobleaching (FRAP) and found that the movement of Arl13b within the cilium is comparable to that of a known cilia membrane protein, SSTR3. These experiments suggest that Arl13b is regulating cilia structure from the cilia membrane, perhaps through interactions with other cilia proteins. Cilia are required for Sonic Hedgehog (Shh) signaling and we previously demonstrated that Arl13bhnn mutants display uniform Shh activity in the neural tube where there is normally a gradient of activity. Here we use mosaic analysis with a floxed Arl13b allele to examine the temporal requirement of maintaining the Shh gradient in neural tube patterning. We define the critical period during which cells respond to changes in Shh activity levels. Amazingly, clones of cells that change their fate upon the loss of Arl13b correct to a wild type pattern over time. However, clones of cells that lack Shh activity do not correct indicating a requirement for the maintenance of Shh activity, but not maintenance of the Shh gradient, in neural tube patterning. doi:10.1016/j.ijdevneu.2010.07.016
645
[S3.1] Imaging synapse remodeling and interactions with glia S. Okabe University of Tokyo, Japan Dendritic spines and the postsynaptic densities (PSDs) are two major structural features of the CNS glutamatergic synapses and their coordinated formation should be important in proper formation of the neuron network. Time sequences of synapse formation and molecular assembly have been well described in dissociated culture of neurons. In this system, accumulation of synaptic vesicles, postsynaptic scaffolding proteins, and protrusion of dendritic filopodia/spines take place within 1–2 h, indicating that formation of dendritic protrusions and their rapid maturation into stable spine structure are essential events in postsynaptic differentiation. To monitor synapse development in more native environment, we utilized both hippocampal slice culture preparations and in vivo imaging to visualize dynamics of dendritic protrusions, accumulation of PSD molecules, and interaction of glial components to dendrites. In slice preparation, we performed time-lapse imaging of both dendritic protrusions and glial components and reported important roles of astrocytes in spine maturation. Visualization of dendritic protrusions and PSDs in the developing mouse neocortex in vivo by two-photon microscopy revealed tight coordination between dendritic protrusive activity and PSD assembly. I will present these imaging data and discuss the sequences of molecular assembly during synapse development. doi:10.1016/j.ijdevneu.2010.07.017 [S3.2] Regulation of synaptic growth signaling at the Drosophila neuromuscular junction J.T. Littleton Massachusetts Institute of Technology, USA The computational power of the brain depends on synaptic connections that link together billions of neurons. The focus of my laboratory’s work is to use the Drosophila model to understand the mechanisms by which neurons form synaptic connections, how synapses transmit information, and how synapses undergo plastic change. Axonal sprouting and synaptic rewiring are key regulators of neuronal plasticity in the developing and adult brain. Similar to many species, modulation of synapse formation in Drosophila has been implicated in learning and memory. The Drosophila larval neuromuscular junction (NMJ) serves as a useful model for synaptic growth, as the muscle surface area expands ∼100-fold over 4 days of larval development, requiring increased input from its innervating motor neuron to drive contraction. The regulation of synapse formation requires coordinated signaling to orchestrate pre- and postsynaptic maturation of synaptic connections. In contrast to synaptic vesicle fusion, the molecular mechanisms that allow postsynaptic targets to transmit retrograde signals are relatively unknown. To define the mechanisms and biological significance of retrograde signaling at synapses, we have performed a genetic dissection of a retrograde signaling pathway that promotes enhanced presynaptic release and synapse-specific growth at Drosophila NMJs. Our studies indicate that a postsynaptic Synaptotagmin isoform (Syt 4) functions as a Ca2+ sensor to control postsynaptic vesicle fusion downstream of Ca2+ influx through glutamate receptors, initiating an acute change in synaptic function that is converted to synapse-specific growth. We have also characterized presynaptic pathways that regulate the activation and
646
Symposium Abstracts / Int. J. Devl Neuroscience 28 (2010) 643–653
trafficking of presynaptic growth receptors that transmit synaptic growth signals. We find synaptic growth signaling output, including through the BMP and Wingless pathways, requires transit through a presynaptic endosomal system controlled by specific F-BAR and PX domain trafficking proteins. Our results suggest a mechanism by which protein traffic between endosomal intermediates at synapses controls the output of signal transduction pathways leading to synaptic growth. This pathway provides an important link between synaptic plasticity and activity-dependent rewiring of neuronal connections. doi:10.1016/j.ijdevneu.2010.07.018 [S3.3] Regulating neurotransmitter release across the synaptic cleft N. Vitureira, Y. Goda ∗ University College London, UK Keywords: Release probability; Central synapse; N-cadherin; Hippocampal cultures The structure and function of synapses are highly heterogeneous, even among synapses formed on single neurons. Several lines of evidence suggest that presynaptic functionality is regulated retrogradely via mechanisms involving postsynaptic components. One potential class of candidate proteins mediating such trans-synaptic modulation are cell adhesion molecules that can coordinate signaling events across the synaptic cleft. In particular, N-cadherin/-catenin complex is a Ca2+ -dependent homophilic adhesion protein with established roles in controlling synapse formation and dendritic spine morphology. We have tested whether the postsynaptic N-cadherin/-catenin complex modulates presynaptic release. Using FM dyes to monitor synaptic vesicle turnover in cultured hippocampal neurons, impairing the adhesion of N-cadherin by postsynaptic overexpression of a dominant negative form of this protein reduces the release probability, the ready-releasable pool and the recycling pool of synaptic vesicles. However, preliminary studies suggest that chronic activity manipulations do no affect the ability of these neurons to undergo adaptive scaling of presynaptic release probability. Our results indicate that the postsynaptic N-cadherin/-catenin complex is an important modulator of presynaptic release probability and plays a basic role in linking presynaptic and postsynaptic functions. doi:10.1016/j.ijdevneu.2010.07.019 [S3.4] microRNAs at the synapse K.S. Kosik University of California, USA A crucial facet of synaptic function is the ability to synthesize and degrade proteins locally. Both of these opposing limbs are necessary for changes in synaptic thresholds related to longterm potentiation. Many mRNAs reside within dendrites and one prominent control system over their translation is microRNAs. By post-transcriptionally regulating gene expression, these ∼21 nucleotide non-coding transcripts, which also reside in dendrites, establish the specificity parameters over a network of functionally related mRNAs. While individual miRNAs confer specificity, the complexes that house the miRNAs, called the RISC, are more generic and determine whether miRNA–mRNA duplexes will undergo translational silencing. An important component of the
RISC is the protein MOV10. MOV10 is present at synapses and is rapidly degraded by the proteasome in an NMDA-receptormediated activity-dependent manner. Its degradation depends upon its recognition by ubiquitin pointing to a role for an as yet undetermined E3 ligase. We designed a translational trap to capture those mRNAs whose spatiotemporal translation is regulated by MOV10. When MOV10 was suppressed by an siRNA, a set of mRNAs—including alpha-CaMKII, Limk1, and the depalmitoylating enzyme lysophospholipase1 (Lypla1)—selectively entered the polysome compartment. We focused upon Lypla1 mRNA (also known as APT1), which is targeted by the brain-enriched microRNA miR-138. This interaction is known to exert control over spine morphogenesis. Using a photoconvertible translation reporter, Kaede, we analyzed the activity-dependent protein synthesis driven by Lypla1 and alpha-CaMKII 3 UTRs. We established this protein synthesis to be MOV10 and proteasome dependent. Thus, synaptic activity triggers the degradation of MOV10 which in turn derepresses the RISC and permits the synthesis of protein. doi:10.1016/j.ijdevneu.2010.07.020 [S4.1] Genetics and genomics of bipolar disorder P. Sklar Harvard Medical School, USA Bipolar disorder (BD) is a severe mood disorder affecting greater than 1% of the population. Classical bipolar disorder is characterized by recurrent manic episodes that often alternate with depression. With onset in late adolescence or early adulthood BD results in a chronic illness with moderate to severe impairment. Numerous epidemiological studies have identified a strong heritable component with family, twin and adoption studies consistently finding markedly elevated relative risks in first-degree relatives and monozygotic co-twins. Over the last 5 years, genetic studies have sought to look at single nucleotide polymorphisms (SNPs) across the genome simultaneously for evidence of association using high-throughput genomic technologies. The current high-density genotyping products are best tailored to the study of common genetic variation and rare structural variation. For BD they have successfully identified specific loci – genes, and in some cases individual variants – strongly and consistently associated with BD. For example, these studies have discovered that genetic variants in ankyrin 3 (ANK3), which was not at all a prior candidate, increase BD risk. Surprisingly, the literature already supports a biological connection since ANK3 is critical for the proper insertion of sodium channels, a known target of the mood stabilizing agent lamotrigine, into the axon initial segment. A critical need for psychiatric genetics continues to be the identification of consistently observed association of particular loci with BD. With what is proving to be a highly complex genetic landscape, existing approaches that model genetic changes in multiple, and complex ways will be needed. Our traditional, somewhat simplistic views of molecular etiology need revision. In the future, research must bring to bare new technologies to develop an understanding of human genetic risk variants on brain function. Powerful tools such as single neuron imaging to probe synapse structure and function, model systems such as Drosophila, Caenorhabditis Elegans and Zebrafish, and investigations of neural networks computationally and biologically already exist to probe neural circuitry. The fast evolving field of stem cell technology – in particular the use of induced pleuripotent stem (iPS) cells created from individual
and MKS3/TMEM67, encoding the proteins MKS1 and meckelin, a novel receptor. Remarkably, MKS is allelic and overlaps in phenotype with the neurodevelopmental disorder Joubert syndrome (JS), with some of the causative genes implicated in regulation of the Hedgehog signalling pathway. However, our recent work has suggested a role for meckelin and some other MKS proteins in modulating non-canonical Wnt signalling and remodelling the actin cytoskeleton. Meckelin is localized at the apical cell surface, basal bodies and ciliary axoneme of ciliated cell lines and tissues, but also interacts with other MKS proteins and the actin-binding proteins nesprin-2 and filamin A. Loss of expression of MKS genes following RNAi-mediated knockdown or in MKS patient fibroblasts: (1) prevents the movement of the basal body to the apical cell surface prior to ciliogenesis; (2) causes hyperactivation of the small GTPase RhoA and Dishevelled, both implicated in the control of apical docking of basal bodies and planar polarization of epithelial cells; and (3) remodels the actin cytoskeleton. These findings are reiterated in the Mks3/Tmem67 knock-out mouse model of MKS/JS. In contrast, MKS1 is implicated in constraining canonical Wnt signalling. These findings therefore underline the critical role of MKS proteins in ciliogenesis and regulation of Wnt signalling, through interactions with apical cell surface proteins associated with the actin cytoskeleton and implicated in basal body docking. doi:10.1016/j.ijdevneu.2010.07.015 [S2.4] Cilia and mammalian hedgehog signaling C.Y. Su, C.E. Larkins, M.J. Hillman, T. Caspary ∗ Emory University School of Medicine, USA Keywords: Sonic Hedgehog; Neural tube patterning; Cilia; Cilia membrane We identified the ciliary protein Arl13b as a novel GTPase of the ARF family through our work with the ENU-induced mouse mutant, hennin (hnn). Arl13b is a 48 kD protein composed of a 20 kD ARF domain and an additional 28 kD C-terminus with no identifiable motifs. Arl13bhnn embryos have short cilia with a structural defect in the microtubule outer doublet. The precise role of Arl13b in cilia is unclear but most of the 30 ARF family proteins have been linked to processes such as vesicle trafficking and microtubule stability. We showed through immunofluorescence that Arl13b is membrane associated. Consistent with this, we used fluorescence recovery after photobleaching (FRAP) and found that the movement of Arl13b within the cilium is comparable to that of a known cilia membrane protein, SSTR3. These experiments suggest that Arl13b is regulating cilia structure from the cilia membrane, perhaps through interactions with other cilia proteins. Cilia are required for Sonic Hedgehog (Shh) signaling and we previously demonstrated that Arl13bhnn mutants display uniform Shh activity in the neural tube where there is normally a gradient of activity. Here we use mosaic analysis with a floxed Arl13b allele to examine the temporal requirement of maintaining the Shh gradient in neural tube patterning. We define the critical period during which cells respond to changes in Shh activity levels. Amazingly, clones of cells that change their fate upon the loss of Arl13b correct to a wild type pattern over time. However, clones of cells that lack Shh activity do not correct indicating a requirement for the maintenance of Shh activity, but not maintenance of the Shh gradient, in neural tube patterning. doi:10.1016/j.ijdevneu.2010.07.016
645
[S3.1] Imaging synapse remodeling and interactions with glia S. Okabe University of Tokyo, Japan Dendritic spines and the postsynaptic densities (PSDs) are two major structural features of the CNS glutamatergic synapses and their coordinated formation should be important in proper formation of the neuron network. Time sequences of synapse formation and molecular assembly have been well described in dissociated culture of neurons. In this system, accumulation of synaptic vesicles, postsynaptic scaffolding proteins, and protrusion of dendritic filopodia/spines take place within 1–2 h, indicating that formation of dendritic protrusions and their rapid maturation into stable spine structure are essential events in postsynaptic differentiation. To monitor synapse development in more native environment, we utilized both hippocampal slice culture preparations and in vivo imaging to visualize dynamics of dendritic protrusions, accumulation of PSD molecules, and interaction of glial components to dendrites. In slice preparation, we performed time-lapse imaging of both dendritic protrusions and glial components and reported important roles of astrocytes in spine maturation. Visualization of dendritic protrusions and PSDs in the developing mouse neocortex in vivo by two-photon microscopy revealed tight coordination between dendritic protrusive activity and PSD assembly. I will present these imaging data and discuss the sequences of molecular assembly during synapse development. doi:10.1016/j.ijdevneu.2010.07.017 [S3.2] Regulation of synaptic growth signaling at the Drosophila neuromuscular junction J.T. Littleton Massachusetts Institute of Technology, USA The computational power of the brain depends on synaptic connections that link together billions of neurons. The focus of my laboratory’s work is to use the Drosophila model to understand the mechanisms by which neurons form synaptic connections, how synapses transmit information, and how synapses undergo plastic change. Axonal sprouting and synaptic rewiring are key regulators of neuronal plasticity in the developing and adult brain. Similar to many species, modulation of synapse formation in Drosophila has been implicated in learning and memory. The Drosophila larval neuromuscular junction (NMJ) serves as a useful model for synaptic growth, as the muscle surface area expands ∼100-fold over 4 days of larval development, requiring increased input from its innervating motor neuron to drive contraction. The regulation of synapse formation requires coordinated signaling to orchestrate pre- and postsynaptic maturation of synaptic connections. In contrast to synaptic vesicle fusion, the molecular mechanisms that allow postsynaptic targets to transmit retrograde signals are relatively unknown. To define the mechanisms and biological significance of retrograde signaling at synapses, we have performed a genetic dissection of a retrograde signaling pathway that promotes enhanced presynaptic release and synapse-specific growth at Drosophila NMJs. Our studies indicate that a postsynaptic Synaptotagmin isoform (Syt 4) functions as a Ca2+ sensor to control postsynaptic vesicle fusion downstream of Ca2+ influx through glutamate receptors, initiating an acute change in synaptic function that is converted to synapse-specific growth. We have also characterized presynaptic pathways that regulate the activation and
646
Symposium Abstracts / Int. J. Devl Neuroscience 28 (2010) 643–653
trafficking of presynaptic growth receptors that transmit synaptic growth signals. We find synaptic growth signaling output, including through the BMP and Wingless pathways, requires transit through a presynaptic endosomal system controlled by specific F-BAR and PX domain trafficking proteins. Our results suggest a mechanism by which protein traffic between endosomal intermediates at synapses controls the output of signal transduction pathways leading to synaptic growth. This pathway provides an important link between synaptic plasticity and activity-dependent rewiring of neuronal connections. doi:10.1016/j.ijdevneu.2010.07.018 [S3.3] Regulating neurotransmitter release across the synaptic cleft N. Vitureira, Y. Goda ∗ University College London, UK Keywords: Release probability; Central synapse; N-cadherin; Hippocampal cultures The structure and function of synapses are highly heterogeneous, even among synapses formed on single neurons. Several lines of evidence suggest that presynaptic functionality is regulated retrogradely via mechanisms involving postsynaptic components. One potential class of candidate proteins mediating such trans-synaptic modulation are cell adhesion molecules that can coordinate signaling events across the synaptic cleft. In particular, N-cadherin/-catenin complex is a Ca2+ -dependent homophilic adhesion protein with established roles in controlling synapse formation and dendritic spine morphology. We have tested whether the postsynaptic N-cadherin/-catenin complex modulates presynaptic release. Using FM dyes to monitor synaptic vesicle turnover in cultured hippocampal neurons, impairing the adhesion of N-cadherin by postsynaptic overexpression of a dominant negative form of this protein reduces the release probability, the ready-releasable pool and the recycling pool of synaptic vesicles. However, preliminary studies suggest that chronic activity manipulations do no affect the ability of these neurons to undergo adaptive scaling of presynaptic release probability. Our results indicate that the postsynaptic N-cadherin/-catenin complex is an important modulator of presynaptic release probability and plays a basic role in linking presynaptic and postsynaptic functions. doi:10.1016/j.ijdevneu.2010.07.019 [S3.4] microRNAs at the synapse K.S. Kosik University of California, USA A crucial facet of synaptic function is the ability to synthesize and degrade proteins locally. Both of these opposing limbs are necessary for changes in synaptic thresholds related to longterm potentiation. Many mRNAs reside within dendrites and one prominent control system over their translation is microRNAs. By post-transcriptionally regulating gene expression, these ∼21 nucleotide non-coding transcripts, which also reside in dendrites, establish the specificity parameters over a network of functionally related mRNAs. While individual miRNAs confer specificity, the complexes that house the miRNAs, called the RISC, are more generic and determine whether miRNA–mRNA duplexes will undergo translational silencing. An important component of the
RISC is the protein MOV10. MOV10 is present at synapses and is rapidly degraded by the proteasome in an NMDA-receptormediated activity-dependent manner. Its degradation depends upon its recognition by ubiquitin pointing to a role for an as yet undetermined E3 ligase. We designed a translational trap to capture those mRNAs whose spatiotemporal translation is regulated by MOV10. When MOV10 was suppressed by an siRNA, a set of mRNAs—including alpha-CaMKII, Limk1, and the depalmitoylating enzyme lysophospholipase1 (Lypla1)—selectively entered the polysome compartment. We focused upon Lypla1 mRNA (also known as APT1), which is targeted by the brain-enriched microRNA miR-138. This interaction is known to exert control over spine morphogenesis. Using a photoconvertible translation reporter, Kaede, we analyzed the activity-dependent protein synthesis driven by Lypla1 and alpha-CaMKII 3 UTRs. We established this protein synthesis to be MOV10 and proteasome dependent. Thus, synaptic activity triggers the degradation of MOV10 which in turn derepresses the RISC and permits the synthesis of protein. doi:10.1016/j.ijdevneu.2010.07.020 [S4.1] Genetics and genomics of bipolar disorder P. Sklar Harvard Medical School, USA Bipolar disorder (BD) is a severe mood disorder affecting greater than 1% of the population. Classical bipolar disorder is characterized by recurrent manic episodes that often alternate with depression. With onset in late adolescence or early adulthood BD results in a chronic illness with moderate to severe impairment. Numerous epidemiological studies have identified a strong heritable component with family, twin and adoption studies consistently finding markedly elevated relative risks in first-degree relatives and monozygotic co-twins. Over the last 5 years, genetic studies have sought to look at single nucleotide polymorphisms (SNPs) across the genome simultaneously for evidence of association using high-throughput genomic technologies. The current high-density genotyping products are best tailored to the study of common genetic variation and rare structural variation. For BD they have successfully identified specific loci – genes, and in some cases individual variants – strongly and consistently associated with BD. For example, these studies have discovered that genetic variants in ankyrin 3 (ANK3), which was not at all a prior candidate, increase BD risk. Surprisingly, the literature already supports a biological connection since ANK3 is critical for the proper insertion of sodium channels, a known target of the mood stabilizing agent lamotrigine, into the axon initial segment. A critical need for psychiatric genetics continues to be the identification of consistently observed association of particular loci with BD. With what is proving to be a highly complex genetic landscape, existing approaches that model genetic changes in multiple, and complex ways will be needed. Our traditional, somewhat simplistic views of molecular etiology need revision. In the future, research must bring to bare new technologies to develop an understanding of human genetic risk variants on brain function. Powerful tools such as single neuron imaging to probe synapse structure and function, model systems such as Drosophila, Caenorhabditis Elegans and Zebrafish, and investigations of neural networks computationally and biologically already exist to probe neural circuitry. The fast evolving field of stem cell technology – in particular the use of induced pleuripotent stem (iPS) cells created from individual