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IL1RAPL1 involved in mental retardation and autism regulates synapse formation
Neuroscience Research 68S (2010) e335–e446
Contents lists available at ScienceDirect
Neuroscience Research journal homepage: www.elsevier.com/locate/neures
Abstracts of the 33rd Annual Meeting of the Japan Neuroscience Society (Neuro 2010) Poster
P3-a01 Cbln1 induces structural changes of parallel fibers at defined sites by interactions with glutamate receptor delta 2 Aya Ito-Ishida 1 , Eriko Miura 2 , Keiko Matsuda 2 , Michisuke Yuzaki 2 , Shigeo Okabe 1 1 Department of Cellular Neurobiology, Graduate School of Medicine, University of Tokyo 2 Department of Physiology, School of Medicine, Keio University, Japan
Cbln1, a secretory protein that belongs to the C1q/tumor necrosis factor superfamily, is a unique synaptic organizer that is required for the formation of synapses between Purkinje cells and granule cells. We recently found that glutamate receptor delta 2 (GluD2) on Purkinje cells is the postsynaptic receptor for Cbln1. Cbln1 together with GluD2, strongly induces synapse formation even in adult mice in vivo. However, how such drastic change occurs remains unknown. In order to clarify the mechanisms, we performed morphological analyses by immunohistochemistry and live-imaging of cultured cerebellar granule cells. First, we induced cDNA encoding hemagglutinin (HA) tagged Cbln1 (HA-Cbln1) and analyzed its distribution at 7DIV. Surface staining revealed that HA-Cbln1 is primarily localized on the plasma membrane. Interestingly, surface HA-Cbln1 was found close to the clusters of synaptic vesicles which were visualized by either synaptophysin-GFP (SypGFP) or immunostaining against synapsin. Since the clusters of Cbln1 and synaptic vesicles were found on isolated granule cells in the absence of Purkinje cells, we hypothesized that these clusters may differentiate into mature presynaptic terminals when they contact GluD2 on Purkinje cells’ spines. In order to test this, we visualized granule cells’ axons by DsRed2 and SypGFP, and observed their changes after direct contact with GluD2-expressing HEK293 cells plated onto neurons. Surprisingly, 1–2 hrs after application of HEK293, new filopodia emerged from Syp-GFP clusters at the site of contact. When there was no Syp-GFP cluster below the HEK293 cell, we did not observe filopodia formation. The number of new filopodia was significantly less when granule cells were prepared from cbln1-null mice. The results show that local interaction between Cbln1 and GluD2 at distinct sites on granule cells’ axons induces structural changes, which may contribute to subsequent steps of synapse development. doi:10.1016/j.neures.2010.07.1482
P3-a02 Regulatory mechanism underlying the dynamics of an actin-binding protein, drebrin in dendritic spines Kenji Hanamura , Yosuke Kamata, Hiroyuki Yamazaki, Tomoaki Shirao Department of Neurobiology and Behavior, Gunma University Graduate School of Medicine, Japan Drebrin A is a side-binding protein of F-actin in dendritic spines of adult brain. During development, the conversion from ubiquitous-isoform drebrin E (DE) to neuron-specific-isoform drebrin A (DA) occurs in parallel with synapse formation. Our previous studies have demonstrated that a knockout of this isoform conversion induces impaired spine formation and contextual fear
0168-0102/$ – see front matter
memory. In this study, to examine the mechanisms underlying these impairments, we measured dynamics of GFP-tagged DE (GFP-DE) and GFP-DA in cultured hippocampal neurons by fluorescence recovery after photobleaching (FRAP) analysis, and found that recovery of GFP-DE was faster than that of GFP-DA. Stable fraction and time constant of GFP-DE were significantly smaller or lower than those of GFP-DA, respectively. Then, we examined the turnover of GFP-s-drebrin A (GFP-sDA), which is a minor short form of drebrin A lacking the C-terminal region including Homer-binding and profilin-binding domains. Although GFP-sDA is shorter than GFP-DE, the difference of turnover between GFP-sDA and GFP-DA was subtle. Stable fraction and time constant of GFP-sDA were comparable to those of GFP-DA. This demonstrates that the binding of Homer and profiling with drebrin does not affect drebrin turnover. Although the role of N-terminal region of drebrin in the dynamics of drebrin has not been clarified, our findings indicate that dendritic spine formation are induced by the stabilization of drebrin dynamics, and suggest that drebrin A isoform-specific domain is directly involved in the regulation of drebrin dynamics in the spine. doi:10.1016/j.neures.2010.07.1483
P3-a03 IL1RAPL1 involved in mental retardation and autism regulates synapse formation Tomoyuki Yoshida , Masayoshi Mishina Department of Mol. Neurobiol. & Pharmacol., Grad. Sch. of Med., University of Tokyo, Japan IL1 receptor accessory protein-like 1 (IL1RAPL1), a member of interleukine1/toll receptor family, is involved in X-linked mental retardation and autism. To examine the possible role of IL1RAPL1 in synapse formation, we employed primary cultures of cortical and hippocampal neurons. Immunohistochemical staining signals for IL1RAPL1 were detected within dendrites of cultured cortical neurons and partially colocalized with those for a postsynaptic marker protein, Shank1. Expression of IL1RAPL1 in cultured neurons stimulated synapse formation as indicated by the increase in dendritic protrusions and accumulation of active zone marker protein, Bassoon, along the dendrites while the expression of its paralog, IL1RAPL2, exerted little effect on the number of dendritic protrusions and accumulation of Bassoon. Consistently, siRNA-mediated knockdown of endogenous IL1RAPL1 decreased dendritic protrusions and accumulation of Bassoon. Swapping the extracellular domain of IL1RAPL1 with that of IL-1 receptor type I (IL-1RI) abolished the stimulatory effect on both number of dendritic protrusions and accumulation of Bassoon. On the other hand, swapping the cytoplasmic domain with that of IL-1RI still had stimulatory effect on accumulation of Bassoon but abolished that on the number of dendritic protrusions. These results suggest that IL1RAPL1 regulates synapse formation by inducing presynaptic differentiation through its extracellular domain. Moreover, IL1RAPL1 controls formation of dendritic protrusions through both extracellular and cytoplasmic domains. doi:10.1016/j.neures.2010.07.1484
Contents lists available at ScienceDirect
Neuroscience Research journal homepage: www.elsevier.com/locate/neures
Abstracts of the 33rd Annual Meeting of the Japan Neuroscience Society (Neuro 2010) Poster
P3-a01 Cbln1 induces structural changes of parallel fibers at defined sites by interactions with glutamate receptor delta 2 Aya Ito-Ishida 1 , Eriko Miura 2 , Keiko Matsuda 2 , Michisuke Yuzaki 2 , Shigeo Okabe 1 1 Department of Cellular Neurobiology, Graduate School of Medicine, University of Tokyo 2 Department of Physiology, School of Medicine, Keio University, Japan
Cbln1, a secretory protein that belongs to the C1q/tumor necrosis factor superfamily, is a unique synaptic organizer that is required for the formation of synapses between Purkinje cells and granule cells. We recently found that glutamate receptor delta 2 (GluD2) on Purkinje cells is the postsynaptic receptor for Cbln1. Cbln1 together with GluD2, strongly induces synapse formation even in adult mice in vivo. However, how such drastic change occurs remains unknown. In order to clarify the mechanisms, we performed morphological analyses by immunohistochemistry and live-imaging of cultured cerebellar granule cells. First, we induced cDNA encoding hemagglutinin (HA) tagged Cbln1 (HA-Cbln1) and analyzed its distribution at 7DIV. Surface staining revealed that HA-Cbln1 is primarily localized on the plasma membrane. Interestingly, surface HA-Cbln1 was found close to the clusters of synaptic vesicles which were visualized by either synaptophysin-GFP (SypGFP) or immunostaining against synapsin. Since the clusters of Cbln1 and synaptic vesicles were found on isolated granule cells in the absence of Purkinje cells, we hypothesized that these clusters may differentiate into mature presynaptic terminals when they contact GluD2 on Purkinje cells’ spines. In order to test this, we visualized granule cells’ axons by DsRed2 and SypGFP, and observed their changes after direct contact with GluD2-expressing HEK293 cells plated onto neurons. Surprisingly, 1–2 hrs after application of HEK293, new filopodia emerged from Syp-GFP clusters at the site of contact. When there was no Syp-GFP cluster below the HEK293 cell, we did not observe filopodia formation. The number of new filopodia was significantly less when granule cells were prepared from cbln1-null mice. The results show that local interaction between Cbln1 and GluD2 at distinct sites on granule cells’ axons induces structural changes, which may contribute to subsequent steps of synapse development. doi:10.1016/j.neures.2010.07.1482
P3-a02 Regulatory mechanism underlying the dynamics of an actin-binding protein, drebrin in dendritic spines Kenji Hanamura , Yosuke Kamata, Hiroyuki Yamazaki, Tomoaki Shirao Department of Neurobiology and Behavior, Gunma University Graduate School of Medicine, Japan Drebrin A is a side-binding protein of F-actin in dendritic spines of adult brain. During development, the conversion from ubiquitous-isoform drebrin E (DE) to neuron-specific-isoform drebrin A (DA) occurs in parallel with synapse formation. Our previous studies have demonstrated that a knockout of this isoform conversion induces impaired spine formation and contextual fear
0168-0102/$ – see front matter
memory. In this study, to examine the mechanisms underlying these impairments, we measured dynamics of GFP-tagged DE (GFP-DE) and GFP-DA in cultured hippocampal neurons by fluorescence recovery after photobleaching (FRAP) analysis, and found that recovery of GFP-DE was faster than that of GFP-DA. Stable fraction and time constant of GFP-DE were significantly smaller or lower than those of GFP-DA, respectively. Then, we examined the turnover of GFP-s-drebrin A (GFP-sDA), which is a minor short form of drebrin A lacking the C-terminal region including Homer-binding and profilin-binding domains. Although GFP-sDA is shorter than GFP-DE, the difference of turnover between GFP-sDA and GFP-DA was subtle. Stable fraction and time constant of GFP-sDA were comparable to those of GFP-DA. This demonstrates that the binding of Homer and profiling with drebrin does not affect drebrin turnover. Although the role of N-terminal region of drebrin in the dynamics of drebrin has not been clarified, our findings indicate that dendritic spine formation are induced by the stabilization of drebrin dynamics, and suggest that drebrin A isoform-specific domain is directly involved in the regulation of drebrin dynamics in the spine. doi:10.1016/j.neures.2010.07.1483
P3-a03 IL1RAPL1 involved in mental retardation and autism regulates synapse formation Tomoyuki Yoshida , Masayoshi Mishina Department of Mol. Neurobiol. & Pharmacol., Grad. Sch. of Med., University of Tokyo, Japan IL1 receptor accessory protein-like 1 (IL1RAPL1), a member of interleukine1/toll receptor family, is involved in X-linked mental retardation and autism. To examine the possible role of IL1RAPL1 in synapse formation, we employed primary cultures of cortical and hippocampal neurons. Immunohistochemical staining signals for IL1RAPL1 were detected within dendrites of cultured cortical neurons and partially colocalized with those for a postsynaptic marker protein, Shank1. Expression of IL1RAPL1 in cultured neurons stimulated synapse formation as indicated by the increase in dendritic protrusions and accumulation of active zone marker protein, Bassoon, along the dendrites while the expression of its paralog, IL1RAPL2, exerted little effect on the number of dendritic protrusions and accumulation of Bassoon. Consistently, siRNA-mediated knockdown of endogenous IL1RAPL1 decreased dendritic protrusions and accumulation of Bassoon. Swapping the extracellular domain of IL1RAPL1 with that of IL-1 receptor type I (IL-1RI) abolished the stimulatory effect on both number of dendritic protrusions and accumulation of Bassoon. On the other hand, swapping the cytoplasmic domain with that of IL-1RI still had stimulatory effect on accumulation of Bassoon but abolished that on the number of dendritic protrusions. These results suggest that IL1RAPL1 regulates synapse formation by inducing presynaptic differentiation through its extracellular domain. Moreover, IL1RAPL1 controls formation of dendritic protrusions through both extracellular and cytoplasmic domains. doi:10.1016/j.neures.2010.07.1484