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Tomoaki Shirao - One of the best experts on this subject based on the ideXlab platform.
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High-content imaging analysis for detecting the loss of Drebrin clusters along dendrites in cultured hippocampal neurons.
Journal of pharmacological and toxicological methods, 2019Co-Authors: Kenji Hanamura, Noriko Koganezawa, Yuko Sekino, Kazumi Kamiyama, Natsume Tanaka, Takero Oka, Mai Yamamura, Tomoaki ShiraoAbstract:Abstract Introduction Detection of drug effects on neuronal synapses is important for predicting their adverse effects. We have used Drebrin as a marker to detect the synaptic changes in cultured neurons. High concentration of glutamate decreases the amount of Drebrin in synapses. To increase the availability of this method for high throughput analysis, we applied the Drebrin-based evaluation of synapses to high-content imaging analysis using microplates. Methods Three weeks old cultured neurons were fixed and processed for immunocytochemistry to visualize Drebrin clusters, dendrites and neuronal cell bodies. After automated image acquisition, total number of Drebrin clusters per fields, linear density of Drebrin cluster along dendrites, dendrite length and neuron number were automatically measured by a custom-designed protocol. Results Automated image acquisition and analysis showed that dendrite length and Drebrin cluster density along dendrites are measured consistently and reproducibly. In addition, application of 10–100 μM glutamate for 10 min or 0.5–50 μM latrunculin A for 5 min significantly decreased Drebrin cluster density without affecting neuron number. These results were consistent with our previous results using manual image acquisition and analysis with regular fluorescence microscope and image analysis software. Furthermore, 0.3 or 1.0 μM staurosporine for 24 h significantly decreased neuron number. Discussion The present study demonstrates that this high-throughput imaging analysis of Drebrin cluster density along dendrites for detecting the effects of substances on synapses is sensitive enough to detect the effects of glutamate receptor activation and latrunculin A treatment, and indicates that this analysis will be useful for safety pharmacology study.
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Drebrin Isoforms Critically Regulate NMDAR- and mGluR-Dependent LTD Induction.
Frontiers in cellular neuroscience, 2018Co-Authors: Hiroki Yasuda, Nobuhiko Kojima, Kenji Sakimura, Hiroyuki Yamazaki, Kenji Hanamura, Tomoaki ShiraoAbstract:Drebrin is an actin-binding protein that is preferentially expressed in the brain. It is highly localized in dendritic spines and regulates spine shapes. The embryonic-type (Drebrin E) is expressed in the embryonic and early postnatal brain and is replaced by the adult-type (Drebrin A) during development. In parallel, NMDA receptor (NMDAR)-dependent long-term depression (LTD) of synaptic transmission, induced by low-frequency stimulation (LFS), is dominant in the immature brain and decreases during development. Here, we report that Drebrin regulates NMDAR-dependent and group 1 metabotropic glutamate receptor (mGluR)-dependent LTD induction in the hippocampus. While LFS induced NMDAR-dependent LTD in the developing hippocampus in wild-type (WT) mice, it did not induce LTD in developing Drebrin E and A double knockout (DXKO) mice, indicating that Drebrin is required for NMDAR-dependent LTD. On the other hand, LFS induced robust LTD dependent on mGluR5, one of group 1 mGluRs, in both developing and adult brains of Drebrin A knockout (DAKO) mice, in which Drebrin E is expressed throughout development and adulthood. Agonist-induced mGluR-dependent LTD was normal in WT and DXKO mice; however, it was enhanced in DAKO mice. Also, mGluR1, another group 1 mGluR, was involved in agonist-induced mGluR-dependent LTD in DAKO mice. These data suggest that abnormal Drebrin E expression in adults promotes group 1 mGluR-dependent LTD induction. Therefore, while Drebrin expression is critical for NMDAR-dependent LTD induction, developmental conversion from Drebrin E to Drebrin A prevents robust group 1 mGluR-dependent LTD.
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CaMKIIβ is localized in dendritic spines as both Drebrin-dependent and Drebrin-independent pools.
Journal of neurochemistry, 2018Co-Authors: Hiroyuki Yamazaki, Yoshio Sasagawa, Hideyuki Yamamoto, Haruhiko Bito, Tomoaki ShiraoAbstract:Drebrin is a major F-actin binding protein in dendritic spines that is critically involved in the regulation of dendritic spine morphogenesis, pathology, and plasticity. In this study, we aimed to identify a novel Drebrin-binding protein involved in spine morphogenesis and synaptic plasticity. We confirmed the beta subunit of Ca2+ /calmodulin-dependent protein kinase II (CaMKIIβ) as a Drebrin-binding protein using a yeast two-hybrid system, and investigated the Drebrin-CaMKIIβ relationship in dendritic spines using rat hippocampal neurons. Drebrin knockdown resulted in diffuse localization of CaMKIIβ in dendrites during the resting state, suggesting that Drebrin is involved in the accumulation of CaMKIIβ in dendritic spines. Fluorescence recovery after photobleaching analysis showed that Drebrin knockdown increased the stable fraction of CaMKIIβ, indicating the presence of Drebrin-independent, more stable CaMKIIβ. NMDA receptor activation also increased the stable fraction in parallel with Drebrin exodus from dendritic spines. These findings suggest that CaMKIIβ can be classified into distinct pools: CaMKIIβ associated with Drebrin, CaMKIIβ associated with post-synaptic density (PSD), and CaMKIIβ free from PSD and Drebrin. CaMKIIβ appears to be anchored to a protein complex composed of Drebrin-binding F-actin during the resting state. NMDA receptor activation releases CaMKIIβ from Drebrin resulting in CaMKIIβ association with PSD.
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Isoform-dependent Regulation of Drebrin Dynamics in Dendritic Spines
Neuroscience, 2018Co-Authors: Kenji Hanamura, Nobuhiko Kojima, Hiroyuki Yamazaki, Yousuke Kamata, Tomoaki ShiraoAbstract:Dendritic spines have stable filamentous actin (F-actin) and dynamic F-actin. The formation of stable F-actin plays a pivotal role in spine formation. Drebrin binds to and stabilizes F-actin in dendritic spines. Interestingly, the conversion of the Drebrin E isoform to Drebrin A occurs in parallel with synapse formation, suggesting that this conversion promotes synapse formation via F-actin accumulation. In this study, we measured the dynamics of GFP-tagged Drebrin E (GFP-DE) and Drebrin A (GFP-DA) in cultured hippocampal neurons by fluorescence recovery after photobleaching analysis. We found that GFP-DA has a larger stable fraction than GFP-DE. The stable Drebrin fraction reflects its accumulation in dendritic spines, therefore the isoform conversion may increase the amount of stable F-actin in dendritic spines. The stable fraction was dependent on the Drebrin A-specific sequence "Ins2", located in the middle of the Drebrin protein. In addition, F-actin depolymerization with latrunculin A significantly reduced the stable GFP-DA fraction. These findings indicate that preferential binding of Drebrin A to F-actin than Drebrin E causes higher stable fraction of Drebrin A in dendritic spines, although the F-actin-binding ability of purified Drebrin E and Drebrin A are comparable. Therefore, we suggest that a Drebrin isoform conversion from Drebrin E to Drebrin A in dendritic spines results in the accumulation of Drebrin-bound stable F-actin, which plays a pivotal role in synapse formation.
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Presentation_1_Drebrin Isoforms Critically Regulate NMDAR- and mGluR-Dependent LTD Induction.pdf
2018Co-Authors: Hiroki Yasuda, Nobuhiko Kojima, Kenji Sakimura, Hiroyuki Yamazaki, Kenji Hanamura, Tomoaki ShiraoAbstract:Drebrin is an actin-binding protein that is preferentially expressed in the brain. It is highly localized in dendritic spines and regulates spine shapes. The embryonic-type (Drebrin E) is expressed in the embryonic and early postnatal brain and is replaced by the adult-type (Drebrin A) during development. In parallel, NMDA receptor (NMDAR)-dependent long-term depression (LTD) of synaptic transmission, induced by low-frequency stimulation (LFS), is dominant in the immature brain and decreases during development. Here, we report that Drebrin regulates NMDAR-dependent and group 1 metabotropic glutamate receptor (mGluR)-dependent LTD induction in the hippocampus. While LFS induced NMDAR-dependent LTD in the developing hippocampus in wild-type (WT) mice, it did not induce LTD in developing Drebrin E and A double knockout (DXKO) mice, indicating that Drebrin is required for NMDAR-dependent LTD. On the other hand, LFS induced robust LTD dependent on mGluR5, one of group 1 mGluRs, in both developing and adult brains of Drebrin A knockout (DAKO) mice, in which Drebrin E is expressed throughout development and adulthood. Agonist-induced mGluR-dependent LTD was normal in WT and DXKO mice; however, it was enhanced in DAKO mice. Also, mGluR1, another group 1 mGluR, was involved in agonist-induced mGluR-dependent LTD in DAKO mice. These data suggest that abnormal Drebrin E expression in adults promotes group 1 mGluR-dependent LTD induction. Therefore, while Drebrin expression is critical for NMDAR-dependent LTD induction, developmental conversion from Drebrin E to Drebrin A prevents robust group 1 mGluR-dependent LTD.
Phillip R Gordonweeks - One of the best experts on this subject based on the ideXlab platform.
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the Drebrin eb3 pathway regulates cytoskeletal dynamics to drive neuritogenesis in embryonic cortical neurons
Journal of Neurochemistry, 2021Co-Authors: Thanushiyan Poobalasingam, Fazal Oozeer, Francesca Bianco, Phillip R GordonweeksAbstract:Co-ordinating the dynamic behaviour of actin filaments (F-actin) and microtubules in filopodia is an important underlying process in neuritogenesis, but the molecular pathways involved are ill-defined. The Drebrin/end-binding protein 3 (EB3) pathway is a candidate pathway for linking F-actin to microtubules in filopodia. Drebrin binds F-actin and, simultaneously, the microtubule-binding protein EB3 when bound to microtubule plus-ends. We assessed the effect on neuritogenesis of gain- or loss-of-function of proteins in the Drebrin/EB3 pathway in rat embryonic cortical neurons in culture. Loss-of-function of Drebrin by gene editing or pharmacological inhibition of Drebrin binding to F-actin reduced the number of dynamic microtubules in the cell periphery and simultaneously delayed the initiation of neuritogenesis, whereas over-expression of Drebrin induced supernumerary neurites. Similarly, loss of EB3 inhibited neuritogenesis, whereas loss of end-binding protein 1 (EB1), a related protein that does not bind to Drebrin, did not affect neuritogenesis. Over-expression of EB3, but not EB1, induced supernumerary neurites. We discovered that EB3 is more proximally located at dynamic microtubule plus-ends than EB1 in growth cone filopodia allowing for continuous microtubule elongation as the Drebrin/EB3 pathway zippers microtubules to F-actin in filopodia. Finally, we showed that preventing the entry of dynamic microtubules into filopodia using a pharmacological inhibitor of microtubule dynamics is associated with a loss of EB3, but not EB1, from microtubule plus-ends and a concurrent attenuation of neuritogenesis. Collectively, these findings support the idea that neuritogenesis depends on microtubule/F-actin zippering in filopodia orchestrated by the Drebrin/EB3 pathway.
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the Drebrin eb3 pathway drives invasive activity in prostate cancer
Oncogene, 2017Co-Authors: A E Dart, Daniel C Worth, G Muir, A Chandra, J D Morris, C Mckee, C Verrill, R J Bryant, Phillip R GordonweeksAbstract:Prostate cancer is the most common cancer in men and the metastatic form of the disease is incurable. We show here that the Drebrin/EB3 pathway, which co-ordinates dynamic microtubule/actin filament interactions underlying cell shape changes in response to guidance cues, plays a role in prostate cancer cell invasion. Drebrin expression is restricted to basal epithelial cells in benign human prostate but is upregulated in luminal epithelial cells in foci of prostatic malignancy. Drebrin is also upregulated in human prostate cancer cell lines and co-localizes with actin filaments and dynamic microtubules in filopodia of pseudopods of invading cells under a chemotactic gradient of the chemokine CXCL12. Disruption of the Drebrin/EB3 pathway using BTP2, a small molecule inhibitor of Drebrin binding to actin filaments, reduced the invasion of prostate cancer cell lines in 3D in vitro assays. Furthermore, gain- or loss-of-function of Drebrin or EB3 by over-expression or siRNA-mediated knockdown increases or decreases invasion of prostate cancer cell lines in 3D in vitro assays, respectively. Finally, expression of a dominant-negative construct that competes with EB3 binding to Drebrin, also inhibited invasion of prostate cancer cell lines in 3D in vitro assays. Our findings show that co-ordination of dynamic microtubules and actin filaments by the Drebrin/EB3 pathway drives prostate cancer cell invasion and is therefore implicated in disease progression.
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the role of the Drebrin eb3 cdk5 pathway in dendritic spine plasticity implications for alzheimer s disease
Brain Research Bulletin, 2016Co-Authors: Phillip R GordonweeksAbstract:The Drebrin/EB3/Cdk5 intracellular signalling pathway couples actin filaments to dynamic microtubules in cellular settings where cells are changing shape. The pathway has been most intensively studied in neuronal development, particularly neuritogenesis and neuronal migration, and in synaptic plasticity at dendritic spines in mature neurons. Drebrin is an actin filament side-binding and bundling protein that stabilises actin filaments. The end-binding (EB) proteins are microtubule plus-end tracking proteins (+TIPs) that localise to the growing plus-ends of dynamic microtubules and regulate their behavior and the binding of other +TIP proteins. EB3 binds specifically to Drebrin when Drebrin is bound to actin filaments, for example at the base of a growth cone filopodium, and EB3 is located at the plus-end of a growing microtubule inserting into the filopodium. This interaction therefore forms the basis for coupling dynamic microtubules to actin filaments in growth cones of developing neurons. Appropriate responses to growth cone guidance cues depend on actin filament/microtubule co-ordination in the growth cone, although the role of the Drebrin/EB3/Cdk5 pathway in this context has not been directly tested. A similar cytoskeleton coupling pathway operates in dendritic spines in mature neurons where the activity-dependent insertion of dynamic microtubules into dendritic spines is facilitated by Drebrin binding to EB3. Microtubule insertion into dendritic spines drives spine maturation during long-term potentiation and therefore has a role in synaptic plasticity and memory formation. In Alzheimer's disease and related chronic neurodegenerative diseases, there is an early and dramatic loss of Drebrin from dendritic spines that precedes synapse loss and neurodegeneration and might contribute to a failure of synaptic plasticity and hence to cognitive decline.
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Drebrin regulates neuroblast migration in the postnatal mammalian brain
PLOS ONE, 2015Co-Authors: Martina Sonego, Sangeetha Gajendra, Carl Hobbs, Michelle Oberoi, Jake Stoddart, Rita Hendricusdottir, Fazal Oozeer, Daniel C Worth, Britta J Eickholt, Phillip R GordonweeksAbstract:After birth, stem cells in the subventricular zone (SVZ) generate neuroblasts that migrate along the rostral migratory stream (RMS) to become interneurons in the olfactory bulb (OB). This migration is crucial for the proper integration of newborn neurons in a pre-existing synaptic network and is believed to play a key role in infant human brain development. Many regulators of neuroblast migration have been identified; however, still very little is known about the intracellular molecular mechanisms controlling this process. Here, we have investigated the function of Drebrin, an actin-binding protein highly expressed in the RMS of the postnatal mammalian brain. Neuroblast migration was monitored both in culture and in brain slices obtained from electroporated mice by time-lapse spinning disk confocal microscopy. Depletion of Drebrin using distinct RNAi approaches in early postnatal mice affects neuroblast morphology and impairs neuroblast migration and orientation in vitro and in vivo. Overexpression of Drebrin also impairs migration along the RMS and affects the distribution of neuroblasts at their final destination, the OB. Drebrin phosphorylation on Ser142 by Cyclin-dependent kinase 5 (Cdk5) has been recently shown to regulate F-actin-microtubule coupling in neuronal growth cones. We also investigated the functional significance of this phosphorylation in RMS neuroblasts using in vivo postnatal electroporation of phosphomimetic (S142D) or non-phosphorylatable (S142A) Drebrin in the SVZ of mouse pups. Preventing or mimicking phosphorylation of S142 in vivo caused similar effects on neuroblast dynamics, leading to aberrant neuroblast branching. We conclude that Drebrin is necessary for efficient migration of SVZ-derived neuroblasts and propose that regulated phosphorylation of Drebrin on S142 maintains leading process stability for polarized migration along the RMS, thus ensuring proper neurogenesis.
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Drebrin contains a cryptic f actin bundling activity regulated by cdk5 phosphorylation
Journal of Cell Biology, 2013Co-Authors: Daniel C Worth, Fazal Oozeer, Catherine N Daly, Sara Geraldo, Phillip R GordonweeksAbstract:Drebrin is an actin filament (F-actin)–binding protein with crucial roles in neuritogenesis and synaptic plasticity. Drebrin couples dynamic microtubules to F-actin in growth cone filopodia via binding to the microtubule-binding +TIP protein EB3 and organizes F-actin in dendritic spines. Precisely how Drebrin interacts with F-actin and how this is regulated is unknown. We used cellular and in vitro assays with a library of Drebrin deletion constructs to map F-actin binding sites. We discovered two domains in the N-terminal half of Drebrin—a coiled-coil domain and a helical domain—that independently bound to F-actin and cooperatively bundled F-actin. However, this activity was repressed by an intramolecular interaction relieved by Cdk5 phosphorylation of serine 142 located in the coiled-coil domain. Phospho-mimetic and phospho-dead mutants of serine 142 interfered with neuritogenesis and coupling of microtubules to F-actin in growth cone filopodia. These findings show that Drebrin contains a cryptic F-actin–bundling activity regulated by phosphorylation and provide a mechanistic model for microtubule–F-actin coupling.
Hiroyuki Yamazaki - One of the best experts on this subject based on the ideXlab platform.
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Drebrin Isoforms Critically Regulate NMDAR- and mGluR-Dependent LTD Induction.
Frontiers in cellular neuroscience, 2018Co-Authors: Hiroki Yasuda, Nobuhiko Kojima, Kenji Sakimura, Hiroyuki Yamazaki, Kenji Hanamura, Tomoaki ShiraoAbstract:Drebrin is an actin-binding protein that is preferentially expressed in the brain. It is highly localized in dendritic spines and regulates spine shapes. The embryonic-type (Drebrin E) is expressed in the embryonic and early postnatal brain and is replaced by the adult-type (Drebrin A) during development. In parallel, NMDA receptor (NMDAR)-dependent long-term depression (LTD) of synaptic transmission, induced by low-frequency stimulation (LFS), is dominant in the immature brain and decreases during development. Here, we report that Drebrin regulates NMDAR-dependent and group 1 metabotropic glutamate receptor (mGluR)-dependent LTD induction in the hippocampus. While LFS induced NMDAR-dependent LTD in the developing hippocampus in wild-type (WT) mice, it did not induce LTD in developing Drebrin E and A double knockout (DXKO) mice, indicating that Drebrin is required for NMDAR-dependent LTD. On the other hand, LFS induced robust LTD dependent on mGluR5, one of group 1 mGluRs, in both developing and adult brains of Drebrin A knockout (DAKO) mice, in which Drebrin E is expressed throughout development and adulthood. Agonist-induced mGluR-dependent LTD was normal in WT and DXKO mice; however, it was enhanced in DAKO mice. Also, mGluR1, another group 1 mGluR, was involved in agonist-induced mGluR-dependent LTD in DAKO mice. These data suggest that abnormal Drebrin E expression in adults promotes group 1 mGluR-dependent LTD induction. Therefore, while Drebrin expression is critical for NMDAR-dependent LTD induction, developmental conversion from Drebrin E to Drebrin A prevents robust group 1 mGluR-dependent LTD.
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CaMKIIβ is localized in dendritic spines as both Drebrin-dependent and Drebrin-independent pools.
Journal of neurochemistry, 2018Co-Authors: Hiroyuki Yamazaki, Yoshio Sasagawa, Hideyuki Yamamoto, Haruhiko Bito, Tomoaki ShiraoAbstract:Drebrin is a major F-actin binding protein in dendritic spines that is critically involved in the regulation of dendritic spine morphogenesis, pathology, and plasticity. In this study, we aimed to identify a novel Drebrin-binding protein involved in spine morphogenesis and synaptic plasticity. We confirmed the beta subunit of Ca2+ /calmodulin-dependent protein kinase II (CaMKIIβ) as a Drebrin-binding protein using a yeast two-hybrid system, and investigated the Drebrin-CaMKIIβ relationship in dendritic spines using rat hippocampal neurons. Drebrin knockdown resulted in diffuse localization of CaMKIIβ in dendrites during the resting state, suggesting that Drebrin is involved in the accumulation of CaMKIIβ in dendritic spines. Fluorescence recovery after photobleaching analysis showed that Drebrin knockdown increased the stable fraction of CaMKIIβ, indicating the presence of Drebrin-independent, more stable CaMKIIβ. NMDA receptor activation also increased the stable fraction in parallel with Drebrin exodus from dendritic spines. These findings suggest that CaMKIIβ can be classified into distinct pools: CaMKIIβ associated with Drebrin, CaMKIIβ associated with post-synaptic density (PSD), and CaMKIIβ free from PSD and Drebrin. CaMKIIβ appears to be anchored to a protein complex composed of Drebrin-binding F-actin during the resting state. NMDA receptor activation releases CaMKIIβ from Drebrin resulting in CaMKIIβ association with PSD.
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Isoform-dependent Regulation of Drebrin Dynamics in Dendritic Spines
Neuroscience, 2018Co-Authors: Kenji Hanamura, Nobuhiko Kojima, Hiroyuki Yamazaki, Yousuke Kamata, Tomoaki ShiraoAbstract:Dendritic spines have stable filamentous actin (F-actin) and dynamic F-actin. The formation of stable F-actin plays a pivotal role in spine formation. Drebrin binds to and stabilizes F-actin in dendritic spines. Interestingly, the conversion of the Drebrin E isoform to Drebrin A occurs in parallel with synapse formation, suggesting that this conversion promotes synapse formation via F-actin accumulation. In this study, we measured the dynamics of GFP-tagged Drebrin E (GFP-DE) and Drebrin A (GFP-DA) in cultured hippocampal neurons by fluorescence recovery after photobleaching analysis. We found that GFP-DA has a larger stable fraction than GFP-DE. The stable Drebrin fraction reflects its accumulation in dendritic spines, therefore the isoform conversion may increase the amount of stable F-actin in dendritic spines. The stable fraction was dependent on the Drebrin A-specific sequence "Ins2", located in the middle of the Drebrin protein. In addition, F-actin depolymerization with latrunculin A significantly reduced the stable GFP-DA fraction. These findings indicate that preferential binding of Drebrin A to F-actin than Drebrin E causes higher stable fraction of Drebrin A in dendritic spines, although the F-actin-binding ability of purified Drebrin E and Drebrin A are comparable. Therefore, we suggest that a Drebrin isoform conversion from Drebrin E to Drebrin A in dendritic spines results in the accumulation of Drebrin-bound stable F-actin, which plays a pivotal role in synapse formation.
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Presentation_1_Drebrin Isoforms Critically Regulate NMDAR- and mGluR-Dependent LTD Induction.pdf
2018Co-Authors: Hiroki Yasuda, Nobuhiko Kojima, Kenji Sakimura, Hiroyuki Yamazaki, Kenji Hanamura, Tomoaki ShiraoAbstract:Drebrin is an actin-binding protein that is preferentially expressed in the brain. It is highly localized in dendritic spines and regulates spine shapes. The embryonic-type (Drebrin E) is expressed in the embryonic and early postnatal brain and is replaced by the adult-type (Drebrin A) during development. In parallel, NMDA receptor (NMDAR)-dependent long-term depression (LTD) of synaptic transmission, induced by low-frequency stimulation (LFS), is dominant in the immature brain and decreases during development. Here, we report that Drebrin regulates NMDAR-dependent and group 1 metabotropic glutamate receptor (mGluR)-dependent LTD induction in the hippocampus. While LFS induced NMDAR-dependent LTD in the developing hippocampus in wild-type (WT) mice, it did not induce LTD in developing Drebrin E and A double knockout (DXKO) mice, indicating that Drebrin is required for NMDAR-dependent LTD. On the other hand, LFS induced robust LTD dependent on mGluR5, one of group 1 mGluRs, in both developing and adult brains of Drebrin A knockout (DAKO) mice, in which Drebrin E is expressed throughout development and adulthood. Agonist-induced mGluR-dependent LTD was normal in WT and DXKO mice; however, it was enhanced in DAKO mice. Also, mGluR1, another group 1 mGluR, was involved in agonist-induced mGluR-dependent LTD in DAKO mice. These data suggest that abnormal Drebrin E expression in adults promotes group 1 mGluR-dependent LTD induction. Therefore, while Drebrin expression is critical for NMDAR-dependent LTD induction, developmental conversion from Drebrin E to Drebrin A prevents robust group 1 mGluR-dependent LTD.
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Drebrin E regulates neuroblast proliferation and chain migration in the adult brain
European Journal of Neuroscience, 2017Co-Authors: Yuki Kajita, Nobuhiko Kojima, Noriko Koganezawa, Kenji Sakimura, Hiroyuki Yamazaki, Tomoaki ShiraoAbstract:F-actin-binding protein Drebrin has two major isoforms: Drebrin A and Drebrin E. Drebrin A is the major isoform in the adult brain and is highly concentrated in dendritic spines, regulating spine morphology and synaptic plasticity. Conversely, Drebrin E is the major isoform in the embryonic brain and regulates neuronal morphological differentiation, but it is also expressed in neurogenic regions of the adult brain. The subventricular zone (SVZ) is one of the brain regions where adult neurogenesis occurs. Neuroblasts migrate to the olfactory bulb (OB) and integrate into existing neuronal networks, after which Drebrin expression changes from E to A, suggesting that Drebrin E plays a specific role in neuroblasts in the adult brain. Therefore, to understand the role of Drebrin E in the adult brain, we immunohistochemically analyzed adult neurogenesis using Drebrin-null-mutant (DXKO) mice. In DXKO mice, the number of neuroblasts and cell proliferation decreased, although cell death remained unchanged. These results suggest that Drebrin E regulates cell proliferation in the adult SVZ. Surprisingly, the decreased number of neuroblasts in the SVZ did not result in less neurons in the OB. This was because the survival rate of newly generated neurons in the OB increased in DXKO mice. Additionally, when neuroblasts reached the OB, the change in the migratory pathway from tangential to radial was partly disturbed in DXKO mice. These results suggest that Drebrin E is involved in a chain migration of neuroblasts.
Gert Lubec - One of the best experts on this subject based on the ideXlab platform.
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Drebrin autoantibodies in patients with seizures and suspected encephalitis
Annals of neurology, 2020Co-Authors: Julika Pitsch, Gert Lubec, Delara Kamalizade, Anna Braun, Julia C. Kuehn, Polina E Gulakova, Theodor Rüber, Dirk Dietrich, Randi Von Wrede, Christoph HelmstaedterAbstract:Objective Assess occurrence of the dendritic spine scaffolding protein Drebrin as a pathophysiologically relevant autoantibody target in patients with recurrent seizures and suspected encephalitis as leading symptoms. Methods Sera of 4 patients with adult onset epilepsy and suspected encephalitis of unresolved etiology and equivalent results in autoantibody screening were subjected to epitope identification. We combined a wide array of approaches, ranging from immunoblotting, immunoprecipitation, mass spectrometry, subcellular binding pattern analyses in primary neuronal cultures, and immunohistochemistry in brains of wild-type and Drebrin knockout mice to in vitro analyses of impaired synapse formation, morphology, and aberrant neuronal excitability by antibody exposure. Results In the serum of a patient with adult onset epilepsy and suspected encephalitis, a strong signal at ∼70kDa was detected by immunoblotting, for which mass spectrometry revealed Drebrin as the putative antigen. Three other patients whose sera also showed strong immunoreactivity around 70kDa on Western blotting were also anti-Drebrin-positive. Seizures, memory impairment, and increased protein content in cerebrospinal fluid occurred in anti-Drebrin-seropositive patients. Alterations in cerebral magnetic resonance imaging comprised amygdalohippocampal T2-signal increase and hippocampal sclerosis. Diagnostic biopsy revealed T-lymphocytic encephalitis in an anti-Drebrin-seropositive patient. Exposure of primary hippocampal neurons to anti-Drebrin autoantibodies resulted in aberrant synapse composition and Drebrin distribution as well as increased spike rates and the emergence of burst discharges reflecting network hyperexcitability. Interpretation Anti-Drebrin autoantibodies define a chronic syndrome of recurrent seizures and neuropsychiatric impairment as well as inflammation of limbic and occasionally cortical structures. Immunosuppressant therapies should be considered in this disorder. ANN NEUROL 2020;87:869-884.
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Drebrin depletion alters neurotransmitter receptor levels in protein complexes dendritic spine morphogenesis and memory related synaptic plasticity in the mouse hippocampus
Journal of Neurochemistry, 2015Co-Authors: Gangsoo Jung, Sunetra Sase, Marion Groger, Fernando J Sialana, Ana Cicvaric, Francisco J. Monje, Johannes Berger, Harald Höger, Gert LubecAbstract:Drebrin an actin-bundling key regulator of dendritic spine genesis and morphology, has been recently proposed as a regulator of hippocampal glutamatergic activity which is critical for memory formation and maintenance. Here, we examined the effects of genetic deletion of Drebrin on dendritic spine and on the level of complexes containing major brain receptors. To this end, homozygous and heterozygous Drebrin knockout mice generated in our laboratory and related wild-type control animals were studied. Level of protein complexes containing dopamine receptor D1/dopamine receptor D2, 5-hydroxytryptamine receptor 1A (5-HT1AR), and 5-hydroxytryptamine receptor 7 (5-HT7R) were significantly reduced in hippocampus of Drebrin knockout mice whereas no significant changes were detected for GluR1, 2, and 3 and NR1 as examined by native gel-based immunoblotting. Drebrin depletion also altered dendritic spine formation, morphology, and reduced levels of dopamine receptor D1 in dendritic spines as evaluated using immunohistochemistry/confocal microscopy. Electrophysiological studies further showed significant reduction in memory-related hippocampal synaptic plasticity upon Drebrin depletion. These findings provide unprecedented experimental support for a role of Drebrin in the regulation of memory-related synaptic plasticity and neurotransmitter receptor signaling, offer relevant information regarding the interpretation of previous studies and help in the design of future studies on dendritic spines. We examined effect of genetic deletion of Drebrin, which an actin-bundling key regulator of dendritic spine genesis and morphology, on dendritic spine density, maturity, level of complexes containing major brain receptors and also, in synaptic plasticity. These findings support for a role of Drebrin in the regulation of memory-related synaptic plasticity and neurotransmitter receptors signaling in dendritic spines.
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Drebrin depletion alters neurotransmitter receptor levels in protein complexes dendritic spine morphogenesis and memory related synaptic plasticity in the mouse hippocampus
Journal of Neurochemistry, 2015Co-Authors: Gangsoo Jung, Sunetra Sase, Marion Groger, Fernando J Sialana, Ana Cicvaric, Francisco J. Monje, Johannes Berger, Harald Höger, Eun Jung Kim, Gert LubecAbstract:Drebrin an actin-bundling key regulator of dendritic spine genesis and morphology, has been recently proposed as a regulator of hippocampal glutamatergic activity which is critical for memory formation and maintenance. Here, we examined the effects of genetic deletion of Drebrin on dendritic spine and on the level of complexes containing major brain receptors. To this end, homozygous and heterozygous Drebrin knockout mice generated in our laboratory and related wild-type control animals were studied. Level of protein complexes containing dopamine receptor D1/dopamine receptor D2, 5-hydroxytryptamine receptor 1A (5-HT1(A)R), and 5-hydroxytryptamine receptor 7 (5-HT7R) were significantly reduced in hippocampus of Drebrin knockout mice whereas no significant changes were detected for GluR1, 2, and 3 and NR1 as examined by native gel-based immunoblotting. Drebrin depletion also altered dendritic spine formation, morphology, and reduced levels of dopamine receptor D1 in dendritic spines as evaluated using immunohistochemistry/confocal microscopy. Electrophysiological studies further showed significant reduction in memory-related hippocampal synaptic plasticity upon Drebrin depletion. These findings provide unprecedented experimental support for a role of Drebrin in the regulation of memory-related synaptic plasticity and neurotransmitter receptor signaling, offer relevant information regarding the interpretation of previous studies and help in the design of future studies on dendritic spines.
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Drebrin a dendritic spine protein is manifold decreased in brains of patients with alzheimer s disease and down syndrome
Neuroscience Letters, 2002Co-Authors: Ki Shuk Shim, Gert LubecAbstract:Abstract Drebrin, located in the dendritic spines of the neuron, plays a role in the synaptic plasticity together with actin filaments. Although Drebrin regulates the morphological changes of spines in neurodegenerative disease such as Alzheimer's disease (AD), Drebrin in Down syndrome (DS) showing AD-like neuropathology has not been studied. We used Western blotting to determine protein levels of Drebrin and F-actin in frontal, temporal cortex and cerebellum from patients with DS and AD as compared to controls. A monoclonal antibody against Drebrin and F-actin was used. Drebrin levels were significantly decreased in frontal (means±standard deviation; DS 0.24±0.52; AD 0.16±0.14; controls 2.56±3.48) and temporal cortex (DS 0.07±0.11; AD 0.07±0.15; controls 1.71±1.51) and Drebrin was also decreased when normalized with F-actin. No changes were observed in cerebellum. Decreased Drebrin could not simply be due to cell loss (F-actin) or neuronal loss (comparable neuron-specific enolase between groups). Reduced Drebrin could be responsible for or representing the loss of spine plasticity in DS and may be a useful indicator for the impaired arborization in neurodegenerative disorders.
Kenji Hanamura - One of the best experts on this subject based on the ideXlab platform.
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High-content imaging analysis for detecting the loss of Drebrin clusters along dendrites in cultured hippocampal neurons.
Journal of pharmacological and toxicological methods, 2019Co-Authors: Kenji Hanamura, Noriko Koganezawa, Yuko Sekino, Kazumi Kamiyama, Natsume Tanaka, Takero Oka, Mai Yamamura, Tomoaki ShiraoAbstract:Abstract Introduction Detection of drug effects on neuronal synapses is important for predicting their adverse effects. We have used Drebrin as a marker to detect the synaptic changes in cultured neurons. High concentration of glutamate decreases the amount of Drebrin in synapses. To increase the availability of this method for high throughput analysis, we applied the Drebrin-based evaluation of synapses to high-content imaging analysis using microplates. Methods Three weeks old cultured neurons were fixed and processed for immunocytochemistry to visualize Drebrin clusters, dendrites and neuronal cell bodies. After automated image acquisition, total number of Drebrin clusters per fields, linear density of Drebrin cluster along dendrites, dendrite length and neuron number were automatically measured by a custom-designed protocol. Results Automated image acquisition and analysis showed that dendrite length and Drebrin cluster density along dendrites are measured consistently and reproducibly. In addition, application of 10–100 μM glutamate for 10 min or 0.5–50 μM latrunculin A for 5 min significantly decreased Drebrin cluster density without affecting neuron number. These results were consistent with our previous results using manual image acquisition and analysis with regular fluorescence microscope and image analysis software. Furthermore, 0.3 or 1.0 μM staurosporine for 24 h significantly decreased neuron number. Discussion The present study demonstrates that this high-throughput imaging analysis of Drebrin cluster density along dendrites for detecting the effects of substances on synapses is sensitive enough to detect the effects of glutamate receptor activation and latrunculin A treatment, and indicates that this analysis will be useful for safety pharmacology study.
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Drebrin Isoforms Critically Regulate NMDAR- and mGluR-Dependent LTD Induction.
Frontiers in cellular neuroscience, 2018Co-Authors: Hiroki Yasuda, Nobuhiko Kojima, Kenji Sakimura, Hiroyuki Yamazaki, Kenji Hanamura, Tomoaki ShiraoAbstract:Drebrin is an actin-binding protein that is preferentially expressed in the brain. It is highly localized in dendritic spines and regulates spine shapes. The embryonic-type (Drebrin E) is expressed in the embryonic and early postnatal brain and is replaced by the adult-type (Drebrin A) during development. In parallel, NMDA receptor (NMDAR)-dependent long-term depression (LTD) of synaptic transmission, induced by low-frequency stimulation (LFS), is dominant in the immature brain and decreases during development. Here, we report that Drebrin regulates NMDAR-dependent and group 1 metabotropic glutamate receptor (mGluR)-dependent LTD induction in the hippocampus. While LFS induced NMDAR-dependent LTD in the developing hippocampus in wild-type (WT) mice, it did not induce LTD in developing Drebrin E and A double knockout (DXKO) mice, indicating that Drebrin is required for NMDAR-dependent LTD. On the other hand, LFS induced robust LTD dependent on mGluR5, one of group 1 mGluRs, in both developing and adult brains of Drebrin A knockout (DAKO) mice, in which Drebrin E is expressed throughout development and adulthood. Agonist-induced mGluR-dependent LTD was normal in WT and DXKO mice; however, it was enhanced in DAKO mice. Also, mGluR1, another group 1 mGluR, was involved in agonist-induced mGluR-dependent LTD in DAKO mice. These data suggest that abnormal Drebrin E expression in adults promotes group 1 mGluR-dependent LTD induction. Therefore, while Drebrin expression is critical for NMDAR-dependent LTD induction, developmental conversion from Drebrin E to Drebrin A prevents robust group 1 mGluR-dependent LTD.
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Isoform-dependent Regulation of Drebrin Dynamics in Dendritic Spines
Neuroscience, 2018Co-Authors: Kenji Hanamura, Nobuhiko Kojima, Hiroyuki Yamazaki, Yousuke Kamata, Tomoaki ShiraoAbstract:Dendritic spines have stable filamentous actin (F-actin) and dynamic F-actin. The formation of stable F-actin plays a pivotal role in spine formation. Drebrin binds to and stabilizes F-actin in dendritic spines. Interestingly, the conversion of the Drebrin E isoform to Drebrin A occurs in parallel with synapse formation, suggesting that this conversion promotes synapse formation via F-actin accumulation. In this study, we measured the dynamics of GFP-tagged Drebrin E (GFP-DE) and Drebrin A (GFP-DA) in cultured hippocampal neurons by fluorescence recovery after photobleaching analysis. We found that GFP-DA has a larger stable fraction than GFP-DE. The stable Drebrin fraction reflects its accumulation in dendritic spines, therefore the isoform conversion may increase the amount of stable F-actin in dendritic spines. The stable fraction was dependent on the Drebrin A-specific sequence "Ins2", located in the middle of the Drebrin protein. In addition, F-actin depolymerization with latrunculin A significantly reduced the stable GFP-DA fraction. These findings indicate that preferential binding of Drebrin A to F-actin than Drebrin E causes higher stable fraction of Drebrin A in dendritic spines, although the F-actin-binding ability of purified Drebrin E and Drebrin A are comparable. Therefore, we suggest that a Drebrin isoform conversion from Drebrin E to Drebrin A in dendritic spines results in the accumulation of Drebrin-bound stable F-actin, which plays a pivotal role in synapse formation.
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Presentation_1_Drebrin Isoforms Critically Regulate NMDAR- and mGluR-Dependent LTD Induction.pdf
2018Co-Authors: Hiroki Yasuda, Nobuhiko Kojima, Kenji Sakimura, Hiroyuki Yamazaki, Kenji Hanamura, Tomoaki ShiraoAbstract:Drebrin is an actin-binding protein that is preferentially expressed in the brain. It is highly localized in dendritic spines and regulates spine shapes. The embryonic-type (Drebrin E) is expressed in the embryonic and early postnatal brain and is replaced by the adult-type (Drebrin A) during development. In parallel, NMDA receptor (NMDAR)-dependent long-term depression (LTD) of synaptic transmission, induced by low-frequency stimulation (LFS), is dominant in the immature brain and decreases during development. Here, we report that Drebrin regulates NMDAR-dependent and group 1 metabotropic glutamate receptor (mGluR)-dependent LTD induction in the hippocampus. While LFS induced NMDAR-dependent LTD in the developing hippocampus in wild-type (WT) mice, it did not induce LTD in developing Drebrin E and A double knockout (DXKO) mice, indicating that Drebrin is required for NMDAR-dependent LTD. On the other hand, LFS induced robust LTD dependent on mGluR5, one of group 1 mGluRs, in both developing and adult brains of Drebrin A knockout (DAKO) mice, in which Drebrin E is expressed throughout development and adulthood. Agonist-induced mGluR-dependent LTD was normal in WT and DXKO mice; however, it was enhanced in DAKO mice. Also, mGluR1, another group 1 mGluR, was involved in agonist-induced mGluR-dependent LTD in DAKO mice. These data suggest that abnormal Drebrin E expression in adults promotes group 1 mGluR-dependent LTD induction. Therefore, while Drebrin expression is critical for NMDAR-dependent LTD induction, developmental conversion from Drebrin E to Drebrin A prevents robust group 1 mGluR-dependent LTD.
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Localization of Drebrin: Light Microscopy Study
Advances in experimental medicine and biology, 2017Co-Authors: Tomoaki Shirao, Noriko Koganezawa, Hiroyuki Yamazaki, Kenji Hanamura, Kazuyuki ImamuraAbstract:Developmental changes in the expression and localization of Drebrin has been mainly analyzed in chick embryo and young rat by various anti-Drebrin polyclonal and monoclonal antibodies. Immunoblot analysis demonstrated that the adult Drebrin isoform (Drebrin A) is restricted to neural tissues, while the embryonic Drebrin isoforms (Drebrin E1 and E2 in chicken and Drebrin E in mammals) are found in a wide variety of tissues. In the developing brain, Drebrin E (including chicken Drebrin E2) is expressed in newly generated neurons. During neuronal migration, Drebrin E is distributed ubiquitously within the neurons. Once Drebrin A is expressed in the developing neuron, Drebrin E is no longer present within the cell soma and accumulates in the growth cone of growing processes, resulting in the cessation of neuronal migration. The limited subcellular localization of Drebrin A, which is possibly regulated by a Drebrin A-specific mechanism, is likely to affect the localization of Drebrin E. In the adult brain, Drebrin is mainly localized in dendritic spines, but in some nuclei, Drebrin can be detected in neuronal somata as well as dendritic spines. The fact that the developmental changes in Drebrin expression highly correlate in time with the sensitive period of visual cortical plasticity in kittens suggests that synaptic plasticity depends on Drebrin.
