A Novel, Brain-Specific Mouse Drebrin: cDNA Cloning, Chromosomal Mapping, Genomic Structure, Expression, and Functional Characterization

doi:10.1006/geno.2002.6764

Genomics

Volume 79, Issue 5, May 2002, Pages 686-692

https://doi.org/10.1006/geno.2002.6764 Get rights and content

Abstract

Drebrin A, a major neuronal actin-binding protein, regulates the dendritic spine shapes of neurons. Here, we have cloned and characterized a novel mouse cDNA clone encoding a truncated form of drebrin A, named s-drebrin A. Analysis of the genomic organization of the mouse drebrin gene (Dbn1), which mapped to the central portion of chromosome 13, revealed that isoforms including s-drebrin A are generated by alternative splicing from a single drebrin gene. The s-drebrin A mRNA was expressed in the brain, but not in non-neuronal tissues. The s-drebrin A expression was barely detected in the embryonic brain, but was upregulated during postnatal development of the brain. Overexpression of GFP-tagged s-drebrin A in fibroblasts showed it to be associated with actin filaments and with changes in actin cytoskeleton organization. These findings suggest that s-drebrin A has a role in spine morphogenesis, possibly by competing the actin-binding activity with drebrin A.

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Cited by (26)

Isoform-dependent Regulation of Drebrin Dynamics in Dendritic Spines
2018, Neuroscience
Citation Excerpt :
To examine this possibility, we compared the dynamics of GFP-DA with GFP-tagged s-drebrin A (sDA). sDA is a short isoform of DA lacking the C-terminal region, including the Homer-binding and profilin-binding motifs (Fig. 5A; Jin et al., 2002). Although GFP-sDA does not have Homer-binding motifs, it accumulated in dendritic spines (Fig. 5B, C; Fig. 6A).
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.
Drebrin A regulates hippocampal LTP and hippocampus-dependent fear learning in adult mice
2016, Neuroscience
Citation Excerpt :
Drebrin has two major isoforms in mammals: drebrin E (Embryonic isoform) and drebrin A (Adult isoform) (Shirao et al., 1989). These isoforms are derived from a single gene by alternative splicing (Kojima et al., 1993; Jin et al., 2002). The amino acid sequences are almost identical between the two isoforms, which include an actin-depolymerizing factor homology domain (Poukkula et al., 2011), an actin-remodeling domain (Hayashi et al., 1999; Grintsevich et al., 2010), a membrane-targeting domain (Xu and Stamnes, 2006), and Homer-binding sites (Shiraishi-Yamaguchi et al., 2009).
Structural plasticity of dendritic spines, which underlies higher brain functions including learning and memory, is dynamically regulated by the actin cytoskeleton and its associated proteins. Drebrin A is an F-actin-binding protein preferentially expressed in the brain and localized in the dendritic spines of mature neurons. Isoform conversion from drebrin E to drebrin A and accumulation of the latter in dendritic spines occurs during synapse maturation. We have previously demonstrated that drebrin A plays a pivotal role in spine morphogenesis and plasticity. However, it is unclear whether drebrin A plays a specific role in processes required for structural plasticity, and whether drebrin E can substitute in this role. To answer these questions, we analyzed mutant mice (named DAKO mice), in which isoform conversion from drebrin E to drebrin A is disrupted. In DAKO mouse brain, drebrin E continues to be expressed throughout life instead of drebrin A. Electrophysiological studies using hippocampal slices revealed that long-term potentiation of CA1 synapses was impaired in adult DAKO mice, but not in adolescents. In parallel with this age-dependent impairment, DAKO mice exhibited impaired hippocampus-dependent fear learning in an age-dependent manner; the impairment was evident in adult mice, but not in adolescents. In addition, histological investigation revealed that the spine length of the apical dendrite of CA1 pyramidal cells was significantly longer in adult DAKO mice than in wild-type mice. Our data indicate that the roles of drebrin E and drebrin A in brain function are different from each other, that the isoform conversion of drebrin is critical, and that drebrin A is indispensable for normal synaptic plasticity and hippocampus-dependent fear memory in the adult brain.
Expression of Dbn1 during mouse brain development and neural stem cell differentiation
2014, Biochemical and Biophysical Research Communications
Citation Excerpt :
Dbn1 is a novel mouse drebrin gene and is a newly discovered gene in the drebrin gene family, which has been mapped to the central portion of chromosome 13. Dbn1 is specifically expressed in the mouse brain and regulates the actin cytoskeleton [16]. In our previous work, we investigated gene expression during CNS development.
Dbn1 is a newly discovered gene in the drebrin gene family of mice. Previous studies have reported that Dbn1 is specifically expressed in the mouse brain suggesting its potential role in brain development. However, a detailed analysis of Dbn1 expression during mouse brain development has not been demonstrated. Here, we describe the expression pattern of Dbn1 and the coexpression of Dbn1 and actin during the development of the mouse brain from embryonic day 14 (E14) to adulthood and during the differentiation of neural stem cells (NSCs), as determined using immunohistochemistry, double-labeling immunofluorescence, and quantitative real-time polymerase chain reaction. During mouse brain development, Dbn1 expression level was high at E14, attenuated postnatally, reached its highest point at postnatal day 7 (P7), and showed a very low level at adulthood. Imaging data showed that Dbn1 was mainly expressed in the hippocampus, ventricular zone, and cortex, where NSCs are densely distributed, and that the intracellular distribution of Dbn1 was predominantly located in the cytoplasm edges and neurites. Moreover, the signal for colocalization of Dbn1 with actin was intense at E14, P0, and P7, but it was weak at adulthood. During NSC differentiation, Dbn1 mRNA expression increased after the onset of differentiation and reached its highest point at 3 days, followed by a decrease in expression. The imaging data showed that Dbn1 was increasingly expressed in the extending neurites in accordance with the cell morphological changes that occur during differentiation. Furthermore, obvious colocalization signals of Dbn1 with actin were found in the neurites and dendritic spines. Collectively, these results suggest that Dbn1 may play a key role in mouse brain development and may regulate NSC differentiation by filamentous actin.
The role of drebrin in glioma migration and invasion
2013, Experimental Cell Research
Citation Excerpt :
To the best of our knowledge, this is the first study that has investigated the implications of drebrin for cell migration, invasion, and morphology in a series of human glioma cell lines. Drebrin was originally identified in neuronal cells [8,9] and three isoforms of drebrin have been found in mammals: embryonic type; drebrin E, adult type; drebrin A, and a C-terminally truncated form of drebrin A; s-drebrin A [10]. Drebrin A is considered to be a neuron-specific isoform involved in the formation of neurites and in the synaptic plasticity [24].
Glioblastoma (GBM) is the most common primary brain tumor in adults. Despite current advances in therapy consisting of surgery followed by chemotherapy and radiation, the overall survival rate still remains poor. Therapeutic failures are partly attributable to the highly infiltrative nature of tumor adjacent to normal brain parenchyma. Recently, evidence is mounting to suggest that actin cytoskeleton dynamics are critical components of the cell invasion process. Drebrin is an actin-binding protein involved in the regulation of actin filament organization, and plays a significant role in cell motility; however, the role of drebrin in glioma cell invasiveness has not yet been fully elucidated. Therefore, this study was aimed to clarify the role of drebrin in glioma cell morphology and cell motility. Here we show that drebrin is expressed in glioma cell lines and in operative specimens of GBM. We demonstrate that stable overexpression of drebrin in U87 cells leads to alterations in cell morphology, and induces increased invasiveness in vitro while knockdown of drebrin in U87 cells by small interfering RNA (siRNA) decreases invasion and migration. In addition, we show that depletion of drebrin by siRNA alters glioma cell morphology in A172 GBM cell line. Our results suggest that drebrin contributes to the maintenance of cell shape, and may play an important role in glioma cell motility.
Functional Diversity of Actin Cytoskeleton in Neurons and its Regulation by Tropomyosin
2012, International Review of Cell and Molecular Biology
Neurons comprise functionally, molecularly, and spatially distinct subcellular compartments which include the soma, dendrites, axon, branches, dendritic spines, and growth cones. In this chapter, we detail the remarkable ability of the neuronal cytoskeleton to exquisitely regulate all these cytoplasmic distinct partitions, with particular emphasis on the microfilament system and its plethora of associated proteins. Importance will be given to the family of actin-associated proteins, tropomyosin, in defining distinct actin filament populations. The ability of tropomyosin isoforms to regulate the access of actin-binding proteins to the filaments is believed to define the structural diversity and dynamics of actin filaments and ultimately be responsible for the functional outcome of these filaments.
Genetic disruption of the alternative splicing of drebrin gene impairs context-dependent fear learning in adulthood
2010, Neuroscience
Citation Excerpt :
The resulting DNA fragments hybridized with drebrin cDNA were subcloned into pBlueScript. In a 1.2 kb HindIII-KpnI DNA fragment containing a drebrin A-specific exon (exon 11A; Jin et al., 2002), two loxP sequences were inserted in the 5′- and 3′-flanking regions of exon 11A. The frt-flanked phosphoglycerate kinase (PGK)-neo cassette was inserted in reverse into exon 11A in the 3′ direction for the positive selection of recombinant embryonic stem (ES) cell clones.
Dendritic spines are postsynaptic structures at excitatory synapses that play important roles in synaptic transmission and plasticity. Dendritic spine morphology and function are regulated by an actin-based cytoskeletal network. Drebrin A, an adult form of drebrin, is an actin-binding protein in dendritic spines, and its decrease is purportedly concerned with synaptic dysfunction in Alzheimer's disease. Rapid conversion of drebrin E, an embryonic form of drebrin, to drebrin A occurs in parallel with synaptic maturation. To understand the physiological role of drebrin isoform conversion in vivo, we generated knockout mice in which a drebrin A-specific exon was deleted from the drebrin gene. Drebrin A-specific knockout (DAKO) mice expressed drebrin E, which substituted for drebrin A. Subcellular fractionation experiment indicated that cytosolic form of drebrin was increased in the brains of DAKO mice. Furthermore, drebrin accumulation in synaptosomes of DAKO mice was much higher than that of wild-type (WT) mice. DAKO mice were viable and showed no apparent abnormalities in their gross brain morphology and general behaviors. However, DAKO mice were impaired in a context-dependent freezing after fear conditioning. These data indicate that drebrin A plays an indispensable role in some processes of generating fear learning and memory.

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^*: Present address: Laboratory for Neurobiology of Emotion, Brain Science Institute of RIKEN, 2-1 Hirosawa, Wako 351-0198, Japan

^†: To whom correspondence and reprint requests should be addressed. Fax: +81-27-220-8053. E-mail: [email protected].

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Regular ArticleA Novel, Brain-Specific Mouse Drebrin: cDNA Cloning, Chromosomal Mapping, Genomic Structure, Expression, and Functional Characterization

Abstract

J. Biol. Chem.

Neurosci. Res.

Neurosci. Res.

Mol. Brain Res.

Dev. Brain Res.

Mol. Brain Res.

Mol. Brain Res.

Brain Res.

Brain Res. Dev. Brain Res.

Eur. J. Cell Biol.

Cell

Brain Res.

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Neuron

Curr. Opin. Neurobiol.

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Cell

The roles of microfilament-associated proteins, drebrins, in brain morphogenesis: a review

J. Biochem. (Tokyo)

Modulatory role of drebrin on the cytoskeleton within dendritic spines in the rat cerebral cortex

J. Neurosci.

Inhibition by drebrin of the actin-bundling activity of brain fascin, a protein localized in filopodia of growth cones

J. Neurochem.

Regular Article
A Novel, Brain-Specific Mouse Drebrin: cDNA Cloning, Chromosomal Mapping, Genomic Structure, Expression, and Functional Characterization