Nobuhiko Kojima

Role of actin cytoskeleton in dendritic spine morphogenesis.

Dendritic spines are the postsynaptic receptive regions of most excitatory synapses, and their morphological plasticity play a pivotal role in higher brain functions, such as learning and memory. The dynamics of spine morphology is due to the actin cytoskeleton concentrated highly in spines. Filopodia, which are thin and headless protrusions, are thought to be precursors of dendritic spines. Drebrin, a spine-resident side-binding protein of filamentous actin (F-actin), is responsible for recruiting F-actin and PSD-95 into filopodia, and is suggested to govern spine morphogenesis. Interestingly, some recent studies on neurological disorders accompanied by cognitive deficits suggested that the loss of drebrin from dendritic spines is a common pathognomonic feature of synaptic dysfunction. In this review, to understand the importance of actin-binding proteins in spine morphogenesis, we first outline the well-established knowledge pertaining to the actin cytoskeleton in non-neuronal cells, such as the mechanism of regulation by small GTPases, the equilibrium between globular actin (G-actin) and F-actin, and the distinct roles of various actin-binding proteins. Then, we review the dynamic changes in the localization of drebrin during synaptogenesis and in response to glutamate receptor activation. Because side-binding proteins are located upstream of the regulatory pathway for actin organization via other actin-binding proteins, we discuss the significance of drebrin in the regulatory mechanism of spine morphology through the reorganization of the actin cytoskeleton. In addition, we discuss the possible involvement of an actin-myosin interaction in the morphological plasticity of spines.

Synaptic dysfunction and disruption of postsynaptic drebrin–actin complex: A study of neurological disorders accompanied by cognitive deficits

Many neurological disorders accompanied by cognitive deficits, including Alzheimers disease (AD) and Down syndrome, exhibit abnormal dendritic spine morphology. Actin-based cytoskeletal network dynamics is critical for the regulation of spine morphology and function. Recent experimental data from an AD animal model revealed that defects in intracellular signaling cascades related to the accumulation of amyloid beta (Abeta) peptide cause disruption of the postsynaptic actin-regulatory machinery, including cofilin and drebrin. The level of postsynaptic drebrin, a major F-actin-binding protein in dendritic spines, correlates well with the severity of cognitive impairment. We propose that an imbalanced regulation of the actin-regulatory machinery (loss of drebrin and increase of dephosphorylated cofilin) results in synaptic dysfunction, which underlies the cognitive impairment accompanying neurological disorders and normal aging.

Cloning of drebrin A and induction of neurite-like processes in drebrin-transfected cells

The developmentally-regulated neuron-specific protein, drebrin A, is expressed first at the time of outgrowth and maturation of dendrites, and is localized within dendrites of the adult brain. A cDNA clone of adult rat drebrin A was isolated and sequenced. There is no overall homology with other reported protein sequences except chicken drebrins. We constructed the expression vector MIW-DA containing the drebrin A cDNA. Transfection of nonneuronal cells with MIW-DA induced the formation of highly branched neurite-like cell processes. In these process-bearing transfectants, expressed debrin A is concentrated in submembraneous regions of the cell. Furthermore, actin concentration is higher in these cells than other fibroblasts. These results suggest a possible role of drebrin A in neurite outgrowth.

Four synaptic vesicle-specific proteins: identification by monoclonal antibodies and distribution in the nervous tissue and the adrenal medulla

Synaptic vesicles from the guinea-pig cerebrum were isolated and administered to mice for the production of monoclonal antibodies (MAb). Four vesicle-associated proteins in the guinea-pig nervous tissue were specifically and differentially recognized by MAbs thus obtained. These proteins had molecular weights of 30,000, 36,000, 38,000 and 65,000 Da and were named SVPs (synaptic vesicle proteins) 30, 36, 38 and 65, respectively. Immunohistochemistry demonstrated that all SVPs were localized in the synaptic regions throughout the central nervous system and in the adrenal medulla. Nerve terminals in skeletal muscle, smooth muscle and sympathetic ganglion contained SVPs 36 and 38. Immunoelectron microscopy of the cerebellar cortex confirmed the localization of SVPs in the synaptic vesicles and the adjacent membranes of the presynaptic nerve terminals. Fractionation of the cerebral tissue and treatment with various agents showed that SVPs were localized in the synaptic vesicles and the synaptic plasma membrane and that SVPs were integrated within the membrane and liberated only after solubilization of the membrane.

Drebrin E is involved in the regulation of axonal growth through actin–myosin interactions

Drebrin is a well‐known side‐binding protein of F‐actin in the brain. Immunohistochemical data suggest that the peripheral parts of growing axons are enriched in the drebrin E isoform and mature axons are not. It has also been observed that drebrin E is concentrated in the growth cones of PC12 cells. These data strongly suggest that drebrin E plays a role in axonal growth during development. In this study, we used primary hippocampal neuronal cultures to analyze the role of drebrin E. Immunocytochemistry showed that within axonal growth cones drebrin E specifically localized to the transitional zone, an area in which dense networks of F‐actins and microtubules overlapped. Over‐expression of drebrin E caused drebrin E and F‐actin to accumulate throughout the growth cone and facilitated axonal growth. In contrast, knockdown of drebrin E reduced drebrin E and F‐actin in the growth cone and prevented axonal growth. Furthermore, inhibition of myosin II ATPase masked the promoting effects of drebrin E over‐expression on axonal growth. These results suggest that drebrin E plays a role in axonal growth through actin–myosin interactions in the transitional zone of axonal growth cones.

Molecular cloning of a cDNA for the developmentally regulated brain protein, drebrin

A lambda gt11 cDNA library from 10-day-old chicken embryo was screened immunologically using an antiserum against drebrins E1, E2 and A, proteins previously designated S5, S6 and S54, respectively. A cDNA clone for a common domain of drebrin was isolated. Northern blot analysis of chicken brain indicated that drebrin mRNAs are about 2.7 kilobases in molecular size and that expression of these proteins is developmentally regulated.

Genetic disruption of the alternative splicing of drebrin gene impairs context-dependent fear learning in adulthood.

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 Alzheimers 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.

Inducible cAMP Early Repressor Acts as a Negative Regulator for Kindling Epileptogenesis and Long-Term Fear Memory

Long-lasting neuronal plasticity as well as long-term memory (LTM) requires de novo synthesis of proteins through dynamic regulation of gene expression. cAMP-responsive element (CRE)-mediated gene transcription occurs in an activity-dependent manner and plays a pivotal role in neuronal plasticity and LTM in a variety of species. To study the physiological role of inducible cAMP early repressor (ICER), a CRE-mediated gene transcription repressor, in neuronal plasticity and LTM, we generated two types of ICER mutant mice: ICER-overexpressing (OE) mice and ICER-specific knock-out (KO) mice. Both ICER-OE and ICER-KO mice show no apparent abnormalities in their development and reproduction. A comprehensive battery of behavioral tests revealed no robust changes in locomotor activity, sensory and motor functions, and emotional responses in the mutant mice. However, long-term conditioned fear memory was attenuated in ICER-OE mice and enhanced in ICER-KO mice without concurrent changes in short-term fear memory. Furthermore, ICER-OE mice exhibited retardation of kindling development, whereas ICER-KO mice exhibited acceleration of kindling. These results strongly suggest that ICER negatively regulates the neuronal processes required for long-term fear memory and neuronal plasticity underlying kindling epileptogenesis, possibly through suppression of CRE-mediated gene transcription.

Drebrin A Content Correlates With Spine Head Size in the Adult Mouse Cerebral Cortex

Synaptic activities alter synaptic strengths at the axospinous junctions, and such changes are often accompanied by changes in the size of the postsynaptic spines. We have been exploring the idea that drebrin A, a neuron‐specific actin‐binding protein localized on the postsynaptic side of excitatory synapses, may be a molecule that links synaptic activity to the shape and content of spines. Here, we performed electron microscopic immunocytochemistry with the nondiffusible gold label to explore the relationship among levels of drebrin A, the NR2A subunit of N‐methyl‐D‐aspartate receptors, and the size of spines in the perirhinal cortex of adult mouse brains. In contrast to the membranous localization within neonatal spines, most immunogold particles for drebrin A were localized to the cytoplasmic core region of spines in mature spines. This distribution suggests that drebrin within adult spines may reorganize the F‐actin network at the spine core, in addition to its known neonatal role in spine formation. Drebrin A‐immunopositive (DIP) spines exhibited larger spine head areas and longer postsynaptic densities (PSDs) than drebrin A‐immunonegative (DIN) spines (P < 0.001). Furthermore, spine head area and PSD lengths correlated positively with drebrin A levels (r = 0.47 and 0.40). The number of synaptic NR2A immunolabels was also higher in DIP spines than in DIN spines, whereas their densities per unit lengths of PSD were not significantly different. These differences between the DIP and the DIN spines indicate that spine sizes and synaptic protein composition of mature brains are regulated, at least in part, by drebrin A levels. J. Comp. Neurol. 503:618–626, 2007.

Nucleotide sequences of two embryonic drebrins, developmentally regulated brain proteins, and developmental change in their mRNAs

Drebrins are developmentally regulated proteins found in chicken brain and are classified into two forms of the embryonic type (E1 and E2) and one form of the adult type (A). Although the time courses of their appearance are different from each other, the structures of the 3 forms are closely related. Two kinds of drebrin cDNA, designated gDcw6 and gDcw17, were isolated from the cDNA library of the chicken embryo and their nucleotide sequences were determined. Their sequences were entirely identical except for a deletion of an internal 129-nucleotide sequence, and the gDcw17 insert contained an open reading frame capable of encoding 607 amino acids. These cDNAs seemed to correspond to two embryonic forms of drebrin mRNAs. The predicted drebrin molecules are highly hydrophilic and have proline-rich sequences and long stretches of glutamate in the carboxyl-terminal region. RNA dot-blot analysis using the drebrin cDNA as a probe demonstrated that the amounts of drebrin mRNAs were also developmentally regulated as those of drebrins. Southern blot analysis showed that the chicken genome has a single copy of the drebrin gene per haploid complement. These findings suggest that the multiple forms of drebrins result from alternative splicing of the single drebrin gene during neural development.