Noriko Koganezawa

The role of drebrin in dendritic spines

ABSTRACT Dendritic spines form typical excitatory synapses in the brain and their shapes vary depending on synaptic inputs. It has been suggested that the morphological changes of dendritic spines play an important role in synaptic plasticity. Dendritic spines contain a high concentration of actin, which has a central role in supporting cell motility, and polymerization of actin filaments (F‐actin) is most likely involved in spine shape changes. Drebrin is an actin‐binding protein that forms stable F‐actin and is highly accumulated within dendritic spines. Drebrin has two isoforms, embryonic‐type drebrin E and adult‐type drebrin A, that change during development from E to A. Inhibition of drebrin A expression results in a delay of synapse formation and inhibition of postsynaptic protein accumulation, suggesting that drebrin A has an important role in spine maturation. In mature synapses, glutamate stimulation induces rapid spine‐head enlargement during long‐term potentiation (LTP) formation. LTP stimulation induces Ca2+ entry through N‐methyl‐d‐aspartate (NMDA) receptors, which causes drebrin exodus from dendritic spines. Once drebrin exits from dendritic spine heads, the dynamic actin pool increases in spine heads to facilitate F‐actin polymerization. To maintain enlarged spine heads, drebrin‐decorated F‐actin is thought to reform within the spine heads. Thus, drebrin plays a pivotal role in spine plasticity through regulation of F‐actin. HighlightsIn dendritic spines, there are distinct dynamic and stable pools of F‐actin.Drebrin, an actin‐binding protein, forms stable F‐actin.Drebrin‐decorated F‐actin may form a platform for postsynaptic protein assembly.Exodus of drebrin‐decorated F‐actin occurs during LTP initiation.Deficits in LTP formation in drebrin‐A knockout mice lead to fear memory impairment.

The role of drebrin in neurons

Drebrin is an actin‐binding protein that changes the helical pitch of actin filaments (F‐actin), and drebrin‐decorated F‐actin shows slow treadmilling and decreased rate of depolymerization. Moreover, the characteristic morphology of drebrin‐decorated F‐actin enables it to respond differently to the same signals from other actin cytoskeletons. Drebrin consists of two major isoforms, drebrin E and drebrin A. In the developing brain, drebrin E appears in migrating neurons and accumulates in the growth cones of axons and dendrites. Drebrin E‐decorated F‐actin links lamellipodium F‐actin to microtubules in the growth cones. Then drebrin A appears at nascent synapses and drebrin A‐decorated F‐actin facilitates postsynaptic molecular assembly. In the adult brain, drebrin A‐decorated F‐actin is concentrated in the central region of dendritic spines. During long‐term potentiation initiation, NMDA receptor‐mediated Ca2+ influx induces the transient exodus of drebrin A‐decorated F‐actin via myosin II ATPase activation. Because of the unique physical characteristics of drebrin A‐decorated F‐actin, this exodus likely contributes to the facilitation of F‐actin polymerization and spine enlargement. Additionally, drebrin reaccumulation in dendritic spines is observed after the exodus. In our drebrin exodus model of structure‐based synaptic plasticity, reestablishment of drebrin A‐decorated F‐actin is necessary to keep the enlarged spine size during long‐term potentiation maintenance. In this review, we introduce the genetic and biochemical properties of drebrin and the roles of drebrin in early stage of brain development, synaptic formation and synaptic plasticity. Further, we discuss the pathological relevance of drebrin loss in Alzheimers disease.

Early-stage development of human induced pluripotent stem cell-derived neurons

Recent advances in human induced pluripotent stem cells (hiPSCs) offer new possibilities for biomedical research and clinical applications. Differentiated neurons from hiPSCs are expected to be useful for developing novel methods of treatment for various neurological diseases. However, the detailed process of functional maturation of hiPSC‐derived neurons (hiPS neurons) remains poorly understood. This study analyzes development of hiPS neurons, focusing specifically on early developmental stages through 48 hr after cell seeding; development was compared with that of primary cultured neurons derived from the rat hippocampus. At 5 hr after cell seeding, neurite formation occurs in a similar manner in both neuronal populations. However, very few neurons with axonal polarization were observed in the hiPS neurons even after 48 hr, indicating that hiPS neurons differentiate more slowly than rat neurons. We further investigated the elongation speed of axons and found that hiPS neuronal axons were slower. In addition, we characterized the growth cones. The localization patterns of skeletal proteins F‐actin, microtubule, and drebrin were similar to those of rat neurons, and actin depolymerization by cytochalasin D induced similar changes in cytoskeletal distribution in the growth cones between hiPS neurons and rat neurons. These results indicate that, during the very early developmental stage, hiPS neurons develop comparably to rat hippocampal neurons with regard to axonal differentiation, but the growth of axons is slower.

Role of Drebrin in Synaptic Plasticity

Synaptic plasticity underlies higher brain function such as learning and memory, and the actin cytoskeleton in dendritic spines composing excitatory postsynaptic sites plays a pivotal role in synaptic plasticity. In this chapter, we review the role of drebrin in the regulation of the actin cytoskeleton during synaptic plasticity, under long-term potentiation (LTP) and long-term depression (LTD). Dendritic spines have two F-actin pools, drebrin-decorated stable F-actin (DF-actin) and drebrin-free dynamic F-actin (FF-actin). Resting dendritic spines change their shape, but are fairly constant over time at steady state because of the presence of DF-actin. Accumulation of DF-actin is inversely regulated by the intracellular Ca2+ concentration. However, LTP and LTD stimulation induce Ca2+ influx through N-methyl-D-aspartate (NMDA) receptors into the potentiated spines, resulting in drebrin exodus via myosin II ATPase activation. The potentiated spines change to excited state because of the decrease in DF-actin and thus change their shape robustly. In LTP, the Ca2+ increase via NMDA receptors soon returns to the basal level, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) expression at the postsynaptic membrane is increased. The Ca2+ recovery and AMPAR increase coordinately induce the re-accumulation of DF-actin and change the dendritic spines from the excited state to steady state during LTP maintenance. During LTD, the prolonged intracellular Ca2+ increase inhibits the re-accumulation of DF-actin, resulting in facilitation of AMPAR endocytosis. Because of the positive feedback loop of the AMPAR decrease and drebrin re-accumulation inhibition, the dendritic spines are instable during LTD maintenance. Taken together, we propose the presence of resilient spines at steady state and plastic spines at excited state and discuss the physiological and pathological relevance of the two-state model to synaptic plasticity.

Drebrin E regulates neuroblast proliferation and chain migration in the adult brain

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.

X Irradiation Induces Acute Cognitive Decline via Transient Synaptic Dysfunction

Cranial X irradiation can severely impair higher brain function, resulting in neurocognitive deficits. Radiation-induced brain injury is characterized by acute, early and late delayed changes, and morbidity is evident more than 6 months after irradiation. While the acute effects of radiation exposure on the brain are known, the underlying mechanisms remain unclear. In this study, we examined the acute effect of X radiation on synaptic function using behavioral analysis and immunohistochemistry. We found that 10 Gy whole-brain irradiation immediately after conditioning (within 30 min) impaired the formation of fear memory, whereas irradiation 24 h prior to conditioning did not. To investigate the mechanisms underlying these behavioral changes, we irradiated one hemisphere of the brain and analyzed synaptic function and adult neurogenesis immunohistochemically. We focused on drebrin, whose loss from dendritic spines is a surrogate marker of synaptopathy. The intensity of drebrin immunoreactivity started to decrease in the irradiated hemisphere 2 h after exposure. The immunostaining intensity recovered to preirradiation levels by 24 h, indicating that X radiation induced transient synaptic dysfunction. Interestingly, the number of newly generated neurons was not changed at 2 h postirradiation, whereas it was significantly decreased at 8 and 24 h postirradiation. Because irradiation 24 h prior to conditioning had no effect on fear memory, our findings suggest that radiation-induced death of newly-generated neurons does not substantially impact fear memory formation. The radiation-induced synaptic dysfunction likely caused a transient memory deficit during the critical period for fear memory formation (approximately 1–3 h after conditioning), which coincides with a change in drebrin immunostaining in the hippocampus, a structure critical for fear memory formation.

The Role of Drebrin-Binding Stable Actin Filaments in Dendritic Spine Morphogenesis

Dendritic spines are the postsynaptic responsive regions of excitatory synapses that play an important role in synaptic transmission or plasticity. They contain high concentrations of actin and vary in their morphology (i.e. spine motility), likely linking the molecular mechanisms of learning and memory. Dendritic spines consist of dynamic and stable F-actin pools. Recent studies suggest that basal motility of dendritic spines occurs due to random supercritical filament nucleation events amplified by autocatalytic branching in the dynamic F-actin pool; drebrin-binding actin filaments in the stable F-actin pool form a cross-linked gel, serving as the structural element for treadmilling of dynamic F-actin to push back against the spine membrane. Increased pitch of helical structures of F-actin by drebrin makes it possible that the drebrin-binding F-actin and the other F-actins respond differently to the same signal within small dendritic spines. Dendritic filopodia serve as the precursor of dendritic spine during neuronal development. Clustering of drebrin-binding F-actin occurs in the dendritic filopodia, resulting in the accumulation of postsynaptic scaffold proteins and NMDA receptors. Interestingly, drebrin changes its isoform from the embryonic-type to the adult type at this time. This conversion is critical for the targeting mechanism of postsynaptic molecules; however, it is not a sufficient condition for postsynaptic formation. Many evidences indicate that AMPA receptors are also needed for the clustering of drebrin at postsynaptic sites. Furthermore, we have recently shown that a new drebrin binding protein, spikar, is involved in the drebrin-mediated spine formation.

An inhibitory pathway controlling the gating mechanism of the mouse lateral amygdala revealed by voltage-sensitive dye imaging

The lateral amygdala nucleus (La) is known as a gateway for emotional learning that interfaces sensory inputs from the cortex and the thalamus. In the La, inhibitory GABAergic inputs control the strength of sensory inputs and interfere with the initial step of the acquisition of fear memory. In the present study, we investigated the spatial and temporal patterns of the inhibitory responses in mouse La using voltage-sensitive dye imaging. Stimulating the external capsule (EC) induced large and long-lasting hyperpolarizing signals in the La. We focused on these hyperpolarizing signals, revealing the origins of the inhibitory inputs by means of surgical cuts on the possible afferent pathways with four patterns. Isolating the medial branch of EC (ECmed), but not the lateral branch of EC (EClat), from the La strongly suppressed the induction of the hyperpolarization. Interestingly, isolating the ECmed from the caudate putamen did not suppress the hyperpolarization, while the surgical cut of the ECmed fiber tract moderately suppressed it. Glutamatergic antagonists completely suppressed the hyperpolarizing signals induced by the stimulation of EC. When directly stimulating the dorsal, middle or ventral part of the ECmed fiber tract in the presence of glutamatergic antagonists, only the stimulation in the middle part of the ECmed caused hyperpolarization. These data indicate that the GABAergic neurons in the medial intercalated cluster (m-ITC), which receive glutamatergic excitatory input from the ECmed fiber tract, send inhibitory afferents to the La. This pathway might have inhibitory effects on the acquisition of fear memory.

Localization of Drebrin: Light Microscopy Study

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.