Increased levels of acidic calponin during dendritic spine plasticity after pilocarpine-induced seizures

We have previously shown that, in HEK 293 cells, overexpression of acidic calponin, an actin-binding protein, induces remodeling of actin filaments, leading to a change in cell morphology. In addition, this protein is found in dendritic spines of adult hippocampal neurons. We hypothesized that this protein plays a role in regulating actin-based filaments during dendritic spine plasticity. To assess this hypothesis, the pilocarpine model of temporal lobe epilepsy was selected because an important reorganization of the glutamatergic network, which includes an aberrant sprouting of granule cell axons, neo-synaptogenesis, and dendritic spine remodeling, is well established in the dentate gyrus. This reorganization begins after the initial period of status epilepticus after pilocarpine injection, during the silent period when animals display a normal behavior, and reaches a plateau at the chronic stage when the animals have developed spontaneous recurrent seizures. Our data show that the intensity of immunolabeling for acidic calponin was clearly increased in the inner one-third of the molecular layer of the dentate gyrus, the site of mossy fiber sprouting, and neo-synaptogenesis, at 1 and 2 weeks after pilocarpine injection (silent period) when the reorganization was taking place. In contrast, in chronic pilocarpine-treated animals, when the reorganization was established, the levels of labeling for acidic calponin in the inner molecular layer were similar to those observed in control rats. In addition, double immunostaining studies suggested that the increase in acidic calponin levels occurred within the dendritic spines. Altogether, these results are consistent with an involvement of acidic calponin in dendritic spine plasticity.     

Actin dynamics in dendritic spines: a form of regulated plasticity at excitatory synapses

Dendritic spines form the postsynaptic element at most excitatory synapses in the brain. The spine cytoskeleton consists of actin filaments which, in time-lapse recordings of living neurons expressing actin labeled with green fluorescent protein, can be seen to undergo rapid, dynamic changes. Because actin dynamics are associated with changes in cell shape, these cytoskeletal rearrangements may form a molecular basis for the morphological plasticity at brain synapses. The rapidity of these dynamic events in dendritic spines raises new questions. First, do the changes in actin cytoskeleton that are visible by light microscopy really correspond to changes in spine morphology, or do they represent changes in the relationship between actin and its many binding partners at postsynaptic sites? Second, how are these changes regulated by synaptic transmission? Third, to what extent do these changes occur in organized brain tissue? Answers to these questions are now beginning to emerge.     

Synaptic Plasticity,a Symphony in GEF

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Abl2:Cortactin Interactions Regulate Dendritic Spine Stability via Control of a Stable Filamentous Actin Pool

Dendritic spines act as the receptive contacts at most excitatory synapses. Spines are enriched in a network of actin filaments comprised of two kinetically distinct pools. The majority of spine actin is highly dynamic and regulates spine size, structural plasticity, and postsynaptic density organization. The remainder of the spine actin network is more stable, but the function of this minor actin population is not well understood, as tools to study it have not been available. Previous work has shown that disruption of the Abl2/Arg nonreceptor tyrosine kinase in mice compromises spine stability and size. Here, using cultured hippocampal neurons pooled from both sexes of mice, we provide evidence that binding to cortactin tethers Abl2 in spines, where Abl2 and cortactin maintain the small pool of stable actin required for dendritic spine stability. Using fluorescence recovery after photobleaching of GFP-actin, we find that disruption of Abl2:cortactin interactions eliminates stable actin filaments in dendritic spines, significantly reducing spine density. A subset of spines remaining after Abl2 depletion retain their stable actin pool and undergo activity-dependent spine enlargement, associated with increased cortactin and GluN2B levels. Finally, tonic increases in synaptic activity rescue spine loss following Abl2 depletion by promoting cortactin enrichment in vulnerable spines. Together, our findings strongly suggest that Abl2:cortactin interactions promote spine stability by maintaining pools of stable actin filaments in spines.SIGNIFICANCE STATEMENT Dendritic spines contain two kinetically distinct pools of actin. The more abundant, highly dynamic pool regulates spine shape, size, and plasticity. The function of the smaller, stable actin network is not well understood, as tools to study it have not been available. We demonstrate here that Abl2 and its substrate and interaction partner, cortactin, are essential to maintain the stable pool in spines. Depletion of the stable actin pool via disruption of Abl2 or cortactin, or interactions between the proteins, significantly reduces spine stability. We also provide evidence that tonic increases in synaptic activity promote spine stability via enrichment of cortactin in spines, suggesting that synaptic activity acts on the stable actin pool to stabilize dendritic spines.     

Interactions between drebrin and Ras regulate dendritic spine plasticity

Dendritic spines are major sites of morphological plasticity in the CNS, but the molecular mechanisms that regulate their dynamics remain poorly understood. Here we show that the association of drebrin with actin filaments plays a major role in regulating dendritic spine stability and plasticity. Overexpressing drebrin or the internal actin-binding site of drebrin in rat hippocampal neurons destabilized mature dendritic spines so that they lost synaptic contacts and came to resemble immature dendritic filopodia. Drebrin-induced spine destabilization was dependent on Ras activation: expression of constitutively active Ras destabilized spine morphology whereas drebrin-induced spine destabilization was rescued by co-expressing dominant negative Ras. Conversely, RNAi-mediated drebrin knockdown prevented Ras-induced destabilization and promoted spine maturation in developing neurons. Together these data demonstrate a novel mechanism in which the balance between stability and plasticity in dendritic spines depends on binding of drebrin to actin filaments in a manner that is regulated by Ras.     

Plasticity of dendritic spines: Molecular function and dysfunction in neurodevelopmental disorders

Dendritic spines are tiny postsynaptic protrusions from a dendrite that receive most of the excitatory synaptic input in the brain. Functional and structural changes in dendritic spines are critical for synaptic plasticity, a cellular model of learning and memory. Conversely, altered spine morphology and plasticity are common hallmarks of human neurodevelopmental disorders, such as intellectual disability and autism. The advances in molecular and optical techniques have allowed for exploration of dynamic changes in structure and signal transduction at single‐spine resolution, providing significant insights into the molecular regulation underlying spine structural plasticity. Here, I review recent findings on: how synaptic stimulation leads to diverse forms of spine structural plasticity; how the associated biochemical signals are initiated and transmitted into neuronal compartments; and how disruption of single genes associated with neurodevelopmental disorders can lead to abnormal spine structure in human and mouse brains. In particular, I discuss the functions of the Ras superfamily of small GTPases in spatiotemporal regulation of the actin cytoskeleton and protein synthesis in dendritic spines. Multiple lines of evidence implicate disrupted Ras signaling pathways in the spine structural abnormalities observed in neurodevelopmental disorders. Both deficient and excessive Ras activities lead to disrupted spine structure and deficits in learning and memory. Dysregulation of spine Ras signaling, therefore, may play a key role in the pathogenesis of multiple neurodevelopmental disorders with distinct etiologies.     

Microanatomy of dendritic spines: emerging principles of synaptic pathology in psychiatric and neurological disease.

Psychiatric and neurologic disorders ranging from mental retardation to addiction are accompanied by structural and functional alterations of synaptic connections in the brain. Such alterations include abnormal density and morphology of dendritic spines, synapse loss, and aberrant synaptic signaling and plasticity. Recent work is revealing an unexpectedly complex biochemical and subcellular organization of dendritic spines. In this review, we highlight the molecular interplay between functional domains of the spine, including the postsynaptic density, the actin cytoskeleton, and membrane trafficking domains. This research points to an emerging level of analysis--a microanatomical understanding of synaptic physiology--that will be critical for discerning how synapses operate in normal physiologic states and for identifying and reversing microscopic changes in psychiatric and neurologic disease.     

Potential role of synaptopodin in spine motility by coupling actin to the spine apparatus

Dendritic spines are dynamic structures that rapidly remodel their shape and size. These morphological adaptations are regulated by changes in synaptic activity, and result from rearrangements of the postsynaptic cytoskeleton. A cytoskeletal molecule preferentially found in mature spines is the actin-associated protein synaptopodin. It is strongly expressed by spine-bearing neurons in the olfactory bulb, striatum, cerebral cortex, and hippocampus. In the hippocampus, principal cells express synaptopodin mRNA and sort the protein to the spine compartment. Within the spine microdomain, synaptopodin is preferentially located in the spine neck and is closely associated with the spine apparatus. On the basis of these data we hypothesize that synaptopodin could affect spine motility by bundling actin filaments in the spine neck. In addition, it could link the actin cytoskeleton of spines to intracellular calcium stores, i.e., the spine apparatus and the smooth endoplasmic reticulum.     

The actin-binding protein profilin I is localized at synaptic sites in an activity-regulated manner

Morphological changes at synaptic specializations have been implicated in regulating synaptic strength. Actin turnover at dendritic spines is regulated by neuronal activity and contributes to spine size, shape and motility. The reorganization of actin filaments requires profilins, which stimulate actin polymerization. Neurons express two independent gene products - profilin I and profilin II. A role for profilin II in activity-dependent mechanisms at spine synapses has recently been described. Although profilin I interacts with synaptic proteins, little is known about its cellular and subcellular localization in neurons. Here, we investigated the subcellular distribution of this protein in brain neurons as well as in hippocampal cultures. Our results indicate that the expression of profilin I varies in different brain regions. Thus, in cerebral cortex and hippocampus profilin I immunostaining was associated predominantly with dendrites and was present in a subset of dendritic spines. In contrast, profilin I in cerebellum was associated primarily with presynaptic structures. Profilin I immunoreactivity was partially colocalized with the synaptic molecules synaptophysin, PSD-95 and gephyrin in cultured hippocampal neurons, indicating that profilin I is present in only a subset of synapses. At dendritic spine structures, profilin I was found primarily in protrusions, which were in apposition to presynaptic terminal boutons. Remarkably, depolarization with KCl caused a moderate but significant increase in the number of synapses containing profilin I. These results show that profilin I can be present at both pre- and postsynaptic sites and suggest a role for this actin-binding protein in activity-dependent remodelling of synaptic structure.     

Regulation of spine morphology and synaptic function by LIMK and the actin cytoskeleton

Filamentous actin (F-actin) is highly enriched in the dendritic spine, a specialized postsynaptic structure on which the great majority of the excitatory synapses are formed in the mammalian central nervous system (CNS). The protein kinases of the Lim-kinase (LIMK) family are potent regulators of actin dynamics in many cell types and they are abundantly expressed in the CNS, including the hippocampus. Using a combination of genetic manipulations and electrophysiological recordings in mice, we have demonstrated that LIMK-1 signaling is important in vivo in the regulation of the actin cytoskeleton, spine morphology, and synaptic function, including hippocampal long-term potentiation (LTP), a prominent form of long lasting synaptic plasticity thought to be critical to memory formation. Our results provide strong genetic evidence that LIMK and its substrate ADF/cofilin are involved in spine morphology and synaptic properties and are consistent with the notion that the Rho family small GTPases and the actin cytoskeleton are critical to spine structure and synaptic regulation.     

Selective localization of high concentrations of F-actin in subpopulations of dendritic spines in rat central nervous system: a three-dimensional electron microscopic study

Dendritic spines differ considerably in their size, shape, and internal organization between brain regions. We examined the actin cytoskeleton in dendritic spines in hippocampus (areas CA1, CA3, and dentate gyrus), neostriatum, and cerebellum at both light and electron microscopic levels by using a novel high-resolution photoconversion method based in the high affinity of phalloidin for filamentous (F)-actin. In all brain regions, labeling was strongest in the heads of dendritic spines, diminishing in the spine neck. The number of labeled spines varied by region. Compared with the cerebellar molecular layer and area CA3, where nearly every dendritic spine was labeled, less than half the spines were labeled in CA1, dentate gyrus, and neostriatum. Serial section reconstructions of spines in these areas indicated that phalloidin labeling was restricted to the largest and most morphologically diverse dendritic spines. The resolution of the photoconversion technique allowed us to examine the localization and organization of actin filaments in the spine. The most intense staining for actin was found in the postsynaptic density and associated with the spines internal membrane system. In mushroom-shaped spines, F-actin staining was particularly strong between the lamellae of the spine apparatus. Three-dimensional reconstruction of labeled spines by using electron tomography showed that the labeled dense material was in continuity with the postsynaptic density. These results highlight differences in the actin cytoskeleton between different spine populations and provide novel information on the organization of the actin cytoskeleton in vivo.     

Chronic Alcohol Alters Dendritic Spine Development in Neurons in Primary Culture

Dendritic spines are specialised membrane protrusions of neuronal dendrites that receive the majority of excitatory synaptic inputs. Abnormal changes in their density, size and morphology have been associated with various neurological and psychiatric disorders, including those deriving from drug addiction. Dendritic spine formation, morphology and synaptic functions are governed by the actin cytoskeleton. Previous in vivo studies have shown that ethanol alters the number and morphology of spines, although the mechanisms underlying these alterations remain unknown. It has also been described how chronic ethanol exposure affects the levels, assembly and cellular organisation of the actin cytoskeleton in hippocampal neurons in primary culture. Therefore, we hypothesised that the ethanol-induced alterations in the number and shape of dendritic spines are due to alterations in the mechanisms regulating actin cytoskeleton integrity. The results presented herein show that chronic exposure to moderate levels of alcohol (30 mM) during the first 2 weeks of culture reduces dendritic spine density and alters the proportion of the different morphologies of these structures in hippocampal neurons, which affects the formation of mature spines. Apparently, these effects are associated with an increase in the G-actin/F-actin ratio due to a reduction of the F-actin fraction, leading to changes in the levels of the different factors regulating the organisation of this cytoskeletal component. The data presented herein indicate that these effects occur between weeks 1 and 2 of culture, an important period in dendritic spines development. These changes may be related to the dysfunction in the memory and learning processes present in children prenatally exposed to ethanol.     

Postsynaptic PDLIM5/Enigma Homolog binds SPAR and causes dendritic spine shrinkage

Dendritic spine morphology is thought to play important roles in synaptic development and plasticity, and morphological derangements in spines are correlated with several neurological disorders. Here, we identified an interaction between Spine-Associated RapGAP (SPAR), a postsynaptic protein that reorganizes actin cytoskeleton and drives dendritic spine head growth, and PDLIM5/Enigma Homolog (ENH), a PDZ-LIM (postsynaptic density-95/Discs large/zona occludens 1-Lin11/Isl-1/Mec3) family member. PDLIM5 has been implicated in susceptibility to bipolar disorder, major depression, and schizophrenia, but its function in neurological disease is poorly understood. We show that PDLIM5 is present in the postsynaptic density, where it promotes decreased dendritic spine head size and longer, filopodia-like morphology. Conversely, RNA interference against PDLIM5 or loss of PDLIM5 interaction with SPAR caused increased spine head diameter. Furthermore, PKC activation promoted delivery of PDLIM5 into dendritic spines and increased its spine colocalization with SPAR. These data reveal new postsynaptic functions for PDLIM5 in shrinkage of dendritic spines that may be relevant to its association with psychiatric illness.     

Spine architecture and synaptic plasticity

Many forms of mental retardation and cognitive disability are associated with abnormalities in dendritic spine morphology. Visualization of spines using live-imaging techniques provides convincing evidence that spine morphology is altered in response to certain forms of LTP-inducing stimulation. Thus, information storage at the cellular level appears to involve changes in spine morphology that support changes in synaptic strength produced by certain patterns of synaptic activity. Because the structure of a spine is determined by its underlying actin cytoskeleton, there has been much effort to identify signaling pathways linking synaptic activity to control of actin polymerization. This review, part of the TINS Synaptic Connectivity series, discusses recent studies that implicate EphB and NMDA receptors in the regulation of actin-binding proteins through modulation of Rho family small GTPases.     

PTEN knockdown alters dendritic spine/protrusion morphology,not density

Mutations in phosphatase and tensin homolog deleted on chromosome 10 (PTEN) are implicated in neuropsychiatric disorders including autism. Previous studies report that PTEN knockdown in neurons in vivo leads to increased spine density and synaptic activity. To better characterize synaptic changes in neurons lacking PTEN, we examined the effects of shRNA knockdown of PTEN in basolateral amygdala neurons on synaptic spine density and morphology by using fluorescent dye confocal imaging. Contrary to previous studies in the dentate gyrus, we find that knockdown of PTEN in basolateral amygdala leads to a significant decrease in total spine density in distal dendrites. Curiously, this decreased spine density is associated with increased miniature excitatory postsynaptic current frequency and amplitude, suggesting an increase in number and function of mature spines. These seemingly contradictory findings were reconciled by spine morphology analysis demonstrating increased mushroom spine density and size with correspondingly decreased thin protrusion density at more distal segments. The same analysis of PTEN conditional deletion in the dentate gyrus demonstrated that loss of PTEN does not significantly alter total density of dendritic protrusions in the dentate gyrus, but does decrease thin protrusion density and increases density of more mature mushroom spines. These findings suggest that, contrary to previous reports, PTEN knockdown may not induce de novo spinogenesis, but instead may increase synaptic activity by inducing morphological and functional maturation of spines. Furthermore, behavioral analysis of basolateral amygdala PTEN knockdown suggests that these changes limited only to the basolateral amygdala complex may not be sufficient to induce increased anxiety‐related behaviors. J. Comp. Neurol. 522:1171–1190, 2014. © 2013 Wiley Periodicals, Inc.     

Myosin IIb controls actin dynamics underlying the dendritic spine maturation

Precise control of the formation and development of dendritic spines is critical for synaptic plasticity. Consequently, abnormal spine development is linked to various neurological disorders. The actin cytoskeleton is a structural element generating specific changes in dendritic spine morphology. Although mechanisms underlying dendritic filopodia elongation and spine head growth are relatively well understood, it is still not known how spine heads are enlarged and stabilized during dendritic spine maturation. By using rat hippocampal neurons, we demonstrate that the size of the stable actin pool increases during the neuronal maturation process. Simultaneously, the treadmilling rate of the dynamic actin pool increases. We further show that myosin IIb controls dendritic spine actin cytoskeleton by regulating these two different pools of F-actin via distinct mechanisms. The findings indicate that myosin IIb stabilizes the stable F-actin pool through actin cross-linking. Simultaneously, activation of myosin IIb contractility increases the treadmilling rate of the dynamic pool of actin. Collectively, these data show that myosin IIb has a major role in the regulation of actin filament stability in dendritic spines, and elucidate the complex mechanism through which myosin IIb functions in this process. These new insights into the mechanisms underlying dendritic spine maturation further the model of dendritic spine morphogenesis.     

Vezatin is essential for dendritic spine morphogenesis and functional synaptic maturation

Vezatin is an integral membrane protein associated with cell-cell adhesion complex and actin cytoskeleton. It is expressed in the developing and mature mammalian brain, but its neuronal function is unknown. Here, we show that Vezatin localizes in spines in mature mouse hippocampal neurons and codistributes with PSD95, a major scaffolding protein of the excitatory postsynaptic density. Forebrain-specific conditional ablation of Vezatin induced anxiety-like behavior and impaired cued fear-conditioning memory response. Vezatin knock-down in cultured hippocampal neurons and Vezatin conditional knock-out in mice led to a significantly increased proportion of stubby spines and a reduced proportion of mature dendritic spines. PSD95 remained tethered to presynaptic terminals in Vezatin-deficient hippocampal neurons, suggesting that the reduced expression of Vezatin does not compromise the maintenance of synaptic connections. Accordingly, neither the amplitude nor the frequency of miniature EPSCs was affected in Vezatin-deficient hippocampal neurons. However, the AMPA/NMDA ratio of evoked EPSCs was reduced, suggesting impaired functional maturation of excitatory synapses. These results suggest a role of Vezatin in dendritic spine morphogenesis and functional synaptic maturation.     

Diversity and fluctuation of spine morphology in CA1 pyramidal neurons after transient global ischemia

Dendritic spines form postsynaptic components of excitatory synapses in CA1 pyramidal neurons and play a key role in excitatory signal transmission. Transient global ischemia is thought to induce excitotoxicity that triggers delayed neuronal death in the CA1 region. However, the mechanism underlying structural changes of excitatory synapses after ischemia is not completely understood. Here, we demonstrate how dendritic spines change in their density and structure at an acute stage after transient global ischemia. Intracellular staining in vivo showed that the total spine density in basal, proximal, and distal apical dendrites increased at 12 hr and 24 hr after ischemia, but returned to control levels at 48 hr after ischemia. Consistent increase of spine density mainly appeared in non-late depolarizing postsynaptic potential neurons, although late depolarizing postsynaptic potential neurons also showed slight increases in spine density in these dendrites at the same intervals after ischemia. Golgi staining showed increased spine density occurred in less swollen dendrites but decreased spine density appeared in severely swollen dendrites at 12 and 24 hr after ischemia. In addition, the density and percentage of stubby spines reduced at 12 hr and 48 hr, whereas the density of thin spines increased at 12 hr after ischemia. The density and percentage of filopodia increased nearly fivefold at 24 hr after ischemia. Moreover, the density of mushroom spines doubled and its percentage increased by 150% at 48 hr after ischemia. These morphological changes of spines may be related to neuronal injury in CA1 pyramidal neurons after ischemia.     

Behaviorally evoked transient reorganization of hippocampal spines

Dendritic spines, microstructures that receive the majority of excitatory synaptic inputs, are fundamental units to integrate and store neuronal information. The morphological reorganization of spines accompanies the functional alterations in synaptic strength underlying memory-relevant modifications of network connectivity. Here we report the rapid dynamics of cell population-selective spine reorganizations related to behavioral experiences. In Thy1-GFP transgenic mice, hippocampal CA1 pyramidal neurons that were putatively activated during environmental explorations were detected with their post hoc immunoreactivity for Arc, an activity-dependent immediately-early gene. Immediately after a 60-min exposure to a familiar environment, the spine densities of Arc-positive and Arc-negative neurons were differently distributed. This density imbalance was due exclusively to changes in the number of small, rather than large, spines. The change disappeared within 60 min after mice were returned to the home cages. Thus, spines possess the ability to rapidly and reversibly alter their morphology in response to a brief environmental change. We propose that these transient spine dynamics represent a latent preliminary stage for longer-term plasticity on demand.