Maintenance Of Dendritic Spines Research Articles

NMDA Receptor GluN2B (GluRε2/NR2B) Subunit Is Crucial for Channel Function, Postsynaptic Macromolecular Organization, and Actin Cytoskeleton at Hippocampal CA3 Synapses

GluN2B (GluRepsilon2/NR2B) subunit is involved in synapse development, synaptic plasticity, and cognitive function. However, its roles in synaptic expression and function of NMDA receptors (NMDARs) in the brain remain mostly unknown because of the neonatal lethality of global knock-out mice. To address this, we generated conditional knock-out mice, in which GluN2B was ablated exclusively in hippocampal CA3 pyramidal cells. By immunohistochemistry, GluN2B disappeared and GluN1 (GluRzeta1/NR1) was moderately reduced, whereas GluN2A (GluRepsilon1/NR2A) and postsynaptic density-95 (PSD-95) were unaltered in the mutant CA3. This was consistent with protein contents in the CA3 crude fraction: 9.6% of control level for GluN2B, 47.7% for GluN1, 90.6% for GluN2A, and 98.0% for PSD-95. Despite the remaining NMDARs, NMDAR-mediated currents and long-term potentiation were virtually lost at various CA3 synapses. Then, we compared synaptic NMDARs by postembedding immunogold electron microscopy and immunoblot using the PSD fraction. In the mutant CA3, GluN1 was severely reduced in both immunogold (20.6-23.6%) and immunoblot (24.6%), whereas GluN2A and PSD-95 were unchanged in immunogold but markedly reduced in the PSD fraction (51.4 and 36.5%, respectively), indicating increased detergent solubility of PSD molecules. No such increased solubility was observed for GluN2B in the CA3 of GluN2A-knock-out mice. Furthermore, significant decreases were found in the ratio of filamentous to globular actin (49.5%) and in the density of dendritic spines (76.2%). These findings suggest that GluN2B is critically involved in NMDAR channel function, organization of postsynaptic macromolecular complexes, formation or maintenance of dendritic spines, and regulation of the actin cytoskeleton.

A lipid kinase controls the maintenance of dendritic spines.

A lipid kinase controls the maintenance of dendritic spines.

Establishing a link between small GTPases and mental health

In Fragile X syndrome (the most common type of inherited mental retardation) cognitive impairment is not associated with brain malformations or neuro‐degeneration, but with deficiency in neuronal connectivity due to aberrant spine morphology. Although abnormalities in spines have been associated with impaired cognition in mental retardation, how they generated is not yet well understood. Evidence is pointing to the Rho family of small GTPases (Rac, Rho, Cdc42), which mediate actin reorganization and neuronal morphogenesis. Neuronal plasticity requires actin remodeling and local protein translation. Rac plays a key role in actin remodeling, formation, maturation and maintenance of dendritic spines. By combining biochemistry, electrophysiology and behavioral studies coupled with the use of a Fmr1 knockout mouse as animal model of Fragile X, our investigations suggest that under normal conditions, FMRP (protein missing in Fragile X syndrome) acts as a regulator on the synthesis of Rac. In contrast, in Fmr1 knockout mice, lack of FMRP induces an increase of Rac in brain, leading to abnormal actin remodeling, generating the anomalies reported in the structure of dendritic spines, and resulting in cognitive deficiencies. Additionally, pharmacological manipulations of Rac improve abnormalities observed in Fmr1 knockout mice.

Synaptic removal of diacylglycerol by DGKζ and PSD-95 regulates dendritic spine maintenance

Diacylglycerol (DAG) is an important lipid signalling molecule that exerts an effect on various effector proteins including protein kinase C. A main mechanism for DAG removal is to convert it to phosphatidic acid (PA) by DAG kinases (DGKs). However, it is not well understood how DGKs are targeted to specific subcellular sites and tightly regulates DAG levels. The neuronal synapse is a prominent site of DAG production. Here, we show that DGKzeta is targeted to excitatory synapses through its direct interaction with the postsynaptic PDZ scaffold PSD-95. Overexpression of DGKzeta in cultured neurons increases the number of dendritic spines, which receive the majority of excitatory synaptic inputs, in a manner requiring its catalytic activity and PSD-95 binding. Conversely, DGKzeta knockdown reduces spine density. Mice deficient in DGKzeta expression show reduced spine density and excitatory synaptic transmission. Time-lapse imaging indicates that DGKzeta is required for spine maintenance but not formation. We propose that PSD-95 targets DGKzeta to synaptic DAG-producing receptors to tightly couple synaptic DAG production to its conversion to PA for the maintenance of spine density.

Regulation of Kalirin by Cdk5

Kalirin, one of the few Rho guanine nucleotide exchange factors (GEFs) that contains spectrin-like repeats, plays a critical role in axon extension and maintenance of dendritic spines. PC12 cells were used to determine whether Cdk5, a critical participant in both processes, regulates the action of Kalirin. Expression of Kalirin-7 in nondifferentiated PC12 cells caused GEF-activity-dependent extension of broad cytoplasmic protrusions; coexpression of dominant-negative Cdk5 largely eliminated this response. The spectrin-like repeat region of Kalirin plays an essential role in this response, which is not mimicked by the GEF domain alone. Thr1590, which follows the first GEF domain of Kalirin, is the only Cdk5 phosphorylation site in Kalirin-7. Although mutant Kalirin-7 with Ala1590 retains GEF activity, it is unable to cause extension of protrusions. Kalirin-7 with an Asp1590 mutation has slightly increased GEF activity and dominant-negative Cdk5 fails to block its ability to cause extension of protrusions. Phosphorylation of Thr1590 causes a slight increase in GEF activity and Kalirin-7 solubility. Dendritic spines formed by cortical neurons in response to the expression of Kalirin-7 with Ala1590 differ in shape from those formed in response to wild-type Kalirin-7 or Kalirin-7 containing Asp1590. The presence of Thr1590 in each major Kalirin isoform would allow Cdk5 to regulate Kalirin function throughout development.

The RhoA‐associated protein Citron‐N controls dendritic spine maintenance by interacting with spine‐associated Golgi compartments

Dendritic spines are highly dynamic protuberances that are thought to be crucial for learning and memory. Although it is well known that actin filaments and membrane dynamics regulate spine plasticity, how these two events are linked locally is less clear. Here, we provide evidence that Citron-N (CIT-N), a binding partner of the small GTPase RhoA, is associated with the actin filaments and Golgi compartments of dendritic spines. We also show that CIT-N is required for recruiting F-actin and Golgi membranes at spines of in vitro-grown neurons. Studies in knockout mice show that this protein is essential for the maturation of dendritic spines. We suggest that CIT-N might function as a scaffold protein in spine organization through its ability to bind to Golgi membranes and by affecting actin remodelling.

The PAR-6 Polarity Protein Regulates Dendritic Spine Morphogenesis through p190 RhoGAP and the Rho GTPase

The majority of excitatory synaptic transmission in the brain occurs at dendritic spines, which are actin-rich protrusions on the dendrites. The asymmetric nature of these structures suggests that proteins regulating cell polarity might be involved in their formation. Indeed, the polarity protein PAR-3 is required for normal spine morphogenesis. However, this function is independent of association with atypical protein kinase C (aPKC) and PAR-6. Here we show that PAR-6 together with aPKC plays a distinct but essential role in spine morphogenesis. Knockdown of PAR-6 inhibits spine morphogenesis, whereas overexpression of PAR-6 increases spine density, and these effects are mediated by aPKC. Using a FRET biosensor, we further show that p190 RhoGAP and RhoA act downstream of the PAR-6/aPKC complex. These results define a role for PAR-6 and aPKC in dendritic spine biogenesis and maintenance, and reveal an unexpected link between the PAR-6/aPKC complex and RhoA activity.

Activation of α2A adrenoceptors alters dendritic spine development and the expression of spinophilin in cultured cortical neurones

α2 adrenoceptors have been shown to regulate the development of dendrites in mammalian cortical neurones. In this study we have investigated how agonists of α2 adrenoceptors affect length and density of dendritic spines in cultured cortical neurones from C57/B6 mice. A twenty-four hour incubation of 14 day old cultured neurones with UK 14304, an α2-adrenoceptor agonist, resulted in a significant increase in the average length and density of dendritic spines. Furthermore, incubation of neurones with the selective α2A agonist guanfacine resulted in 1.2-fold increase in spine length and 1.8-fold increase in spine density. These effects were blocked by RX 821002 and BRL 44408, α2- and α2A-adrenoceptor antagonists, respectively. The observed changes in the density and length of dendritic spines were correlated with increased expression of spinophilin, a key cytoskeletal protein in the formation and maintenance of dendritic spines, and a decrease in the phosphorylation of spinophilin on serine residues. The latter finding points to a possible mechanism by which adrenoceptors may regulate spinophilin function in dendritic spine development and structure in cortical neurones in vitro.

Synaptic modifications at the CA3–CA1 synapse after chronic AMPA receptor blockade in rat hippocampal slices

Maintenance of dendritic spines, the postsynaptic elements of most glutamatergic synapses in the central nervous system, requires continued activation of AMPA receptors. In organotypic hippocampal slice cultures, chronic blockade of AMPA receptors for 14 days induces a substantial loss of dendritic spines on CA1 pyramidal neurons. Here, using serial section electron microscopy, we show that loss of dendritic spines is paralleled by a significant reduction in synapse density. In contrast, we observed an increased number of asymmetric synapses onto the dendritic shaft, suggesting that spine retraction does not inevitably lead to synapse elimination. Functional analysis of the remaining synapses revealed that hippocampal circuitry compensates for the anatomical loss of synapses by increasing synaptic efficacy. Moreover, we found that the observed morphological and functional changes were associated with altered bidirectional synaptic plasticity. We conclude that continued activation of AMPA receptors is necessary for maintaining structure and function of central glutamatergic synapses.

TrkB but not trkC receptors are necessary for postnatal maintenance of hippocampal spines

Dendritic spines are major sites of excitatory synaptic transmission and changes in their densities have been linked to alterations in learning and memory. The neurotrophins brain-derived neurotrophic factor and neurotrophin-3 and their receptors, trkB and trkC, are thought to be involved in learning, memory and long-term potentiation (LTP). LTP is known to induce trkB and trkC gene expression as well as spinogenesis in the hippocampus. In the aging hippocampus, declines in trkB and trkC mRNA levels may underlie, at least in part, impairments in spatial memory and reductions in spine densities. To determine the significance of trkB and trkC for the maintenance of dendritic spines, we have analyzed Golgi-impregnated hippocampi of adult and aged mice heterozygous for trkB, trkC, or both along with respective wildtype littermates. Deletion of one allele of trkB, but not trkC, significantly reduces spine densities of CA1 pyramidal neurons in both adult and aged mice, as compared to age-matched controls. This indicates that trkB, but not trkC, receptors are necessary for the maintenance of hippocampal spines during postnatal life.

Liprinα1 Degradation by Calcium/Calmodulin-Dependent Protein Kinase II Regulates LAR Receptor Tyrosine Phosphatase Distribution and Dendrite Development

Neural activity regulates dendrite and synapse development, but the underlying molecular mechanisms are unclear. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is an important sensor of synaptic activity, and the scaffold protein liprinalpha1 is involved in pre- and postsynaptic maturation. Here we show that synaptic activity can suppress liprinalpha1 protein level by two pathways: CaMKII-mediated degradation and the ubiquitin-proteasome system. In hippocampal neurons, liprinalpha1 mutants that are immune to CaMKII degradation impair dendrite arborization, reduce spine and synapse number, and inhibit dendritic targeting of receptor tyrosine phosphatase LAR, which is important for dendrite development. Thus, regulated degradation of liprinalpha1 is important for proper LAR receptor distribution, and could provide a mechanism for localized control of dendrite and synapse morphogenesis by activity and CaMKII.

Many faces of drebrin: from building dendritic spines and stabilizing gap junctions to shaping neurite-like cell processes

In this review we consider the multiple functions of developmentally regulated brain protein (drebrin), an actin-binding protein, in the formation of cellular polarity in different cell types. Drebrin has a well-established role in the morphogenesis, patterning and maintenance of dendritic spines in neurons. We have recently shown that drebrin also stabilizes Connexin-43 containing gap junctions at the plasma membrane. The latest literature and our own data suggest that drebrin may be broadly involved in shaping cell processes and in the formation of stabilized plasma membrane domains, an effect that is likely to be of crucial significance for formation of cell polarity in both neuronal and non-neuronal types.

ARF6 and EFA6A Regulate the Development and Maintenance of Dendritic Spines

The cellular and molecular mechanisms underlying the development and maintenance of dendritic spines are not fully understood. ADP-ribosylation factor 6 (ARF6) is a small GTPase known to regulate actin remodeling and membrane traffic. Here, we report involvement of ARF6 and exchange factor for ARF6 (EFA6A) in the regulation of spine development and maintenance. An active form of ARF6 promotes the formation of dendritic spines at the expense of filopodia. EFA6A promotes spine formation in an ARF6 activation-dependent manner. Knockdown of ARF6 and EFA6A by small interfering RNA decreases spine formation. Live imaging indicates that ARF6 knockdown decreases the conversion of filopodia to spines and the stability of early spines. The spine-promoting effect of ARF6 is partially blocked by Rac1. ARF6 and EFA6A protect mature spines from inactivity-induced destabilization. These results suggest that ARF6 and EFA6A may regulate the conversion of filopodia to spines and the stability of both early and mature spines.

Cocaine-induced dendritic spine formation in D1 and D2 dopamine receptor-containing medium spiny neurons in nucleus accumbens.

Psychostimulant-induced alteration of dendritic spines on dopaminoceptive neurons in nucleus accumbens (NAcc) has been hypothesized as an adaptive neuronal response that is linked to long-lasting addictive behaviors. NAcc is largely composed of two distinct subpopulations of medium-sized spiny neurons expressing high levels of either dopamine D1 or D2 receptors. In the present study, we analyzed dendritic spine density after chronic cocaine treatment in distinct D1 or D2 receptor-containing medium-sized spiny neurons in NAcc. These studies made use of transgenic mice that expressed EGFP under the control of either the D1 or D2 receptor promoter (Drd1-EGFP or Drd2-EGFP). After 28 days of cocaine treatment and 2 days of withdrawal, spine density increased in both Drd1-EGFP- and Drd2-EGFP-positive neurons. However, the increase in spine density was maintained only in Drd1-EGFP-positive neurons 30 days after drug withdrawal. Notably, increased DeltaFosB expression also was observed in Drd1-EGFP- and Drd2-EGFP-positive neurons after 2 days of drug withdrawal but only in Drd1-EGFP-positive neurons after 30 days of drug withdrawal. These results suggest that the increased spine density observed after chronic cocaine treatment is stable only in D1-receptor-containing neurons and that DeltaFosB expression is associated with the formation and/or the maintenance of dendritic spines in D1 as well as D2 receptor-containing neurons in NAcc.

The brain-specific double-stranded RNA-binding protein Staufen2 is required for dendritic spine morphogenesis

Mammalian Staufen2 (Stau2) is a member of the double-stranded RNA-binding protein family. Its expression is largely restricted to the brain. It is thought to play a role in the delivery of RNA to dendrites of polarized neurons. To investigate the function of Stau2 in mature neurons, we interfered with Stau2 expression by RNA interference (RNAi). Mature neurons lacking Stau2 displayed a significant reduction in the number of dendritic spines and an increase in filopodia-like structures. The number of PSD95-positive synapses and miniature excitatory postsynaptic currents were markedly reduced in Stau2 down-regulated neurons. Akin effects were caused by overexpression of dominant-negative Stau2. The observed phenotype could be rescued by overexpression of two RNAi cleavage-resistant Stau2 isoforms. In situ hybridization revealed reduced expression levels of β-actin mRNA and fewer dendritic β-actin mRNPs in Stau2 down-regulated neurons. Thus, our data suggest an important role for Stau2 in the formation and maintenance of dendritic spines of hippocampal neurons.

Role of Spikar in the maintenance of dendritic spines

Role of Spikar in the maintenance of dendritic spines

MARCKS for Maintenance in Dendritic Spines

Synapses in the brain must maintain a balance between learning-related plasticity and the stability necessary for reliable function. In this issue of Neuron, Calabrese and Halpain describe cell-transfection experiments implicating MARCKS, a protein that binds to both the cell surface and actin cytoskeleton, in the maintenance of dendritic spines.

Essential Role for the PKC Target MARCKS in Maintaining Dendritic Spine Morphology

Spine morphology is regulated by intracellular signals, like PKC, that affect cytoskeletal and membrane dynamics. We investigated the role of MARCKS (myristoylated, alanine-rich C-kinase substrate) in dendrites of 3-week-old hippocampal cultures. MARCKS associates with membranes via the combined action of myristoylation and a polybasic effector domain, which binds phospholipids and/or F-actin, unless phosphorylated by PKC. Knockdown of endogenous MARCKS using RNAi reduced spine density and size. PKC activation induced similar effects, which were prevented by expression of a nonphosphorylatable mutant. Moreover, expression of pseudophosphorylated MARCKS was, by itself, sufficient to induce spine loss and shrinkage, accompanied by reduced F-actin content. Nonphosphorylatable MARCKS caused spine elongation and increased the mobility of spine actin clusters. Surprisingly, it also decreased spine density via a novel mechanism of spine fusion, an effect that required the myristoylation sequence. Thus, MARCKS is a key factor in the maintenance of dendritic spines and contributes to PKC-dependent morphological plasticity.

Inhibition of Rho-Kinase in the Nucleus Tractus Solitarius Enhances Glutamate Sensitivity in Rats

The Rho/Rho–kinase pathway in the central nervous system is involved in the maintenance of dendritic spines, which form the postsynaptic contact sites of excitatory synapses. Inhibition of the Rho–kinase pathway in neuron promotes dendritic spines or branches. In contrast, activation of the Rho/Rho–kinase pathway reduces dendritic spines or branches. Recent studies suggest that morphological changes of dendritic spines occur rapidly, and spine morphology is associated with glutamate sensitivity. The aim of the present study was to determine whether Rho-kinase activity affects glutamate sensitivity in the nucleus tractus solitarii (NTS) of Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). We first examined the effects of unilateral glutamate injection in the NTS. There was a significantly smaller decrease in arterial pressure in SHR than in WKY. We then examined the depressor responses evoked by unilateral glutamate injection into the NTS after preinjection of Y-27632, a specific Rho-kinase inhibitor. Preinjection of Y-27632 enhanced the glutamate response in both strains. However, the magnitude of the augmentation was significantly greater in SHR than in WKY. Furthermore, we recorded single-unit activity of NTS neurons from medulla brain slice preparations. N -methyl- d -aspartate (NMDA) or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) was applied iontophoretically to the recorded neurons, and neuronal activity was recorded before and after Y-27632 perfusion. Y-27632 perfusion increased the response to NMDA and AMPA. These results suggest that inhibition of Rho-kinase activity in the NTS enhances glutamate sensitivity in WKY and SHR and might improve impaired glutamate sensitivity in SHR.

The Rho-Specific GEF Lfc Interacts with Neurabin and Spinophilin to Regulate Dendritic Spine Morphology

Neurabin and spinophilin are homologous protein phosphatase 1 and actin binding proteins that regulate dendritic spine function. A yeast two-hybrid analysis using the coiled-coil domain of neurabin revealed an interaction with Lfc, a Rho GEF. Lfc was highly expressed in brain, where it interacted with either neurabin or spinophilin. In neurons, Lfc was largely found in the shaft of dendrites in association with microtubules but translocated to spines upon neuronal stimulation. Moreover, expression of Lfc resulted in reduction in spine length and size. Both the translocation and the effect on spine morphology depended on the coiled-coil domain of Lfc. Coexpression of neurabin or spinophilin with Lfc resulted in their clustering together with F-actin, a process that depended on Rho activity. Thus, interaction between Lfc and neurabin/spinophilin selectively regulates Rho-dependent organization of F-actin in spines and is a link between the microtubule and F-actin cytoskeletons in dendrites.