Molecular mechanisms of dendritic spine morphogenesis

Excitatory synapses are formed on dendritic spines, postsynaptic structures that change during development and in response to synaptic activity. Once mature, however, spines can remain stable for many months. The molecular mechanisms that control the formation and elimination, motility and stability, and size and shape of dendritic spines are being revealed. Multiple signaling pathways, particularly those involving Rho and Ras family small GTPases, converge on the actin cytoskeleton to regulate spine morphology and dynamics bidirectionally. Numerous cell surface receptors, scaffold proteins and actin binding proteins are concentrated in spines and engaged in spine morphogenesis.     

ROCK, PAK, and Toll of synapses in Alzheimer's disease

Alzheimer’s disease is a neurodegenerative disorder where the cognitive deficit is the hallmark symptom reflecting the progression of the disease. Synaptic dysfunction is a sensitive parameter of the AD pathology. Rho GTPases and the Rho kinases, ROCK1/2, and PAK1-3, are important regulators of synaptic plasticity, especially in maintaining the actin cytoskeleton of dendritic spines. Recent studies have revealed that β-amyloid oligomers can inhibit PAK and stimulate ROCK-mediated signaling. Both of these effects enhance the disassembly of synaptic actin filaments and ultimately evoke synaptic loss. Brain tissue in AD recognizes the β-amyloid peptide oligomers as foreign protein particles and mounts an inflammatory defense via Toll-like receptor (TLR) signaling which causes synaptic impairment. We will review here the dysfunction of ROCK, PAK, and Toll signaling associated with AD pathology. The protection of synapses in AD may provide new therapeutic approaches to combatting the cognitive impairment in AD.     

alpha5 integrin signaling regulates the formation of spines and synapses in hippocampal neurons

The actin-based dynamics of dendritic spines play a key role in synaptic plasticity, which underlies learning and memory. Although it is becoming increasingly clear that modulation of actin is critical for spine dynamics, the upstream molecular signals that regulate the formation and plasticity of spines are poorly understood. In non-neuronal cells, integrins are critical modulators of the actin cytoskeleton, but their function in the nervous system is not well characterized. Here we show that alpha5 integrin regulates spine morphogenesis and synapse formation in hippocampal neurons. Knockdown of alpha5 integrin expression using small interfering RNA decreased the number of dendritic protrusions, spines, and synapses. Expression of constitutively active or dominant negative alpha5 integrin also resulted in alterations in the number of dendritic protrusions, spines, and synapses. alpha5 integrin signaling regulates spine morphogenesis and synapse formation by a mechanism that is dependent on Src kinase, Rac, and the signaling adaptor GIT1. Alterations in the activity or localization of these molecules result in a significant decrease in the number of spines and synapses. Thus, our results point to a critical role for integrin signaling in regulating the formation of dendritic spines and synapses in hippocampal neurons.     

Rho GTPase signaling at the synapse: Implications for intellectual disability

Intellectual disability (ID) imposes a major medical and social–economical problem in our society. It is defined as a global reduction in cognitive and intellectual abilities, associated with impaired social adaptation. The causes of ID are extremely heterogeneous and include non-genetic and genetic changes. Great progress has been made over recent years towards the identification of ID-related genes, resulting in a list of approximately 450 genes. A prominent neuropathological feature of patients with ID is altered dendritic spine morphogenesis. These structural abnormalities, in part, reflect impaired cytoskeleton remodeling and are associated with synaptic dysfunction. The dynamic, actin-rich nature of dendritic spines points to the Rho GTPase family as a central contributor, since they are key regulators of actin dynamics and organization. It is therefore not surprising that mutations in genes encoding regulators and effectors of the Rho GTPases have been associated with ID. This review will focus on the role of Rho GTPase signaling in synaptic structure/function and ID.     

Induction of spine growth and synapse formation by regulation of the spine actin cytoskeleton

We explored the relationship between regulation of the spine actin cytoskeleton, spine morphogenesis, and synapse formation by manipulating expression of the actin binding protein NrbI and its deletion mutants. In pyramidal neurons of cultured rat hippocampal slices, NrbI is concentrated in dendritic spines by binding to the actin cytoskeleton. Expression of one NrbI deletion mutant, containing the actin binding domain, dramatically increased the density and length of dendritic spines with synapses. This hyperspinogenesis was accompanied by enhanced actin polymerization and spine motility. Synaptic strengths were reduced to compensate for extra synapses, keeping total synaptic input per neuron constant. Our data support a model in which synapse formation is promoted by actin-powered motility.     

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.     

The GTPase Rem2 regulates synapse development and dendritic morphology

Rem2 is a member of the Rad/Rem/Rem2/Gem/Kir subfamily of small Ras-like GTPases that was identified as an important mediator of synapse development. We performed a comprehensive, loss- of-function analysis of Rem2 function in cultured hippocampal neurons using RNAi to substantially decrease Rem2 protein levels. We found that knockdown of Rem2 decreases the density and maturity of dendritic spines, the primary site of excitatory synapses onto pyramidal neurons in the hippocampus. Knockdown of Rem2 also alters the gross morphology of dendritic arborizations, increasing the number of dendritic branches without altering total neurite length. Thus, Rem2 functions to inhibit dendritic branching and promote the development of dendritic spines and excitatory synapses. Interestingly, binding to the calcium-binding protein calmodulin is required for the Rem2 regulation of dendritic branching. However, this interaction is completely dispensable for synapse development. Overall, our results suggest that Rem2 regulates dendritic branching and synapse development via distinct and overlapping signal transduction pathways.     

Combinatorial morphogenesis of dendritic spines and filopodia by SPAR and α-actinin2

Rap small GTPases regulate excitatory synaptic strength and morphological plasticity of dendritic spines. Changes in spine structure are mediated by the F-actin cytoskeleton, but the link between Rap activity and actin dynamics is unclear. Here, we report a novel interaction between SPAR, a postsynaptic inhibitor of Rap, and α-actinin, a family of actin-cross-linking proteins. SPAR and α-actinin engage in bidirectional structural plasticity of dendritic spines: SPAR promotes spine head enlargement, whereas increased α-actinin2 expression favors dendritic spine elongation and thinning. Surprisingly, SPAR and α-actinin2 can function in an additive rather than antagonistic fashion at the same dendritic spine, generating combination spine/filopodia hybrids. These data identify a molecular pathway bridging the actin cytoskeleton and Rap at synapses, and suggest that formation of spines and filopodia are not necessarily opposing forms of structural plasticity.     

Synapse formation is regulated by the signaling adaptor GIT1

Dendritic spines in the central nervous system undergo rapid actin-based shape changes, making actin regulators potential modulators of spine morphology and synapse formation. Although several potential regulators and effectors for actin organization have been identified, the mechanisms by which these molecules assemble and localize are not understood. Here we show that the G protein-coupled receptor kinase-interacting protein (GIT)1 serves such a function by targeting actin regulators and locally modulating Rac activity at synapses. In cultured hippocampal neurons, GIT1 is enriched in both pre- and postsynaptic terminals and targeted to these sites by a novel domain. Disruption of the synaptic localization of GIT1 by a dominant-negative mutant results in numerous dendritic protrusions and a significant decrease in the number of synapses and normal mushroom-shaped spines. The phenotype results from mislocalized GIT1 and its binding partner PIX, an exchange factor for Rac. In addition, constitutively active Rac shows a phenotype similar to the GIT1 mutant, whereas dominant-negative Rac inhibits the dendritic protrusion formation induced by mislocalized GIT1. These results demonstrate a novel function for GIT1 as a key regulator of spine morphology and synapse formation and point to a potential mechanism by which mutations in Rho family signaling leads to decreased neuronal connectivity and cognitive defects in nonsyndromic mental retardation.     

Activity-dependent synaptogenesis: regulation by a CaM-kinase kinase/CaM-kinase I/betaPIX signaling complex

Neuronal activity augments maturation of mushroom-shaped spines to form excitatory synapses, thereby strengthening synaptic transmission. We have delineated a Ca(2+)-signaling pathway downstream of the NMDA receptor that stimulates calmodulin-dependent kinase kinase (CaMKK) and CaMKI to promote formation of spines and synapses in hippocampal neurons. CaMKK and CaMKI form a multiprotein signaling complex with the guanine nucleotide exchange factor (GEF) betaPIX and GIT1 that is localized in spines. CaMKI-mediated phosphorylation of Ser516 in betaPIX enhances its GEF activity, resulting in activation of Rac1, an established enhancer of spinogenesis. Suppression of CaMKK or CaMKI by pharmacological inhibitors, dominant-negative (dn) constructs and siRNAs, as well as expression of the betaPIX Ser516Ala mutant, decreases spine formation and mEPSC frequency. Constitutively-active Pak1, a downstream effector of Rac1, rescues spine inhibition by dnCaMKI or betaPIX S516A. This activity-dependent signaling pathway can promote synapse formation during neuronal development and in structural plasticity.     

Multiple EphB receptor tyrosine kinases shape dendritic spines in the hippocampus

Here, using a genetic approach, we dissect the roles of EphB receptor tyrosine kinases in dendritic spine development. Analysis of EphB1, EphB2, and EphB3 double and triple mutant mice lacking these receptors in different combinations indicates that all three, although to varying degrees, are involved in dendritic spine morphogenesis and synapse formation in the hippocampus. Hippocampal neurons lacking EphB expression fail to form dendritic spines in vitro and they develop abnormal spines in vivo. Defective spine formation in the mutants is associated with a drastic reduction in excitatory glutamatergic synapses and the clustering of NMDA and AMPA receptors. We show further that a kinase-defective, truncating mutation in EphB2 also results in abnormal spine development and that ephrin-B2-mediated activation of the EphB receptors accelerates dendritic spine development. These results indicate EphB receptor cell autonomous forward signaling is responsible for dendritic spine formation and synaptic maturation in hippocampal neurons.     

N-wasp and the arp2/3 complex are critical regulators of actin in the development of dendritic spines and synapses

Changes in the number, size, and shape of dendritic spines are associated with synaptic plasticity, which underlies cognitive functions such as learning and memory. This plasticity is attributed to reorganization of actin, but the molecular signals that regulate this process are poorly understood. In this study, we show neural Wiskott-Aldrich syndrome protein (N-WASP) regulates the formation of dendritic spines and synapses in hippocampal neurons. N-WASP localized to spines and active, functional synapses as shown by loading with FM4-64 dye. Knock down of endogenous N-WASP expression by RNA interference or inhibition of its activity by treatment with a specific inhibitor, wiskostatin, caused a significant decrease in the number of spines and excitatory synapses. Deletion of the C-terminal VCA region of N-WASP, which binds and activates the actin-related protein 2/3 (Arp2/3) complex, dramatically decreased the number of spines and synapses, suggesting activation of the Arp2/3 complex is critical for spine and synapse formation. Consistent with this, Arp3, like N-WASP, was enriched in spines and excitatory synapses and knock down of Arp3 expression impaired spine and synapse formation. A similar defect in spine and synapse formation was observed when expression of an N-WASP activator, Cdc42, was knocked down. Thus, activation of N-WASP and, subsequently, the Arp2/3 complex appears to be an important molecular signal for regulating spines and synapses. Arp2/3-mediated branching of actin could be a mechanism by which dendritic spine heads enlarge and subsequently mature. Collectively, our results point to a critical role for N-WASP and the Arp2/3 complex in spine and synapse formation.     

The novel synaptogenic protein Farp1 links postsynaptic cytoskeletal dynamics and transsynaptic organization

Synaptic adhesion organizes synapses, yet the signaling pathways that drive and integrate synapse development remain incompletely understood. We screened for regulators of these processes by proteomically analyzing synaptic membranes lacking the synaptogenic adhesion molecule SynCAM 1. This identified FERM, Rho/ArhGEF, and Pleckstrin domain protein 1 (Farp1) as strongly reduced in SynCAM 1 knockout mice. Farp1 regulates dendritic filopodial dynamics in immature neurons, indicating roles in synapse formation. Later in development, Farp1 is postsynaptic and its 4.1 protein/ezrin/radixin/moesin (FERM) domain binds SynCAM 1, assembling a synaptic complex. Farp1 increases synapse number and modulates spine morphology, and SynCAM 1 requires Farp1 for promoting spines. In turn, SynCAM 1 loss reduces the ability of Farp1 to elevate spine density. Mechanistically, Farp1 activates the GTPase Rac1 in spines downstream of SynCAM 1 clustering, and promotes F-actin assembly. Farp1 furthermore triggers a retrograde signal regulating active zone composition via SynCAM 1. These results reveal a postsynaptic signaling pathway that engages transsynaptic interactions to coordinate synapse development.     

Rho GTPases, dendritic structure, and mental retardation

A consistent feature of neurons in patients with mental retardation is abnormal dendritic structure and/or alterations in dendritic spine morphology. Deficits in the regulation of the dendritic cytoskeleton affect both the structure and function of dendrites and synapses and are believed to underlie mental retardation in some instances. In support of this, there is good evidence that alterations in signaling pathways involving the Rho family of small GTPases, key regulators of the actin and microtubule cytoskeletons, contribute to both syndromic and nonsyndromic mental retardation disorders. Because the Rho GTPases have been shown to play increasingly well-defined roles in determining dendrite and dendritic spine development and morphology, Rho signaling has been suggested to be important for normal cognition. The purpose of this review is to summarize recent data on the Rho GTPases pertaining to dendrite and dendritic spine morphogenesis, as well as to highlight their involvement in mental retardation resulting from a variety of genetic mutations within regulators and effectors of these molecules.     

Activation of Rho GTPases by synaptic transmission in the hippocampus

Ras-related GTPases of the Rho family, such as RhoA and RhoB, are well-characterised mediators of morphological change in peripheral tissues via their effects on the actin cytoskeleton. We tested the hypothesis that Rho family GTPases are involved in synaptic transmission in the CA1 region of the hippocampus. We show that GTPases are activated by synaptic transmission. RhoA and RhoB were activated by low frequency stimulation, while the induction of long-term potentiation (LTP) by high frequency stimulation was associated with specific activation of RhoB via NMDA receptor stimulation. This illustrates that these GTPases are potential mediators of synaptic transmission in the hippocampus, and raises the possibility that RhoB may play a role in plasticity at hippocampal synapses during LTP.     

SWAP70 functions as a Rac/Rop guanine nucleotide-exchange factor in rice

Rho family small GTPases are involved in diverse signaling processes including immunity, growth, and development. The activity of Rho GTPases is regulated by cycling between guanosine diphosphate (GDP)-bound inactive and guanosine triphosphate (GTP)-bound active forms, in which guanine nucleotide exchange factors (GEFs) predominantly function to promote activation of the GTPases. In animals, most Rho GEFs possess a Dbl (diffuse B-cell lymphoma) homology (DH) domain which functions as a GEF-catalytic domain. However, no proteins with the DH domain have been identified in plants so far. Instead, plant-specific Rho GEFs with the PRONE domain responsible for GEF activity have been found to constitute a large family in plants. In this study, we found rice homologs of human SWAP70, Oryza sativa (Os) SWAP70A and SWAP70B, containing the DH domain. OsSWAP70A interacted with rice Rho GTPase OsRac1, an important signaling factor for immune responses. The DH domain of OsSWAP70A exhibited the GEF-catalytic activity toward OsRac1 as found in animal Rho GEFs, indicating that plants have the functional DH domains. Transient expression of OsSWAP70A enhanced OsRac1-mediated production of reactive oxygen species in planta. Reduction of OsSWAP70A and OsSWAP70B mRNA levels by RNA interference resulted in the suppression of chitin elicitor-induced defense gene expression and ROS production. Thus, it is likely that OsSWAP70 regulates immune responses through activation of OsRac1.     

Spinophilin is phosphorylated by Ca2+/calmodulin-dependent protein kinase II resulting in regulation of its binding to F-actin

Spinophilin is a protein phosphatase-1- and actin-binding protein that modulates excitatory synaptic transmission and dendritic spine morphology. We have recently shown that the interaction of spinophilin with the actin cytoskeleton depends upon phosphorylation by protein kinase A. We have now found that spinophilin is phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in neurons. Ca(2+)/calmodulin-dependent protein kinase II, located within the post-synaptic density of dendritic spines, is known to play a role in synaptic plasticity and is ideally positioned to regulate spinophilin. Using tryptic phosphopeptide mapping, site-directed mutagenesis and microsequencing analysis, we identified two sites of CaMKII phosphorylation (Ser-100 and Ser-116) within the actin-binding domain of spinophilin. Phosphorylation by CaMKII reduced the affinity of spinophilin for F-actin. In neurons, phosphorylation at Ser-100 by CaMKII was Ca(2+) dependent and was associated with an enrichment of spinophilin in the synaptic plasma membrane fraction. These results indicate that spinophilin is phosphorylated by multiple kinases in vivo and that differential phosphorylation may target spinophilin to specific locations within dendritic spines.     

Vasodilator-stimulated Phosphoprotein (VASP) Induces Actin Assembly in Dendritic Spines to Promote Their Development and Potentiate Synaptic Strength

Dendritic spines are small actin-rich structures that receive the majority of excitatory synaptic input in the brain. The actin-based dynamics of spines are thought to mediate synaptic plasticity, which underlies cognitive processes, such as learning and memory. However, little is known about the molecular mechanisms that regulate actin dynamics in spines and synapses. In this study we show the multifunctional actin-binding protein vasodilator-stimulated phosphoprotein (VASP) regulates the density, size, and morphology of dendritic spines by inducing actin assembly in these structures. Knockdown of endogenous VASP by siRNA led to a significant decrease in the density of spines and synapses, whereas expression of siRNA-resistant VASP rescued this defect. The ability of VASP to modulate spine and synapse formation, maturation, and spine head enlargement is dependent on its actin binding Ena/VASP homology 2 (EVH2) domain and its EVH1 domain, which contributes to VASP localization to actin-rich structures. Moreover, VASP increases the amount of PSD-scaffolding proteins and the number of surface GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) in spines. VASP knockdown results in a reduction in surface AMPAR density, suggesting a role for this protein in regulating synaptic strength. Consistent with this, VASP significantly enhances the retention of GluR1 in spines as determined by fluorescence recovery after photobleaching and increases AMPAR-mediated synaptic transmission. Collectively, our results suggest that actin polymerization and bundling by VASP are critical for spine formation, expansion, and modulating synaptic strength.     

Rho-GTPase-activating Protein Interacting with Cdc-42-interacting Protein 4 Homolog 2 (Rich2): A NEW Ras-RELATED C3 BOTULINUM TOXIN SUBSTRATE 1 (Rac1) GTPase-ACTIVATING PROTEIN THAT CONTROLS DENDRITIC SPINE MORPHOGENESIS*

Development of dendritic spines is important for synaptic function, and alteration in spine morphogenesis is often associated with mental disorders. Rich2 was an uncharacterized Rho-GAP protein. Here we searched for a role of this protein in spine morphogenesis. We found that it is enriched in dendritic spines of cultured hippocampal pyramidal neurons during early stages of development. Rich2 specifically stimulated the Rac1 GTPase in these neurons. Inhibition of Rac1 by EHT 1864 increased the size and decreased the density of dendritic spines. Similarly, Rich2 overexpression increased the size and decreased the density of dendritic spines, whereas knock-down of the protein by specific si-RNA decreased both size and density of spines. The morphological changes were reflected by the increased amplitude and decreased frequency of miniature EPSCs induced by Rich2 overexpression, while si-RNA treatment decreased both amplitude and frequency of these events. Finally, treatment of neurons with EHT 1864 rescued the phenotype induced by Rich2 knock-down. These results suggested that Rich2 controls dendritic spine morphogenesis and function via inhibition of Rac1.     

Spatially restricted actin-regulatory signaling contributes to synapse morphology

The actin cytoskeleton in dendritic spines is organized into microdomains, but how signaling molecules that regulate actin are spatially governed is incompletely understood. Here we examine how the localization of the RacGEF kalirin-7, a well-characterized regulator of actin in spines, varies as a function of post-synaptic density area and spine volume. Using serial section electron microscopy, we find that extrasynaptic, but not synaptic, expression of kalirin-7 varies directly with synapse size and spine volume. Moreover, we find that overall expression levels of kalirin-7 differ in spines bearing perforated and non-perforated synapses, due primarily to extrasynaptic pools of kalirin-7 expression in the former. Overall, our findings indicate that kalirin-7 is differentially compartmentalized in spines as a function of both synapse morphology and spine size.