The actin binding domain of βI-spectrin regulates the morphological and functional dynamics of dendritic spines

Actin microfilaments regulate the size, shape and mobility of dendritic spines and are in turn regulated by actin binding proteins and small GTPases. The βI isoform of spectrin, a protein that links the actin cytoskeleton to membrane proteins, is present in spines. To understand its function, we expressed its actin-binding domain (ABD) in CA1 pyramidal neurons in hippocampal slice cultures. The ABD of βI-spectrin bundled actin in principal dendrites and was concentrated in dendritic spines, where it significantly increased the size of the spine head. These effects were not observed after expression of homologous ABDs of utrophin, dystrophin, and α-actinin. Treatment of slice cultures with latrunculin-B significantly decreased spine head size and decreased actin-GFP fluorescence in cells expressing the ABD of α-actinin, but not the ABD of βI-spectrin, suggesting that its presence inhibits actin depolymerization. We also observed an increase in the area of GFP-tagged PSD-95 in the spine head and an increase in the amplitude of mEPSCs at spines expressing the ABD of βI-spectrin. The effects of the βI-spectrin ABD on spine size and mEPSC amplitude were mimicked by expressing wild-type Rac3, a small GTPase that co-immunoprecipitates specifically with βI-spectrin in extracts of cultured cortical neurons. Spine size was normal in cells co-expressing a dominant negative Rac3 construct with the βI-spectrin ABD. We suggest that βI-spectrin is a synaptic protein that can modulate both the morphological and functional dynamics of dendritic spines, perhaps via interaction with actin and Rac3.     

The Polarity Protein Partitioning-defective 1 (PAR-1) Regulates Dendritic Spine Morphogenesis through Phosphorylating Postsynaptic Density Protein 95 (PSD-95)

The polarity protein PAR-1 plays an essential role in many cellular contexts, including embryogenesis, asymmetric cell division, directional migration, and epithelial morphogenesis. Despite its known importance in different cellular processes, the role of PAR-1 in neuronal morphogenesis is less well understood. In particular, its role in the morphogenesis of dendritic spines, which are sites of excitatory synaptic inputs, has been unclear. Here, we show that PAR-1 is required for normal spine morphogenesis in hippocampal neurons. We further show that PAR-1 functions through phosphorylating the synaptic scaffolding protein PSD-95 in this process. Phosphorylation at a conserved serine residue in the KXGS motif in PSD-95 regulates spine morphogenesis, and a phosphomimetic mutant of this site can rescue the defects of kinase-dead PAR-1. Together, our findings uncover a role of PAR-1 in spine morphogenesis in hippocampal neurons through phosphorylating PSD-95.     

Casein Kinase 2 Phosphorylation of Protein Kinase C and Casein Kinase 2 Substrate in Neurons (PACSIN) 1 Protein Regulates Neuronal Spine Formation

The PACSIN (protein kinase C and casein kinase 2 substrate in neurons) adapter proteins couple components of the clathrin-mediated endocytosis machinery with regulators of actin polymerization and thereby regulate the surface expression of specific receptors. The brain-specific PACSIN 1 is enriched at synapses and has been proposed to affect neuromorphogenesis and the formation and maturation of dendritic spines. In studies of how phosphorylation of PACSIN 1 contributes to neuronal function, we identified serine 358 as a specific site used by casein kinase 2 (CK2) in vitro and in vivo. Phosphorylated PACSIN 1 was found in neuronal cytosol and membrane fractions. This localization could be modulated by trophic factors such as brain-derived neurotrophic factor (BDNF). We further show that expression of a phospho-negative PACSIN 1 mutant, S358A, or inhibition of CK2 drastically reduces spine formation in neurons. We identified a novel protein complex containing the spine regulator Rac1, its GTPase-activating protein neuron-associated developmentally regulated protein (NADRIN), and PACSIN 1. CK2 phosphorylation of PACSIN 1 leads to a dissociation of the complex upon BDNF treatment and induces Rac1-dependent spine formation in dendrites of hippocampal neurons. These findings suggest that upon BDNF signaling PACSIN 1 is phosphorylated by CK2 which is essential for spine formation.     

The polarity protein PAR-3 and TIAM1 cooperate in dendritic spine morphogenesis

PAR-3 (partitioning-defective gene 3) is essential for cell polarization in many contexts, including axon specification. However, polarity proteins have not been implicated in later steps of neuronal differentiation, such as dendritic spine morphogenesis. Here, we show that PAR-3 is necessary for normal spine development in primary hippocampal neurons. Depletion of PAR-3 causes the formation of multiple filopodia- and lamellipodia-like dendritic protrusions - a phenotype similar to neurons expressing activated Rac. PAR-3 regulates spine formation by binding the Rac guanine nucleotide-exchange factor (GEF) TIAM1, and spatially restricting it to dendritic spines. Thus, a balance of PAR-3 and TIAM1 is essential to modulate Rac-GTP levels and to allow spine morphogenesis.     

The Guanine Nucleotide Exchange Factor (GEF) Asef2 Promotes Dendritic Spine Formation via Rac Activation and Spinophilin-dependent Targeting

Dendritic spines are actin-rich protrusions that establish excitatory synaptic contacts with surrounding neurons. Reorganization of the actin cytoskeleton is critical for the development and plasticity of dendritic spines, which is the basis for learning and memory. Rho family GTPases are emerging as important modulators of spines and synapses, predominantly through their ability to regulate actin dynamics. Much less is known, however, about the function of guanine nucleotide exchange factors (GEFs), which activate these GTPases, in spine and synapse development. In this study we show that the Rho family GEF Asef2 is found at synaptic sites, where it promotes dendritic spine and synapse formation. Knockdown of endogenous Asef2 with shRNAs impairs spine and synapse formation, whereas exogenous expression of Asef2 causes an increase in spine and synapse density. This effect of Asef2 on spines and synapses is abrogated by expression of GEF activity-deficient Asef2 mutants or by knockdown of Rac, suggesting that Asef2-Rac signaling mediates spine development. Because Asef2 interacts with the F-actin-binding protein spinophilin, which localizes to spines, we investigated the role of spinophilin in Asef2-promoted spine formation. Spinophilin recruits Asef2 to spines, and knockdown of spinophilin hinders spine and synapse formation in Asef2-expressing neurons. Furthermore, inhibition of N-methyl-d-aspartate receptor (NMDA) activity blocks spinophilin-mediated localization of Asef2 to spines. These results collectively point to spinophilin-Asef2-Rac signaling as a novel mechanism for the development of dendritic spines and synapses.     

SNX26, a GTPase-activating Protein for Cdc42, Interacts with PSD-95 Protein and Is Involved in Activity-dependent Dendritic Spine Formation in Mature Neurons

SNX26, a brain-enriched RhoGAP, plays a key role in dendritic arborization during early neuronal development in the neocortex. In mature neurons, it is localized to dendritic spines, but little is known about its role in later stages of development. Our results show that SNX26 interacts with PSD-95 in dendritic spines of cultured hippocampal neurons, and as a GTPase-activating protein for Cdc42, it decreased the F-actin content in COS-7 cells and in dendritic spines of neurons. Overexpression of SNX26 resulted in a GTPase-activating protein activity-dependent decrease in total protrusions and spine density together with dramatic inhibition of filopodia-to-spine transformations. Such effects of SNX26 were largely rescued by a constitutively active mutant of Cdc42. Consistently, an shRNA-mediated knockdown of SNX26 significantly increased total protrusions and spine density, resulting in an increase in thin or stubby type spines at the expense of the mushroom spine type. Moreover, endogenous expression of SNX26 was shown to be bi-directionally modulated by neuronal activity. Therefore, we propose that in addition to its key role in neuronal development, SNX26 also has a role in the activity-dependent structural change of dendritic spines in mature neurons.     

EphB receptors regulate dendritic spine morphogenesis through the recruitment/phosphorylation of focal adhesion kinase and RhoA activation

Dendritic filopodia are small protrusions on the surface of neuronal dendrites that transform into dendritic spines upon synaptic contact with axon terminals. The formation of dendritic spines is a critical aspect of synaptic development. Dendritic spine morphogenesis is characterized by filopodia shortening followed by the formation of mature mushroom-shaped spines. Here we show that activation of the EphB receptor tyrosine kinases in cultured hippocampal neurons by their ephrinB ligands induces morphogenesis of dendritic filopodia into dendritic spines. This appears to occur through assembly of an EphB-associated protein complex that includes focal adhesion kinase (FAK), Src, Grb2, and paxillin and the subsequent activations of FAK, Src, paxillin, and RhoA. Furthermore, Cre-mediated knock-out of loxP-flanked fak or RhoA inhibition blocks EphB-mediated morphogenesis of dendritic filopodia. Finally, EphB-mediated RhoA activation is disrupted by FAK knock-down. These data suggest that EphB receptors are upstream regulators of FAK in dendritic filopodia and that FAK-mediated RhoA activation contributes to assembly of actin filaments in dendritic spines.     

The RhoG/ELMO1/Dock180 signaling module is required for spine morphogenesis in hippocampal neurons

Dendritic spines are actin-rich structures, the formation and plasticity of which are regulated by the Rho GTPases in response to synaptic input. Although several guanine nucleotide exchange factors (GEFs) have been implicated in spine development and plasticity in hippocampal neurons, it is not known how many different Rho GEFs contribute to spine morphogenesis or how they coordinate the initiation, establishment, and maintenance of spines. In this study, we screened 70 rat Rho GEFs in cultured hippocampal neurons by RNA interference and identified a number of candidates that affected spine morphogenesis. Of these, Dock180, which plays a pivotal role in a variety of cellular processes including cell migration and phagocytosis, was further investigated. We show that depletion of Dock180 inhibits spine morphogenesis, whereas overexpression of Dock180 promotes spine morphogenesis. ELMO1, a protein necessary for in vivo functions of Dock180, functions in a complex with Dock180 in spine morphogenesis through activating the Rac GTPase. Moreover, RhoG, which functions upstream of the ELMO1/Dock180 complex, is also important for spine formation. Together, our findings uncover a role for the RhoG/ELMO1/Dock180 signaling module in spine morphogenesis in hippocampal neurons.     

Rapid induction of dendritic spine morphogenesis by trans-synaptic ephrinB-EphB receptor activation of the Rho-GEF kalirin

The morphogenesis of dendritic spines, the major sites of excitatory synaptic transmission in the brain, is important in synaptic development and plasticity. We have identified an ephrinB-EphB receptor trans-synaptic signaling pathway which regulates the morphogenesis and maturation of dendritic spines in hippocampal neurons. Activation of the EphB receptor induces translocation of the Rho-GEF kalirin to synapses and activation of Rac1 and its effector PAK. Overexpression of dominant-negative EphB receptor, catalytically inactive kalirin, or dominant-negative Rac1, or inhibition of PAK eliminates ephrin-induced spine development. This novel signal transduction pathway may be critical for the regulation of the actin cytoskeleton controlling spine morphogenesis during development and plasticity.     

ROCK1 and 2 differentially regulate actomyosin organization to drive cell and synaptic polarity

RhoGTPases organize the actin cytoskeleton to generate diverse polarities, from front–back polarity in migrating cells to dendritic spine morphology in neurons. For example, RhoA through its effector kinase, RhoA kinase (ROCK), activates myosin II to form actomyosin filament bundles and large adhesions that locally inhibit and thereby polarize Rac1-driven actin polymerization to the protrusions of migratory fibroblasts and the head of dendritic spines. We have found that the two ROCK isoforms, ROCK1 and ROCK2, differentially regulate distinct molecular pathways downstream of RhoA, and their coordinated activities drive polarity in both cell migration and synapse formation. In particular, ROCK1 forms the stable actomyosin filament bundles that initiate front–back and dendritic spine polarity. In contrast, ROCK2 regulates contractile force and Rac1 activity at the leading edge of migratory cells and the spine head of neurons; it also specifically regulates cofilin-mediated actin remodeling that underlies the maturation of adhesions and the postsynaptic density of dendritic spines.     

ApoE receptor 2 regulates synapse and dendritic spine formation

Background

Apolipoprotein E receptor 2 (ApoEr2) is a postsynaptic protein involved in long-term potentiation (LTP), learning, and memory through unknown mechanisms. We examined the biological effects of ApoEr2 on synapse and dendritic spine formation—processes critical for learning and memory.

Methodology/Principal Findings

In a heterologous co-culture synapse assay, overexpression of ApoEr2 in COS7 cells significantly increased colocalization with synaptophysin in primary hippocampal neurons, suggesting that ApoEr2 promotes interaction with presynaptic structures. In primary neuronal cultures, overexpression of ApoEr2 increased dendritic spine density. Consistent with our in vitro findings, ApoEr2 knockout mice had decreased dendritic spine density in cortical layers II/III at 1 month of age. We also tested whether the interaction between ApoEr2 and its cytoplasmic adaptor proteins, specifically X11α and PSD-95, affected synapse and dendritic spine formation. X11α decreased cell surface levels of ApoEr2 along with synapse and dendritic spine density. In contrast, PSD-95 increased cell surface levels of ApoEr2 as well as synapse and dendritic spine density.

Conclusions/Significance

These results suggest that ApoEr2 plays important roles in structure and function of CNS synapses and dendritic spines, and that these roles are modulated by cytoplasmic adaptor proteins X11α and PSD-95.     

DNA damage response (DDR) induced by topoisomerase II poisons requires nuclear function of the small GTPase Rac

Here, we investigated the influence of Rac family small GTPases on mechanisms of the DNA damage response (DDR) stimulated by topoisomerase II poisons. To this end, we examined the influence of the Rac-specific small molecule inhibitor EHT1864 on Ser139 phosphorylation of histone H2AX, a widely used marker of the DDR triggered by DNA double-strand breaks. EHT1864 attenuated the doxorubicin-stimulated DDR in a subset of cell lines tested, including HepG2 hepatoma cells. EHT1864 reduced the level of DNA strand breaks and increased viability following treatment of HepG2 cells with topo II poisons. Protection by EHT1864 was observed in both p53 wildtype (HepG2) and p53 deficient (Hep3B) human hepatoma cells and, furthermore, remained unaffected upon pharmacological inhibition of p53 in HepG2. Apparently, the impact of Rac on the DDR is independent of p53. Protection from doxorubicin-induced DNA damage by EHT1864 comprises both S and G2 phase cells. The inhibitory effect of EHT1864 on doxorubicin-stimulated DDR was mimicked by pharmacological inhibition of various protein kinases, including JNK, ERK, PI3K, PAK and CK1. EHT1864 and protein kinase inhibitors also attenuated the formation of the topo II-DNA cleavable complex. Moreover, EHT1864 mitigated the constitutive phosphorylation of topoisomerase IIα at positions S1106, S1213 and S1247. Doxorubicin transport, nuclear import/export of topoisomerase II and Hsp90-related mechanisms are likely not of relevance for doxorubicin-stimulated DDR impaired by EHT1864. We suggest that multiple kinase-dependent but p53- and heat shock protein-independent Rac-regulated nuclear mechanisms are required for activation of the DDR following treatment with topo II poisons.     

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.     

The RhoGEF DOCK10 is essential for dendritic spine morphogenesis

By regulating actin cytoskeleton dynamics, Rho GTPases and their activators RhoGEFs are implicated in various aspects of neuronal differentiation, including dendritogenesis and synaptogenesis. Purkinje cells (PCs) of the cerebellum, by developing spectacular dendrites covered with spines, represent an attractive model system in which to decipher the molecular signaling underlying these processes. To identify novel regulators of dendritic spine morphogenesis among members of the poorly characterized DOCK family of RhoGEFs, we performed gene expression profiling of fluorescence-activated cell sorting (FACS)-purified murine PCs at various stages of their postnatal differentiation. We found a strong increase in the expression of the Cdc42-specific GEF DOCK10. Depleting DOCK10 in organotypic cerebellar cultures resulted in dramatic dendritic spine defects in PCs. Accordingly, in mouse hippocampal neurons, depletion of DOCK10 or expression of a DOCK10 GEF-dead mutant led to a strong decrease in spine density and size. Conversely, overexpression of DOCK10 led to increased spine formation. We show that DOCK10 function in spinogenesis is mediated mainly by Cdc42 and its downstream effectors N-WASP and PAK3, although DOCK10 is also able to activate Rac1. Our global approach thus identifies an unprecedented function for DOCK10 as a novel regulator of dendritic spine morphogenesis via a Cdc42-mediated pathway.     

NESH regulates dendritic spine morphology and synapse formation

Background

Dendritic spines are small membranous protrusions on the neuronal dendrites that receive synaptic input from axon terminals. Despite their importance for integrating the enormous information flow in the brain, the molecular mechanisms regulating spine morphogenesis are not well understood. NESH/Abi-3 is a member of the Abl interactor (Abi) protein family, and its overexpression is known to reduce cell motility and tumor metastasis. NESH is prominently expressed in the brain, but its function there remains unknown.

Methodology/Principal Findings

NESH was strongly expressed in the hippocampus and moderately expressed in the cerebral cortex, cerebellum and striatum, where it co-localized with the postsynaptic proteins PSD95, SPIN90 and F-actin in dendritic spines. Overexpression of NESH reduced numbers of mushroom-type spines and synapse density but increased thin, filopodia-like spines and had no effect on spine density. siRNA knockdown of NESH also reduced mushroom spine numbers and inhibited synapse formation but it increased spine density. The N-terminal region of NESH co-sedimented with filamentous actin (F-actin), which is an essential component of dendritic spines, suggesting this interaction is important for the maturation of dendritic spines.

Conclusions/Significance

NESH is a novel F-actin binding protein that likely plays important roles in the regulation of dendritic spine morphogenesis and synapse formation.     

Specificity and mechanism of action of EHT 1864, a novel small molecule inhibitor of Rac family small GTPases

There is now considerable experimental evidence that aberrant activation of Rho family small GTPases promotes the uncontrolled proliferation, invasion, and metastatic properties of human cancer cells. Therefore, there is considerable interest in the development of small molecule inhibitors of Rho GTPase function. However, to date, most efforts have focused on inhibitors that indirectly block Rho GTPase function, by targeting either enzymes involved in post-translational processing or downstream protein kinase effectors. We recently determined that the EHT 1864 small molecule can inhibit Rac function in vivo. In this study, we evaluated the biological and biochemical specificities and biochemical mechanism of action of EHT 1864. We determined that EHT 1864 specifically inhibited Rac1-dependent platelet-derived growth factor-induced lamellipodia formation. Furthermore, our biochemical analyses with recombinant Rac proteins found that EHT 1864 possesses high affinity binding to Rac1, as well as the related Rac1b, Rac2, and Rac3 isoforms, and this association promoted the loss of bound nucleotide, inhibiting both guanine nucleotide association and Tiam1 Rac guanine nucleotide exchange factor-stimulated exchange factor activity in vitro. EHT 1864 therefore places Rac in an inert and inactive state, preventing its engagement with downstream effectors. Finally, we evaluated the ability of EHT 1864 to block Rac-dependent growth transformation, and we determined that EHT 1864 potently blocked transformation caused by constitutively activated Rac1, as well as Rac-dependent transformation caused by Tiam1 or Ras. Taken together, our results suggest that EHT 1864 selectively inhibits Rac downstream signaling and transformation by a novel mechanism involving guanine nucleotide displacement.     

Extracellular interactions between GluR2 and N-cadherin in spine regulation

Via its extracellular N-terminal domain (NTD), the AMPA receptor subunit GluR2 promotes the formation and growth of dendritic spines in cultured hippocampal neurons. Here we show that the first N-terminal 92 amino acids of the extracellular domain are necessary and sufficient for GluR2's spine-promoting activity. Moreover, overexpression of this extracellular domain increases the frequency of miniature excitatory postsynaptic currents (mEPSCs). Biochemically, the NTD of GluR2 can interact directly with the cell adhesion molecule N-cadherin, in cis or in trans. N-cadherin-coated beads recruit GluR2 on the surface of hippocampal neurons, and N-cadherin immobilization decreases GluR2 lateral diffusion on the neuronal surface. RNAi knockdown of N-cadherin prevents the enhancing effect of GluR2 on spine morphogenesis and mEPSC frequency. Our data indicate that in hippocampal neurons N-cadherin and GluR2 form a synaptic complex that stimulates presynaptic development and function as well as promoting dendritic spine formation.     

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.     

A kinase anchor protein 150 (AKAP150)-associated protein kinase A limits dendritic spine density

The A kinase anchor protein AKAP150 recruits the cAMP-dependent protein kinase (PKA) to dendritic spines. Here we show that in AKAP150 (AKAP5) knock-out (KO) mice frequency of miniature excitatory post-synaptic currents (mEPSC) and inhibitory post-synaptic currents (mIPSC) are elevated at 2 weeks and, more modestly, 4 weeks of age in the hippocampal CA1 area versus litter mate WT mice. Linear spine density and ratio of AMPAR to NMDAR EPSC amplitudes were also increased. Amplitude and decay time of mEPSCs, decay time of mIPSCs, and spine size were unaltered. Mice in which the PKA anchoring C-terminal 36 residues of AKAP150 are deleted (D36) showed similar changes. Furthermore, whereas acute stimulation of PKA (2-4 h) increases spine density, prolonged PKA stimulation (48 h) reduces spine density in apical dendrites of CA1 pyramidal neurons in organotypic slice cultures. The data from the AKAP150 mutant mice show that AKAP150-anchored PKA chronically limits the number of spines with functional AMPARs at 2-4 weeks of age. However, synaptic transmission and spine density was normal at 8 weeks in KO and D36 mice. Thus AKAP150-independent mechanisms correct the aberrantly high number of active spines in juvenile AKAP150 KO and D36 mice during development.