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.     

Regulation of rho GTPases by crosstalk and neuronal activity in vivo

Proper development of neurons depends on synaptic activity, but the mechanisms of activity-dependent neuronal growth are not well understood. The small GTPases, RhoA, Rac, and Cdc42, regulate neuronal morphogenesis by controlling the assembly and stability of the actin cytoskeleton. We report an in situ method to determine endogenous Rho GTPase activity in intact Xenopus brain. We use this method to provide evidence for crosstalk between Rho GTPases in optic tectal cells. Moreover, crosstalk between the Rho GTPases appears to affect dendritic arbor development in vivo. Finally, we demonstrate that optic nerve stimulation regulates Rho GTPase activity in a glutamate receptor-dependent manner. These data suggest a link between glutamate receptor function, Rho GTPase activity, and dendritic arbor growth in the intact animal.     

Essential roles of Drosophila RhoA in the regulation of neuroblast proliferation and dendritic but not axonal morphogenesis

The pleiotropic functions of small GTPase Rho present a challenge to its genetic analysis in multicellular organisms. We report here the use of the MARCM (mosaic analysis with a repressible cell marker) system to analyze the function of RhoA in the developing Drosophila brain. Clones of cells homozygous for null RhoA mutations were specifically labeled in the mushroom body (MB) neurons of mosaic brains. We found that RhoA is required for neuroblast (Nb) proliferation but not for neuronal survival. Surprisingly, RhoA is not required for MB neurons to establish normal axon projections. However, neurons lacking RhoA overextend their dendrites, and expression of activated RhoA causes a reduction of dendritic complexity. Thus, RhoA is an important regulator of dendritic morphogenesis, while distinct mechanisms are used for axonal morphogenesis.     

Convergent CaMK and RacGEF signals control dendritic structure and function

Structural plasticity of excitatory synapses is a vital component of neuronal development, synaptic plasticity and behavior, and its malfunction underlies many neurodevelopmental and psychiatric disorders. However, the molecular mechanisms that control dendritic spine morphogenesis have only recently emerged. We summarize recent work that has revealed an important connection between calcium/calmodulin-dependent kinases (CaMKs) and guanine-nucleotide-exchange factors (GEFs) that activate the small GTPase Rac (RacGEFs) in controlling dendritic spine morphogenesis. These two groups of molecules function in neurons as a unique signaling cassette that transduces calcium influx into small GTPase activity and, thence, actin reorganization and spine morphogenesis. Through this pathway, CaMKs and RacGEFs amplify calcium signals and translate them into spatially and temporally regulated structural remodeling of dendritic spines.     

A mosaic genetic screen for genes necessary for Drosophila mushroom body neuronal morphogenesis

Neurons undergo extensive morphogenesis during development. To systematically identify genes important for different aspects of neuronal morphogenesis, we performed a genetic screen using the MARCM system in the mushroom body (MB) neurons of the Drosophila brain. Mutations on the right arm of chromosome 2 (which contains approximately 20% of the Drosophila genome) were made homozygous in a small subset of uniquely labeled MB neurons. Independently mutagenized chromosomes (4600) were screened, yielding defects in neuroblast proliferation, cell size, membrane trafficking, and axon and dendrite morphogenesis. We report mutations that affect these different aspects of morphogenesis and phenotypically characterize a subset. We found that roadblock, which encodes a dynein light chain, exhibits reduced cell number in neuroblast clones, reduced dendritic complexity and defective axonal transport. These phenotypes are nearly identical to mutations in dynein heavy chain Dhc64 and in Lis1, the Drosophila homolog of human lissencephaly 1, reinforcing the role of the dynein complex in cell proliferation, dendritic morphogenesis and axonal transport. Phenotypic analysis of short stop/kakapo, which encodes a large cytoskeletal linker protein, reveals a novel function in regulating microtubule polarity in neurons. MB neurons mutant for flamingo, which encodes a seven transmembrane cadherin, extend processes beyond their wild-type dendritic territories. Overexpression of Flamingo results in axon retraction. Our results suggest that most genes involved in neuronal morphogenesis play multiple roles in different aspects of neural development, rather than performing a dedicated function limited to a specific process.     

Cyclin-Dependent Kinase 5 Links Extracellular Cues to Actin Cytoskeleton During Dendritic Spine Development

Emerging evidence has indicated a regulatory role of cyclin-dependent kinase 5 (Cdk5) in synaptic plasticity as well as in higher brain functions, such as learning and memory. However, the molecular and cellular mechanisms underlying the actions of Cdk5 at synapses remain unclear. Recent findings demonstrate that Cdk5 regulates dendritic spine morphogenesis through modulating actin dynamics. Ephexin1 and WAVE-1, two important regulators of the actin cytoskeleton, have both been recently identified as substrates for Cdk5. Importantly, phosphorylation of these proteins by Cdk5 leads to dendritic spine loss, revealing a potential mechanism by which Cdk5 regulates synapse remodeling. Furthermore, Cdk5-dependent phosphorylation of ephexin1 is required for the ephrin-A1 mediated spine retraction, pointing to a critical role of Cdk5 in conveying signals from extracellular cues to actin cytoskeleton at synapses. Taken together, understanding the precise regulation of Cdk5 and its downstream targets at synapses would provide important insights into the multi-regulatory roles of Cdk5 in actin remodeling during dendritic spine development.     

Molecular and Synaptic Bases of CDKL5 Disorder

The X‐linked gene cyclin‐dependent kinase‐like 5 (CDKL5) encodes a serine/threonine kinase abundantly expressed in the brain. Mutations in CDKL5 have been associated with neurodevelopmental disorders characterized by early‐onset epileptic encephalopathy and severe intellectual disability, suggesting that CDKL5 plays important roles in brain development and function. Recent studies using cultured neurons, knockout mice, and human iPSC‐derived neurons have demonstrated that CDKL5 regulates axon outgrowth, dendritic morphogenesis, and synapse formation. The role of CDKL5 in maintaining synaptic function in the mature brain has also begun to emerge. Moreover, mouse models that are deficient for CDKL5 recapitulate some of the key clinical phenotypes in human patients. Here we review these findings related to the function of CDKL5 in the brain and discuss the underlying molecular and cellular mechanisms.     

Actin filament assembly by myristoylated alanine-rich C kinase substrate-phosphatidylinositol-4,5-diphosphate signaling is critical for dendrite branching

Dendrites undergo extensive growth and branching at early stages, but relatively little is known about the molecular mechanisms underlying these processes. Here, we show that increasing the level of myristoylated, alanine-rich C kinase substrate (MARCKS), a prominent substrate of protein kinase C and a phosphatidylinositol-4,5-diphosphate [PI(4,5)P2] sequestration protein highly expressed in the brain, enhanced branching and growth of dendrites both in vitro and in vivo. Conversely, knockdown of endogenous MARCKS by RNA interference reduced dendritic arborization. Results from expression of different mutants indicated that membrane binding is essential for MARCKS-induced dendritic morphogenesis. Furthermore, MARCKS increased the number and length of filamentous actin-based filopodia along neurites, as well as the motility of filopodia, in a PI(4,5)P2-dependent manner. Time-lapse imaging showed that MARCKS increased frequency of filopodia initiation but did not affect filopodia longevity, suggesting that MARCKS may increase dendritic branching through its action on filopodia initiation. These findings demonstrate a critical role for MARCKS–PI(4,5)P2 signaling in regulating dendrite development.     

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.     

Initial formation of zebrafish brain ventricles occurs independently of circulation and requires the nagie oko and snakehead/atp1a1a.1 gene products

The mechanisms by which the vertebrate brain develops its characteristic three-dimensional structure are poorly understood. The brain ventricles are a highly conserved system of cavities that form very early during brain morphogenesis and that are required for normal brain function. We have initiated a study of zebrafish brain ventricle development and show here that the neural tube expands into primary forebrain, midbrain and hindbrain ventricles rapidly, over a 4-hour window during mid-somitogenesis. Circulation is not required for initial ventricle formation, only for later expansion. Cell division rates in the neural tube surrounding the ventricles are higher than between ventricles and, consistently, cell division is required for normal ventricle development. Two zebrafish mutants that do not develop brain ventricles are snakehead and nagie oko. We show that snakehead is allelic to small heart, which has a mutation in the Na+K+ ATPase gene atp1a1a.1. The snakehead neural tube undergoes normal ventricle morphogenesis; however, the ventricles do not inflate, probably owing to impaired ion transport. By contrast, mutants in nagie oko, which was previously shown to encode a MAGUK family protein, fail to undergo ventricle morphogenesis. This correlates with an abnormal brain neuroepithelium, with no clear midline and disrupted junctional protein expression. This study defines three steps that are required for brain ventricle development and that occur independently of circulation: (1) morphogenesis of the neural tube, requiring nok function; (2) lumen inflation requiring atp1a1a.1 function; and (3) localized cell proliferation. We suggest that mechanisms of brain ventricle development are conserved throughout the vertebrates.     

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.     

BIG2-ARF1-RhoA-mDia1 Signaling Regulates Dendritic Golgi Polarization in Hippocampal Neurons

Proper dendrite development is essential for establishing neural circuitry, and Rho GTPases play key regulatory roles in this process. From mouse brain lysates, we identified Brefeldin A-inhibited guanine exchange factor 2 (BIG2) as a novel Rho GTPase regulatory protein involved in dendrite growth and maintenance. BIG2 was highly expressed during early development, and knockdown of the ARFGEF2 gene encoding BIG2 significantly reduced total dendrite length and the number of branches. Expression of the constitutively active ADP-ribosylation factor 1 ARF1 Q71L rescued the defective dendrite morphogenesis of ARFGEF2-null neurons, indicating that BIG2 controls dendrite growth and maintenance by activating ARF1. Moreover, BIG2 co-localizes with the Golgi apparatus and is required for Golgi deployment into major dendrites in cultured hippocampal neurons. Simultaneous overexpression of BIG2 and ARF1 activated RhoA, and treatment with the RhoA activator lysophosphatidic acid in neurons lacking BIG2 or ARF1 increased the number of cells with dendritic Golgi, suggesting that BIG2 and ARF1 activate RhoA to promote dendritic Golgi polarization. mDia1 was identified as a downstream effector of BIG2-ARF1-RhoA axis, mediating Golgi polarization and dendritic morphogenesis. Furthermore, in utero electroporation of ARFGEF2 shRNA into the embryonic mouse brain confirmed an in vivo role of BIG2 for Golgi deployment into the apical dendrite. Taken together, our results suggest that BIG2-ARF1-RhoA-mDia1 signaling regulates dendritic Golgi polarization and dendrite growth and maintenance in hippocampal neurons.     

Protocadherin-19 is essential for early steps in brain morphogenesis

One of the earliest stages of brain morphogenesis is the establishment of the neural tube during neurulation. While some of the cellular mechanisms responsible for neurulation have been described in a number of vertebrate species, the underlying molecular processes are not fully understood. We have identified the zebrafish homolog of protocadherin-19, a member of the cadherin superfamily, which is expressed in the anterior neural plate and is required for brain morphogenesis. Interference with Protocadherin-19 function with antisense morpholino oligonucleotides leads to a severe disruption in early brain morphogenesis. Despite these pronounced effects on neurulation, axial patterning of the neural tube appears normal, as assessed by in situ hybridization for otx2, pax2.1 and krox20. Characterization of embryos early in development by in vivo 2-photon timelapse microscopy reveals that the observed disruption of morphogenesis results from an arrest of cell convergence in the anterior neural plate. These results provide the first functional data for protocadherin-19, demonstrating an essential role in early brain development.     

EphB/syndecan-2 signaling in dendritic spine morphogenesis

We previously reported that the cell surface proteoglycan syndecan-2 can induce dendritic spine formation in hippocampal neurons. We demonstrate here that the EphB2 receptor tyrosine kinase phosphorylates syndecan-2 and that this phosphorylation event is crucial for syndecan-2 clustering and spine formation. Syndecan-2 is tyrosine phosphorylated and forms a complex with EphB2 in mouse brain. Dominant-negative inhibition of endogenous EphB receptor activities blocks clustering of endogenous syndecan-2 and normal spine formation in cultured hippocampal neurons. This is the first evidence that Eph receptors play a physiological role in dendritic spine morphogenesis. Our observations suggest that spine morphogenesis is triggered by the activation of Eph receptors, which causes tyrosine phosphorylation of target molecules, such as syndecan-2, in presumptive spines.     

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.     

Molecular Architecture of Synaptic Actin Cytoskeleton in Hippocampal Neurons Reveals a Mechanism of Dendritic Spine Morphogenesis

Excitatory synapses in the brain play key roles in learning and memory. The formation and functions of postsynaptic mushroom-shaped structures, dendritic spines, and possibly of presynaptic terminals, rely on actin cytoskeleton remodeling. However, the cytoskeletal architecture of synapses remains unknown hindering the understanding of synapse morphogenesis. Using platinum replica electron microscopy, we characterized the cytoskeletal organization and molecular composition of dendritic spines, their precursors, dendritic filopodia, and presynaptic boutons. A branched actin filament network containing Arp2/3 complex and capping protein was a dominant feature of spine heads and presynaptic boutons. Surprisingly, the spine necks and bases, as well as dendritic filopodia, also contained a network, rather than a bundle, of branched and linear actin filaments that was immunopositive for Arp2/3 complex, capping protein, and myosin II, but not fascin. Thus, a tight actin filament bundle is not necessary for structural support of elongated filopodia-like protrusions. Dynamically, dendritic filopodia emerged from densities in the dendritic shaft, which by electron microscopy contained branched actin network associated with dendritic microtubules. We propose that dendritic spine morphogenesis begins from an actin patch elongating into a dendritic filopodium, which tip subsequently expands via Arp2/3 complex-dependent nucleation and which length is modulated by myosin II-dependent contractility.     

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.     

Defining mechanisms of actin polymerization and depolymerization during dendritic spine morphogenesis

Dendritic spines are small protrusions along dendrites where the postsynaptic components of most excitatory synapses reside in the mature brain. Morphological changes in these actin-rich structures are associated with learning and memory formation. Despite the pivotal role of the actin cytoskeleton in spine morphogenesis, little is known about the mechanisms regulating actin filament polymerization and depolymerization in dendritic spines. We show that the filopodia-like precursors of dendritic spines elongate through actin polymerization at both the filopodia tip and root. The small GTPase Rif and its effector mDia2 formin play a central role in regulating actin dynamics during filopodia elongation. Actin filament nucleation through the Arp2/3 complex subsequently promotes spine head expansion, and ADF/cofilin-induced actin filament disassembly is required to maintain proper spine length and morphology. Finally, we show that perturbation of these key steps in actin dynamics results in altered synaptic transmission.     

Right place,right time - Spatial guidance of neuronal morphogenesis by septin GTPases

Neuronal morphogenesis is guided by outside-in signals and inside-out mechanisms, which require spatiotemporal precision. How the intracellular mechanisms of neuronal morphogenesis are spatiotemporally controlled is not well understood. Septins comprise a unique GTPase module, which consists of complexes with differential localizations and functions. Septins demarcate distinct membrane domains in neural precursor cells, orienting the axis of cell division and the sites of neurite formation. By controlling the localization of membrane and cytoskeletal proteins, septins promote axon-dendrite formation and polarity. Furthermore, septins modulate vesicle exocytosis at pre-synaptic terminals, and stabilize dendritic spines and post-synaptic densities in a phospho-regulatable manner. We posit that neuronal septins are topologically and functionally specialized for the spatiotemporal regulation of neuronal morphogenesis and plasticity.