Signaling mechanisms of axon guidance and early synaptogenesis.

Signaling Mechanisms of Axon Guidance and Early Synaptogenesis Michael A. Robichaux and Christopher W. Cowan

Abstract The development of the vertebrate nervous system, including the brain and spinal cord, progresses in a step-wise fashion that involves the function of thousands of genes. The birth of new neurons (also known as neurogenesis) and their subsequent migration to appropriate locations within the developing brain mark the earliest stages of CNS development. Subsequently, these newborn neurons extend axons and dendrites to make stereotyped synaptic connections within the developing brain, which is a complex process involving cell intrinsic mechanisms that respond to specific extracellular signals. The extension and navigation of the axon to its appropriate target region in the brain and body is dependent upon many cell surface proteins that detect extracellular cues and transduce signals to the inside of the cell. In turn, intracellular signaling mechanisms orchestrate axon structural reorganization and appropriate turning toward or away from a guidance cue. Once the target region is reached, chemical synapses are formed between the axon and target cell, and again, this appears to involve cell surface proteins signaling to the inside of the neuron to stabilize and mature a synapse. Here, we describe some of the key convergent and, in some cases, divergent molecular pathways that regulate axon guidance and synaptogenesis in early brain development. Mutations in genes involved in early brain wiring and synapse formation and pruning increase the risk for developing autism, further highlighting the relevance of brain development factors in the pathophysiology of neurodevelopmental disorders.

Keywords Axon guidance Synaptogenesis Growth cone Filopodia Spine Cytoskeleton Rho-GTPase

M. A. Robichaux C. W. Cowan (&) Department of Psychiatry, Harvard Medical School, McLean Hospital, 115 Mill Street, Belmont, MA 02478, USA e-mail: [email protected] M. A. Robichaux Neuroscience Graduate Program, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA

Curr Topics Behav Neurosci (2014) 16: 19–48 DOI: 10.1007/7854_2013_255 Springer-Verlag Berlin Heidelberg 2013 Published Online: 8 December 2013

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M. A. Robichaux and C. W. Cowan

Abbreviations 4EBP ABL BDNF CAM CAMKII CAN cAMP cGMP CH CNS DCC DRG EVH1 FAK GAP GEF GDP GIT GSK-3b GTP Hh MTOR NRP OTK PAK PKA PP1 PSD RER RGC ROCK SEMA SFK Shh SMO SPAR TRP VDCC WNT ZBP

eIF-4E Binding Protein Abelson Brain-Derived Neurotrophic Factor Cell Adhesion Molecule Calcium/Calmodulin Kinase II Calcineurin Cyclic Adenosine Monophosphate Cyclic Guanosine Monophosphate Calponin Homology Central Nervous System Deleted in Colorectal Cancer Dorsal Root Ganglia Ena/VASP Homology 1 Focal Adhesion Kinase GTPase Activating Protein Guanine Nucleotide Exchange Factor Guanosine Diphosphate G-Protein Coupled Receptor Kinase Interacting Protein Glycogen Synthase Kinase 3Beta Guanosine Triphosphate Hedgehog Mammalian Target of Rapamycin Neuropilin Off-Track P21-Associated Kinase Protein Kinase A Protein Phosphatase 1 Postsynaptic Density Rough Endoplasmic Reticulum Retinal Ganglion Cell Rho-Kinase Semaphorin Src Family Kinase Sonic Hedgehog Smoothened Spine-Associated Rap GAP Transient Receptor Potential Voltage-Dependent Calcium Channel Wingless Integration Zipcode-Binding Protein

Signaling Mechanisms of Axon Guidance and Early Synaptogenesis

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Contents 1 2

Introduction: Circuit Formation in the Developing Brain ................................................. Axon Guidance .................................................................................................................... 2.1 Overview ..................................................................................................................... 2.2 Guidance Receptor Signaling..................................................................................... 2.3 Morphogenic Signaling .............................................................................................. 2.4 Calcium and Cyclic Nucleotide Signaling................................................................. 2.5 Local Translation ........................................................................................................ 3 Early Synaptogenesis........................................................................................................... 3.1 Overview ..................................................................................................................... 3.2 Axon Guidance Molecules in Synapse Formation and Plasticity............................. 4 Conclusion ........................................................................................................................... References..................................................................................................................................

21 22 22 25 33 34 35 37 37 37 40 41

1 Introduction: Circuit Formation in the Developing Brain The mammalian brain first emerges from the neural tube as a morphologically distinct structure at around gestational day 26 in humans (embryonic day 10 in mice). At this early stage, the future forebrain, midbrain, and hindbrain are patterned as the prosencephalon, mesencephalon, and rhombencephalon, respectively (for review, see Keynes and Lumsden 1990). Within this rudimentary brain structure, numerous sequential and simultaneous developmental processes occur that enable expansion, stereotyped organization, connectivity that characterizes a mature, functional brain. Developmental processes, such as cell division, neurogenesis, gliogenesis, and cell migration, are required for the growth and basic cellular organization of the brain; however, brain morphology is also defined by the white matter axon fibers that interconnect neurons and hardwire brain circuitry. Indeed, one the most fascinating and complex aspects of brain development is the process by which neurons interconnect in a highly stereotyped fashion. The proper establishment of brain circuitry requires the precise navigation of growing axons to their appropriate target region(s) within the brain and periphery. Once axons arrive at their terminal destination, they will form chemical synapses with target neurons or effector cells (e.g., muscles). These two developmental processes are often referred to as axon guidance and synaptogenesis, respectively. The importance and complexity of these processes becomes clear when one considers that billions of neurons will form trillions of targeted, stereotyped synaptic connections within the developing brain. Decades of neurobiological research have contributed to our understanding of the ‘‘decisions’’ made by neurons in the developing brain that drive circuit formation. In most cases, the navigation of axons to appropriate target regions, and eventual formation and remodeling of synapses, is determined by complex cell signaling events initiated when cell surface receptors encounter, and are activated

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by, extracellular ligands (guidance cues or synaptogenic ligands). In some cases, the same cell surface receptors that mediate axon guidance are known to play dual roles in synapse formation (reviewed, Shen and Cowan 2010). For axon guidance, the binding of an extracellular guidance cue to a cell surface guidance receptor initiates local, intracellular signaling cascades that change the axonal morphology, substrate adhesion properties, and motility of the axon. Activated axon guidance receptors can signal long distances back to the soma and nucleus to facilitate axon outgrowth and guidance, but local signaling events within the axon’s distal tip—a structure known as the growth cone—are thought to be most critical for proper axon navigation. Once an axon reaches its appropriate target region, chemical synapses are established between the axon and a target cell (e.g., on a neuronal dendrite or on a target cell’s plasma membrane), and again, it is the binding and/or activation of cell surface receptors that initiate the formation of functional, chemical synapses. In this chapter, we will describe current knowledge about some of the key local signaling processes that are critical for axon guidance and synaptogenesis, and discuss how these events function to orchestrate proper neuronal connectivity in the brain (Table 1).

2 Axon Guidance 2.1 Overview A newly born neuron establishes a cell polarity and extends a morphologically distinct axon that is supported by microtubules within its axon shaft and a morphologically complex structure, called the growth cone, at its distal tip. Subsequent guidance of the axon to its local or distant target region is coordinated by numerous extracellular cues that push or pull the distal growth cone. The growth cone is a complex, dynamic, and motile structure that is supported by cytoskeletal proteins, such as filamentous actin (F-actin) and microtubules. F-actin is a linear polymer of actin proteins that generates microfilaments, whereas microtubules are tubular polymers of tubulin proteins. Together with these cytoskeletal components, numerous axon guidance receptors and cell adhesion molecules (e.g., integrins) are expressed at its surface and interact with the underlying cytoskeleton to control growth cone shape and extension/retraction. Growth cones are tasked with interpreting extrinsic environmental signals, known as axon guidance cues, and responding properly through rapid cytoskeletal remodeling that can effectively steer the extending axon toward its stereotyped destination. Guidance cues are broadly classified as ‘‘attractive’’ or ‘‘repulsive’’ based on the growth cone response when presented with the guidance cue. However, many guidance factors, including ephrins and semaphorins, can promote either growth cone attraction or repulsion based on axon guidance receptor composition and other factors, many of which are still poorly understood. In normal development, navigating axon growth

Netrin-1

Ephrin

Semaphorin (3B, 3F), Neuropilin (2) Wnt (5A, 7A)

Wnt (4, 5A)

Hedgehog

DCC

Eph (A4, B2)

Nr-CAM Frizzled

Ryk

Patched

Axonal extension Axonal extension Axonal repulsion Spine stabilization Axonal extension Axonal repulsion Axonal extension Axonal repulsion

Axonal extension Axonal repulsion Dendritic filopodial activity Spine stabilization

Axonal extension

Table 1 Summary of receptor proteins and functions described in this chapter Surface receptor Ligand Function

(continued)

Hutchins et al. (2011), Li et al. (2009), Wouda et al. (2008) Charron and Tessier-Lavigne (2007), Tenzen et al. (2006), Trousse et al. (2001)

Briancon-Marjollet et al. (2008), Corset et al. (2000), Gitai et al. (2003), Kennedy et al. (1994), Leung et al. (2006), Li et al. (2004), Liu et al. (2004), Meriane et al. (2004), Ren et al. (2004), Serafini et al. (1996), Shekarabi and Kennedy (2002), Tcherkezian et al. (2010), Wu et al. (2006), Yao et al. (2006) Cowan et al. (2005), Dalva et al. (2000), Egea and Klein (2007), Harbott and Nobes (2005), Henkemeyer et al. (2003), Kayser et al. (2006, 2008), Malartre et al. (2010), Pasquale (2005), Petros et al. (2010), Sahin et al. (2005), Shamah et al. (2001), Shi et al. (2007), Tolias et al. (2007), Wahl et al. (2000), Yu et al. (2001), Castellani et al. (1998), Dodelet et al. (1999), Hansen et al. (2004), Iwasato et al. (2007), Kao et al. (2009), Knoll and Drescher (2004), Mohamed et al. (2012), Richter et al. (2007), Srivastava et al. (2013), Yates et al. (2001) Castellani et al. (2002), Falk et al. (2005) Ciani et al. (2011), Farias et al. (2009), Li et al. (2009), Wolf et al. (2008)

References

Signaling Mechanisms of Axon Guidance and Early Synaptogenesis 23

BDNF

Semaphorin (1A, 3A, 4D), Neuropilin (1 & 2), Off-track

Slit (2)

Semaphorin (3A), Neuropilin (1)

EphB

Cadherin

Trk (A, B)

Plexin (A1, B1)

Robo

L1-CAM

Ephrin-B (B1, B3)

Cadherin (Ncadherin)

Table 1 (continued) Surface receptor Ligand Function

Spine stabilization

Spine stabilization

Axonal repulsion

Axonal repulsion

Axonal extension Dendritic filopodial motility Spine stabilization Axonal repulsion Spine stabilization

References

Hu et al. (2001), Hung et al. (2011), Ito et al. 2006), Lin et al. (2007), Oinuma et al. (2004a, b), Swiercz et al. (2002), Turner et al. (2004), Yang and Terman (2012) Bashaw et al. (2000), Dickson and Gilestro (2006), Yu et al. (2002), Piper et al. (2006), Wu et al. (2005) Castellani et al. (2000, 2002), Maness and Schachner (2007), Raper (2000) Aoto et al. (2007), Lai and Ip (2009), Segura et al. (2007), Zhang et al. (2005) Abe et al. (2004), Paradis et al. (2007), Togashi et al. (2008), Xie et al. (2007)

Hale et al. (2011), Luikart et al. (2008), Miyamoto et al. (2006), Yao et al. (2006)

24 M. A. Robichaux and C. W. Cowan


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cones encounter guidance cues in one of two forms: (1) secreted molecular cues, often presented in a concentration gradient or (2) cell membrane-associated cues, often requiring axon:cell contact. Axons navigating long distances will often encounter multiple, different guidance cues at multiple guidance ‘‘choice points’’ en route. These ‘‘intermediate targets’’ provide important guidance information to keep navigating axons directed toward their ultimate target destinations. In addition, axons of the same origin and general destination will often bind together in large white matter bundles through an axon:axon adhesive phenomenon known as fasciculation. Fasciculated axon bundles often develop in vivo along the route established by pioneer axons earlier in development (reviewed in Raper and Mason 2010), and often remain large white matter brain structures, such as the corpus callosum, thalamocortical tract, or the anterior commissure (e.g., Fig. 1). As such, pioneer axon guidance relies upon extracellular guidance cues for proper guidance, and errors made by pioneer axons can result in aberrant axon guidance by subsequent fasciculating axons that follow the misprojected pioneer axon path. To this end, the axon growth cone must be equipped with the appropriate guidance cue detection and cell structure (cytoskeleton) remodeling machinery. Growth cones often express multiple axon guidance receptor proteins, the combinations of which are dependent upon, and somewhat specific to, the cell type and axon navigation path. In addition, navigating growth cones must possess the local signaling machinery to rapidly respond to these cues and orchestrate a change in the direction of axon growth. In some cases, distinct axon guidance receptor classes can share several common downstream signaling cascades and effector molecules (e.g., F-actin remodeling enzymes, cell adhesion molecules, etc.), but also signal through unique signaling partners, or unique combinations of signaling proteins (e.g., Fig. 2), to accomplish growth cone turning. Furthermore, a developing axon typically responds to many different classes of guidance cues during its navigation, so the necessary signaling machinery, common and unique, must be available in the vicinity of the guidance receptors. In general, all guidance receptors intracellular signaling cascades appear to converge on F-actin cytoskeletal remodeling. Understanding how these distinct guidance receptor pathways recruit and coordinate critical cell signaling factors to orchestrate proper axon guidance is a critical and challenging aspect of modern brain development research.

2.2 Guidance Receptor Signaling 2.2.1 Rho-GTPase Signaling Ras and Rho-GTPase signaling molecules are two of the most commonly employed signaling classes of molecules in the developing axon growth cone, and both families fall into the Ras GTPase superfamily of nearly 200 proteins. Not surprisingly, this protein superfamily functions redundantly in a broad range of

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E13.5

P0

Cortex

Cortex Th Th

VTel

Fig. 1 Pioneer axons establish the projection path for subsequent axon projections. In early brain development, pioneer axons respond to attractive (+) and repulsive (-) guidance cues to navigate to their appropriate target regions in the immature and dynamically changing brain environment. In this example, a mouse embryonic day 13.5 (E13.5) brain and postnatal day zero (P0) mouse brain are depicted. The brain dramatically increases in size between these periods through the processes of cell division, neurogenesis, gliogenesis, and cell migration. (middle) Coronal sections of E13.5 and P0 mouse brains are labeled with a marker of early thalamic and cortical axons (L1-CAM. At E13.5), pioneer axons from the thalamus (Th) descend from the dorsal thalamus and penetrate the ventral telencephalon (VTel) as a loosely organized population, and then turn dorsally toward the developing cerebral cortex. At P0, the L1-positive axons represent reciprocal, co-mingled axons from the thalamus and cortex that are observed in bundled arrays through the striatum region. Descending cortical axons likely require interaction with reciprocal thalamic axons to navigate to their appropriate target fields in the thalamus and other subcortical target zones


(b)

(c)

EphB2

Robo

Vav2

Cdc42

DOCK

Nck1

EphrinB2

Slit

netrin-1

DCC

(a)

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Nck1

DOCK

RhoA

Rac1

Rac1

Pak1

Pak1

Pak1

ROCK

LimK N-WASP

cofilin

Attraction

Repulsion

Repulsion

Fig. 2 Distinct axon guidance receptors differentially regulate PAK to achieve either attractive or repulsive axon guidance. a Netrin-1 binds and activates the DCC receptor in spinal commissural axons. Activated DCC recruits Rac1 and Cdc42 GTPases. PAK1 is also recruited to DCC via the Nck1 docking protein and is activated by Rac1/Cdc42-GTP. Activated PAK1 phosphorylates Lim kinase (LimK), which in turn phosphorylates and inhibits the F-actin severing activity of cofilin, which promotes axon outgrowth (Shekarabi and Kennedy 2002). b In Drosophila photoreceptor axons, Slit proteins bind to the Robo receptor, which recruits both Rac1 and PAK1 via the DOCK Drosophila adaptor protein (ortholog of Nck). Rac1 and PAK1 activities are both necessary for Slit-induced repulsion in these neurons (Fan et al. 2003). c EphrinB2 activation of the EphB2 receptor induces PAK1 kinase-dependent growth cone repulsion of embryonic cortical neurons. In this case, Nck recruits PAK1 to the activated EphB2 receptor to promote growth cone collapse independent of Rac-GTP activation (Srivastava et al. 2013)

cellular and developmental processes. Small GTPases, like other G proteins, are active when bound to guanosine triphosphate (GTP), and are inactivated following hydrolysis of bound GTP to guanosine diphosphate (GDP). This molecular switch is highly regulated by guanine nucleotide exchange factors (GEFs), which promote release of bound GDP and allow for binding to GTP. In contrast, inactivation of the GTPases is regulated by GTPase activating proteins (GAPs), which bind to and accelerate GTP hydrolysis (reviewed in Colicelli 2004; Jaffe and Hall 2005; Schmidt and Hall 2002). Rho-family GTPases are key regulators of the F-actin cytoskeleton, and function by promoting the formation, branching, or disassembly of F-actin scaffolds. These F-actin scaffolds support the cell’s shape, and they provide dynamic control over cell membrane protrusion, retraction, or internalization—key mechanisms of cell migration. Rho-GTPases, of which there are over 20 different genes, play important roles in the dynamics, forward movement, and retraction of the F-actin cytoskeleton in a navigating axon growth cone (reviewed in Dickson 2001; Hall and Lalli 2010). Rho-family GTPases are divided into three major subgroups: Rho, Rac, and Cdc42. In general, Rac-GTP promotes the formation of highly branched F-actin structures called lamellapodia, while Cdc42-GTP promotes the formation of unbranched F-actin structures called filopodia. Finally, RhoA-GTP is reported to regulate F-actin severing and contractility via regulation of F-actin and type-II

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myosin structures (reviewed in Hall 1998). Interestingly, numerous studies report roles for these Rho-family GTPases in axon guidance that do not seemingly fit within the ascribed F-actin regulatory function in non-neuronal cells. For example, F-actin severing, which promotes destruction of F-actin structures and is often associated with axon repulsion, also generates new F-actin branch points that can promote axon outgrowth and remodeling (Hall and Lalli 2010), and Rac-GTP (usually associated with axon outgrowth) is required to mediate axon repulsion downstream of several axon guidance receptors. As such, within the same growth cone, these GTPases can be differentially regulated to control complex axon guidance events. Netrin1 is a secreted axon guidance protein that binds to the Deleted in Colorectal Cancer (DCC) receptor in growth cones and induces attraction and axon growth cone extension during the development of various mammalian white matter tracts, including commissural axons in the spinal cord (Kennedy et al. 1994) and the cerebral cortex (Serafini et al. 1996). Netrin binding to the DCC receptor promotes the generation of active Rac1-GTP and Cdc42-GTP, while RhoA and its downstream effector, ROCK (Rho-kinase), are switched off (Li et al. 2002a, b; Shekarabi and Kennedy 2002). The coordinated increase in Rac/Cdc42 and the decrease in RhoA activity promote axon outgrowth (Hall 1998; Hall and Lalli 2010). For axon growth cone repulsion, many ligand-activated guidance receptors activate, or at least require, RhoA-GTP to initiate F-actin disassembly and retraction (e.g., Hu et al. 2001; Petros et al. 2010; Swiercz et al. 2002; Wahl et al. 2000). Thus, RhoA-mediated growth cone remodeling is a common downstream signaling cascade resulting in growth cone repulsion or collapse. RhoA-ROCK signaling not only triggers the breakdown of the cytoskeleton, but it also appears to function by coordinating the remodeling of F-actin through bundling of myosin-II protein in DRG growth cones (Gallo 2006) and by regulating F-actin arc structures within the navigating axon growth cone (Zhang et al. 2003). Considering the functional importance of Rho-GTPases in the axon growth cone, much research has focused on identifying the GEFs and GAPs that link the ligand-bound axon guidance receptors to the activation or inactivation, respectively, of the Rho-family GTPases. Many Rho-family GEFs possess a dibble homology (DH) domain, a critical enzymatic domain needed to facilitate the release of bound GDP on Rho-GTPases. There are over 70 DH-containing proteins in the vertebrate genome, and several of these DH-family GEFs are recruited to, and/or activated by, various axon guidance receptors. For example, the DH-family GEF, Trio, functions downstream of netrin-DCC in mammalian spinal cord axons to promote axon attraction (Briancon-Marjollet et al. 2008). Trio selectively activates Rac1, but not Cdc42 and RhoA. In contrast, the LARG GEFs, which activate RhoA, function downstream of the PlexinB1 receptor to facilitate growth cone repulsion in hippocampal neurons (Swiercz et al. 2002). For some axon guidance receptors, like the Eph receptor family of receptor tyrosine kinases, multiple GEFs and GAPs have been linked to their axon guidance functions. Eph receptors become activated upon binding to their ‘‘ligands,’’ the ephrins. However, since both ephrins and Eph receptors are membrane proteins,


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their binding occurs largely via cell–cell interactions during axon guidance (reviewed in Egea and Klein 2007; Pasquale 2005) and elicits both Eph receptor ‘‘forward’’ signaling and ephrin-mediated ‘‘reverse’’ signaling. Depending on the context, either forward or reverse signaling is most crucial, but in some cases, bidirectional signaling is needed. Eph receptors possess intrinsic tyrosine kinase activity within a conserved Src-like kinase domain, and this activity is increased dramatically upon ephrin binding and lateral clustering of Eph receptors. This clustering induces the autophosphorylation of highly conserved juxtamembrane tyrosines, which create high-affinity binding sites for the recruitment of several signaling proteins (Wybenga-Groot et al. 2001), including a number of Rho-family GEFs and adaptor proteins, such as Nck, that recruit additional F-actin regulating proteins. EphA receptors, which represent a subclass of Eph receptors that preferentially bind the ephrin-A subclass ligands, can recruit the Rho-GEF, Ephexin-1 (Shamah et al. 2001). In its basal state, Ephexin-1 activates RhoA, Rac, and Cdc42, but upon EphA receptor activation, Ephexin-1 becomes tyrosine phosphorylated and switches to a strong RhoA activator (Sahin et al. 2005; Shamah et al. 2001). The switch to RhoA-GTP activation is important for ephrinA-induced growth cone collapse. In addition to Ephexin-1, multiple Eph receptors recruit Vav family GEFs—DH family members capable of activating Rho, Rac, and Cdc42. Vav GEFs are necessary for ephrin-induced growth cone collapse and are important for proper formation of the ipsilateral optic nerve tract (Cowan et al. 2005), a process involving EphB1 receptor repulsive signaling at the optic chiasm (Chenaux and Henkemeyer 2011; Petros et al. 2009; Williams et al. 2003). Vav GEFs appear to mediate the internalization of the activated Eph receptors (Cowan et al. 2005), which is necessary for EphB receptor-mediated cell–cell repulsion (Zimmer et al. 2003). In addition to Rho-family GEFs, Eph receptors also interact with Rho-family GAPs. EphA4 requires the Rac-GAP, a-chimaerin, to mediate ephrin-induced hippocampal growth cone collapse (Shi et al. 2007), and a-chimaerin is required for corticospinal axon guidance in vivo (Iwasato et al. 2007). Like EphA4 receptor-deficient mice, a-chimaerin-deficient mice display a hopping gait as a result of abnormal spinal cord axon guidance and wiring. These findings suggest rapid inactivation of Rac-GTP may be important for Eph Receptor-mediated repulsive axon guidance. The activation of the Rho-family GTPases initiates several downstream signaling events that ultimately control growth cone behavior. One of the best-studied effectors of Rac-GTP and Cdc42-GTP is the actin-regulating factor, PAK (p21activated kinase). The PAK genes are conserved from flies to humans, and regulate F-actin dynamics indirectly via phosphorylation and regulation of additional downstream factors. PAK1 is recruited to the netrin1-activated DCC receptors to stimulate axonal outgrowth of spinal commissural axons (Shekarabi et al. 2005). In this context, PAK1 appears to function ‘‘traditionally’’ by activating LIM kinase, which in turn phosphorylates cofilin. Cofilin’s F-actin severing activities are inhibited upon direct phosphorylation by LIM Kinase, which results in stable F-actin and axon outgrowth (Edwards et al. 1999). In Drosophila, dPAK is

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necessary for the proper development of photoreceptor axons (Hing et al. 1999). dPAK is also activated downstream of Rac1 through an interaction with Dock, an adaptor protein, to signal growth cone repulsion downstream of the Robo receptor (Fan et al. 2003). Similarly, ephrinB2 stimulation of cortical neurons triggers an EphB2 receptor-dependent growth cone collapse. Like dPAK, Nck binds to the activated EphB2 receptor and recruits PAK, but unlike dPAK in the fly photoreceptor cells, EphB2 receptor-mediated cortical growth cone collapse does not require activated Rac or Cdc42 (Fig. 2) (Srivastava et al. 2013). Together, these examples illustrate the complexity of axon guidance signaling and highlight how the same signaling molecules can be uniquely regulated to generate complex axon guidance responses. In addition to guidance receptor-dependent activation of Rho-family GEFs and GAPs, some receptors directly interact with the Rho-GTPases to regulate their function in axon guidance. For example, PlexinB1 binds directly to Rac1-GTP, and this association is thought to antagonize Rac function (Hu et al. 2001). In contrast to PlexinB1, Sema3A/PlexinA1-mediated axonal repulsion requires direct binding of Rac-GTP and PlexinA1, suggesting the Rac may play a local, regulatory role for PlexinA1 receptor-mediated growth cone repulsion (Turner et al. 2004). These examples emphasize the complex regulation mechanisms employed by distinct guidance receptors, even within the same receptor subfamily.

2.2.2 Ras GTPase Signaling Ras and Rho-GTPase signaling pathways represent distinct molecular networks that generally have opposing functions in developing axons. Classically, Ras GTPases promote axon outgrowth by regulating the stability of microtubules in the axon. Ras activation inactivates GSK-3b (glycogen synthase kinase 3beta), a microtubule de-stabilizing protein, through the activation of the PI3-kinase/Akt signaling pathway (Downward 2004; Ito et al. 2006; Marte et al. 1997; Suire et al. 2002; Yoshimura et al. 2006). Typically, when an axon growth cone encounters a repulsive cue, Ras signaling is decreased while Rho-GTPase signaling is increased to reorganize the F-actin cytoskeleton (reviewed in Polleux and Snider 2010). Although the role of Ras signaling is crucial in coordinating guidance and outgrowth, its precise regulation and function in repulsive axon guidance is less well understood. One series of studies on the Sema4D/PlexinB1 receptor revealed coordinated regulation of the Rho and Ras signaling pathways in repulsive axon guidance (Oinuma et al. 2004a). Plexins possess intracellular Ras-GAP functional domains, C1 and C2, and upon Sema4D binding to PlexinB1 receptor, these GAP domains become activated by the Rho-GTPase, Rnd1 (Oinuma et al. 2004b). PlexinB1 activation results in a decrease in R-Ras activity and destabilization of microtubules in the axon (Ito et al. 2006). Similar coordinated Rho/Ras signaling has been reported for the Eph Receptors, such that activated EphB receptors recruit p120Ras-GAP, which in turn facilitates the inhibition of R-Ras signaling by


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accelerating its GTP hydrolysis (Dail et al. 2006). Similarly, EphA4 receptors recruit spine-associated RapGAP (SPAR), which inactivates Rap1-GTP, a Rasfamily GTPase (Richter et al. 2007). The interplay of the Rho/Ras GTPases in axon guidance is obviously complex, but it highlights the importance of these signaling factors for axon guidance.

2.2.3 Alternative Cytoskeletal Regulatory Signaling While Rho-GTPases are a common signaling component of axon guidance signaling, some receptors regulate the F-actin cytoskeleton through association with actin-modifying proteins. F-actin cytoskeletal changes occur largely in the outer peripheral zone of the axon growth cone, where F-actin polymers are structured in bundles and in interweaving meshworks. Actin-modifying proteins generate and/or protect pointed, or ‘‘barbed,’’ F-actin ends that are required for polymer elongation and axonal extension. Other accessory proteins sever or depolymerize F-actin filaments to break down the F-actin cytoskeleton. While this F-actin severing process is most commonly associated with growth cone repulsion, the severing of actin filaments also creates new sites for actin filament polymerization and membrane protrusion, which is an important part of axonal growth cone extension (reviewed in Dent et al. 2011). It is interesting to note that the presumed axon guidance functions of many of the actin-modifying or associating proteins are based, at least in part, on their roles in nonneuronal cell morphology and cell migration. Over a dozen actin-regulating proteins have been identified in growth cones (see Dent et al. 2011), but their precise roles and regulation in axon growth cones are less well understood. Ena/VASP is an important actin-regulatory protein that functions during axon guidance by binding barbed F-actin polymers and promoting polymerization during axon outgrowth or by coordinating F-actin remodeling during axon repulsion (Drees and Gertler 2008). In the nematode, C. elegans, the Ena/VASP ortholog, Unc-34, functions downstream of the UNC-40/DCC receptor to mediate netrin-dependent axonal attraction (Gitai et al. 2003). In contrast, the fruitfly Ena/ VASP ortholog, enabled, functions downstream of the Robo receptor to mediate slit-induced axonal repulsion at the embryonic midline (Bashaw et al. 2000; Dickson and Gilestro 2006). Similarly, Unc-34 is essential for ventral midline repulsion mediated by the Sax-3 gene (the C. elegans Robo ortholog) (Yu et al. 2002), and it plays a role downstream of activated Eph receptors in axon repulsion (Mohamed et al. 2012). Thus, the same F-actin regulatory protein can be essential for either axonal attraction/outgrowth or repulsion, depending on the guidance receptor and developmental context. F-actin severing and depolymerizing proteins promote the disassembly of Factin polymers into G-actin monomers, a process thought to be critical for axon guidance. Cofilin and gelsolin are two well-studied F-actin severing proteins that play critical roles in axon guidance (Aizawa et al. 2001; Lu et al. 1997). More recently, the fruitfly gene, MICAL, a flavoprotein mono-oxygenase, was shown to

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bind to the PlexinA1 receptor and promote F-actin depolymerization by directly interacting with F-actin polymers and catalyzing oxidation of actin (Hung et al. 2010, 2011; Terman et al. 2002). This serves to sever F-actin and block reincorporation of oxidized actin into new F-actin structures. MICAL is one of the few examples of a direct molecular link between activated guidance receptors and a direct regulator of F-actin, and it seems likely that similar genes mediate an analogous function downstream of other guidance receptors.

2.2.4 Kinase Signaling Kinases, such as PAK, are enzymes that catalyze the covalent attachment of organic phosphate to hydroxyl groups on serine, threonine, or tyrosine residues of target proteins. Many kinases are detected in the peripheral zone of axon growth cones and have been implicated in regulation of axon guidance. There are a number of tyrosine kinases that are involved in axon guidance. For example, one major tyrosine kinase, Abelson (Abl), functions in both Eph receptor signaling and the Drosophila Robo receptor signaling (Bashaw et al. 2000; Harbott and Nobes 2005; Yu et al. 2001). In addition, the Src-family of tyrosine kinases (SFKs), which include Src, Fyn, and focal adhesion kinase (FAK), have well-characterized signaling functions during axon guidance (reviewed in Parsons 2003; Thomas and Brugge 1997). For example, netrin-1/DCC receptor signaling involves Src, Fyn, and FAK (Li et al. 2004; Liu et al. 2004; Meriane et al. 2004; Ren et al. 2004). Specifically, the netrin-bound DCC receptor recruits Src to phosphorylate a critical C-terminal tyrosine on DCC, which in turn activates the DCC receptor. After receptor activation, Fyn and FAK function downstream of netrin-DCC through the phosphorylation of p130CAS (Cork-associated substrate), a signaling adaptor protein that can activate Rac and Cdc42 (Liu et al. 2007). Therefore, SFKs can function at multiple levels in the same signaling cascade to promote axon guidance. Similarly, SFKs phosphorylate the ephrinA-bound EphA receptors and two downstream signaling molecules, Ephexin1 and Cortactin, that are needed for growth cone repulsion (Kao et al. 2009; Knoll and Drescher 2004). Similar to RhoGTPases, SFKs appear to play roles in both attractive and repulsive axon guidance, suggesting that activated guidance receptors help specify which SFKs are recruited and regulate which SFK substrates are phosphorylated. Protein kinase A (PKA), a major serine/threonine kinase that is activated upon binding to the 2nd messenger, cAMP, plays a key role in axon guidance (see below). During Sema-1A-induced growth cone repulsion, PKA phosphorylates a 14-3-3 protein, which then binds to and inhibits the Ras-GAP signaling domain of the PlexinA1 receptor, which is required during Drosophila motor axon repulsion in vivo (Yang and Terman 2012). In contrast, 14-3-3 directly binds to PKA in dorsal root ganglion (DRG) growth cones and regulates PKA activity in a cAMPindependent manner in response to chemotropic cues (Kent et al. 2010). 14-3-3 plays a similar role upstream of PKA upon Sonic Hedgehog (Shh)-mediated axon guidance across the developing spinal cord midline in vivo (Yam et al. 2012).


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2.2.5 CAM and Co-receptor Signaling Cell adhesion molecules, or CAMs, are transmembrane proteins on the cell surface that adhere to substrates in the extracellular matrix, including other CAMs. CAMmediated adhesion is necessary for proper axonal outgrowth in many different neuronal cell types (reviewed in Raper and Mason 2010). CAMs are tethered to the underlying, intracellular actin cytoskeleton within a growth cone, and they cooperate with guidance receptors to recruit and activate signaling proteins during axon guidance. L1-CAM is a surface protein that contains extracellular immunoglobulin-like and fibronectin-type domains that form a cis association with Neuropilin-1 (Nrp1), a guidance receptor that binds Sema-3A (Maness and Schachner 2007; Raper 2000). This L1-CAM/Nrp1 co-receptor interaction is required for Sema3A binding and growth cone repulsion in spinal cord axons (Castellani et al. 2000). Interestingly, L1-CAM can also bind to Nrp1 in trans, and this stable co-receptor complex binds Sema3A and promotes growth cone attraction rather than repulsion (Castellani et al. 2002). Similarly, another CAM protein, Nr-CAM, associates in cis with Neuropilin-2 (Nrp2) to convert the axonal response from Sema3B and Sema3F from repulsion to attraction (Falk et al. 2005). Indeed, CAMs are thought to initially attract commissural axons to the ventral midline of the forebrain, but then switch to promote repulsion after the axons cross the midline region (Falk et al. 2005). PlexinA receptors form co-receptor complexes with Nrp1 & Nrp2 and the tyrosine kinase-like transmembrane protein, Off-track (OTK). These co-receptor complexes are necessary for growth cone repulsion in response to class 3 semaphorins in different developing neuronal cell types (reviewed in Tran et al. 2007). As with CAM complexes, PlexinA co-receptor complexes likely function as docking stations that recruit additional and specific signaling proteins to orchestrate axonal repulsion through F-actin remodeling. For example, FARP2 is a RacGEF that binds to the PlexinA1/Nrp1 co-receptor complex, but not PlexinA1 alone (Toyofuku et al. 2005). In addition, Sema3A binding to the PlexinA1/Nrp1 complex triggers recruitment of Rnd1, a Rho-family GTPase that regulates PlexinA Ras-GAP activity (Toyofuku et al. 2005). As a collective group, these signaling components are required for Sema3A-dependent DRG axonal repulsion.

2.3 Morphogenic Signaling In addition to ‘‘traditional’’ axon guidance molecules, a class of proteins called morphogens, including Hedgehog (Hh) and Wnt (short for Wingless Integration), regulate axon guidance through a unique set of morphogenic receptors (reviewed in Charron and Tessier-Lavigne 2007). The membrane-bound Hh protein interacts with the Patched (Ptc) receptor to manipulate the axon growth cone through cAMP and SFK-dependent local signaling pathways (Charron and Tessier-Lavigne 2007;

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Tenzen et al. 2006; Trousse et al. 2001; Yam et al. 2009). Wnt proteins are secreted factors, and they bind to either the Frizzled family of G-protein coupled receptors or to Ryk receptor tyrosine kinases, which are both capable of initiating an array of signaling pathways (Mulligan and Cheyette 2012; Salinas and Zou 2008). Wnt4 is expressed at the ventral midline of the developing spinal cord, and it plays a critical role in directing the anterior growth of spinal cord axons after they cross the midline. In the crossed axons, Wnt4 binds to the Frizzled receptor and initiates a heterotrimeric G-protein signaling cascade that ultimately activates the atypical protein kinase C (aPKC) to promote anterior growth of crossed axons (Wolf et al. 2008). Interestingly, Wnt5A can promote either axon outgrowth or axonal repulsion in developing cortical axons, depending on the manner in which the Wnt is presented (Li et al. 2009). Addition of a uniform concentration of Wnt5A stimulates axonal outgrowth through binding and activating the Ryk receptor, which in turn activates phosphoinositide signaling and release of intracellular Ca2+ stores. However, when Wnt5A is presented to the neurons as a concentration gradient, it triggered axonal repulsion through a TRP (Transient Receptor Potential) channel-mediated calcium rise activated by both the Ryk and Frizzled receptors (Li et al. 2009). Thus, the specific mechanisms by which Wnt5A triggers a cytoplasmic Ca2+ rise determines whether the ligand-receptor binding promotes attractive or repulsive axonal responses. This Wnt5A-mediated switch between attraction and repulsion appears to be critical for midline crossing of axons forming the corpus callosum (Hutchins et al. 2011), a major interhemispheric cortical fiber tract in the mammalian brain.

2.4 Calcium and Cyclic Nucleotide Signaling As mentioned above, a rise in intracellular Ca2+ concentration regulates axon guidance processes. In general, moderate Ca2+ levels signal attractive growth cone responses, whereas very high or very low Ca2+ levels promote axonal repulsion (reviewed in Gomez and Zheng 2006). In addition, the spatiotemporal rise in Ca2+ instructs directional growth cone turning, such that stimulation of a calcium rise on one side of the growth cone is sufficient to induce axon outgrowth toward the stimulated side (Zheng 2000). Calcium triggers growth cone remodeling in large part by binding and activating calcium-binding proteins, such as the calciumcalmodulin kinase II (CaMKII) or the calcium-activated protein phosphatases, calcineurin (CaN), and protein phosphatase-1 (PP1). Activation of CaMKII often promotes axon outgrowth, whereas activation of CaN and PP1 often promotes axonal repulsion (Wen et al. 2004). For example, Sema5B addition to DRG neurons triggers Ca2+ influx into the growth cone, which in turn, activates CaN and calpain, a Ca2+-dependent protease, both of which are required for rapid growth cone collapse (To et al. 2007). It is thought that most of the calcium-activated


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signaling pathways ultimately control the activity of the Rho-GTPases to regulate the F-actin cytoskeleton. For example, the artificial release of intracellular Ca2+ in cerebellar granule cells induces axonal outgrowth through activation of Rac and Cdc42 GTPases, and the inhibition of RhoA GTPases. Similar Rho-family GTPase mechanisms have been proposed for attractive axon guidance cues, netrin-1 or brain-derived neurotrophic factor (BDNF) (Jin et al. 2005). Netrin binding to the DCC receptor triggers a calcium influx as a result of TRP-C channel activation, which in turn depolarizes the growth cone membrane and activates voltagedependent calcium channels (VDCCs). The activated VDCCs promote the influx of extracellular Ca2+, which in turn facilitates the turning of the growth cone toward the source of netrin (Hong et al. 2000; Wang and Poo 2005). The cyclic nucleotide molecules, cAMP and cGMP, also regulate growth cone guidance. Generally, a change in cAMP/cGMP levels is sufficient to define a growth cone response. Increased cAMP levels induce attractive growth cone turning, while elevated cGMP levels induce repulsive growth cone turning (Song et al. 1998). Netrin-1 signaling through the DCC receptor increases cAMP levels in the growth cone, which in turn stimulates PKA activity (Corset et al. 2000). Netrin-1 binding to DCC increases the activity of a soluble form of adenylyl cyclase (sAC), the enzyme that produces cAMP (Wu et al. 2006). In addition to PKA, netrin stimulation of embryonic DRGs triggers the cAMP-dependent activation of the Rap-GEF, Epac, to promote attractive axon guidance (Murray et al. 2009). In comparison, Sema1A binding to PlexinA receptors in fly motor neurons stimulates the recruitment of Gyc7GC, a receptor guanylyl cyclase, to increase cGMP levels and promote axonal repulsion (Ayoob et al. 2004). Together, these and other studies illustrate how regulation of cAMP and cGMP levels in growth cones controls axon guidance during early neural development.

2.5 Local Translation While most protein synthesis is thought to occur in the neuronal cell body (soma), the local synthesis of some proteins also occurs in or near axon growth cones. Indeed, ribosomes, rough endoplasmic reticulum (RER), and various mRNA transcripts are transported into the limited, sequestered space of the growth cone and new proteins can be generated locally and on demand (reviewed in Jung et al. 2012). The ability to generate new proteins at a long distance from the neuronal cell body is advantageous for axon guidance and maintenance/remodeling of the dynamic growth cone. Many studies have shown the necessity of local protein synthesis for attractive and repulsive axon guidance (Leung et al. 2006; Piper et al. 2006; Wu et al. 2005; Yao et al. 2006). mTOR (mammalian target of rapamycin) is also necessary for proper growth cone response to extrinsic guidance cues, and these studies collectively conclude that regulation of the mTOR kinase pathway,

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which activates the protein synthesis machinery (Campbell and Holt 2001), is a common signaling mechanism that links guidance receptors to local protein synthesis and growth cone responses. Attractive guidance cues, including netrin and BDNF, stimulate the local protein synthesis of b-actin, and the local generation of new G-actin monomers contributes to the continued polymerization of the F-actin cytoskeleton in the direction of the chemotropic source (Leung et al. 2006; Yao et al. 2006). Indeed, focal netrin application triggers the asymmetric translocation of b-actin mRNA toward the stimulated side of the growth cone (Leung et al. 2006). Netrin stimulation also activates the translation initiation regulator, 4EBP (eIF-4E binding protein), a substrate of mTOR kinase activity. Furthermore, either netrin- or BDNF-induced growth cone attraction requires new b-actin protein synthesis (Leung et al. 2006; Yao et al. 2006). Interestingly, the asymmetric translocation of b-actin mRNA occurs in conjunction with both Zipcode-binding protein 1 (ZBP1), the protein chaperone of axonal b-actin mRNA, and phosphorylated Src (Yao et al. 2006), suggesting that SFKs may work in concert with local protein synthesis machinery to control outgrowth polarity. Another study reported that netrin1 binding to the DCC receptor regulates an interaction between DCC and a subunit of the 60S ribosomal complex (Tcherkezian et al. 2010). Thus, ribosomes may be differentially tethered to guidance receptors to control rapid and local synthesis of b-actin and other key axon guidance proteins. The role of local protein synthesis is not restricted to growth cone attraction or outgrowth processes. The repulsive cues, Sema3A and Slit2, stimulate the local protein synthesis of proteins critical for axonal repulsion (Piper et al. 2006; Wu et al. 2005). Specifically, Sema3A stimulates the local protein synthesis of the RhoA GTPase, which is critical for F-actin remodeling in repulsive axon guidance (Wu et al. 2005). This evidence suggests that newly synthesized RhoA is needed immediately and locally to facilitate axonal repulsion. Similarly, Slit2 stimulates the local protein synthesis of cofilin, an actin-severing protein critical for F-actin cytoskeletal disassembly in growth cones (Piper et al. 2006). Interestingly, not all axon guidance receptors appear to require local protein synthesis for growth cone repulsion or collapse (Mann et al. 2003; Nedelec et al. 2012), and in chick neurons, protein synthesis is not necessary for axon growth cone attraction, outgrowth, or repulsion triggered by many different classes of guidance cues, including Slit, Sema3A, ephrinA, or nerve growth factor. In addition, one study showed that low concentrations of Sema3A required local protein synthesis for growth cone collapse, but high concentrations of Sema3A did not (Nedelec et al. 2012). Obviously, more work is needed to fully understand the neuronal, developmental, and receptor signaling contexts in which local protein synthesis is required, and since these studies are largely performed in cell culture systems, analysis of local protein synthesis in axons in developing animals will be critical in the future.


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3 Early Synaptogenesis 3.1 Overview Axon guidance is largely completed when the navigating axons reach a stereotyped target location in the brain or periphery. In many cases, the axons ‘‘overshoot’’ the eventual region where synapses will form, and pruning of axons is not uncommon in the maturing brain. Once the axons reach the terminal zone, they form transient chemical synapses with target postsynaptic cells (neurons or other target cells like muscles), and it is these chemical synapses that constitute the basic mode of communication between excitable cells. Some of the initial axon-effector cell contacts will be stabilized and matured into functional synapses, and others will be disassembled and lost. A number of processes are involved in the formation and maintenance of a new chemical synapse, and since this chapter is largely focused on axon guidance signaling, this section will give only a high-altitude view. For neuron–neuron synapses, these tend to form between the axon of one neuron and the extensively branched regions of a neuron known as the dendrites. Most glutamatergic (the major excitatory neurotransmitter in the brain), excitatory synapses in the central nervous system form onto small dendritic membrane protrusions known as dendritic spines. Spines are the sites of *90 % of excitatory synapses in the vertebrate brain, and are also the sites of local postsynaptic signaling pathways necessary for maintaining, strengthening, and eliminating functional synapses between mature neurons. However, excitatory synapses can form along the dendritic shaft, so spines are not strictly required for normal synaptic transmission or plasticity. Nonetheless, the formation of dendritic spines appears to be an integral part of the formation of new excitatory synapses, and axon guidance molecules have been reported to play key roles in the formation and maintenance of dendritic spines and excitatory synapses.

3.2 Axon Guidance Molecules in Synapse Formation and Plasticity There are a number of striking similarities between an axon growth cone and a dendritic spine synapse. For example, dendritic spines are rich in dynamic F-actin, are constantly changing shape, and they contain many of the axon guidance molecules (and their downstream signaling partners). As reviewed in more detail elsewhere (reviewed in Shen and Cowan 2010), many axon guidance molecules appear to play a secondary role in synapse formation and plasticity, which seems an efficient way for neurons to utilize existing proteins that might otherwise become dispensable after the period of axon guidance is completed. ‘‘Guidance molecules,’’ as described in the above sections, are often utilized during early

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synaptogenesis to activate and regulate intracellular signaling events that remodel the local F-actin cytoskeleton of dendritic spines. Dendritic filopodia are thin membrane protrusions from the dendritic shaft that are largely unbranched and lack a bulbous region at the tip. These bundled F-actinrich structures are highly abundant and dynamic during early synaptogenesis, and ultrastructural analysis demonstrated that synapses form on these structures. In contrast, mature neurons have fewer filopodia and more dendritic spines at the sites of functional synapses (Fiala et al. 1998). Dendritic filopodia extend in a dynamic fashion to putatively ‘‘seek out’’ presynaptic axon partners (Trachtenberg et al. 2002; Ziv and Smith 1996). As such, the F-actin-dependent extension and motility of dendritic filopodia likely increase the frequency of contact with passing axons and thereby increase the chance of forming a synapse. Several ‘‘guidance’’ molecules contribute to filopodial motility and synaptogenesis. For example, BDNF is a soluble protein that binds to the Trk family of receptor tyrosine kinases and has a role in the regulation of synapse formation (Lu et al. 2008; Luikart and Parada 2006). BDNF binding to TrkB receptors triggers autophosphorylation of several tyrosine residues on the intracellular portion of the receptor. Similar to Eph receptors (described above), the TrkB phosphotyrosines function as binding sites to recruit downstream signaling factors, including PI3kinase and ERK signaling cascades and the Rac-GEFs, Vav2/3, and Tiam1 (Hale et al. 2011; Huang and Reichardt 2003). TrkB receptor kinase activity is required for hippocampal CA1 synapse formation, at least in part, by promoting dynamic motility of dendritic filopodia, a process dependent upon PI3 K signaling pathway and activation of Rac-GTP (Luikart et al. 2008). The Rac GEFs, Vav2, and Tiam1, are also activated by BDNF/TrkB signaling, and they regulate dendritic spine growth and synapse strengthening (Hale et al. 2011; Miyamoto et al. 2006). These Rho-family GEFs are a direct link from BDNF/TrkB to remodeling of the F-actin cytoskeleton in dendritic spines (Fig. 3). The EphB2 receptor, a member of the Eph family of axon guidance receptor tyrosine kinases, is critical for synaptogenesis in developing hippocampal neurons, in part, through the cell–cell adhesive properties of the ephrin-Eph receptor highaffinity binding interaction (Dalva et al. 2000; Henkemeyer et al. 2003; Kayser et al. 2006, 2008). In addition to its role in forming axodendritic contacts, EphB2deficient hippocampal neurons have impaired dendritic filopodial motility, which also appears to contribute to the deficit in synaptogenesis (Kayser et al. 2008). EphB2 receptors are reported to activate the serine-threonine kinase and F-actin regulator, PAK (Kayser et al. 2008; Penzes et al. 2003; Srivastava et al. 2013), as well as several Rho-family GEFs, including Intersectin-1 (Irie and Yamaguchi 2002), Kalirin-7 (Penzes et al. 2003), and Tiam1 (Tolias et al. 2007). One or more of these GEF pathways might contribute to EphB2 kinase-dependent filopodial dynamics during synaptogenesis. However, it is interesting to note that chemical inhibition of EphB receptor kinase activity did not disrupt synaptogenesis, suggesting that EphB2-dependent enhancement filopodial motility and synaptogenesis might involve EphB2 signaling functions independent of its intrinsic kinase activity (Soskis et al. 2012). Finally, the EphB2 receptor was demonstrated to


(a)

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(b)

TrkB

P

P

EphB2

EphrinB

TrkB

P

P

EphB2

NMDA

P

Tiam1

Vav2

Grb4/GIT1 Rho GTPases

Rho GTPases

PAK

RO

CK

PI3K P P

F-actin Stabilization

Myosin II

dendrite

dendrite F-actin Dynamics

Dendritic Filopodium

Dendritic Spine

Fig. 3 Dual roles for several ‘‘axon guidance’’ receptors and associated signaling cascades in axodendritic synaptogenesis. a During early synaptogenesis, dynamic filopodial motility is regulated in part by TrkB receptor and EphB2 receptor signaling. These receptors converge on signaling pathways, such as Rho and PAK that regulate F-actin cytoskeletal dynamics in the dendritic filopodium. b Dendritic spine maturation and plasticity are regulated by axon guidance receptors, such as TrkB, EphB2, and ephrin-B. In conjunction with synaptic activity, these receptors stimulate several downstream signaling pathways to regulate F-actin cytoskeletal dynamics and promote dendritic spine maturation or plasticity

recruit both the NMDA and AMPA ionotropic glutamatergic receptors to nascent postsynaptic sites through direct cis-interactions (Fig. 3) (Dalva et al. 2000; Kayser et al. 2006), demonstrating the multifaceted roles of this ‘‘guidance’’ receptors in the formation and maturation of an excitatory synapse. The ephrin-B proteins, which bind to the EphB receptors, are also necessary for postsynaptic spine maturation and stabilization through reverse signaling mechanisms (reviewed in Lai and Ip 2009). Although ephrinBs lack their own intrinsic kinase domain, they are able to activate signaling cascades by recruiting signaling partners, such as SFKs, to their short cytoplasmic region. This region contains several critical phosphorylated tyrosine residues and a C-terminal PDZ binding motif. Blocking these ephrin-B reverse signaling residues increases immature dendritic filopodia and reduces mature dendritic spines (Segura et al. 2007). The adaptor protein, Grb4, binds directly to tyrosine phosphorylated ephrin-Bs, and recruits additional signaling factors such as GIT-1 (G-protein-coupled receptor kinase interacting protein 1), a Rho-family GEF (Fig. 3) (Segura et al. 2007; Zhang et al. 2005). In addition, the ephrin-B PDZ-binding motif binds to GRIP-1 (glutamate receptor interacting protein 1), a scaffold protein necessary for dendritic spine formation and stabilization (Aoto et al. 2007).

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Cadherins, a superfamily of postsynaptic surface proteins, are also necessary for proper dendritic spine formation (Paradis et al. 2007). N-cadherin, in particular, plays a critical role by supporting the axodendritic contact adhesion and by enhancing dendritic spine formation through the activation of the Rac-GEF, Kalirin-7 (Abe et al. 2004; Togashi et al. 2002; Xie et al. 2007). Similarly, Tiam-1, another postsynaptic Rac-GEF, is necessary for proper dendritic spine formation (Tolias et al. 2005, 2007; Zhang and Macara 2006). Interestingly, Tiam-1 is activated by the postsynaptic NMDA receptors in a calcium-dependent manner (Tolias et al. 2005), emphasizing the critical interplay among synaptic activity, calcium and synapse formation/maturation. The morphogenic Wnt proteins are also reported to regulate dendritic spine formation and stabilization (reviewed in Mulligan and Cheyette 2012). Wnt7a, through binding the Frizzled/Disheveled co-receptor complex, stimulates a calcium- and CaMKII-dependent clustering of PSD-95 (postsynaptic density 95), a postsynaptic scaffold protein, at axodendritic contacts (Ciani et al. 2011). Alternatively, Wnt5a increases dendritic spine density of hippocampal neurons by activating the JNK pathway, which ultimately activates the Rho-GTPases and F-actin cytoskeletal dynamics (Farias et al. 2009).

4 Conclusion Over the last few decades, research in the fields of axon guidance and synaptogenesis has revealed a highly complex network of signaling events and cell biological processes that control proper brain wiring during development. While much is known about individual receptor signaling pathways, and the presumed roles of the downstream proteins on controlling neuronal cell shape and function, there remain many unknowns. Many different proteins regulate the F-actin cytoskeleton and contribute important aspects to axon guidance and synapse formation. In the future, it will be critical to better understand how precisely these signaling proteins alter the F-actin cytoskeleton and how diverse receptors can converge through unique signaling pathways to common cytoarchitectural changes. For example, the activity of RhoA is necessary for most forms of repulsive axon guidance, but precisely how RhoA functions in axonal repulsion, and whether its steady-state activity levels must be altered by axon guidance receptors or whether it is simply required as a parallel process, remain to be resolved. Additionally, while we can pinpoint which signaling factors become activated in an axon or dendrite in response to a specific guidance cue, these factors must still carry out the daunting task of assembling and disassembling the vast cytoskeletal structure, which invariably must require the orchestrated support of many other signaling partners and enzymes. Nonetheless, the current findings strongly suggest that diverse axon guidance receptors do converge on some common downstream factors, and that dynamic regulation of the F-actin cytoskeleton plays a critical role in both axon guidance and synapse formation.


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Finally, recent genetic studies have revealed that mutations, deletions, or duplications of genes involved in early brain wiring and synaptogenesis increase the risk for neurodevelopmental disorders such as autism. Of particular note, a de novo mutation in the EPHB2 gene results in a truncated receptor protein that lacks forward signaling capacity (Sanders et al. 2012) (unpublished findings, C. Cowan). As discussed, this gene in mice plays a critical role in cortical axon guidance, synapse formation and pruning, and synapse plasticity, highlighting the importance of these basic processes for normal brain function and emphasizing the relevance of these molecules as potential pharmacotherapeutic targets for the treatment of neurodevelopmental disorders, like autism, and possibly for other adult-onset disorders with links to neurodevelopmental dysfunction (e.g., schizophrenia).

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Molecular mechanisms of axon growth and guidance.

Semaphorin-Plexin signaling influences early ventral telencephalic development and thalamocortical axon guidance.

Canonical wnt signaling is required for commissural axon guidance.

Axon guidance: FLRTing promotes attraction.

Transcriptional coordination of synaptogenesis and neurotransmitter signaling.

Celsr3 and Fzd3 in axon guidance.

Independent signaling by Drosophila insulin receptor for axon guidance and growth.

Signaling mechanism of the netrin-1 receptor DCC in axon guidance.

Where does axon guidance lead us?

Axon guidance proteins in neurological disorders.

Exosomes mediate cell contact-independent ephrin-Eph signaling during axon guidance.

Coevolution of axon guidance molecule Slit and its receptor Robo.

Using Xenopus laevis retinal and spinal neurons to study mechanisms of axon guidance in vivo and in vitro.

Partial interchangeability of Fz3 and Fz6 in tissue polarity signaling for epithelial orientation and axon growth and guidance.

GSK-3β activation in cerebral ischemia model.

Stat Pathway into Phosphotyrosine-Based Signaling Circuitries in Drosophila.

Control of axon-axon attraction by Semaphorin reverse signaling.

Netrin signaling and Rac GTPases.

Glypican Is a Modulator of Netrin-Mediated Axon Guidance.

Morphogenesis of the C. elegans Intestine Involves Axon Guidance Genes.

Mechanisms of glutamate activation of axon-to-Schwann cell signaling in the squid.

BMP signaling in axon regeneration.

EphA4 receptor shedding regulates spinal motor axon guidance.

Axon guidance: Setting up for seeing in slow motion.