Wnt Signaling

C H A P T E R

32 Wnt Signaling P.C. Salinas University College London, London, UK

O U T L I N E 32.1 Introduction

623

32.2 Wnts and Their Signaling Pathways 32.2.1 Wnt Secretion 32.2.2 Wnt Signaling Pathways

624 624 625

32.3 Regulation of Presynaptic Differentiation 32.3.1 Axon Remodeling and Synaptic Bouton Formation 32.3.2 Cytoskeleton Reorganization at the Presynaptic Terminal 32.3.3 Recruitment of Presynaptic Components

626

32.4 Regulation of Postsynaptic Organization 32.4.1 Postsynaptic Assembly of Invertebrate Peripheral Synapses 32.4.2 Vertebrate Neuromuscular Synapse 32.4.3 Prepatterning of AChRs in Muscle Cells 32.4.4 Assembly of the Neuromuscular Synapse 32.4.5 Postsynaptic Assembly at Central Synapses

629

Abbreviations AChR Acetylcholine receptor APC Adenomatous polyposis coli CaMK Calcium–calmodulin-dependent protein kinase CNS Central nervous system CRD Cystein-rich domain CREB cAMP response element-binding protein Dvl Dishevelled Fz Frizzled receptor GFP Green fluorescent protein Gsk3 Glycogen synthase kinase-3 IP3 Inositol 1,4,5-triphosphate JNK Jun-N-terminal kinase LRP Lipoprotein receptor-related protein MAP1B Microtubule-associated protein 1B MuSK Muscle-specific kinase

Cellular Migration and Formation of Neuronal Connections: Comprehensive Developmental Neuroscience, Volume 2 http://dx.doi.org/10.1016/B978-0-12-397266-8.00106-X

626 627 628

629 629 630 630

32.5 Wnt Proteins as Antisynaptogenic Factors 32.5.1 Regulation of Synaptic Distribution 32.5.2 Wnt/b-Catenin Signaling at the Vertebrate NMJ 32.5.3 Inhibition of Synapse Formation at Central Synapses 32.6 Wnt Signaling and Activity-Mediated Synaptic Remodeling 32.6.1 Neuronal Activity, Wnts and Their Receptors 32.6.2 Synaptic Remodeling in the Adult Hippocampus 32.6.3 Emerging Roles for Wnts

631 631 632 633 633 633 634 635

Glossary

635

Acknowledgments

636

References

636

Relevant Websites

638

631

NMDA N-methyl-D-aspartic acid NMJ Neuromuscular junction PCP Planar cell polarity Sgg Shaggy (Drosophila Gsk3) TCF T-cell factor Wg Wingless protein Wls Wntless protein

32.1 INTRODUCTION Wnt factors, a large family of secreted proteins, play central roles in many aspects of development. Importantly, mutations in these factors or in components of the signaling pathway have been implicated in a range of pathological conditions including cancer, bone density defects, type II diabetes, familial exudative

623

# 2013 Elsevier Inc. All rights reserved.

624

32. WNT SIGNALING

vitreoretinopathy, neural tube defects, and late-onset Alzheimer’s disease (Reya and Clevers, 2005; Clevers, 2006; De Ferrari et al., 2007; Milat and Ng, 2009; Nikopoulos et al., 2010; Welters and Kulkarni, 2008). In the nervous system, Wnts are well established as important regulators of early patterning of the neural tube, neuronal differentiation, cell migration, axon guidance, dendritic development, target specificity, and synapse formation (Fradkin et al., 2010; Inestrosa and Arenas, 2009; Korkut and Budnik, 2009; Salinas and Zou, 2008; Wu et al., 2010). Moreover, the presence of Wnts and their receptors in the adult nervous system (http://www.brainmap.org) indicate that they also play a role later in life. Indeed, Wnts can regulate adult neurogenesis and survival of neuronal progenitors (Kuwabara et al., 2009; Lie et al., 2005), as well as synaptic remodeling (Gogolla et al., 2009). This chapter focuses on the role of Wnts in the formation of central and peripheral synapses and their emerging role in synaptic modulation in the mature nervous system.

32.2 WNTs AND THEIR SIGNALING PATHWAYS 32.2.1 Wnt Secretion Studies on Wnts have been challenging due to the large number of Wnt genes, the range of receptors, and the several signaling pathways they activate. In the mouse genome, 19 Wnts and 13 Wnt receptors have been identified. Initial studies on Wnt proteins were

G

slow due to the difficulty in isolating these proteins. After more than a decade of studies on Wnts, the nature of their posttranslational modifications revealed why these secreted proteins are difficult to purify. Wnts are glycoproteins that are further modified by the addition of a fatty acid, a palmitate group. Importantly, enzymatic removal of the palmitate moiety or mutations that impair palmitoylation render the protein inactive (Willert et al., 2003). Palmitoylation increases the hydrophobicity of Wnts and, therefore, influences subcellular trafficking and association with the plasma membrane and other proteins once in the extracellular space. Given these properties, how are Wnt proteins released into the extracellular space? How far do they diffuse? Due to their hydrophobic nature, Wnts do not diffuse far in the extracellular space without assistance. Although it was initially thought that Wnt proteins were released from expressing cells as free molecules, recent studies at the Drosophila neuromuscular junction (NMJ) revealed that Wingless (Wg), the fly Wnt protein, is released within exosome-like vesicles (Korkut et al., 2009). Genetic analyses led to the identification of Wntless (Wls/Evi), a conserved multipass membrane protein that promotes the secretion of Wnt proteins (Banziger et al., 2006; Bartscherer et al., 2006; Franch-Marro et al., 2008). Wntless deficiency leads to the retention of Wg in the producing cells, resulting in profound developmental defects (Bartscherer et al., 2006). Moreover, Wls is not only involved in the transport of Wg from the endoplasmic reticulum to the plasma membrane, but also across the synaptic cleft at the Drosophila NMJ (Korkut et al., 2009) (Figure 32.1). The finding that

FIGURE 32.1 Proposed model for the secre-

Wg Evi-pre Bouton Muscle DFz2

Lysosome

Evi-post

GRIP Golgi

ER

Nucleus Secretory pathway

III. SYNAPTOGENESIS

tion of Wg. The multipass membrane protein Evi/Wntless functions in both sides of the synapse. Wg expressed by motoneurons is transported across the synapse in Evi-containing vesicles. On the postsynaptic membrane, Wg binds to DFz2 resulting in its internalization within a complex-containing Evi and dGRIP. Reproduced from Korkut C, Ataman B, Ramachandran P, et al. (2009) Trans-synaptic transmission of vesicular Wnt signals through Evi/Wntless. Cell 139: 393–404.

625

32.2 WNTs AND THEIR SIGNALING PATHWAYS

cascade is the canonical or b-catenin pathway. Binding of Wnts to the Fz and coreceptor LRP5 or LRP6 activates Dishevelled (Dvl) by a yet unclear mechanism. Dvl is a cytoplasmic scaffold protein that functions as a hub that brings components of the pathway together for an efficient signaling (Gao and Chen, 2009). In addition, Dvl could activate specifically the pathway within confined subcellular compartments (Salinas, 2007). Dvl is phosphorylated when cells are exposed to Wnts; this posttranslational modification may be important for its functions (Chen et al., 2003; Spiegel, 2003). Once activated, Dvl induces the disassembly of the ‘destruction complex’ consisting of the serine/threonine kinase glycogen synthase-3 (Gsk3), the scaffold proteins axin, and adenomatous polyposis coli (APC) protein. The main function of this complex, as its name implies, is to induce the degradation of the cytoplasmic protein b-catenin. Sequential phosphorylation of b-catenin by casein kinase-1 and Gsk3 earmarks b-catenin for ubiquitination and degradation. In the presence of Wnts, the destruction complex disassembles, resulting in the recruitment of axin to the plasma membrane where it binds to the intracellular domain of the coreceptor LRP5/6, resulting in the inability of Gsk3 to phosphorylate b-catenin (Mao et al., 2001; Tamai et al., 2004; Zeng et al., 2005). Nonphosphorylated b-catenin accumulates in the cytoplasm and translocates to the nucleus where, in association with the transcription factors T-cell factor (TCF) or lymphocyte enhancer factor (LEF) and

Wnt proteins can be transported in vesicles between cells raises many questions. For example, what is the transport rate of these vesicles? Do the vesicles contain other molecules resulting in the coordinated action of several signaling molecules?

32.2.2 Wnt Signaling Pathways The ability of Wnts to bind four structurally different classes of receptors results in the activation of distinct signaling cascades (Angers and Moon, 2009; van Amerongen and Nusse, 2009). The most studied and characterized receptors are the seven transmembrane receptors, Frizzled (Fz), which can activate several pathways. Although Fzs can signal on their own, they can also form a complex with the single pass transmembrane coreceptor lipoprotein receptor-related protein (LRP) 5/6 (Semenov et al., 2001; Tamai et al., 2000) or with the FRL1/Crypto coreceptor (Tao et al., 2005). Finally, two other tyrosine kinase receptors, the RYK/Derailed (Drl) and ROR, have been identified as Wnt receptors (for review see Fradkin et al., 2010; Green et al., 2008). Given the large number of Wnt ligands, Fz isoforms, and additional receptors and coreceptors, it is not surprising that Wnts can activate a large range of signaling cascades with distinct cellular outcomes. Three main Wnt signaling cascades have been studied in detail (Figure 32.2). The best characterized signaling

Canonical/β-catenin pathway

Wnt

Calcium pathway

Wnt

Wnt

Fz

LRP5/6

PCP pathway

Fz

Fz

Dvl

Dvl

Dvl

Axin APC

GSK-3β

MACF1

Axon remodeling presynaptic assembly

Rho

Rac

Ca2+

PKC

β-catenin Rock

Cell fate decisions

Nucleus

CamKII

JNK

Cytoskeleton

Planar cell polarity dendritogenesis postsynaptic assembly

Nucleus Cell fate determination cell movement

FIGURE 32.2 Wnt proteins signal through three main pathways. In the canonical or b-catenin pathway, Wnts act through LRP5/6 and Fz receptors to activate a signaling cascade resulting in the inhibition of Gsk3 and elevation of b-catenin. Entry of b-catenin to the nucleus increases the transcription of target genes. This pathway can diverge downstream of Gsk3 to regulate axon remodeling and presynaptic differentiation. In the planar cell polarity pathway, Wnts signals through Fz and Dvl to activate Rho GTPases resulting in changes in the cytoskeleton. This pathway regulates cell polarity, dendrite morphogenesis, and postsynaptic assembly. In the calcium pathway, Wnts, through Fz and Dvl, activate protein kinase C and calcium–calmodulin -dependent protein kinase II resulting in changes in cell fate determination and cell movement.

III. SYNAPTOGENESIS

626

32. WNT SIGNALING

other molecules, it activates the transcription of Wnt target genes. Thus, the final outcome of the canonical signaling is the transcriptional regulation of specific genes. However, activation of this pathway does not always result in transcriptional changes. A pathway that diverges downstream of Gsk3 or b-catenin directly regulates the cytoskeleton (Cabello et al., 2010; Ciani et al., 2004; Miech et al., 2008; Purro et al., 2008). Although several b-catenin-independent or noncanonical pathways have been identified, two specific cascades have been particularly studied. The so-called planar cell polarity (PCP) pathway is activated when Wnts bind to their Fz receptors but the LRP5/6 receptors seem to be dispensable (Gordon and Nusse, 2006). As in the canonical signaling pathway, Dvl is required downstream of the receptors. Here, Dvl activates one or both of the small GTPases Rac1 and Rho, resulting in the subsequent activation of c-Jun-N-terminal kinase (JNK) and Rock, respectively (Habas et al., 2001, 2003). The modulation of the activity and/or localization of these key molecules result in changes in the cytoskeleton with profound effects on cell division, tissue polarity and cell shape (Carmona-Fontaine et al., 2008; Gros et al., 2009; Segalen and Bellaiche, 2009). In the nervous system, the PCP pathway plays a key role in cell differentiation (Andersson et al., 2008; De Calisto et al., 2005), neuronal migration (Mapp et al., 2010; Vivancos et al., 2009), and dendritic growth and branching (Rosso et al., 2005). The second most-studied, noncanonical pathway is the calcium/calmodulin cascade. As in the other signaling pathways, both Fz and Dvl are required. Here, Wnt–Fz– Dvl signaling activates phospholipase C with the subsequent increase of inositol 1,4,5-triphosphate and the activation of calcium channels, which result in the rise of intracellular calcium. Increased calcium levels can trigger several cascades including the activation of protein kinase C, calcium–calmodulin-dependent protein kinase II (CaMKII), and calmodulin (Kohn and Moon, 2005). However, the sequential steps in this cascade have not been fully characterized. The calcium pathway has been implicated in the control of cell fate and cell migration during early development (Kuhl et al., 2000). In neurons, Wnt5a has been reported to activate this pathway resulting in profound changes in axon outgrowth (Li et al., 2009).

32.3 REGULATION OF PRESYNAPTIC DIFFERENTIATION 32.3.1 Axon Remodeling and Synaptic Bouton Formation The formation of synapses requires the coordinated assembly of the machinery required for neurotransmitter release, on the presynaptic side, and the recruitment of

ion channels, neurotransmitter receptors, and signaling complexes, on the postsynaptic side. This assembly is achieved through a dialogue between the presynaptic axon and the postsynaptic cell. A combination of anterograde or retrograde signals and transmembrane proteins contribute to the assembly of pre- and postsynaptic components in perfect apposition. During the initial stages of synaptic assembly, presynaptic axons are remodeled to form synaptic boutons along the axon shaft or at the growth cones (see Chapter 27). The first study that demonstrated that Wnts influence axon behavior came from gain of function experiments in Drosophila. Overexpression of DWnt3, now renamed DWnt5, resulted in increased axon fasciculation (Fradkin et al., 1995). Subsequent studies in rodent neurons demonstrated that Wnt7a induces axon remodeling, a process characterized by increased growth of cone size and complexity, and increased branching; while at the same time, axon outgrowth is decreased (Lucas and Salinas, 1997). The behavior of the axons was distinct from what would have been expected for an attractive or repulsive axon guidance molecule. Instead, the remodeling induced by Wnt is reminiscent of when a growth cone encounters its synaptic target and is converted into a synaptic bouton. In the cerebellum, granule cells secrete Wnt7a at the time when these neurons make synaptic contacts with their presynaptic targets, the mossy fiber axons that come from outside the cerebellum (Hall et al., 2000). These axons, as their name implies, acquire a mossy appearance upon contact with their postsynaptic granule cells (Hamori and Somogyi, 1983). Consistent with the view that granule cells contribute to the characteristic morphology of mossy fibers, conditioned medium from granule cells induces remodeling of mossy axons in culture. Importantly, the secreted Wnt antagonist, Sfrp1, blocks this axon remodeling. This data suggests that Wnts secreted by granule cells contribute to the remodeling of mossy axons. Indeed, Wnt7a is required in vivo as the Wnt7a null mutant mice exhibit defects in mossy fiber remodeling, manifested by less complex glomerular rosettes, multisynaptic structures formed between a mossy fiber terminal, and several granule cell dendrites (Hall et al., 2000; Figure 32.3). Thus, Wnt7a from granule cells acts retrogradely on mossy fibers to induce axon remodeling, a process that contributes to the formation of complex synaptic structures. At the Drosophila NMJ, Wg regulates the morphology and size of synaptic boutons. In contrast to Wnt7a in the cerebellum, however, Wg is secreted by presynaptic motoneuron axons and acts on both sides of the synapse (Packard et al., 2002; see Chapter 28). Temperaturesensitive allele of wg leads to a reduction in the total number of boutons and those containing active zones and postsynaptic specializations. At the ultrastructural level, boutons have disorganized active zones; some

III. SYNAPTOGENESIS

32.3 REGULATION OF PRESYNAPTIC DIFFERENTIATION

Cerebellar cortex

32.3.2 Cytoskeleton Reorganization at the Presynaptic Terminal MF

Wnt7a

GC

GC

Wnt7a

Maturation

MF MF

FIGURE 32.3 Wnt7a regulate axon terminal remodeling and presynaptic assembly. Cerebellar granule cells secrete Wnt7a when these neurons make contact with mossy fibers (depicted in red). Wnt7a induces the remodeling of mossy fiber axons resulting in the formation of complex synaptic terminals. Wnt7a also regulates the accumulation of presynaptic proteins in these axons.

Motoneuron

Synaptic bouton DFz-2 Arrow Wg-containing vesicle

Muscle

Sgg/Gsk3 Drl DWnt5

627

Nucleus

T bars at active zones Futsch-labeled microtubules

FIGURE 32.4 Role of Wg and DWnt5 at the Drosophila NMJ. Wg is released from the motoneurons as vesicles and acts on the pre- and postsynaptic sides of the synapses. Presynaptically, Wg activates DFz2 and Arrow/LRP6 to regulate the assembly of presynaptic structures such as the T bars and the reorganization of bouton microtubules through the inhibition of Shaggy (Sgg)/Gsk3. Postsynaptically, Wg stimulates the internalization and cleavage of DFz2. The truncated receptor is then transported to the nucleus to regulate postsynaptic assembly. Another Wnt, DWnt5 also regulates synapse assembly by acting through the Drl receptor.

boutons lack T bars (presynaptic structures where synaptic vesicles are present); or, the T bars have an amorphous appearance (Figure 32.4). Thus, loss of Wg function results in changes in bouton number and morphology as well as in the ultrastructure of the synapse.

Studies in both vertebrates and in Drosophila have demonstrated that Wnts induce axon remodeling and change synaptic bouton morphology by inducing profound changes in the cytoskeleton. Gain of function of Wnt7a increases the amount of stable microtubules along the axon shaft and in the growth cone, suggesting an increase in microtubule stability (Hall et al., 2000). Indeed, axonal microtubules are more stable in the presence of Wnts. In contrast, loss of Dvl1 function renders axonal microtubules more unstable (Purro et al., 2008). Further studies demonstrated that activation of a divergent canonical pathway that requires inhibition of Gsk3, but not transcriptional activity, regulates microtubule stability (Ciani et al., 2004). Although it remains to be determined whether b-catenin is required in this process, these studies suggest that Wnt signaling directly affects the cytoskeleton through changes in Gsk3 activity in axons. Several Gsk3 targets that modulate microtubules have been identified including microtubule-associated proteins Tau, MAP2, and microtubule-associated protein 1B (MAP1B) (Lucas et al., 1998; Sanchez et al., 2000; Sperber et al., 1995). Critically, Wnt decreases the phosphorylation of MAP1B by Gsk3 in axons (Lucas et al., 1998). Biochemical characterization indicates that Gsk3-mediated phosphorylation of MAP1B increases the amount of dynamic microtubules (Goold et al., 1999). Thus, Wnt signaling increases microtubule stability through inhibition of Gsk3 and the subsequent decrease in MAP1B phosphorylation. Do changes in Gsk3-mediated phosphorylation affect bouton structure and morphology? The answer to this question came from studies in the Drosophila peripheral synapse. At the Drosophila NMJ, a clear role for the Wg-Gsk3 pathway and MAP1B on synaptic microtubule organization has been demonstrated. In wg mutant embryos, boutons exhibit defects in the organization of microtubules with the presence of fewer looped microtubules that contain decreased MAP1B/Futsch levels (Packard et al., 2002). Accordantly, futsch mutants exhibit defects in the morphology of synaptic boutons and in the organization of synaptic microtubules (Roos et al., 2000), as observed in wg mutants. Similar to the situation in cultured mouse neurons, the Drosophila Shaggy (sgg), the Gsk3 orthologue, regulates synaptic bouton morphology (Miech et al., 2008) and phosphorylates Futsch/MAP1B to regulate microtubule organization and stability at the fly NMJ (Franco et al., 2004). Together, these findings strongly support a model where Wnt signaling, through inhibition of Gsk3, regulates the phosphorylation of MAP1B/ Futsch, resulting in changes in the organization of axonal or synaptic microtubules. These events, in turn, modify

III. SYNAPTOGENESIS

628

32. WNT SIGNALING

the morphology of axons and synaptic boutons (Figure 32.4). In addition to MAP1B, Wnt signaling through Gsk3 also affects other microtubule-associated proteins, resulting in profound changes in microtubule growth direction. Close examination of the effects of Wnts on axons of dorsal root ganglia (DRG) neurons revealed that the formation of looped microtubules correlates with the slow rate of axonal extension (Purro et al., 2008). To examine the behavior of microtubules in real time, the end of microtubules were labeled with the plus-end microtubule-binding protein EB3 fused to green fluorescent protein (GFP). Time-lapse recordings of neurons expressing EB3-GFP showed that when axons encounter Wnt3a, microtubules lose their directional growth within the growth cone. Rather than splaying towards the leading edge of the growth cone, many microtubules move across the growth cones and in a retrograde direction. Moreover, a large number of microtubules do not have a persistent trajectory. Thus, loss of directional growth of polymerizing microtubules seems to contribute to the formation of microtubule loops, to a decreased rate of growth cone translocation, and to the increase in growth cone size. Analyses of the proteins present at the plus-end of microtubules showed that Wnt3a induces the loss of APC, which is a component of the canonical Wnt pathway but also functions as a microtubule plus-end-binding protein (for review see Etienne-Manneville, 2009; McCartney and Nathke, 2008). Knockdown of APC with short hairpin RNA (ShRNA) in neurons fully mimics the phenotype induced by Wnts (Purro et al., 2008). Although it is unclear how Wnt3a induces the loss of APC from the plus-ends of microtubules, these studies demonstrate that Wnt signaling can directly modulate the directionality of microtubule growth through the loss of APC. The findings discussed above provide strong evidence that Wnt signaling modulates the phosphorylation and localization of cytoskeletal proteins through the inhibition of Gsk3. Moreover, several components of the canonical pathway are associated to the cytoskeleton. For example, Dvl, Axin, Gsk3, APC, and b-catenin bind to microtubules (Salinas, 2007) and some of them also bind to the actin cytoskeleton. The ability of these molecules to bind to the cytoskeleton suggests that activation of the Wnt pathway within a cellular compartment could ensure that changes occur locally rather than globally within the cell.

32.3.3 Recruitment of Presynaptic Components The first evidence that Wnt signaling modulates synapse formation came from studies showing that, in cultured cerebellar neurons, Wnt7a increases the clustering of the presynaptic protein synapsin I (Lucas and Salinas,

1997), a hallmark of presynaptic assembly. A later study provided clear evidence for a role of Wnt in vivo. In the mouse cerebellum, Wnt7a is highly expressed in granule cells when these neurons make synaptic contacts with mossy fiber axons. Granule cells secrete factors with a strong synaptogenic activity for mossy fiber axons, and this activity is severely blocked by the secreted Wnt antagonists. Importantly, Wnt7a increases the clustering of several presynaptic proteins in mossy fibers. Conversely, the Wnt7a mutant mice exhibit defects in the accumulation of presynaptic proteins in mossy fibers in vivo (Hall et al., 2000). These findings, together with those of axon remodeling, demonstrate that Wnt7a from granule cells acts retrogradely on the presynaptic mossy axons to regulate both the terminal remodeling of axons and the clustering of presynaptic components (Figure 32.3). The effect of Wnts is not restricted to the cerebellum, as Wnts can stimulate presynaptic differentiation in DRG (Krylova et al., 2002) and hippocampal neurons (Davis et al., 2008; Sahores et al., 2010; Varela-Nallar et al., 2009). Which signaling pathway regulates presynaptic differentiation? The Dvl1 mutant mice have similar presynaptic defects in the cerebellum to the Wnt7a mutant (AhmadAnnuar et al., 2006). Moreover, double mutant Wnt7a/ Dvl1 mice exhibit a stronger defect than single mutants, indicating a genetic interaction between Wnt7a and Dvl1 (Ahmad-Annuar et al., 2006). Further studies demonstrated that as in axon remodeling, Wnt signals through Gsk3 to stimulate the clustering of presynaptic proteins (Davis et al., 2008; Hall et al., 2000). The mechanism by which inhibition of Gsk3 induces presynaptic clustering remains poorly understood. Wnts could signal to the nucleus and thereby influence synaptic clustering, or they could more directly induce the recruitment or transport of synaptic components to future synaptic sites. One central question in the quest to understand the mechanisms that control synaptic assembly is ‘what determines the site where synaptic assembly is initiated?’ Studies in vertebrate cells and in the fly demonstrate that Fz receptors are crucial in this process. At the Drosophila NMJ, DFz2 is localized to, and required at, both sides of the synapse (Ataman et al., 2006). Interestingly, different signaling pathways are activated at the pre- and postsynaptic sides. On the presynaptic terminal, Wg binding to Dfz2 and Arrow/LRP6 receptors leads to the activation of the canonical Wnt pathway resulting in the inhibition of Sgg/Gsk3 and changes in the cytoskeleton (Miech et al., 2008; Packard et al., 2002). However, it is still unclear how presynaptic components are recruited to the presynaptic site. At vertebrate central synapses, the role of Fz receptors in presynaptic differentiation has also been demonstrated. In hippocampal neurons, several Fz receptors are expressed (Davis et al., 2008; Sahores et al., 2010; Varela-Nallar et al., 2009). Axonal expression of Fz1,

III. SYNAPTOGENESIS

32.4 REGULATION OF POSTSYNAPTIC ORGANIZATION

which localizes to synapses, increases the number of clusters for the active zone protein Bassoon and the number of vesicle recycling sites. Importantly, blockade of Fz1 function by expression of the cystein-rich domain (CRD) of Fz1 results in a decreased number of presynaptic sites (Varela-Nallar et al., 2009). These results demonstrate that Fz1 contributes to presynaptic assembly in hippocampal neurons. Another study also showed that Fz5 regulates synaptic assembly in hippocampal neurons. Fz5 is expressed in the hippocampus during the peak of synaptogenesis and is present in axons and dendrites (Sahores et al., 2010). As neurons mature, a pool of this receptor localizes to both sides of the synapse with preference to the presynaptic membrane. Expression of Fz5 on axons stimulates the formation of presynaptic sites. Conversely, exposure to the CRD domain or shRNA knockdown of Fz5 significantly decreases the number of presynaptic sites. Binding assays demonstrated that Wnt7a, which is also expressed in the hippocampus, binds to the CRD domain of Fz5 (Carmon and Loose, 2008; Sahores et al., 2010). Importantly, shRNA knockdown of Fz5 suppresses the synaptogenic activity of Wnt7a (Sahores et al., 2010). Thus, Fz5 is a key receptor for the synaptic organizer Wnt7a. In summary, both Fz1 and Fz5 play a role in synaptogenesis in hippocampal neurons. Further studies are needed to elucidate the in vivo role of these receptors at central synapses. In addition to Fz receptors, Wnts stimulate presynaptic differentiation in hippocampal neurons through Ror1 and Ror2 receptors. RNA knockdown of these receptors by RNAi or by antisense oligonucleotides decreases the number of synaptophysin puncta in cultured hippocampal neurons. Biochemical analyses revealed that Wnt5a binds to Ror receptors, and suppression of Ror1 or Ror2 expression blocks the ability of Wnt5a to stimulate presynaptic differentiation in cultured neurons (Paganoni et al., 2010). Thus, signaling through two different Wnt ligands and different receptors contributes to the assembly of presynaptic sites in hippocampal neurons. Why are two Wnts involved in synapse formation? Do these signals have a cooperative effect? Future studies will address these questions.

32.4 REGULATION OF POSTSYNAPTIC ORGANIZATION

629

subsequently cleaved. The truncated C-terminus domain is subsequently imported to the nucleus to regulate postsynaptic assembly (Mathew et al., 2005; Figure 32.1). The transport of the receptor to the nucleus is regulated by dGRIP, an anchoring protein that directly interacts with DFz2 through two of its PDZ (a domain found in the PSD-96, Disc Large, and ZO1 proteins) domains. A combination of genetic and tissue-specific RNA interference experiments demonstrated that loss of dgrip function results in the formation of fewer synaptic boutons with abnormal morphology (Ataman et al., 2006). These defects are similar to those observed in wg mutants and when expression of dfz2 is reduced at the postsynaptic side (Packard et al., 2002). Importantly, selective knockdown of dgrip in the postsynaptic cell results in defects in the accumulation of postsynaptic markers and in the formation of postsynaptic specializations such as the subsynaptic reticulum. However, postsynaptic loss of dgrip also results in defects on the presynaptic side. In summary, these results demonstrate that dGRIP is crucial for the transport of DFz2 from the postsynaptic membrane to the nucleus and for synaptic assembly (Ataman et al., 2006). The exact mechanism by which the truncated DFz2 regulates postsynaptic assembly remains to be determined. Whether a similar mechanism operates in vertebrates is currently unknown. In Drosophila, Wnt5 also regulates NMJ formation. In contrast to Wg, Wnt5 signals through Drl, an atypical tyrosine kinase receptor (Liebl et al., 2008), and the fly orthologue of the RYK receptor (Hovens et al., 1992). The wnt5 mutant exhibits a reduction in the frequency of excitatory junctional currents and in the amplitude of the evoked junctional currents. Consistent with these electrophysiological defects, the density of the active zones and the number of boutons is reduced. Mutations in drl also result in a reduced number of synaptic boutons and suppress the effect of synaptic overgrowth induced by overexpression of Wnt5. Several experiments support the model where Wnt5 from motoneurons signals across the synaptic cleft on Drl, expressing muscle cells to regulate NMJ formation (Liebl et al., 2008). Thus, two Wnt signaling pathways, one through the DFz2 receptor and another through the Drl, regulate NMJ formation in Drosophila (Figure 32.4). This conclusion raises a number of questions. Why are two pathways required for NMJ formation? Do both pathways function in parallel or do they influence each other?

32.4.1 Postsynaptic Assembly of Invertebrate Peripheral Synapses Wnts also signal to the postsynaptic side of the synapse. The first evidence came from studies at the Drosophila NMJ. Dfz2 is present at the pre and postsynaptic membranes, but, on the postsynaptic muscle cell, Dfz2 is endocytosed, transported to the perinuclear area and

32.4.2 Vertebrate Neuromuscular Synapse At the vertebrate NMJ, Wnts also regulate postsynaptic organization. In contrast to the Drosophila glutamatergic synapse, the vertebrate NMJ is cholinergic. Here, acetylcholine receptors (AChRs) cluster in perfect

III. SYNAPTOGENESIS

630

32. WNT SIGNALING

apposition to the presynaptic axon terminal. However, early in development and before the arrival of motoneuron axons, AChRs form aneural clusters in the central domain of muscles, a process called prepatterning (for review see Wu et al., 2010). Later, with the arrival of motoneurons to the muscle field, bidirectional signaling between the presynaptic motor axon and the postsynaptic muscle cell regulates the assembly of NMJ. It is well established that the heparan sulfate proteoglycan agrin is released from the motoneurons and stimulates the clustering of AChRs on the muscle (McMahan, 1990) through muscle-specific kinase (MuSK), a tyrosine kinase receptor and the scaffold protein rapsyn (for review see Wu et al., 2010). However, agrin plays not only a role in the formation but in the maintenance of the NMJ (for review see Kummer et al., 2006; Wu et al., 2010; see Chapter 29). Recent studies demonstrate that Wnts regulate both early prepatterning of AChRs and the formation of the NMJ proper.

Wnt11r

Aneural clusters

?

LRP4

MuSK AChR Dvl

?

Non canonical signaling

Muscle fiber Motor axon

Wnt

?

Agrin

Microclusters

Clusters

MuSK AChR

LRP4 Dvl

Rac1

Rho

Muscle fiber

32.4.3 Prepatterning of AChRs in Muscle Cells Before the arrival of motor axons, aneural AChRs cluster in the central domain of the muscle, and this process is under the regulation of MuSK (Lin et al., 2001). Although MuSK functions as the receptor for agrin, during prepatterning, MuSK can induce AChR clusters independent of agrin and of the presence of motor axons (Lin et al., 2001). These findings suggest that MuSK is likely to be activated by a ligand coming from the muscle or surrounding tissues to regulate prepatterning (see Chapter 29). Using zebrafish embryos, Granato and colleagues demonstrated that Wnt acts through MuSK to regulate prepatterning. Morpholino knockdown of Wnt11r results in severe defects in the clustering of aneural AChRs (Jing et al., 2009). Wnt11r binds to Unplugged/MuSK receptors and requires unplugged for its function. Deletion analyses showed that Dvl, through a noncanonical pathway, regulates this process (Jing et al., 2009). Thus, Wnt11r by binding to Unplugged activates a signaling cascade that stimulates the clustering of aneural AChRs in the central region of the muscle before the arrival of the motoneuron axons (Figure 32.5).

32.4.4 Assembly of the Neuromuscular Synapse Although prepatterning of AChRs in the central muscle region is crucial for the appropriate targeting of motor axons, it is not required for the formation of neuromuscular synapses later in development (Jing et al., 2009; Lomo, 2003). These findings raise the question: what controls the latter formation of proper synapses. It is well established that the motoneuron-derived,

FIGURE 32.5 Wnt signaling at the vertebrate neuromuscular junction. (a) Before the arrival of the nerve, Wnt11r binds to MuSK to regulate the formation of aneural clusters and Dvl is required for this process. It remains to be determined whether Wnt11r also signals through LRP4 or Fz (depicted in yellow). (b) During formation of the NMJ, agrin and Wnt collaborate to regulate the formation of AChR clusters. Wnt through the activation of Rac1 stimulate the formation of unstable microclusters, which are then converted to larger and stable clusters in the presence of agrin and activation of Rho.

secreted protein agrin plays a crucial role in NMJ formation and maintenance (for review see Kummer et al., 2006; Wu et al., 2010). Through a combination of genetic and cell culture studies, a model has emerged in which agrin-MuSK signaling triggers the clustering of AChRs through the scaffold protein rapsyn (Apel et al., 1997; Gautam et al., 1995) and activation of Rho GTPases (Weston et al., 2000, 2003). However, other signals could influence agrin function. A recent study revealed that Wnts collaborate with agrin to regulate clustering of AChRs during the formation of the NMJ. Ectopic expression of Wnt3, which is normally expressed by motoneurons, stimulates the formation of AChR clusters in the chick limb. Conversely, blockade of endogenous Wnts with the secreted Wnt antagonist Sfrp1 substantially decreases the formation of AChR clusters in the limb (Henriquez et al., 2008). Experiments using cultured myotubes showed that Wnt3 increases the formation of small and unstable clusters of AChRs. In the presence of agrin, in contrast, these microclusters get converted to large stable clusters. Thus, Wnt3 increases the clustering activity of agrin by increasing the number of microclusters. How does Wnt signaling regulate AChR clustering? Dvl has been shown to bind to MuSK and is required

III. SYNAPTOGENESIS

32.5 WNT PROTEINS AS ANTISYNAPTOGENIC FACTORS

for agrin-mediated clustering of AChRs in culture myotubes (Luo et al., 2002). Moreover, Dvl1 knockout mutant mice exhibit defects similar to those caused by loss of agrin, albeit mild, in the clustering of AChRs in the diaphragm (Henriquez et al., 2008). Moreover, Dvl seems to signal through a noncanonical pathway to regulate AChR clustering (Luo et al., 2002). Earlier studies had demonstrated that agrin induces clustering through the activation of both Rac and Rho (Weston et al., 2000, 2003). Interestingly, Wnt3 alone activates Rac1, whereas agrin activates both Rac and Rho. Moreover, blockade of Rac activity suppresses the ability of Wnt3 to induce the formation of microclusters in myotubes (Henriquez et al., 2008). The combined results in the chick and mouse systems strongly support a role for Wnts in collaborating with agrin to regulate the clustering of AChRs, a prerequisite for postsynaptic assembly (Figure 32.5). However, it remains to be determined whether Wnt3 or other Wnts from motoneurons play a role in agrin-mediated synaptic assembly. Although the data strongly suggest that Wnt3 collaborates with agrin by further increasing Rac activity, the question as to how Wnts enhance agrin activity remains open. Although the receptors that mediate Wnt function during NMJ formation remain poorly characterized, some studies point to the agrin receptor MuSK and its coreceptor LRP4. The finding that Wnt11r binds to MuSK raises the possibility that, in higher vertebrates, Wnts might bind to MuSK to stimulate agrin function. However, other models are also possible. For example, agrin does not bind directly to MuSK but binds to LRP4, a single pass membrane receptor (Kim et al., 2008; Zhang et al., 2008). LRP4 is expressed in muscle cells and required for agrin function and NMJ formation (Kim et al., 2008; Weatherbee et al., 2006; Zhang et al., 2008). Both receptors, MuSK and LRP4, share homology with the extracellular domains of Fz (Masiakowski and Yancopoulos, 1998; Saldanha et al., 1998; Stiegler et al., 2009) and LRP5/6 receptors, respectively (Schneider and Nimpf, 2003). As Wnts stimulate the formation of a complex between Fzs and LRP5/6, Wnt could collaborate with agrin by increasing the binding of agrin to its receptors, LRP4 and MuSK, resulting in the formation of a functional ligand–receptor complex. Further studies are needed to establish the mechanism by which Wnts collaborate with agrin at the NMJ.

32.4.5 Postsynaptic Assembly at Central Synapses At the vertebrate central nervous system (CNS), much less is known about the role of Wnt signaling in postsynaptic organization. Studies using cultured hippocampal neurons have demonstrated that short-term (1 h)

631

exposure to Wnt5a increases the number of puncta of PSD-95 (Farias et al., 2009), a protein crucial for the synaptic localization of glutamate receptors and a hallmark of postsynaptic differentiation (Kim and Sheng, 2004; Sheng and Hoogenraad, 2007). Interestingly, Wnt5a does not affect the clustering of synaptophysin during this short exposure, suggesting that this Wnt factor directly affects the organization of the postsynaptic side without affecting the presynaptic terminal. Rather than affecting the total level of PSD-95, clustering occurs through the recruitment from an existing pool. Consistent with an effect on postsynaptic organization, Wnt5a increases the amplitude of fEPSPs when Schaeffer collaterals are stimulated. Wnt5a regulates PSD-95 clustering through the activation of a noncanonical pathway. Wnt5a increases JNK activation, whereas blockade of JNK abolishes the effect of Wnt5a (Farias et al., 2009). Interestingly, previous studies have shown that JNK1 phosphorylates PSD-95 at serine 295 and that this posttranslational modification regulates PSD-95 localization to synapses (Kim et al., 2007). Although the in vivo role of Wnt5a in hippocampal synaptogenesis needs to be examined, these studies strongly suggest that Wnt5a–JNK signaling regulates PSD-95 localization through changes in PSD-95 phosphorylation.

32.5 WNT PROTEINS AS ANTISYNAPTOGENIC FACTORS 32.5.1 Regulation of Synaptic Distribution Wnt proteins can also inhibit the formation of synapses. In Caenorhabditis elegans, Lin-44, a Wnt protein expressed in the posterior region of the worm, negatively regulates the formation of synaptic boutons between presynaptic dorsal A (DA) motoneurons and their postsynaptic muscle cells (Klassen and Shen, 2007). In this system, the cell body of the DA neuron is located ventrally and its axon extends posteriorly and then moves dorsally to continue in a caudal to rostral direction. Synapses are primarily formed on the most dorsoanterior part of the DA axons but not in the most posterior region (Figure 32.6). In the lin-44 mutant, however, synapses appear on the posterior region though, interestingly, the total number of synapses is the same in both wild type and lin-44 mutant. These results suggest that Lin-44 locally inhibits synapse formation within a section of the axon resulting in the redistribution of synaptic boutons. Further studies demonstrated that Fz is required for Lin-44 function as mutants, as lin-17/Fz exhibit a similar phenotype to the lin-44 mutant. Lin-44 positively regulates the localization of Lin-17 receptor along the DA axon (Klassen and Shen, 2007). Thus, a Wnt ligand through the regulation of Fz receptor localization determines the distribution of synapses.

III. SYNAPTOGENESIS

632

32. WNT SIGNALING

Phenotype of lin-44 mutant

Model Wild type Lin-44 gradient

Asynaptic area

Wnt/Lin-44

Axon Wnt/lin-44-/-

Synaptic area

Asynaptic area

Reduced asynaptic area

Lin-17 Lin-44

DA9 (a)

Synaptic boutons

(b)

Synaptic boutons

FIGURE 32.6 Wnt restricts the localization of synapses. (a) In wild type C. elegans, cholinergic DA9 motoneurons do not assemble synapses at the most posterior domain (asynaptic area) of the axon due to the presence of Wnt/Lin-44, which is expressed in the posterior hypodermis. In the lin-44 mutant, synaptic boutons now form in most posterior regions. However, the number of synapses remains the same as wild type animals. (b) Model illustrating the effect of Lin-44 on the distribution of Lin-17. The receptor Lin-17 is not present in part of the axon away from the source of Lin-44 resulting in the assembly of synaptic boutons.

Studies in Drosophila have provided evidence that Wnts, with antisynaptogenic activity, regulate the formation of synapses between specific motoneuron axons with their appropriate target muscles. Two muscles, M12 and M13, are innervated by distinct sets of motoneurons, MN12s and RP1, respectively (Chiba, 1999; Keshishian et al., 1996). Their motor axons run in parallel but then branch out to innervate different muscles. For example, axons from MN12s pass through M13 muscle to then synapse with M12 muscle. How is this specificity achieved? A single cell expression profiling of the different muscles revealed that Wnt4 is highly expressed in M13 (Inaki et al., 2007). Functional studies revealed that inhibition of Wnt4 function results in the ectopic formation of synapses between MN12 axons and M13 and the concomitant decrease of nerve endings into M12. This aberrant innervation is due to the loss of specificity by MN12 motoneurons. Further studies into the signaling pathway showed that Wnt4, through Fz2 and Drl together with Dvl, regulates synaptic inhibition at the M13 muscle. However, the downstream signaling pathway remains to be fully characterized. These findings, together with those in C. elegans, demonstrate that Wnts can shape the formation of neuronal circuits by promoting or inhibiting the formation of synapses at specific sites.

32.5.2 Wnt/b-Catenin Signaling at the Vertebrate NMJ Wnts might also play a negative role at the vertebrate NMJ. Wnt3a, a protein that shares a high level of homology with Wnt3, is expressed during the peak of

synaptogenesis in skeletal muscles. In contrast to Wnt3, Wnt3a decreases the ability of agrin to induce AChR clustering in cultured myotubes. When myotubes are exposed to agrin, and then subsequently to Wnt3a, AChRs become dispersed (Wang et al., 2008). Wnt3a usually activates the canonical Wnt signaling in a number of cell types and tissues including muscle cells. Consistent with this, activation of the canonical pathway by inhibition of Gsk3 or by expression of b-catenin in muscle cells results in the dispersal of AChRs. Conversely, inhibition of canonical Wnt signaling with the secreted Wnt antagonist Dkk1 increases AChR clustering (Wang et al., 2008). A possible mechanism by which Wnt3a induces AchR dispersal is by negatively regulating the transcription of rapsyn, a critical scaffold protein required for AChR clustering. Interestingly, regulation of rapsyn expression is dependent on b-catenin but not TCF. In vivo experiments showed that overexpression of Wnt3a or b-catenin decreases the size of AChR clusters in adult muscles when the NMJ has already formed (Wang et al., 2008). The in vivo role of b-catenin was further examined by specifically deleting b-catenin in muscle or in motoneurons using a conditional knockout mutant. Loss of muscle b-catenin results in a wide end-plate band containing larger AChR clusters (Li et al., 2008). These findings are consistent with the view that canonical Wnt signaling negatively regulates the assembly and/or maintenance of the vertebrate NMJ. Another study suggested that b-catenin has a different role at the NMJ. First, b-catenin directly interacts with rapsyn to form a complex with AChRs. Second,

III. SYNAPTOGENESIS

32.6 WNT SIGNALING AND ACTIVITY-MEDIATED SYNAPTIC REMODELING

agrin increases the interaction of b-catenin with AChR receptors. Third, shRNA knockdown of b-catenin in cultured myotubes significantly reduces the number of AChR clusters induced by agrin. As shown in another study (Wang et al., 2008), TCF function is not required for the effect of b-catenin (Zhang et al., 2007). How can one explain the apparent discrepancy on the role of b-catenin on AChR clustering? Although b-catenin is crucial for canonical Wnt signaling, it also plays a central role in cell adhesion, a process independent of Wnt signaling (Nelson and Nusse, 2004). Indeed, the positive effect of b-catenin on agrin function is dependent on a-catenin, a key molecule that regulates cell adhesion (Zhang et al., 2007). Further studies are needed to resolve the function of b-catenin at the NMJ. However, the studies on Wnt3 and Wnt3a strongly support a model where noncanonical Wnt signaling promotes AChR clustering, whereas canonical signaling disperses clustering by affecting agrin function.

32.5.3 Inhibition of Synapse Formation at Central Synapses Further evidence that Wnts can inhibit synapse formation comes from studies in cultured hippocampal neurons. Unexpectedly, Wnt5a inhibits the clustering of presynaptic marker vGlut1 (Davis et al., 2008). These results differ from a more recent study which showed that Wnt5a does not affect presynaptic differentiation (Farias et al., 2009). Possible difference in the maturity of the cultured neurons or in the timing of exposure to Wnts could explain these apparently conflicting results. Further studies are needed to elucidate the role of Wnt5a. However, the finding that Wnt5a can inhibit presynaptic differentiation under certain conditions raises the interesting possibility that Wnts act both as antiand prosynaptogenic factors in the vertebrate system. The balance of molecules with opposite effects on synapse formation could play a crucial role in determining the number and location of synapses within the neuron, resulting in profound changes in the circuit.

32.6 WNT SIGNALING AND ACTIVITYMEDIATED SYNAPTIC REMODELING 32.6.1 Neuronal Activity, Wnts and Their Receptors Many of the processes regulated by Wnts, such as terminal remodeling of axons and dendritic development, are also modulated by neuronal activity. These findings raise the possibility that activity could regulate the expression and/or release of Wnt proteins or the localization of their signaling components.

633

The first evidence that neuronal activity regulates Wnts came from studies looking at factors released upon depolarization of cultured hippocampal neurons. KCl depolarization results in an increased level of Wnt activity in the culture medium (Yu and Malenka, 2003). In addition, Wnt3a protein levels increase upon tetanic stimulation (Chen et al., 2006). Further studies into the function of Wnts and neuronal activity led to the discovery that N-methyl-D-aspartic acid (NMDA) receptor activation increases the expression of Wnt2 in the hippocampus (Wayman et al., 2006). Indeed, blockade of excitatory transmission or CaMKI activity suppresses the effect of neuronal activity on dendrite growth. Importantly, activation of NMDA receptors triggers a signaling cascade through the CaMKK pathway, resulting in the subsequent activation of Ras/MEK/ERK and cAMP response element-binding protein (CREB). Knockdown of CREB with siRNA blocks the dendritic arborization induced by neuronal activity. To identify the targets of CREB, serial analyses of chromatin occupancy (SACO) were performed. SACO revealed that Wnt2 is a key target. Consistent with this result, depolarization of cultured neurons with KCl results in a significant increase in the level of Wnt2 mRNA. Importantly, the secreted Wnt antagonist Wif blocks activity-mediated dendritic outgrowth whereas Wnt2 stimulates dendritic development (Wayman et al., 2006). Combined, these results demonstrate that neuronal activity stimulates the expression of Wnts in the hippocampus, which in turn induces profound changes in neuronal connectivity. It remains to be determined if Wnt2 plays a role in synapse formation or function. Neuronal activity plays a critical role in the formation and function of neuronal circuits as the number, morphology, and efficacy of synapses are dramatically modified by the pattern of activity. Studies at the Drosophila larval NMJ have demonstrated that activity through Wg induces structural changes at synapses. Time-lapse recordings of GFP-labeled motor axons demonstrated that depolarization induces structural changes manifested by increased number of synaptic boutons and filopodia (Ataman et al., 2008). The initial boutons do not contain many of the typical synaptic markers and, therefore, they represent ‘ghost’ boutons. With time, however, these ghost boutons develop into mature boutons containing the characteristic pre- and postsynaptic markers. Consistent with increased synapse number, potentiation of miniature excitatory junctional potentials was also observed. As neuronal activity increases the level of endogenous Wg protein at the NMJ synapse, its role was further examined. Importantly, the formation of ghost boutons induced by patterned activity is suppressed in a temperature-sensitive mutant of Wg. Thus, Wg protein is required for the structural changes induced by activity.

III. SYNAPTOGENESIS

634

32. WNT SIGNALING

How does Wg regulate activity-mediated synaptic changes? Wg signals bidirectionally to activate a divergent canonical Wnt pathway, on the presynaptic side, and a noncanonical pathway, on the postsynaptic muscle cell in which the DFz2 receptor is cleaved and subsequently translocated to the nucleus (Mathew et al., 2005; Miech et al., 2008; Packard et al., 2002). Careful analyses by overexpressing Sgg/Gsk3 on motoneurons or by interfering with the nuclear import of DFz2 revealed that Wg signals to both sides of the synapse to induce structural changes (Ataman et al., 2008). Thus, patterned activity induces the release of Wg, which then signals to both sides of the synapse to regulate structural changes at stimulated synapses. Wg expression or release is also regulated by activity in the Drosophila adult olfactory system. Rodrigues and colleagues have demonstrated that Wg regulates the maintenance of adult olfactory sensory neurons (OSNs). These neurons are located in the antennae and maxillary palps and project to glomeruli in the antennal lobe where they make synaptic contacts (Laissue and Vosshall, 2008). The maintenance of these OSN in the adult is regulated by cell-autonomous neuronal activity. Although activity is not required for formation of the neuronal circuit during development, blockade of neuronal activity in the adult results in axon and synaptic elimination (Chiang et al., 2009). Spaced depolarization with KCl increases the levels of endogenous Wg protein in the antennal lobes, suggesting that neuronal activity regulates the maintenance of neuronal circuits through Wg. In the vertebrate, neuronal activity also regulates the localization of Fz receptors. In hippocampal neurons, activity increases the level of surface Fz5, a receptor for Wnt7a, which mediates its presynaptic assembly activity. A fraction of the receptor pool is localized to excitatory synapses as determined by its colocalization with the pre and postsynaptic markers, vGlut1 and NR1, respectively. Neuronal activity also increases the surface Control

HFS

level of Fz5 localized to synapses without affecting the total level of the protein (Sahores et al., 2010). These results demonstrate that activity increases the trafficking of Fz5 to the surface and particularly to synapses. Interestingly, different type of neuronal activity has different effects. While high-frequency stimulation (HFS) increases surface Fz5 receptor to synapses, low-frequency stimulation decreases the level of surface receptors. What is the mechanism by which neuronal activity regulates Fz5 mobilization? As neuronal activity regulates the expression of Wnts (Ataman et al., 2008) and Wnt ligands regulate the distribution of Fz receptors (Klassen and Shen, 2007; Wu and Herman, 2007), the effect of endogenous Wnts on Fz5 was examined. Blockade of endogenous Wnts with the secreted Wnt antagonist Sfrp1 suppresses the recruitment of Fz5 to synapses induced by HFS. Similarly, the soluble form of the extracellular domain of the Fz5 receptor blocks HFS-mediated mobilization of Fz5. These findings strongly suggest that activity regulates the level of endogenous Wnts and that these Wnts induce the mobilization of surface Fz5 receptor to synapses (Figure 32.7). As Fz5 mediates the synaptogenic activity of Wnt7a, the increased mobilization of Fz5 will lead to positive feedback loop resulting in a further sensitization of the cells to endogenous Wnts. An important finding that emerged from these studies was that blockade of Wnt signaling suppresses the ability of HFS to increase the number of synapses.

32.6.2 Synaptic Remodeling in the Adult Hippocampus It is well documented that the environment induces morphological and functional changes to synaptic connections (for review see Feldman, 2009; Holtmaat and Svoboda, 2009; Lomo, 2003; Sanes and Bao, 2009; see Chapter 37). In the CNS, for example, sensory

HFS + Fz5 blockade

Presynapse

Postsynapse

Fz-5

Wnt

Fz5-CRD

III. SYNAPTOGENESIS

FIGURE 32.7 Neuronal activity regulates the mobilization of surface and synaptic Fz5. In control conditions, surface Fz5 (sFz5) is present in the pre- and postsynaptic membranes. High-frequency stimulation (HFS) increases the levels of surface Fz5 and the amount of sFz5 at synapses. This is correlated with an increase in the number of synapses. Blockade of endogenous Wnts with the secreted CRD domain of Fz5 suppresses the increase of sFz5 and the synaptogenic effect of HFS. The proposed model suggests that neuronal activity increases the level of endogenous Wnts, which in turn increase the accumulation of surface Fz5 at the synapse. Signaling through Fz5 contributes to activity-mediated synapse formation.

32.6 WNT SIGNALING AND ACTIVITY-MEDIATED SYNAPTIC REMODELING

stimulation regulates the formation and stability of dendritic spines (Alvarez and Sabatini, 2007; Trachtenberg et al., 2002). However, the mechanisms that control the structural changes induced by neuronal activity remain poorly understood. In the hippocampus, mossy fiber axons from the dentate gyrus granule cells form complex synapses with the dendrites of pyramidal neurons in the CA3 region (Claiborne et al., 1986; Henze et al., 2000). Exposure to an enriched environment (EE) induces the remodeling of the large mossy fiber terminals (LMTs) in the CA3 region by increasing the complexity of these terminals and the number of satellite LMTs (Gogolla et al., 2009). There are striking similarities between the terminal remodeling of the hippocampal mossy fibers and pontine mossy fiber terminals in the cerebellum (Hamori and Somogyi, 1983). As mentioned above, Wnt7a regulates terminal remodeling of mossy axons during synapse formation in the cerebellum (Hall et al., 2000). These findings led to the suggestion that Wnts might also regulate the activity-mediated changes in the LMTs. Indeed, the level of endogenous Wnt7a/7b proteins significantly increases in the hippocampus when animals are exposed to an EE. Importantly, local blockade with the secreted Wnt antagonist Sfrp1 during the exposure to EE suppresses the terminal remodeling induced by EE (Gogolla et al., 2009). Thus, behavioral experience regulates synapse remodeling in the hippocampus through Wnt signaling.

32.6.3 Emerging Roles for Wnts Several studies suggest that Wnt signaling may also play a role in synaptic function. Electrophysiological recordings at the mossy fiber-granule cell synapse of the double Wnt7a;Dvl1 mutant have suggested that Wnt signaling might regulate neurotransmitter release (Ahmad-Annuar et al., 2006). In addition, recordings of hippocampal brain slices exposed to Wnt7a or to the pharmacological Wnt agonist WASP revealed a decrease in paired-pulse facilitation, suggesting an increase in neurotransmitter release (Cerpa et al., 2008) and an increase in excitatory transmission, respectively (Beaumont et al., 2007). Moreover, blockade of Wnt signaling impairs, whereas Wnts increase long-term potentiation in brain slices (Chen et al., 2006). These findings support the notion that Wnt signaling modulates synaptic function. However, further studies using loss of functional approaches are needed to determine the precise function of Wnt signaling in synaptic transmission and plasticity. Future studies will also reveal whether the interplay between Wnts with synaptic promoting and inhibiting activities collaborate with other synaptic organizers to regulate the formation of functional and complex neuronal circuits.

635

Glossary Active zone Area close to the plasma membrane on the presynaptic terminal where neurotransmitter-containing synaptic vesicles are localized and where neurotransmitters are released. Allele A specific form of a gene. Individual alleles of the gene can result in the complete loss of function (null allele), a partial loss of function (hypomorphic allele) or a gain of function of the gene. Antagonist A molecule that blocks or prevents the action of another molecule. Anterograde signal A signal released by the presynaptic terminal that acts on the postsynaptic side. Axon A long process that emanates from the cell body of a neuron and has the ability to propagate action potentials resulting in the release of neurotransmitters. Axon guidance The process by which axons are guided to their appropriate synaptic targets. During guidance, axons extend, branch and retract along their trajectory. Bouton A specialized area in the axon that contains the machinery for the release of neurotransmitters. Boutons are present in both central and peripheral nervous system. Calcium–calmodulin-dependent kinase (CaMK) A serine/threonine kinase that is activated by rise in intracellular calcium. Coreceptor A transmembrane or a membrane-associated protein that facilitates the function of a receptor by forming a complex with the main receptor. Dendrite Branched projections that emanate from the cell body of a neuron with the ability to receive and conduct electrical stimuli from the end of the process to the cell body of the neuron. Dendritogenesis A process that involves the formation, growth and branching of dendrites. Glycogen synthase kinase-3 (Gsk3) A serine/threonine kinase that is inhibited by either insulin or Wnt proteins. Long-term potentiation (LTP) A long lasting enhancement in synaptic transmission between neurons, which is generated by coincident activity in pre- and postsynaptic neurons. LTP is believed to mediate the storage of information in the brain. Microtubule organization Distribution of microtubules within the cell. Some microtubules are organized as straight parallel bundles whereas others bend and form complex loops. Microtubule stability Microtubules are dynamic polymers that are constantly polymerizing and depolymerizing. Stable microtubules are less susceptible to microtubule depolymerising drugs. Microtubules Large polymers of tubulin that regulate the movement and segregation of chromosomes, the transport of vesicle and molecules in the cell and that determine cell shape. Miniature excitatory postsynaptic currents (mEPSCs) Postsynaptic currents at excitatory synapses elicited by the spontaneous fusion of synaptic vesicles on the presynaptic terminal. Muscle-specific kinase (MuSK) A tyrosine kinase receptor present in the surface of muscle cells and regulates the assembly of the postsynaptic terminal. Neuromuscular junction (NMJ) The peripheral synapse formed between a motor axon and a muscle fiber. Neurotransmitter A chemical released from a nerve terminal that carries signals from a neuron to another neuron or muscle cell. Neurotransmitters are packed within synaptic vesicles in the presynaptic cell; these vesicles fused to the plasma membrane and release the neurotransmitters within the synaptic cleft. Postsynaptic density A multiprotein complex containing scaffold proteins, signaling molecules and cytoskeleton proteins located in very close proximity to the plasma membrane of the postsynaptic cell. Under the electron microscope is visible for its electron dense material. Receptor Receptor is a protein embedded within the plasma membrane that has the ability to bind an extracellular molecule resulting in structural changes, which trigger a cellular response.

III. SYNAPTOGENESIS

636

32. WNT SIGNALING

Retrograde signal A signal released by the postsynaptic side that acts on the presynaptic terminal. Short hairpin RNA (ShRNA) A ShRNA that has the ability to silence the expression of a specific gene. Synapse A specialized cellular junction that connects neurons with one another or neurons with their peripheral targets. At synapses, an electrical stimulus is converted into a chemical signal released by the presynaptic cell. This chemical signal is then converted back into an electrical impulse on the postsynaptic cell. Central and peripheral synapses are located within the central nervous system or in the periphery (muscles or glands). Synaptic vesicle A vesicle where neurotransmitters are stored. Synaptic vesicles fuse to the plasma membrane to release their contents into the synaptic space. Synaptogenesis A process that involves the formation of new synapses. Pre- and postsynaptic differentiation is defined as the specific assembly of the pre- and postsynaptic sides of the synapse. Temperature-sensitive allele Mutant allele that encodes for a protein that is functional only under low or high temperatures. Wnts A family of lipid-modified secreted glycoproteins that play a crucial role in cell–cell communication in embryonic patterning, cell migration and neuronal connectivity. Zebrafish Danio rerio, a tropical freshwater fish with characteristic stripes along the anterior to posterior axis. It is the prominent model organism for the study of early developmental processes due to its transparency and fast development.

Acknowledgments Our work is funded by the MRC, the Wellcome Trust, EU, Alzheimer’s Research UK, and Parkinson’s UK.

References Ahmad-Annuar, A., Ciani, L., Simeonidis, I., et al., 2006. Signaling across the synapse: A role for Wnt and Dishevelled in presynaptic assembly and neurotransmitter release. Journal of Cell Biology 174, 127–139. Alvarez, V.A., Sabatini, B.L., 2007. Anatomical and physiological plasticity of dendritic spines. Annual Review of Neuroscience 30, 79–97. Andersson, E.R., Prakash, N., Cajanek, L., et al., 2008. Wnt5a regulates ventral midbrain morphogenesis and the development of A9-A10 dopaminergic cells in vivo. PLoS One 3, e3517. Angers, S., Moon, R.T., 2009. Proximal events in Wnt signal transduction. Nature Reviews Molecular Cell Biology 10, 468–477. Apel, E.D., Glass, D.J., Moscoso, L.M., Yancopoulos, G.D., Sanes, J.R., 1997. Rapsyn is required for MuSK signaling and recruits synaptic components to a MuSK-containing scaffold. Neuron 18, 623–635. Ataman, B., Ashley, J., Gorczyca, D., et al., 2006. Nuclear trafficking of Drosophila Frizzled-2 during synapse development requires the PDZ protein dGRIP. Proceedings of the National Academy of Sciences of the United States of America 103, 7841–7846. Ataman, B., Ashley, J., Gorczyca, M., et al., 2008. Rapid activitydependent modifications in synaptic structure and function require bidirectional Wnt signaling. Neuron 57, 705–718. Banziger, C., Soldini, D., Schutt, C., Zipperlen, P., Hausmann, G., Basler, K., 2006. Wntless, a conserved membrane protein dedicated to the secretion of Wnt proteins from signaling cells. Cell 125, 509–522. Bartscherer, K., Pelte, N., Ingelfinger, D., Boutros, M., 2006. Secretion of Wnt ligands requires Evi, a conserved transmembrane protein. Cell 125, 523–533. Beaumont, V., Thompson, S.A., Choudhry, F., et al., 2007. Evidence for an enhancement of excitatory transmission in adult CNS by Wnt

signaling pathway modulation. Molecular and Cellular Neurosciences 35, 513–524. Cabello, J., Neukomm, L.J., Gunesdogan, U., et al., 2010. The Wnt pathway controls cell death engulfment, spindle orientation, and migration through CED-10/Rac. PLoS Biology 8, e1000297. Carmon, K.S., Loose, D.S., 2008. Wnt7a interaction with Fzd5 and detection of signaling activation using a split eGFP. Biochemical and Biophysical Research Communications 368, 285–291. Carmona-Fontaine, C., Matthews, H., Mayor, R., 2008. Directional cell migration in vivo: Wnt at the crest. Cell Adhesion and Migration 2, 240–242. Cerpa, W., Godoy, J.A., Alfaro, I., et al., 2008. Wnt-7a modulates the synaptic vesicle cycle and synaptic transmission in hippocampal neurons. Journal of Biological Chemistry 283, 5918–5927. Chen, J., Park, C.S., Tang, S.J., 2006. Activity-dependent synaptic Wnt release regulates hippocampal long term potentiation. Journal of Biological Chemistry 281, 11910–11916. Chen, W., ten Berge, D., Brown, J., et al., 2003. Dishevelled 2 recruits beta-arrestin 2 to mediate Wnt5A-stimulated endocytosis of Frizzled 4. Science 301, 1391–1394. Chiang, A., Priya, R., Ramaswami, M., Vijayraghavan, K., Rodrigues, V., 2009. Neuronal activity and Wnt signaling act through Gsk3-beta to regulate axonal integrity in mature Drosophila olfactory sensory neurons. Development 136, 1273–1282. Chiba, A., 1999. Early development of the Drosophila neuromuscular junction: A model for studying neuronal networks in development. International Review of Neurobiology 43, 1–24. Ciani, L., Krylova, O., Smalley, M.J., Dale, T.C., Salinas, P.C., 2004. A divergent canonical WNT-signaling pathway regulates microtubule dynamics: Dishevelled signals locally to stabilize microtubules. Journal of Cell Biology 164, 243–253. Claiborne, B.J., Amaral, D.G., Cowan, W.M., 1986. A light and electron microscopic analysis of the mossy fibers of the rat dentate gyrus. Journal of Comparative Neurology 246, 435–458. Clevers, H., 2006. Wnt/beta-catenin signaling in development and disease. Cell 127, 469–480. Davis, E.K., Zou, Y., Ghosh, A., 2008. Wnts acting through canonical and noncanonical signaling pathways exert opposite effects on hippocampal synapse formation. Neural Development 3, 32. De Calisto, J., Araya, C., Marchant, L., Riaz, C.F., Mayor, R., 2005. Essential role of non-canonical Wnt signalling in neural crest migration. Development 132, 2587–2597. De Ferrari, G.V., Papassotiropoulos, A., Biechele, T., et al., 2007. Common genetic variation within the low-density lipoprotein receptorrelated protein 6 and late-onset Alzheimer’s disease. Proceedings of the National Academy of Sciences of the United States of America 104, 9434–9439. Etienne-Manneville, S., 2009. APC in cell migration. Advances in Experimental Medicine and Biology 656, 30–40. Farias, G.G., Alfaro, I.E., Cerpa, W., et al., 2009. Wnt-5a/JNK signaling promotes the clustering of PSD-95 in hippocampal neurons. Journal of Biological Chemistry 284, 15857–15866. Feldman, D.E., 2009. Synaptic mechanisms for plasticity in neocortex. Annual Review of Neuroscience 32, 33–55. Fradkin, L.G., Dura, J.M., Noordermeer, J.N., 2010. Ryks: New partners for Wnts in the developing and regenerating nervous system. Trends in Neurosciences 33, 84–92. Fradkin, L.G., Noordermeer, J.N., Nusse, R., 1995. The Drosophila Wnt protein DWnt-3 is a secreted glycoprotein localized on the axon tracts of the embryonic CNS. Developmental Biology 168, 202–213. Franch-Marro, X., Wendler, F., Guidato, S., et al., 2008. Wingless secretion requires endosome-to-Golgi retrieval of Wntless/Evi/Sprinter by the retromer complex. Nature Cell Biology 10, 170–177. Franco, B., Bogdanik, L., Bobinnec, Y., et al., 2004. Shaggy, the homolog of glycogen synthase kinase 3, controls neuromuscular junction growth in Drosophila. Journal of Neuroscience 24, 6573–6577.

III. SYNAPTOGENESIS

32.6 WNT SIGNALING AND ACTIVITY-MEDIATED SYNAPTIC REMODELING

Gao, C., Chen, Y.G., 2009. Dishevelled: The hub of Wnt signaling. Cellular Signalling 22 (5), 717–727. Gautam, M., Noakes, P.G., Mudd, J., et al., 1995. Failure of postsynaptic specialization to develop at neuromuscular junctions of rapsyndeficient mice. Nature 377, 232–236. Gogolla, N., Galimberti, I., Deguchi, Y., Caroni, P., 2009. Wnt signaling mediates experience-related regulation of synapse numbers and mossy fiber connectivities in the adult hippocampus. Neuron 62, 510–525. Goold, R.G., Owen, R., Gordon-Weeks, P.R., 1999. Glycogen synthase kinase 3beta phosphorylation of microtubule-associated protein 1B regulates the stability of microtubules in growth cones. Journal of Cell Science 112 (Pt 19), 3373–3384. Gordon, M.D., Nusse, R., 2006. Wnt signaling: multiple pathways, multiple receptors, and multiple transcription factors. Journal of Biological Chemistry 281, 22429–22433. Green, J.L., Kuntz, S.G., Sternberg, P.W., 2008. Ror receptor tyrosine kinases: Orphans no more. Trends in Cell Biology 18, 536–544. Gros, J., Serralbo, O., Marcelle, C., 2009. WNT11 acts as a directional cue to organize the elongation of early muscle fibres. Nature 457, 589–593. Habas, R., Dawid, I.B., He, X., 2003. Coactivation of Rac and Rho by Wnt/Frizzled signaling is required for vertebrate gastrulation. Genes and Development 17, 295–309. Habas, R., Kato, Y., He, X., 2001. Wnt/Frizzled activation of Rho regulates vertebrate gastrulation and requires a novel Formin homology protein Daam1. Cell 107, 843–854. Hall, A.C., Lucas, F.R., Salinas, P.C., 2000. Axonal remodeling and synaptic differentiation in the cerebellum is regulated by WNT-7a signaling. Cell 100, 525–535. Hamori, J., Somogyi, J., 1983. Differentiation of cerebellar mossy fiber synapses in the rat: A quantitative electron microscope study. Journal of Comparative Neurology 220, 365–377. Henriquez, J.P., Webb, A., Bence, M., et al., 2008. Wnt signaling promotes AChR aggregation at the neuromuscular synapse in collaboration with agrin. Proceedings of the National Academy of Sciences of the United States of America 105, 18812–18817. Henze, D.A., Urban, N.N., Barrionuevo, G., 2000. The multifarious hippocampal mossy fiber pathway: A review. Neuroscience 98, 407–427. Holtmaat, A., Svoboda, K., 2009. Experience-dependent structural synaptic plasticity in the mammalian brain. Nature Reviews Neuroscience 10, 647–658. Hovens, C.M., Stacker, S.A., Andres, A.C., Harpur, A.G., Ziemiecki, A., Wilks, A.F., 1992. RYK, a receptor tyrosine kinase-related molecule with unusual kinase domain motifs. Proceedings of the National Academy of Sciences of the United States of America 89, 11818–11822. Inaki, M., Yoshikawa, S., Thomas, J.B., Aburatani, H., Nose, A., 2007. Wnt4 is a local repulsive cue that determines synaptic target specificity. Current Biology 17, 1574–1579. Inestrosa, N.C., Arenas, E., 2009. Emerging roles of Wnts in the adult nervous system. Nature Reviews Neuroscience 11, 77–86. Jing, L., Lefebvre, J.L., Gordon, L.R., Granato, M., 2009. Wnt signals organize synaptic prepattern and axon guidance through the zebrafish unplugged/MuSK receptor. Neuron 61, 721–733. Keshishian, H., Broadie, K., Chiba, A., Bate, M., 1996. The Drosophila neuromuscular junction: A model system for studying synaptic development and function. Annual Review of Neuroscience 19, 545–575. Kim, M.J., Futai, K., Jo, J., Hayashi, Y., Cho, K., Sheng, M., 2007. Synaptic accumulation of PSD-95 and synaptic function regulated by phosphorylation of serine-295 of PSD-95. Neuron 56, 488–502. Kim, E., Sheng, M., 2004. PDZ domain proteins of synapses. Nature Reviews Neuroscience 5, 771–781. Kim, N., Stiegler, A.L., Cameron, T.O., et al., 2008. Lrp4 is a receptor for Agrin and forms a complex with MuSK. Cell 135, 334–342. Klassen, M.P., Shen, K., 2007. Wnt signaling positions neuromuscular connectivity by inhibiting synapse formation in C. elegans. Cell 130, 704–716.

637

Kohn, A.D., Moon, R.T., 2005. Wnt and calcium signaling: Beta-cateninindependent pathways. Cell Calcium 38, 439–446. Korkut, C., Ataman, B., Ramachandran, P., et al., 2009. Trans-synaptic transmission of vesicular Wnt signals through Evi/Wntless. Cell 139, 393–404. Korkut, C., Budnik, V., 2009. WNTs tune up the neuromuscular junction. Nature Reviews Neuroscience 10, 627–634. Krylova, O., Herreros, J., Cleverley, K.E., et al., 2002. WNT-3, expressed by motoneurons, regulates terminal arborization of neurotrophin-3responsive spinal sensory neurons. Neuron 35, 1043–1056. Kuhl, M., Sheldahl, L.C., Malbon, C.C., Moon, R.T., 2000. Ca(2þ)/ calmodulin-dependent protein kinase II is stimulated by Wnt and Frizzled homologs and promotes ventral cell fates in Xenopus. Journal of Biological Chemistry 275, 12701–12711. Kummer, T.T., Misgeld, T., Sanes, J.R., 2006. Assembly of the postsynaptic membrane at the neuromuscular junction: paradigm lost. Current Opinion in Neurobiology 16, 74–82. Kuwabara, T., Hsieh, J., Muotri, A., et al., 2009. Wnt-mediated activation of NeuroD1 and retro-elements during adult neurogenesis. Nature Neuroscience 12, 1097–1105. Laissue, P.P., Vosshall, L.B., 2008. The olfactory sensory map in Drosophila. Advances in Experimental Medicine and Biology 628, 102–114. Li, X.M., Dong, X.P., Luo, S.W., et al., 2008. Retrograde regulation of motoneuron differentiation by muscle beta-catenin. Nature Neuroscience 11, 262–268. Li, L., Hutchins, B.I., Kalil, K., 2009. Wnt5a induces simultaneous cortical axon outgrowth and repulsive axon guidance through distinct signaling mechanisms. Journal of Neuroscience 29, 5873–5883. Lie, D.C., Colamarino, S.A., Song, H.J., et al., 2005. Wnt signalling regulates adult hippocampal neurogenesis. Nature 437, 1370–1375. Liebl, F.L., Wu, Y., Featherstone, D.E., Noordermeer, J.N., Fradkin, L., Hing, H., 2008. Derailed regulates development of the Drosophila neuromuscular junction. Developmental Neurobiology 68, 152–165. Lin, W., Burgess, R.W., Dominguez, B., Pfaff, S.L., Sanes, J.R., Lee, K.F., 2001. Distinct roles of nerve and muscle in postsynaptic differentiation of the neuromuscular synapse. Nature 410, 1057–1064. Lomo, T., 2003. What controls the position, number, size, and distribution of neuromuscular junctions on rat muscle fibers? Journal of Neurocytology 32, 835–848. Lucas, F.R., Goold, R.G., Gordon-Weeks, P.R., Salinas, P.C., 1998. Inhibition of GSK-3beta leading to the loss of phosphorylated MAP-1B is an early event in axonal remodelling induced by WNT-7a or lithium. Journal of Cell Science 111 (Pt 10), 1351–1361. Lucas, F.R., Salinas, P.C., 1997. WNT-7a induces axonal remodeling and increases synapsin I levels in cerebellar neurons. Developmental Biology 192, 31–44. Luo, Z.G., Wang, Q., Zhou, J.Z., et al., 2002. Regulation of AChR clustering by Dishevelled interacting with MuSK and PAK1. Neuron 35, 489–505. Mao, J., Wang, J., Liu, B., et al., 2001. Low-density lipoprotein receptorrelated protein-5 binds to Axin and regulates the canonical Wnt signaling pathway. Molecular Cell 7, 801–809. Mapp, O.M., Wanner, S.J., Rohrschneider, M.R., Prince, V.E., 2010. Prickle1b mediates interpretation of migratory cues during zebrafish facial branchiomotor neuron migration. Developmental Dynamics 239, 1596–1608. Masiakowski, P., Yancopoulos, G.D., 1998. The Wnt receptor CRD domain is also found in MuSK and related orphan receptor tyrosine kinases. Current Biology 8, R407. Mathew, D., Ataman, B., Chen, J., Zhang, Y., Cumberledge, S., Budnik, V., 2005. Wingless signaling at synapses is through cleavage and nuclear import of receptor DFrizzled2. Science 310, 1344–1347. McCartney, B.M., Nathke, I.S., 2008. Cell regulation by the Apc protein Apc as master regulator of epithelia. Current Opinion in Cell Biology 20, 186–193.

III. SYNAPTOGENESIS

638

32. WNT SIGNALING

McMahan, U.J., 1990. The agrin hypothesis. Cold Spring Harbor Symposia on Quantitative Biology 55, 407–418. Miech, C., Pauer, H.U., He, X., Schwarz, T.L., 2008. Presynaptic local signaling by a canonical wingless pathway regulates development of the Drosophila neuromuscular junction. Journal of Neuroscience 28, 10875–10884. Milat, F., Ng, K.W., 2009. Is Wnt signalling the final common pathway leading to bone formation? Molecular and Cellular Endocrinology 310, 52–62. Nelson, W.J., Nusse, R., 2004. Convergence of Wnt, beta-catenin, and cadherin pathways. Science 303, 1483–1487. Nikopoulos, K., Venselaar, H., Collin, R.W., et al., 2010. Overview of the mutation spectrum in familial exudative vitreoretinopathy and Norrie disease with identification of 21 novel variants in FZD4, LRP5, and NDP. Human Mutation 31, 656–666. Packard, M., Koo, E.S., Gorczyca, M., Sharpe, J., Cumberledge, S., Budnik, V., 2002. The Drosophila Wnt, wingless, provides an essential signal for pre- and postsynaptic differentiation. Cell 111, 319–330. Paganoni, S., Bernstein, J., Ferreira, A., 2010. Ror1-Ror2 complexes modulate synapse formation in hippocampal neurons. Neuroscience 165, 1261–1274. Purro, S.A., Ciani, L., Hoyos-Flight, M., Stamatakou, E., Siomou, E., Salinas, P.C., 2008. Wnt regulates axon behavior through changes in microtubule growth directionality: A new role for adenomatous polyposis coli. Journal of Neuroscience 28, 8644–8654. Reya, T., Clevers, H., 2005. Wnt signalling in stem cells and cancer. Nature 434, 843–850. Roos, J., Hummel, T., Ng, N., Klambt, C., Davis, G.W., 2000. Drosophila Futsch regulates synaptic microtubule organization and is necessary for synaptic growth. Neuron 26, 371–382. Rosso, S.B., Sussman, D., Wynshaw-Boris, A., Salinas, P.C., 2005. Wnt signaling through Dishevelled, Rac and JNK regulates dendritic development. Nature Neuroscience 8, 34–42. Sahores, M., Gibb, A., Salinas, P.C., 2010. Frizzled-5, a receptor for the synaptic organizer Wnt7a, regulates activity-mediated synaptogenesis. Development 137, 2215–2225. Saldanha, J., Singh, J., Mahadevan, D., 1998. Identification of a Frizzled-like cysteine rich domain in the extracellular region of developmental receptor tyrosine kinases. Protein Science 7, 1632–1635. Salinas, P.C., 2007. Modulation of the microtubule cytoskeleton: A role for a divergent canonical Wnt pathway. Trends in Cell Biology 17, 333–342. Salinas, P.C., Zou, Y., 2008. Wnt signaling in neural circuit assembly. Annual Review of Neuroscience 31, 339–358. Sanchez, C., Perez, M., Avila, J., 2000. GSK3beta-mediated phosphorylation of the microtubule-associated protein 2C (MAP2C) prevents microtubule bundling. European Journal of Cell Biology 79, 252–260. Sanes, D.H., Bao, S., 2009. Tuning up the developing auditory CNS. Current Opinion in Neurobiology 19, 188–199. Schneider, W.J., Nimpf, J., 2003. LDL receptor relatives at the crossroad of endocytosis and signaling. Cellular and Molecular Life Sciences 60, 892–903. Segalen, M., Bellaiche, Y., 2009. Cell division orientation and planar cell polarity pathways. Seminars in Cell and Developmental Biology 20, 972–977. Semenov, M.V., Tamai, K., Brott, B.K., Kuhl, M., Sokol, S., He, X., 2001. Head inducer Dickkopf-1 is a ligand for Wnt coreceptor LRP6. Current Biology 11, 951–961. Sheng, M., Hoogenraad, C.C., 2007. The postsynaptic architecture of excitatory synapses: A more quantitative view. Annual Review of Biochemistry 76, 823–847. Sperber, B.R., Leight, S., Goedert, M., Lee, V.M., 1995. Glycogen synthase kinase-3 beta phosphorylates tau protein at multiple sites in intact cells. Neuroscience Letters 197, 149–153.

Spiegel, A., 2003. Cell signaling: Beta-arrestin – Not just for G proteincoupled receptors. Science 301, 1338–1339. Stiegler, A.L., Burden, S.J., Hubbard, S.R., 2009. Crystal structure of the frizzled-like cysteine-rich domain of the receptor tyrosine kinase MuSK. Journal of Molecular Biology 393, 1–9. Tamai, K., Semenov, M., Kato, Y., et al., 2000. LDL-receptor-related proteins in Wnt signal transduction. Nature 407, 530–535. Tamai, K., Zeng, X., Liu, C., et al., 2004. A mechanism for Wnt coreceptor activation. Molecular Cell 13, 149–156. Tao, Q., Yokota, C., Puck, H., et al., 2005. Maternal wnt11 activates the canonical wnt signaling pathway required for axis formation in Xenopus embryos. Cell 120, 857–871. Trachtenberg, J.T., Chen, B.E., Knott, G.W., et al., 2002. Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex. Nature 420, 788–794. van Amerongen, R., Nusse, R., 2009. Towards an integrated view of Wnt signaling in development. Development 136, 3205–3214. Varela-Nallar, L., Grabowski, C.P., Alfaro, I.E., Alvarez, A.R., Inestrosa, N.C., 2009. Role of the Wnt receptor Frizzled-1 in presynaptic differentiation and function. Neural Development 4, 41. Vivancos, V., Chen, P., Spassky, N., et al., 2009. Wnt activity guides facial branchiomotor neuron migration, and involves the PCP pathway and JNK and ROCK kinases. Neural Development 4, 7. Wang, J., Ruan, N.J., Qian, L., Lei, W.L., Chen, F., Luo, Z.G., 2008. Wnt/ beta-catenin signaling suppresses Rapsyn expression and inhibits acetylcholine receptor clustering at the neuromuscular junction. Journal of Biological Chemistry 283, 21668–21675. Wayman, G.A., Impey, S., Marks, D., et al., 2006. Activity-dependent dendritic arborization mediated by CaM-kinase I activation and enhanced CREB-dependent transcription of Wnt-2. Neuron 50, 897–909. Weatherbee, S.D., Anderson, K.V., Niswander, L.A., 2006. LDLreceptor-related protein 4 is crucial for formation of the neuromuscular junction. Development 133, 4993–5000. Welters, H.J., Kulkarni, R.N., 2008. Wnt signaling: Relevance to betacell biology and diabetes. Trends in Endocrinology and Metabolism 19, 349–355. Weston, C., Gordon, C., Teressa, G., Hod, E., Ren, X.D., Prives, J., 2003. Cooperative regulation by Rac and Rho of agrin-induced acetylcholine receptor clustering in muscle cells. Journal of Biological Chemistry 278, 6450–6455. Weston, C., Yee, B., Hod, E., Prives, J., 2000. Agrin-induced acetylcholine receptor clustering is mediated by the small guanosine triphosphatases Rac and Cdc42. Journal of Cell Biology 150, 205–212. Willert, K., Brown, J.D., Danenberg, E., et al., 2003. Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 423, 448–452. Wu, M., Herman, M.A., 2007. Asymmetric localizations of LIN-17/Fz and MIG-5/Dsh are involved in the asymmetric B cell division in C. elegans. Developmental Biology 303, 650–662. Wu, H., Xiong, W.C., Mei, L., 2010. To build a synapse: Signaling pathways in neuromuscular junction assembly. Development 137, 1017–1033. Yu, X., Malenka, R.C., 2003. Beta-catenin is critical for dendritic morphogenesis. Nature Neuroscience 6, 1169–1177. Zeng, X., Tamai, K., Doble, B., et al., 2005. A dual-kinase mechanism for Wnt co-receptor phosphorylation and activation. Nature 438, 873–877. Zhang, B., Luo, S., Dong, X.P., et al., 2007. Beta-catenin regulates acetylcholine receptor clustering in muscle cells through interaction with rapsyn. Journal of Neuroscience 27, 3968–3973. Zhang, B., Luo, S., Wang, Q., Suzuki, T., Xiong, W.C., Mei, L., 2008. LRP4 serves as a coreceptor of agrin. Neuron 60, 285–297.

Relevant Websites The Wnt Homepages: http://www.stanford.edu/rnusse/wntwindow. html.

III. SYNAPTOGENESIS