Epigenetic repression of Wnt receptors in AD: a role for Sirtuin2-induced H4K16ac deacetylation of Frizzled1 and Frizzled7 promoters.

Growing evidence supports a role for deficient Wnt signalling in Alzheimer's disease (AD). First, the Wnt antagonist DKK1 is elevated in AD brains and is required for amyloid-ß-induced synapse loss. Second, LRP6 Wnt co-receptor is required for synapse integrity and three variants of this receptor are linked to late-onset AD. However, the expression/role of other Wnt signalling components remain poorly explored in AD. Wnt receptors Frizzled1 (Fzd1), Fzd5, Fzd7 and Fzd9 are of interest due to their role in synapse formation/plasticity. Our analyses showed reduced FZD1 and FZD7 mRNA levels in the hippocampus of human early AD stages and in the hAPPNLGF/NLGF mouse model. This transcriptional downregulation was accompanied by reduced levels of the pro-transcriptional histone mark H4K16ac and a concomitant increase of its deacetylase Sirt2 at Fzd1 and Fzd7 promoters in AD. In vitro and in vivo inhibition of Sirt2 rescued Fzd1 and Fzd7 mRNA expression and H4K16ac levels at their promoters. In addition, we showed that Sirt2 recruitment to Fzd1 and Fzd7 promoters is dependent on FoxO1 activity in AD, thus acting as a co-repressor. Finally, we found reduced levels of SIRT2 inhibitory phosphorylation in nuclear samples from human early AD stages with a concomitant increase in the SIRT2 phosphatase PP2C. This results in hyperactive nuclear Sirt2 and favours Fzd1 and Fzd7 repression in AD. Collectively, our findings define a novel role for nuclear hyperactivated SIRT2 in repressing Fzd1 and Fzd7 expression via H4K16ac deacetylation in AD. We propose SIRT2 as an attractive target to ameliorate AD pathology.

Clathrin light chain diversity regulates membrane deformation in vitro and synaptic vesicle formation in vivo.

Clathrin light chain (CLC) subunits in vertebrates are encoded by paralogous genes CLTA and CLTB, and both gene products are alternatively spliced in neurons. To understand how this CLC diversity influences neuronal clathrin function, we characterized the biophysical properties of clathrin comprising individual CLC variants for correlation with neuronal phenotypes of mice lacking either CLC-encoding gene. CLC splice variants differentially influenced clathrin knee conformation within assemblies, and clathrin with neuronal CLC mixtures was more effective in membrane deformation than clathrin with single neuronal isoforms nCLCa or nCLCb. Correspondingly, electrophysiological recordings revealed that neurons from mice lacking nCLCa or nCLCb were both defective in synaptic vesicle replenishment. Mice with only nCLCb had a reduced synaptic vesicle pool and impaired neurotransmission compared to WT mice, while nCLCa-only mice had increased synaptic vesicle numbers, restoring normal neurotransmission. These findings highlight differences between the CLC isoforms and show that isoform mixing influences tissue-specific clathrin activity in neurons, which requires their functional balance.

Rational design of a conformation-specific antibody for the quantification of Aß oligomers.

Protein misfolding and aggregation is the hallmark of numerous human disorders, including Alzheimer's disease. This process involves the formation of transient and heterogeneous soluble oligomers, some of which are highly cytotoxic. A major challenge for the development of effective diagnostic and therapeutic tools is thus the detection and quantification of these elusive oligomers. Here, to address this problem, we develop a two-step rational design method for the discovery of oligomer-specific antibodies. The first step consists of an "antigen scanning" phase in which an initial panel of antibodies is designed to bind different epitopes covering the entire sequence of a target protein. This procedure enables the determination through in vitro assays of the regions exposed in the oligomers but not in the fibrillar deposits. The second step involves an "epitope mining" phase, in which a second panel of antibodies is designed to specifically target the regions identified during the scanning step. We illustrate this method in the case of the amyloid ß (Aß) peptide, whose oligomers are associated with Alzheimer's disease. Our results show that this approach enables the accurate detection and quantification of Aß oligomers in vitro, and in Caenorhabditis elegans and mouse hippocampal tissues.

Correction: Loss of Bardet-Biedl syndrome proteins causes synaptic aberrations in principal neurons.

[This corrects the article DOI: 10.1371/journal.pbio.3000414.].

Wnt7b signalling through Frizzled-7 receptor promotes dendrite development by coactivating CaMKII and JNK.

The formation of complex dendritic arbors is crucial for the assembly of functional networks as abnormal dendrite formation underlies several neurodevelopmental and psychiatric disorders. Many extracellular factors have been postulated as regulators of dendritic growth. Wnt proteins play a critical role in neuronal development and circuit formation. We previously demonstrated that Wnt7b acts through the scaffold protein dishevelled 1 (Dvl1) to modulate dendrite arborisation by activating a non-canonical Wnt signalling pathway. Here, we identify the seven-transmembrane frizzled-7 (Fz7, also known as FZD7) as the receptor for Wnt7b-mediated dendrite growth and complexity. Importantly, Fz7 is developmentally regulated in the intact hippocampus, and is localised along neurites and at dendritic growth cones, suggesting a role in dendrite formation and maturation. Fz7 loss-of-function studies demonstrated that Wnt7b requires Fz7 to promote dendritic arborisation. Moreover, in vivo Fz7 loss of function results in dendritic defects in the intact mouse hippocampus. Furthermore, our findings reveal that Wnt7b and Fz7 induce the phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and JNK proteins, which are required for dendritic development. Here, we demonstrate that Wnt7b-Fz7 signals through two non-canonical Wnt pathways to modulate dendritic growth and complexity.

Activity-dependent spine morphogenesis: a role for the actin-capping protein Eps8.

Neuronal activity regulates the formation and morphology of dendritic spines through changes in the actin cytoskeleton. However, the molecular mechanisms that regulate this process remain poorly understood. Here we report that Eps8, an actin-capping protein, is required for spine morphogenesis. In rat hippocampal neurons, gain- and loss-of-function studies demonstrate that Eps8 promotes the formation of dendritic spines but inhibits filopodium formation. Loss of function of Eps8 increases actin polymerization and induces fast actin turnover within dendritic spines, as revealed by free-barbed end and FRAP assays, consistent with a role for Eps8 as an actin-capping protein. Interestingly, Eps8 regulates the balance between excitatory synapses on spines and on the dendritic shaft, without affecting the total number of synapses or basal synaptic transmission. Importantly, Eps8 loss of function impairs the structural and functional plasticity of synapses induced by long-term potentiation. These findings demonstrate a novel role for Eps8 in spine formation and in activity-mediated synaptic plasticity.

Rac1 acts in conjunction with Nedd4 and dishevelled-1 to promote maturation of cell-cell contacts.

The Rho-GTPase Rac1 promotes actin polymerization and membrane protrusion that mediate initial contact and subsequent maturation of cell-cell junctions. Here we report that Rac1 associates with the ubiquitin-protein ligase neural precursor cell expressed developmentally down-regulated 4 (Nedd4). This interaction requires the hypervariable C-terminal domain of Rac1 and the WW domains of Nedd4. Activated Rac1 colocalises with endogenous Nedd4 at epithelial cell-cell contacts. Reduction of Nedd4 expression by shRNA results in reduced transepithelial electrical resistance (TER) and concomitant changes in the distribution of adherens and tight junction markers. Conversely, expression of Nedd4 promotes TER, suggesting that Nedd4 cooperates with Rac1 in the induction of junctional maturation. We found that Nedd4, but not Nedd4-2, mediates the ubiquitylation and degradation of the adapter protein dishevelled-1 (Dvl1), the expression of which negatively regulates cell-cell contact. Nedd4-mediated ubiquitylation requires its binding to the C-terminal domain of Dvl1, comprising the DEP domain, and targets an N-terminal lysine-rich region upstream of the Dvl1 DIX domain. We found that endogenous Rac1 colocalises with endogenous Dvl1 in intracellular puncta as well as on cell-cell junctions. Finally, activated Rac1 was found to stimulate Nedd4 activity, resulting in increased ubiquitylation of Dvl1. Together, these data reveal a novel Rac1-dependent signalling pathway that, through Nedd4-mediated ubiquitylation of Dvl1, stimulates the maturation of epithelial cell-cell contacts.

Neurotrophin and Wnt signaling cooperatively regulate dendritic spine formation.

Dendritic spines are major sites of excitatory synaptic transmission and changes in their numbers and morphology have been associated with neurodevelopmental and neurodegenerative disorders. Brain-derived Neurotrophic Factor (BDNF) is a secreted growth factor that influences hippocampal, striatal and neocortical pyramidal neuron dendritic spine density. However, the mechanisms by which BDNF regulates dendritic spines and how BDNF interacts with other regulators of spines remain unclear. We propose that one mechanism by which BDNF promotes dendritic spine formation is through an interaction with Wnt signaling. Here, we show that Wnt signaling inhibition in cultured cortical neurons disrupts dendritic spine development, reduces dendritic arbor size and complexity, and blocks BDNF-induced dendritic spine formation and maturation. Additionally, we show that BDNF regulates expression of Wnt2, and that Wnt2 is sufficient to promote cortical dendrite growth and dendritic spine formation. Together, these data suggest that BDNF and Wnt signaling cooperatively regulate dendritic spine formation.

Wnt7a signaling promotes dendritic spine growth and synaptic strength through Ca²âº/Calmodulin-dependent protein kinase II.

The balance between excitatory and inhibitory synapses is crucial for normal brain function. Wnt proteins stimulate synapse formation by increasing synaptic assembly. However, it is unclear whether Wnt signaling differentially regulates the formation of excitatory and inhibitory synapses. Here, we demonstrate that Wnt7a preferentially stimulates excitatory synapse formation and function. In hippocampal neurons, Wnt7a increases the number of excitatory synapses, whereas inhibitory synapses are unaffected. Wnt7a or postsynaptic expression of Dishevelled-1 (Dvl1), a core Wnt signaling component, increases the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs), but not miniature inhibitory postsynaptic currents (mIPSCs). Wnt7a increases the density and maturity of dendritic spines, whereas Wnt7a-Dvl1-deficient mice exhibit defects in spine morphogenesis and mossy fiber-CA3 synaptic transmission in the hippocampus. Using a postsynaptic reporter for Ca(2+)/Calmodulin-dependent protein kinase II (CaMKII) activity, we demonstrate that Wnt7a rapidly activates CaMKII in spines. Importantly, CaMKII inhibition abolishes the effects of Wnt7a on spine growth and excitatory synaptic strength. These data indicate that Wnt7a signaling is critical to regulate spine growth and synaptic strength through the local activation of CaMKII at dendritic spines. Therefore, aberrant Wnt7a signaling may contribute to neurological disorders in which excitatory signaling is disrupted.

Downregulation of Dickkopf-3, a Wnt antagonist elevated in Alzheimer's disease, restores synapse integrity and memory in a disease mouse model.

Increasing evidence supports a role for deficient Wnt signaling in Alzheimer's disease (AD). Studies reveal that the secreted Wnt antagonist Dickkopf-3 (DKK3) colocalizes to amyloid plaques in AD patients. Here, we investigate the contribution of DKK3 to synapse integrity in healthy and AD brains. Our findings show that DKK3 expression is upregulated in the brains of AD subjects and that DKK3 protein levels increase at early stages in the disease. In hAPP-J20 and hAPPNL-G-F/NL-G-F mouse AD models, extracellular DKK3 levels are increased and DKK3 accumulates at dystrophic neuronal processes around plaques. Functionally, DKK3 triggers the loss of excitatory synapses through blockade of the Wnt/GSK3ß signaling with a concomitant increase in inhibitory synapses via activation of the Wnt/JNK pathway. In contrast, DKK3 knockdown restores synapse number and memory in hAPP-J20 mice. Collectively, our findings identify DKK3 as a novel driver of synaptic defects and memory impairment in AD.

Alzheimer's disease is the most common form of dementia worldwide. The cognitive decline typically observed in this condition is associated with the weakening and eventually the loss of synapses, the structures that allow neurons to communicate. Increasing evidence points to this deterioration being linked to deficiency in the Wnt signalling pathway, a cascade of molecular events crucial for brain function and development. The DKK protein family helps to tightly regulate the Wnt pathway by dampening its activity. Previous work suggests that DKK proteins could also be connected to Alzheimer's disease. For example, an elevated amount of DKK1 leads to synapse and memory defects in mice, while brain production of DKK1 is increased in individuals with late Alzheimer's. More recent studies show high levels of another DKK protein, DKK3, in Alzheimer's patients. This protein is also present in the harmful amyloid-ß aggregates, named 'plaques', that typically form in the brain in this condition. Despite these findings, how DKK3 participates in synaptic health remains unclear. To address this question, Martin-Flores, Podpolny et al. tracked DKK3 levels in the brains of Alzheimer's patients, revealing that they increase early in the disease. Additional experiments in Alzheimer's mouse models suggested that DKK3 secretion rise before amyloid-ß plaques form, with the protein then accumulating in abnormal neuronal structures present in the surroundings of these toxic deposits. Martin-Flores, Podpolny et al. then examined the impact of DKK3 on the Wnt pathway, and ultimately, on the balance between synapses that control neuronal activity. These experiments showed that elevated DKK3 levels are linked to a loss of synapses which are excitatory, with a concomitant increase in those that are inhibitory. Crucially, reducing DKK3 levels in a mouse model of Alzheimer's restored this synaptic balance and improved memory, highlighting DKK3 as a potential driver of cognitive impairment. Overall, these findings help to refine our understanding of the molecular mechanisms that contribute to synaptic impairment in Alzheimer's disease. They may also be relevant for researchers studying other conditions that involve aberrant activity of the Wnt pathway, such as cancer.

The secreted Wnt antagonist Dickkopf-1 is required for amyloid ß-mediated synaptic loss.

Extensive evidence supports a central role for amyloid-ß (Aß) in the pathogenesis of Alzheimer's disease (AD). Synaptic loss mediated by Aß in early stages of the disease might contribute to cognitive impairments. However, little is known about the mechanism by which Aß induces the loss of synapses. The expression of the Wnt antagonist Dickkopf-1 (Dkk1) is increased in brains of AD patients and in AD transgenic mouse models, suggesting that dysfunction of Wnt signaling could contribute to AD pathology. Here we report that acute exposure to Aß oligomers induces Dkk1 expression together with the loss of synaptic sites. Importantly, Dkk1-neutralizing antibodies suppress Aß-induced synapse loss in mouse brain slices. In mature rat hippocampal neurons, Dkk1 decreases the number of synapses without affecting cell viability. Ultrastructural analyses revealed that Wnt blockade decreases the size of presynaptic and postsynaptic terminals. Time-lapse recordings of RFP-labeled stable synaptic sites demonstrate that Dkk1 induces the dispersal of synaptic components. These findings identify Dkk1 as a potential therapeutic target for the treatment of AD.

Frizzled-5, a receptor for the synaptic organizer Wnt7a, regulates activity-mediated synaptogenesis.

Wnt proteins play a crucial role in several aspects of neuronal circuit formation. Wnts can signal through different receptors including Frizzled, Ryk and Ror2. In the hippocampus, Wnt7a stimulates the formation of synapses; however, its receptor remains poorly characterized. Here, we demonstrate that Frizzled-5 (Fz5) is expressed during the peak of synaptogenesis in the mouse hippocampus. Fz5 is present in synaptosomes and colocalizes with the pre- and postsynaptic markers vGlut1 and PSD-95. Expression of Fz5 during early stages of synaptogenesis increases the number of presynaptic sites in hippocampal neurons. Conversely, Fz5 knockdown or the soluble Fz5-CRD domain (Fz5CRD), which binds to Wnt7a, block the ability of Wnt7a to stimulate synaptogenesis. Increased neuronal activity induced by K+ depolarization or by high-frequency stimulation (HFS), known to induce synapse formation, raises the levels of Fz5 at the cell surface. Importantly, both stimuli increase the localization of Fz5 at synapses, an effect that is blocked by Wnt antagonists or Fz5CRD. Conversely, low-frequency stimulation, which reduces the number of synapses, decreases the levels of surface Fz5 and the percentage of synapses containing the receptor. Interestingly, Fz5CRD abolishes HFS-induced synapse formation. Our results indicate that Fz5 mediates the synaptogenic effect of Wnt7a and that its localization to synapses is regulated by neuronal activity, a process that depends on endogenous Wnts. These findings support a model where neuronal activity and Wnts increase the responsiveness of neurons to Wnt signalling by recruiting Fz5 receptor at synaptic sites.

A genetic variant of the Wnt receptor LRP6 accelerates synapse degeneration during aging and in Alzheimer's disease.

Synapse loss strongly correlates with cognitive decline in Alzheimer's disease (AD), but the underlying mechanisms are poorly understood. Deficient Wnt signaling contributes to synapse dysfunction and loss in AD. Consistently, a variant of the LRP6 receptor, (LRP6-Val), with reduced Wnt signaling, is linked to late-onset AD. However, the impact of LRP6-Val on the healthy and AD brain has not been examined. Knock-in mice, generated by gene editing, carrying this Lrp6 variant develop normally. However, neurons from Lrp6-val mice do not respond to Wnt7a, a ligand that promotes synaptic assembly through the Frizzled-5 receptor. Wnt7a stimulates the formation of the low-density lipoprotein receptor-related protein 6 (LRP6)-Frizzled-5 complex but not if LRP6-Val is present. Lrp6-val mice exhibit structural and functional synaptic defects that become pronounced with age. Lrp6-val mice present exacerbated synapse loss around plaques when crossed to the NL-G-F AD model. Our findings uncover a previously unidentified role for Lrp6-val in synapse vulnerability during aging and AD.

Microglial Expression of the Wnt Signaling Modulator DKK2 Differs between Human Alzheimer's Disease Brains and Mouse Neurodegeneration Models.

Wnt signaling is crucial for synapse and cognitive function. Indeed, deficient Wnt signaling is causally related to increased expression of DKK1, an endogenous negative Wnt regulator, and synapse loss, both of which likely contribute to cognitive decline in Alzheimer's disease (AD). Increasingly, AD research efforts have probed the neuroinflammatory role of microglia, the resident immune cells of the CNS, which have furthermore been shown to be modulated by Wnt signaling. The DKK1 homolog DKK2 has been previously identified as an activated response and/or disease-associated microglia (DAM/ARM) gene in a mouse model of AD. Here, we performed a detailed analysis of DKK2 in mouse models of neurodegeneration, and in human AD brain. In APP/PS1 and APPNL-G-F AD mouse model brains as well as in SOD1G93A ALS mouse model spinal cords, but not in control littermates, we demonstrated significant microgliosis and microglial Dkk2 mRNA upregulation in a disease-stage-dependent manner. In the AD models, these DAM/ARM Dkk2+ microglia preferentially accumulated close to ßAmyloid plaques. Furthermore, recombinant DKK2 treatment of rat hippocampal primary neurons blocked WNT7a-induced dendritic spine and synapse formation, indicative of an anti-synaptic effect similar to that of DKK1. In stark contrast, no such microglial DKK2 upregulation was detected in the postmortem human frontal cortex from individuals diagnosed with AD or pathologic aging. In summary, the difference in microglial expression of the DAM/ARM gene DKK2 between mouse models and human AD brain highlights the increasingly recognized limitations of using mouse models to recapitulate facets of human neurodegenerative disease.

S-acylation of the Wnt receptor Frizzled-5 by zDHHC5 controls its cellular localization and synaptogenic activity in the rodent hippocampus.

Proper localization of receptors for synaptic organizing factors is crucial for synapse formation. Wnt proteins promote synapse assembly through Frizzled (Fz) receptors. In hippocampal neurons, the surface and synaptic localization of Fz5 is regulated by neuronal activity, but the mechanisms involved remain poorly understood. Here, we report that all Fz receptors can be post-translationally modified by S-acylation and that Fz5 is S-acylated on three C-terminal cysteines by zDHHC5. S-acylation is essential for Fz5 localization to the cell surface, axons, and presynaptic sites. Notably, S-acylation-deficient Fz5 is internalized faster, affecting its association with signalosome components at the cell surface. S-acylation-deficient Fz5 also fails to activate canonical and divergent canonical Wnt pathways. Fz5 S-acylation levels are regulated by the pattern of neuronal activity. In vivo studies demonstrate that S-acylation-deficient Fz5 expression fails to induce presynaptic assembly. Our studies show that S-acylation of Frizzled receptors is a mechanism controlling their localization and function.

From neuronal activity to the actin cytoskeleton: a role for CaMKKs and betaPIX in spine morphogenesis.

Electrical activity plays a crucial role in neuronal circuit assembly. Activation of NMDA receptors induces the elevation of intracellular calcium, resulting in the modulation of calcium-calmodulin-dependent protein kinases (CaMKs). The CaMK pathway regulates synaptogenesis by driving the formation of dendritic spines. However, the molecular effectors downstream of this pathway have remained poorly defined. In this issue of Neuron, Saneyoshi et al. identify a new signaling complex containing CaMKK/CaMKI/betaPIX/Rac that regulates the morphogenesis of spines in an activity-dependent manner.

Design of a Potent, Selective, and Brain-Penetrant Inhibitor of Wnt-Deactivating Enzyme Notum by Optimization of a Crystallographic Fragment Hit.

Notum is a carboxylesterase that suppresses Wnt signaling through deacylation of an essential palmitoleate group on Wnt proteins. There is a growing understanding of the role Notum plays in human diseases such as colorectal cancer and Alzheimer's disease, supporting the need to discover improved inhibitors, especially for use in models of neurodegeneration. Here, we have described the discovery and profile of 8l (ARUK3001185) as a potent, selective, and brain-penetrant inhibitor of Notum activity suitable for oral dosing in rodent models of disease. Crystallographic fragment screening of the Diamond-SGC Poised Library for binding to Notum, supported by a biochemical enzyme assay to rank inhibition activity, identified 6a and 6b as a pair of outstanding hits. Fragment development of 6 delivered 8l that restored Wnt signaling in the presence of Notum in a cell-based reporter assay. Assessment in pharmacology screens showed 8l to be selective against serine hydrolases, kinases, and drug targets.

Structural Analysis and Development of Notum Fragment Screening Hits.

The Wnt signaling suppressor Notum is a promising target for osteoporosis, Alzheimer's disease, and colorectal cancers. To develop novel Notum inhibitors, we used an X-ray crystallographic fragment screen with the Diamond-SGC Poised Library (DSPL) and identified 59 fragment hits from the analysis of 768 data sets. Fifty-eight of the hits were found bound at the enzyme catalytic pocket with potencies ranging from 0.5 to >1000 µM. Analysis of the fragments' diverse binding modes, enzymatic inhibitory activities, and chemical properties led to the selection of six hits for optimization, and five of these resulted in improved Notum inhibitory potencies. One hit, 1-phenyl-1,2,3-triazole 7, and its related cluster members, have shown promising lead-like properties. These became the focus of our fragment development activities, resulting in compound 7d with IC50 0.0067 µM. The large number of Notum fragment structures and their initial optimization provided an important basis for further Notum inhibitor development.

Signaling across the synapse: a role for Wnt and Dishevelled in presynaptic assembly and neurotransmitter release.

Proper dialogue between presynaptic neurons and their targets is essential for correct synaptic assembly and function. At central synapses, Wnt proteins function as retrograde signals to regulate axon remodeling and the accumulation of presynaptic proteins. Loss of Wnt7a function leads to defects in the localization of presynaptic markers and in the morphology of the presynaptic axons. We show that loss of function of Dishevelled-1 (Dvl1) mimics and enhances the Wnt7a phenotype in the cerebellum. Although active zones appear normal, electrophysiological recordings in cerebellar slices from Wnt7a/Dvl1 double mutant mice reveal a defect in neurotransmitter release at mossy fiber-granule cell synapses. Deficiency in Dvl1 decreases, whereas exposure to Wnt increases, synaptic vesicle recycling in mossy fibers. Dvl increases the number of Bassoon clusters, and like other components of the Wnt pathway, it localizes to synaptic sites. These findings demonstrate that Wnts signal across the synapse on Dvl-expressing presynaptic terminals to regulate synaptic assembly and suggest a potential novel function for Wnts in neurotransmitter release.

Wnt signaling promotes AChR aggregation at the neuromuscular synapse in collaboration with agrin.

Wnt proteins regulate the formation of central synapses by stimulating synaptic assembly, but their role at the vertebrate neuromuscular junction (NMJ) is unclear. Wnt3 is expressed by lateral motoneurons of the spinal cord during the period of motoneuron-muscle innervation. Using gain- and loss-of-function studies in the chick wing, we demonstrate that Wnt signaling is necessary for the formation of acetylcholine receptor (AChR) clusters without affecting muscle growth. Similarly, diaphragms from Dishevelled-1 mutant mice with deficiency in Wnt signaling exhibit defects in cluster distribution. In cultured myotubes, Wnt3 increases the number and size of AChR clusters induced by agrin, a nerve-derived signal critical for NMJ development. Wnt3 does not signal through the canonical Wnt pathway to induce cluster formation. Instead, Wnt3 induces the rapid formation of unstable AChR micro-clusters through activation of Rac1, which aggregate into large clusters only in the presence of agrin. Our data reveal a role for Wnts in post-synaptic assembly at the vertebrate NMJ by enhancing agrin function through Rac1 activation.