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.Wnt7a | Dvl1 mutant | plasticity T he formation of functional neuronal circuits requires the assembly of different types of synapses with great specificity. The development of an appropriate balance of glutamatergic excitatory and GABAergic inhibitory synapses (E/I ratio) is essential for proper circuit function (1, 2) because an imbalance in the E/I ratio can result in neurological disorders (3-5). Some synaptogenic factors regulate both excitatory and inhibitory synapses (6, 7), whereas other synaptic organizers are more specific (8-10). However, the precise mechanisms by which synaptic organizing signals regulate excitatory and inhibitory synapses remain poorly understood.In the central nervous system, most excitatory inputs are located on dendritic spines, postsynaptic protrusions that function as domains where synaptic activity is regulated in a compartmentalized manner (11-13). Several intracellular molecules have been implicated in the formation and maturation of dendritic spines (14-16), but the mechanisms by which extracellular factors signal through intracellular regulators to promote spine development and maturation remain poorly characterized.Wnt secreted proteins are synaptic organizers that stimulate the formation of central and peripheral synapses (17-19) by promoting presynaptic assembly (20) and the clustering of postsynaptic components (19,(21)(22)(23)(24). In cultured neurons, Wnt5a regulates postsynaptic development of both GABAergic and glutamatergic synapses (24,25). However, it is unclear whether other Wnts play a...

SUMMARYWnt 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.

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BackgroundThe urokinase receptor (uPAR/CD87) is highly expressed in malignant tumours. uPAR, as a GPI anchored protein, is preferentially located at the cell surface, where it interacts with its ligands urokinase (uPA) and the extracellular matrix protein vitronectin, thus promoting plasmin generation, cell-matrix interactions and intracellular signalling events. Interaction with a complex formed by uPA and its inhibitor PAI-1 induces cell surface down regulation and recycling of the receptor via the clathrin-coated pathway, a process dependent on the association to LRP-1.Methodology/Principal FindingsIn this study, we have found that along with the ligand-induced down-regulation, uPAR also internalizes and recycles constitutively through a second pathway that is independent of LRP-1 and clathrin but shares some properties with macropinocytosis. The ligand-independent route is amiloride-sensitive, does not require uPAR partitioning into lipid rafts, is independent of the activity of small GTPases RhoA, Rac1 and Cdc42, and does not require PI3K activity. Constitutively endocytosed uPAR is found in EEA1 positive early/recycling endosomes but does not reach lysosomes in the absence of ligands. Electron microscopy analysis reveals the presence of uPAR in ruffling domains at the cell surface, in macropinosome-like vesicles and in endosomal compartments.Conclusions/SignificanceThese results indicate that, in addition to the ligand-induced endocytosis of uPAR, efficient surface expression and membrane trafficking might also be driven by an uncommon macropinocytic mechanism coupled with rapid recycling to the cell surface.