Wnt Signaling in Presynaptic Assembly
Wnt signaling has also been reported to have a function in presynaptic assembly during development. Studies in the neuromuscular junction (NMJ) of Drosophila have demonstrated a function of Wg, the prototypical Drosophila Wnt, in synaptogenesis, where its loss leads to a reduction in target-dependent synapse formation (Packard et al.,2002). Conversely, in C. elegans, it has been observed that Wnt signaling inhibits synapse formation, suggesting a regulation in the patterning of synaptic connections (Klassen and Shen,2007).
In mammals, Wnt-7a increases the levels of the synaptic vesicle protein synapsin I in developing cerebellar neurons (Lucas and Salinas,1997). Moreover, in Wnt-7a mutant mice, it has been observed a delay in the accumulation of synapsin I (Hall et al.,2000), suggesting a role for this ligand in presynaptic assembly during nervous system development (Fig. 1C). Besides Wnt-7a, Wnt-3a and Wnt-7b also increase the number of excitatory presynaptic puncta in cultured hippocampal neurons (Davis et al.,2008).
Recently, a role for Wnt signaling in presynaptic assembly in the mature central nervous system (CNS) has also been reported. Hippocampal neurons incubated with Wnt-7a show increased numbers of clusters of synaptic vesicle proteins, such as synapsin I, synaptophysin, SV-2, and synaptotagmin (Farías et al.,2007; Cerpa et al.,2008). Of interest, both Wnt-5a and Wnt-7a are highly expressed in the mature CNS, but only Wnt-7a acts presynaptically (Cerpa et al.,2008). A role for Wnt signaling in the clustering of presynaptic receptors also has been studied. Wnt-7a increases the expression of the α7-nicotinic acetylcholine receptor (α7-nAChR), as well as the number and size of α7-nAChR clusters in rat hippocampal neurons (Farías et al.,2007). These studies suggest that specific Wnt ligands can modulate the assembly of the presynaptic terminal in the mature CNS (Fig. 2A).
Figure 2. Wnt in synaptic function in mature hippocampal neurons. A: Scheme summarizing the Wnt-7a effects on neurotransmitter release. Wnt-7a induces the exocytosis and recycling of vesicles proteins. A possible mechanism involve adenomatous polyposis coli (APC) protein, that in the presence of Wnt-7a ligand, dissociates the β-catenin destruction complex and it associates to the α7-nicotinic acetylcholine receptor (α7-nAChR). It is possible that APC functions as a cargo protein that interacts with microtulules to transport another protein. α7-nAChR localized in the plasma membrane can allow the entry of calcium to modulate the exocytosis of synaptic vesicles and finally to regulate the synaptic transmission. B: Wnt-5a increases synaptic transmission through a postsynaptic mechanism. New PSD-95 clusters are localized in dendritic spines, and they are formed through recruitment from the cytosolic PSD-95 pool. Activation of JNK by Wnt-5a is required for the clustering of PSD-95. Morever, glutamate receptors are anchored to the membrane and can explain the increase in the amplitude of mEPSP.
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In the context of these observations, an interesting question arises: which Wnt signaling components are involved in the presynaptic effect? In neurons, Wnt-7a,Wnt-3a, and Wnt-7b activate the canonical Wnt/β-catenin pathway. Moreover, treatment with Dickkopf-1, which promotes internalization of the LRP5/6 co-receptor required for canonical signal activation but not for noncanonical Wnt signaling, resulted in decreased excitatory presynaptic puncta number, indicating that activation of the endogenous canonical pathway contributes to synapse formation (Davis et al.,2008). Following this evidence, it has been proposed that Wnt signaling regulates synapse formation indirectly by promoting neuronal maturation through gene transcription (Waites et al.,2005). However, work of other groups, suggests that the mechanism involved in presynaptic assembly is transcription-independent. In cultured neurons, Wnt signaling increases the number and the size of synaptic vesicle proteins without affecting presynaptic protein expression, before the stabilization of β-catenin and its translocation into the nucleus (Cerpa et al.,2008). In vivo, Wnt deficiency also affects the localization of presynaptic proteins without affecting their levels (Ahmad-Annuar et al.,2006). Similarly, it was observed in conditional mutant mice for β-catenin that this protein is required for the proper localization of synaptic vesicles along the axon, but through a mechanism independent of TCF-mediated transcription (Bamji et al.,2003). In addition, the presynaptic clustering of α7-nAChR induced by Wnt-7a depends on APC but is independent of β-catenin stabilization (Farías et al.,2007). Thus, there appears to be a consensus regarding a transcription-independent mechanism involved in the effect of the Wnt signaling in the presynaptic assembly.
Wnt Signaling in the Postsynaptic Assembly
The functional maturation of the postsynaptic region requires a gradual recruitment of scaffold proteins to anchor receptors to the postsynaptic membrane. Specialized signaling machinery is necessary to cluster receptors and their respective scaffold proteins in the postsynaptic region to form functional synapses (Goda and Davis,2003). Studies on the Drosophila glutamatergic NMJ indicated that loss of Wg results in aberrant development of postsynaptic specializations (Packard et al.,2002). In zebrafish cholinergic NMJ, Wnt-11r organizes the central muscle zone before NMJ formation, affecting the initial prepatterning of AChRs (Jing et al.,2009). Agrin is a classic factor that controls the distribution of the muscle-specific AChR in the mammalian postsynaptic region (Nitkin et al.,1987; Gautam et al.,1996). Diverse components of the various Wnt signaling pathways have been involved in the clustering of this muscle-type receptor. The participation of Dvl and APC in the clustering of the AChR induced by agrin has been demonstrated (Luo et al.,2002; Wang et al.,2003). Recently, it was determined that Wnt-3 induces the rapid formation of unstable AChR micro-clusters during early stages of NMJ assembly in chick wing muscles, which aggregate into large clusters only in the presence of agrin (Henriquez et al.,2008). These results indicate that Wnt-3 acts as a modulator of postsynaptic differentiation at NMJ synapses by collaborating with agrin.
Classic factors that specifically regulate the neuronal postsynaptic region have not been identified. In the peripheral nervous system (PNS), the participation of APC in the localization and anchoring of α3-nAChRs and their scaffold protein PSD-93 at the postsynaptic membrane has been demonstrated (Temburni et al.,2004); however, this study did not elucidate whether Wnt ligands act as synaptogenic factors to modulate postsynaptic assembly.
We have recently found that, in the mature CNS, Wnt-5a but not Wnt-7a increases the number of PSD-95 clusters in dendritic spines (Fig. 1D; Farías et al.,2009). Whether the new clusters of PSD-95 are contained in newly formed protrusions or whether they are formed before, concomitantly, or after the protrusion formation, is being intensively studied. Regarding this issue, an interesting question is how PSD-95 clusters are formed. Four possibilities have been considered: increased expression of PSD-95 protein, decreased levels of PSD-95 per cluster, splitting of pre-existent PSD-95 clusters, and recruitment of PSD-95 from a diffuse pool. Wnt-5a does not decrease the mean intensity or size of PSD-95 clusters, eliminating the possibility of the splitting of pre-existent PSD-95 clusters into new clusters, as previously described (Gerrow et al.,2006). Short-term exposure to Wnt-5a (2 hr), does not increase total PSD-95 levels, indicating that Wnt-5a induces the number of PSD-95 clusters through redistribution of an existing PSD-95 pool. It has been reported that new clusters of PSD-95 can be formed through recruitment of PSD-95 from a cytosolic PSD-95 pool (Bresler et al.,2001). This is the most feasible alternative, considering that Wnt-5a treatment reduces the dendritic diffuse pool of PSD-95 while increasing the membrane attached pool (Farías et al.,2009). In addition to its effect on PSD-95 clustering, Wnt-5a increases the insertion of glutamate receptors in the cell surface (Inestrosa et al.,2007; Cerpa et al.,2009).
How does the Wnt signaling pathway regulate postsynaptic assembly in the mature CNS? Reports indicate that, in the excitatory postsynaptic region, phosphorylation of PSD-95 on Ser-295 by JNK-1 (p46 isoform), modulates the anchoring of PSD-95 to the postsynaptic membrane and the synaptic accumulation of PSD-95 (Kim et al.,2007). Considering this evidence, we wanted to evaluate the participation of Wnt/JNK signaling pathway in the clustering of PSD-95. Studies in mature cultured hippocampal neurons exposed to a specific JNK inhibitor showed that the clustering of PSD-95 induced by Wnt-5a is inhibited by the modulation of JNK activity (Farías et al.,2009). These findings suggest that Wnt-5a modulates the assembly of the excitatory postsynaptic region in mature CNS through activation of the Wnt/JNK signaling pathway (Fig. 2B).