BIOLOGICAL OVERVIEW

The recent confirmation of frizzled2 as the long sought Wingless receptor is illustrative of the detective work flavor of developmental biology. The discovery of a large family of transmembrane receptors homologous to the Drosophila frizzled gene left a large hole in receptor research, just waiting to be filled. In fact, the only family of ligands known to be as large as the frizzled family, for which no receptors had been identified, is the Wnt family (Wang, 1996), for which the Drosophila signaling protein Wingless is the prototype. This was circumstantial evidence, but only that, for assigning a frizzled family member a role in Wingless reception.

The second piece of circumstantial evidence that frizzled family members serve as Wingless receptors was the discovery that dishevelled is required for the proper functioning of both the wingless and frizzled pathways (Krasnow, 1995). Dishevelled, a phosphoprotein, is synthesized uniformly throughout the embryo, but its level of phosphorylation varies with the level of wingless activity (Yanagawa, 1995). Genetics experiments had already discerned a biochemical hierarchy of protein action in response to wingless signals. porcupine and dishevelled act upstream, prior to the action of shaggy/zeste white 3, while armadillo is required downstream of shaggy. In fact Porcupine acts in cells secreting Wingless, while Dishevelled acts in cells that receive wingless signals (Siegfried, 1994).

Wingless signals function to inactivate Shaggy, the glycogen synthase kinase 3 also known as Zeste white 3 that phosphorylates Armadillo protein. In response to WG protein, the cytoplasmic pool of Armadillo protein increases, whereas the membrane-bound form of ARM is less affected. ARM protein is present in two different forms, with two different patterns of migration, a result of differences in phosphorylation. Both forms are associated almost exclusively with the membrane fraction. In cells that have been exposed to WG protein, a dramatic increase in the faster migrating (underphosphorylated) form of ARM is seen in the soluble cytoplasmic fraction and, to a lesser extent, in the membrane fraction. The slower migrating (phosphorylated) form of ARM, detectable only in the membrane fraction, is not affected by WG (Yanagawa, 1995).

With all the evidence pointing to frizzled family members as Wingless receptors, the search began in earnest for Frizzled homologs in Drosophila, and indirectly, for the putative Wingless receptor. fz2 was cloned on the basis of its homology to the frizzled family. Experiments then ensued to find out whether it could in fact act biologically as a Wingless receptor.

Schneider cultured cells (also known as S2 cells) fail respond to Wingless protein, that is they fail to stabilize ARM in response to added WG. It is possible that S2 cells don't respond because they do not express fz2. To test this possibility, a plasmid carrying the fz2 gene was transfected into S2 cells. When fz2-transfected cells are incubated in the presence of WG, the level of faster migrating (hypophosphorylated) ARM increases. In addition, transfected S2 cells show strong surface staining when incubated with WG and anti-WG antibodies, indicating a direct effect of fz2-transfection on WG binding (Bhanot, 1996).

How do wingless signals get to the nucleus? The pathway is beginning to be understood in vertebrates. A component of the wingless pathway has been identified in Xenopus. A maternally expressed Xenopus homolog of mammalian HMG box factors (XTcf-3) binds to ß-catenin, the vertebrate homolog of Armadillo. XTcf-3 is a transcription factor that mediates ß-Catenin-induced axis formation in Xenopus embryos. It has been proposed that the ßcatenin-XTcf-3 complex is responsible for activation of targets genes in response to upstream Wnt (Wnt is the vertebrate homolog of Wingless) signals. By an as yet unknown mechanism, Wnt signals allow cytoplasmic ß-catenin to interact with XTcf-3 (Molenaar, 1996). The discovery of the involvement of an HMG-domain protein in vertebrate Wnt signaling encouraged a search for a similar HMG-domain protein in Drosophila. Isolated as a homolog of the vertebrate HMG-domain proteins described above, Drosophila Pangolin is found to be able to physically interact with vertebrate beta-catenin (Brunner, 1997).

Frizzled is involved in tissue polarity in Drosophila. A method was devised to induce a gradient of fz expression with the highest levels near the distal wing tip. The result is a large area of proximally pointing hairs in this region. This reversal of polarity is seen when fz expression is induced just before the start of hair morphogenesis, at the time polarity is established, suggesting that the gradient of Fz protein acts fairly directly to reverse hair polarity. A similar induction of the dishevelled (dsh) gene, which acts cell autonomously and functions downstream of fz in the generation of tissue polarity, results in a distinct tissue polarity phenotype, but indicates no reversal of polarity; this argues that fz signaling is required for polarity reversal. The finding that functional dsh is required for the reversal of polarity argues that the reversal requires normal fz signal transduction. The data suggest that cells sense the level of Fz protein on neighboring cells and use this information in order to polarize themselves. A polarizing signal is transmitted from cells with higher Fz levels to cells with lower levels. These observations enable the proposal of a general mechanism to explain how Wnts polarize target cells (Adler, 1997).

Interaction of Wnt and a Frizzled homologue triggers G-protein-linked phosphatidylinositol signalling

Recent evidence points to a role for lipid signaling downstream of Frizzled proteins. Stimulation of a G-protein-linked receptor initiates the hydrolysis of a membrane-bound inositol lipid, generating at least two second messengers: diacylglycerol and inositol-1,4,5-trisphosphate (InsP3). Diacylglycerol stimulates protein kinase C while InsP3 promotes the release of intracellular calcium (see Drosophila InsP3 receptor). The rat protein Frizzled-2 causes an increase in the release of intracellular calcium, which is enhanced by Xwnt-5a, a member of the Wnt family. Pertussis toxin (PTX) (which is a specific inhibitor of G alpha0 and G alphai subunits of G proteins that act by preventing the catalysis of GDP-GTP exchange stimulated by receptors) inhibits rat protein Frizzled-2 modulation of calcium flux. A nonhydrolysable GDP analog that irreversibly inactives G-protein-coupled events, inhibits rat FZ-2 induced Ca2+ transients. The release of intracellular calcium is suppressed by an inhibitor of the enzyme inositol monophosphatase, and hence of the phosphatidylinositol signaling pathway. This suppression can be rescued by injection of the compound myo-inositol, which overcomes the decrease in this intermediate caused by the inhibitor. These results indicate that some Wnt proteins work through specific Frizzled homologs to stimulate the phosphatidylinositol signalling pathway via heterotrimeric G-protein subunits, and that FZ-2 stimulates the phosphatidylinositol cycle through the betagamma subunits of pertussis-toxin-sensitive G proteins, leading to release of intracellular Ca2+ and diverse cellular responses. Since Gbetagamma subunits also activate protein kinase C, which may be involved in Wnt signaling, the responses by cells and embryos to signaling through Frizzled homologs could involve the stimulation of multiple cytoplasmic pathways. In early vertebrate embryos, regulation of the phosphatidylinositol pathway may be important for establishing the embryonic mesoderm and in other processes (Slusarski, 1997).

Signaling specificity by Frizzled receptors in Drosophila

How is signaling specificity achieved by two closely related receptors? Both Frizzled and Fz2 signaling pathways use Dishevelled (Dsh) as a transduction component, raising the intriguing question of how two structurally related receptors signal through a common protein into distinct effector pathways. Fz2 has a higher affinity for Wg than Fz, and removal of either Fz or Fz2 has subtle, but different, effects on the patterning of the embryonic nervous system. Moreover, only Fz is specifically required for epithelial planar polarity by signaling through a Wg-Arm-independent pathway (Boutros, 2000).

Fz overexpression during Drosophila eye development causes a gain-of-function (GOF) planar polarity phenotype. Overexpression of Fz2 in the developing wing activates Wg-Arm targets. To compare the functional equivalence of Fz and Fz2 (Fz will be referred to as Fz1) for activating either the planar polarity or Wg-Arm pathways, Fz1 and Fz2 were expressed with tissue-specific enhancers in imaginal discs during Drosophila development. Whereas Fz1 overexpression in eye and wing discs results in planar polarity phenotypes, Fz2 expression leads to planar polarity defects with only very low penetrance. Conversely, overexpression of Fz2 in wing imaginal discs leads to formation of ectopic bristles (a wg GOF phenotype), whereas Fz1 overexpression does not affect bristle formation. Thus, Fz receptors have distinct signaling abilities in imaginal discs, despite their redundant role for Wg-Arm signaling in loss-of-function (LOF) analysis (Boutros, 2000).

To assess Wnt-beta-cat signaling in a quantifiable in vitro assay, both receptors were injected into Xenopus embryos and Wnt target induction in animal cap explants was analyzed. In this heterologous assay, Fz2 induces strong activation of the Wnt-beta-cat targets Xnr-3 and Siamois (Sia), whereas Fz1 induces a much weaker response. Thus, taken together with the imaginal disc phenotypes, Fz2 is a strong activator of Wnt-beta-cat signaling, and Fz1 is a potent activator of the planar polarity pathway. However, both receptors retain a low intrinsic potential to cross-activate either pathway (Boutros, 2000).

Differential Dsh localization may determine Fz signaling specificity, whereby Fz1, but not Fz2, can induce recruitment of Dsh to the membrane in Xenopus. At normalized protein levels for Fz1 and Fz2, however, no differences were observed in their ability to recruit Dsh. Titration experiments with Fz1 and Fz2 RNA concentrations show very similar threshold levels for either receptor in Dsh membrane localization. Thus, differential Dsh recruitment is unlikely to be the mechanism by which specificity between these Fz receptors is generated (Boutros, 2000).

Fz receptors are serpentine transmembrane proteins composed of an extracellular ligand-sequestering domain (CRD), a seven-pass transmembrane segment, and a COOH-terminal cytosolic tail. To determine which domains in Fz1 and Fz2 are required for directing signaling into either pathway, chimeric and truncated receptors were constructed. These chimeric proteins were tested for their signaling potential in Drosophila imaginal disc development in wings and nota, eyes, and legs for their ability to induce either GOF Wg-Arm signaling or planar polarity phenotypes (Boutros, 2000).

Both Fz1-2 and Fz1-1-2 chimeric proteins activate a Wg-Arm target (Achaete) in the wing imaginal disc, induce ectopic marginal bristles, and show wg-associated effects in the leg. However, they have no significant effect on planar polarity signaling in the eye, the wing, or the notum. Thus, the Wg-Arm signaling outcome corresponds with the presence of the Fz2 cytoplasmic tail. In contrast, GOF planar polarity phenotypes are observed with chimeric Fz2-1 receptors in the wing, the notum, and the eye that are indistinguishable from those caused by Fz1. Both Fz2-1 and Fz2-2-1 show a (mild) dominant-negative phenotype for Wg-Arm signaling, as judged by a reduction in Ac expression, (partial) absence of marginal bristles and notches in the wing margin, and the presence of stunted legs. These data suggest that the chimeric receptors containing the high-affinity Fz2 CRD, but lacking intracellular Fz2 sequences, can sequester Wg efficiently without eliciting an efficient signal transduction response. To test this possibility directly, stabilization of Wg was analyzed in wing discs. Whereas Fz1 or Fz1-2 type chimeras have no significant effect on Wg stability, all chimeric receptors with the Fz2 CRD strongly sequester Wg. The importance of cytoplasmic sequences for efficient Wnt target activation was confirmed in Xenopus animal cap assays (exchanging cytoplasmic domains switches the signaling efficiency). All chimeric receptors are able to recruit Dsh indistinguishably from wild-type Fz1 and Fz2 (Boutros, 2000).

The observation that the Fz1-2 chimera does not dominantly interfere with planar polarity signaling in the eye and because as yet no "planar polarity" ligand has been identified raises the question of whether planar polarity signaling depends on the presumed ligand binding Fz1 CRD. This hypothesis was tested by rescue of the fz-/- polarity mutant with sev-Fz1 and chimeric transgenes. In contrast to Fz1, however, neither Fz1-2 and Fz2-1 chimeras nor Fz2 rescue the fz mutant, indicating that the Fz1 CRD, coupled to its signaling unit, is required for correct levels of activation of planar polarity signaling in the eye in vivo. Thus, although overexpression of Fz2-1 induces a GOF planar polarity phenotype, identical to that induced by Fz1, it cannot replace Fz1 in a LOF background. Although both receptor isoforms, Fz1 and Fz2-1, when overexpressed, are capable of activating planar polarity signaling and perturbing correct polarity determination, the pathway is activated to the correct level only by the Fz1 CRD (and ligand)-dependent regulation of the receptor. Because the establishment of correct ommatidial polarity results from small differences in Fz signaling levels between neighboring R3 and R4 cells, the ability to precisely respond to the ligand in a spatially and temporally controlled manner is essential. Presumably, only Fz1 is appropriately regulated through its CRD to instruct correct ommatidial polarity (Boutros, 2000).

Fz1 and Fz2 appear to have different abilities to activate Wg-Arm and planar polarity signaling in the GOF assays in imaginal discs. Whereas Fz2 induces a Wg-Arm GOF phenotype, Fz1 causes GOF planar polarity phenotypes. The chimeric receptors define the respective cytoplasmic tail (Fz2) or the cytoplasmic domains (Fz1) as largely, but not solely, responsible for mediating these differences in the GOF assays. The Wg-Arm GOF phenotype is ligand- and CRD domain-dependent, because it can only be observed close to the source of Wg. Also, Fz2 has a stronger effect than Fz1-2 or Fz1-1-2. The planar polarity ligand is, possibly, a member of the Wnt family with a different CRD binding affinity from that of Wg. The mechanism by which the ligand-CRD interaction regulates Fz signaling is unclear. The present data cannot distinguish between an activating (conformational) change, or alternatively, a constitutive signaling capacity by Fz's that is inhibited by another factor and needs to be antagonized by the ligand (similar to Smoothened/Patched signaling with Hedgehog) (Boutros, 2000).

How can one reconcile the Fz1 and Fz2 redundancy for Wg signaling in LOF analysis and the dominant-negative behavior of the Fz2-1 chimeras? Fz2 is a high-affinity Wg receptor, and fz2 transcription is down-regulated by Wg, whereas Fz1 (a low-affinity receptor) is expressed fairly uniformly. Thus, Fz2 might be the primary Wg receptor, and Fz1 substitutes only in its absence. Moreover, another Drosophila Fz family member, Fz3, acts as a negative attenuator of Wg signaling and is positively regulated by Wg, suggesting that the expression patterns of Fz2 and Fz3 shape the Wg response, whereas Fz1 does not contribute to this effect. In this context, overexpression of Fz2-1, consisting of a high-affinity Wg-binding CRD fused to a low-efficiency signaling unit, adversely affects the signaling outcome and causes a dominant-negative phenotype (Boutros, 2000).

These experiments provide a model for how signaling specificity can be achieved by closely related receptors, and they demonstrate that LOF studies, like GOF experiments, might only provide a partial answer in case of redundancies. Quantitative differences in ligand affinity and signal transduction efficiency of Fz receptors could provide overlapping and nonoverlapping functions in different cells, depending on the threshold needed to induce targets and expression levels of the various members of the receptor family. Thus, the relative ratio of the different Fz receptors on the cell surface and their degree of occupancy could be an important factor determining the signaling outcome. Additional factors such as coreceptors could influence the signaling outcome: for example, the heparan sulfate proteoglycan Dally has been identified as a coreceptor in Wg signaling. Fz1 and Fz2 signaling preferences provide an example of how quantitative differences in signaling levels can lead to redundant and specific roles for these receptors during development and evolution (Boutros, 2000).

Subcellular localization of frizzled receptors, mediated by their cytoplasmic tails, regulates signaling pathway specificity

The Frizzled (Fz; called here Fz1) and Fz2 receptors have distinct signaling specificities activating either the canonical Wnt/beta-catenin pathway or Fz/planar cell polarity (PCP) signaling in Drosophila. The regulation of signaling specificity remains largely obscure. This study shows that Fz1 and Fz2 have different subcellular localizations in imaginal disc epithelia, with Fz1 localizing preferentially to apical junctional complexes, and Fz2 being evenly distributed basolaterally. The subcellular localization difference directly contributes to the signaling specificity outcome. Whereas apical localization favors Fz/PCP signaling, it interferes with canonical Wnt/beta-catenin signaling. Receptor localization is mediated by sequences in the cytoplasmic tail of Fz2 that appear to block apical accumulation. Based on these data, it is proposed that subcellular Fz localization, through the association with other membrane proteins, is a critical aspect in regulating the signaling specificity within the Wnt/Fz signaling pathways (Wu, 2004).

Frizzled chimeras described below are denoted by three numbers, the first being the source of the N-terminal Wnt-interacting cysteine-rich domain (CRD), the second being the source of the remaining proximal extracellular domain and 7 transmembrane region and loop region (collectively referred to as 7-TM), and the third refering to the source of the intracellular C-tail (Wu, 2004).

It was asked whether the apical localization of Fz is required for PCP signaling? The Fz1-1-2 chimera, which is distributed ubiquitously within the apical-basolateral membrane, only partially rescues the fz^- eye phenotype, and it can also cause defects related to canonical Wg/Arm signaling. In contrast, apically localized Fz1-1-2S ('S' for 'short') fully rescues the fz^- phenotype and has no additional effects. The Fz1-1-2 chimera also shows much weaker PCP phenotypes in the GOF assay. Taken together, these results suggest that a reduction in the apical localization of Fz leads to a reduction in PCP signaling activity. However, about 80% of the chirality defects in fz^- eyes are rescued by tub-fz1-1-2, and in the wing tub-fz1-1-2 rescues the fz^- mutant to a similar extent as tub-fz1-1-1 and tub-fz1-1-2S, suggesting that Fz1-1-2 contains substantial PCP signaling activity (Wu, 2004).

Because both GOF and loss-of-function studies indicate that the Fz1 7-TM region is critical for Fz1 function, Fz1-1-2 is expected to have Fz/PCP signaling activity, although with altered subcellular distribution. Thus, the remaining PCP signaling activity of Fz1-1-2 seen is probably due to the presence of some of this protein in apical regions. It is difficult to determine how much of Fz1-1-2 is actually localized to this membrane region. Since the immunohistochemical staining indicates that it is not excluded apically, it is assumed that Fz1-1-2 has enough apical localization to participate when PCP signaling is initiated. It has been suggested that wing cell orientation does not depend on absolute Fz levels, but instead depends on relative Fz/PCP activity differences in a Fz activity gradient across a field. Thus, although the absolute activity of Fz1-1-2 is reduced (based on weaker GOF phenotypes and weaker rescue of fz^- in the eye), the relative difference might be sufficient for the partial rescue (Wu, 2004).

In this context, it is worth noting that tub-fz1-1-2 rescues the fz^- phenotype better in the wing than in the eye, whereas there is no apparent difference in rescue activity between the eye and the wing for tub-fz1-1-1 or tub-fz1-1-2S. The difference could be due to the observed nonautonomous PCP signaling effects in the wing, where neighboring cells affect each other's planar polarization. Fz1-1-2 may allow some wing cells to adopt the correct orientation, which then in turn influences many of the remaining wing cells to also orient themselves correctly through nonautonomous interactions (Wu, 2004).

It has been shown that Fz1 localization is affected in flamingo (fmi) mutant clones at about 30 h APF, leading to the proposal that Fmi recruits Fz1 into apical junctions. However, this study found that Fz1 is localized normally in fmi null mutant clones earlier in the third instar wing disc. What causes the difference between these two observations? PCP signaling in the wing is thought to act in two phases (one 6-24 h APF and the second 24-32 h APF [, and it results in the distal enrichment and maintenance of Fz1. Since Fz1/PCP signaling is modulated by Fmi, Fmi-dependent changes in Fz1 localization likely result from effects on PCP signaling activity. At the same time, Fmi localization is also dependent on Fz1 activity and becomes also less apically localized in fz^- tissue at 30-36 h APF, suggesting that the regulation of apical localization between Fz1 and Fmi is complicated and mutual at these late stages (Wu, 2004).

This study has shown that initial apical localization of Fz1, preceding both stages of PCP signaling, is not fmi dependent. This result suggests that Fmi and Fz1 get recruited to apical junctions independently. During later stages, Fmi and Fz1 then affect each other's localization through PCP signaling. At this point, it remains unclear which molecules initially recruit Fz1 into the apical junctional region (Wu, 2004).

Secreted Wg mainly binds to Fz2 at basolateral membrane regions of the wing epithelium, indirectly suggesting that canonical signaling occurs in the basolateral membrane compartment. The current experiments show that overexpression of Fz1-1-1 or Fz2-1-1 leads to a cell-autonomous loss of wing margin bristles and associated tissue, suggesting that these molecules act like dominant negatives, inhibiting Wnt/β-cat signaling. As these molecules are enriched apically and sequester Dsh there, Fz-Dsh complexes at apical junctions may be largely inactive for canonical Wnt signaling. This result suggests that canonical Wnt signaling and PCP signaling occur in different subcellular compartments. Basolateral Wnt/β-cat signaling is also suggested by the fact that (1) secreted Wg binds to Fz2 at the basolateral membrane and that (2) apical Wg secretion and signaling could lead to mis-specification in disc folds and cells in the peripodial membrane (Wu, 2004).

Both Fz1 and Fz2 are capable of canonical Wnt/β-cat signaling. Consistently, different Fz1/2 chimeras, including related versions of Fz2-1-1 and Fz2-2-1, are capable of rescuing the fz, fz2 double mutant phenotype. However, when Fz1-1-1, Fz2-1-1, or Fz2-2-1 is expressed at high levels, Dsh accumulates at apical junctions, thus decreasing cytosolic Dsh levels. Since the chimeric receptors can rescue the fz, fz2 double mutant when expressed at low levels (under the control of the tub promoter), the relative level of each receptor together with its subcellular localization appear critical for the signaling outcome (Wu, 2004).

In conclusion, this study has shown that subcellular localization contributes to Fz signaling specificity. The data indicate that the localization of Fz1 at apical junctions promotes Fz/PCP signaling, whereas this localization can inhibit canonical Wnt/β-cat signaling. The localization is mediated through sequences in the C-tail (Wu, 2004).

Heterotrimeric Go protein links Wnt-Frizzled signaling with ankyrins to regulate the neuronal microtubule cytoskeleton

Drosophila neuromuscular junctions (NMJs) represent a powerful model system with which to study glutamatergic synapse formation and remodeling. Several proteins have been implicated in these processes, including components of canonical Wingless (Drosophila Wnt1) signaling and the giant isoforms of the membrane-cytoskeleton linker Ankyrin 2, but possible interconnections and cooperation between these proteins were unknown. This study demonstrates that the heterotrimeric G protein G_o functions as a transducer of Wingless-Frizzled 2 signaling in the synapse. Ankyrin 2 was identified as a target of Go signaling required for NMJ formation. Moreover, the G_o-ankyrin interaction is conserved in the mammalian neurite outgrowth pathway. Without ankyrins, a major switch in the G_o-induced neuronal cytoskeleton program is observed, from microtubule-dependent neurite outgrowth to actin-dependent lamellopodial induction. These findings describe a novel mechanism regulating the microtubule cytoskeleton in the nervous system. This work in Drosophila and mammalian cells suggests that this mechanism might be generally applicable in nervous system development and function (Luchtenborg, 2014).

Ankyrins (Ank) are highly abundant modular proteins that mediate protein-protein interactions, mainly serving as adaptors for linking the cytoskeleton to the plasma membrane. Mammalian genomes encode three Ank genes [AnkR (Ank1), AnkB (Ank2) and AnkG (Ank3)], whereas Drosophila has two [Ank1 (also known as Ank - FlyBase) and Ank2]. Ank2 is expressed exclusively in neurons and exists in several splicing variants. The larger isoforms (Ank2M, Ank2L and Ank2XL) are localized to axons and play important roles in NMJ formation and function. The C-terminal part of Ank2L can bind to microtubules. Despite the well-established role of Ank2 in NMJ formation, its function has been considered somewhat passive and its mode of regulation has not been clarified. This study shows that Gαo binds to Ank2 and that these proteins and the Wg pathway components Wg, Fz2, and Sgg jointly coordinate the formation of the NMJ. The functional Gαo-Ank interaction is conserved from insects to mammals (Luchtenborg, 2014).

Synaptic plasticity underlies learning and memory. Both in invertebrates and vertebrates, activation of Wnt signaling is involved in several aspects of synapse formation and remodeling, and defects in this pathway may be causative of synaptic loss and neurodegeneration. Thus, understanding the molecular mechanisms of synaptic Wnt signaling is of fundamental as well as medical importance. The Drosophila NMJ is a powerful model system with which to study glutamatergic synapses, and the Wnt pathway has been widely identified as one of the key regulators of NMJ formation.

This study provides important mechanistic insights into Wnt signal transduction in the NMJ, identifying the heterotrimeric G_o protein as a crucial downstream transducer of the Wg-Fz2 pathway in the presynapse. It was further demonstrated that Ank2, a known player in the NMJ, is a target of Gαo in this signaling (Luchtenborg, 2014).

This study found that the α subunit of G_o is strongly expressed in the presynaptic cell, and that under- or overactivation of this G protein leads to neurotransmission and behavioral defects. At the level of NMJ morphology, presynaptic downregulation or Ptx-mediated inactivation of Gαo was found to recapitulate the phenotypes obtained by similar silencing of wg and fz2. These data confirm that presynaptic Wg signaling, in addition to the Wg pathway active in the muscle, is crucial for proper NMJ formation, and that G_o is required for this process. Furthermore, neuronal Gαo overexpression can rescue the wg and fz2 loss-of-function phenotypes, demonstrating that, as in other contexts of Wnt/Fz signaling, G_o acts as a transducer of Wg/Fz2 in NMJ formation. In contrast to its evident function and clear localization in the presynapse, Gαo localization on the muscle side of the synapse is much less pronounced or absent. Unlike Gαo, the main Drosophila Gβ subunit is strongly expressed in both the pre- and postsynapse. Thus, a heterotrimeric G protein other than G_o might be involved in the postsynaptic Fz2 transduction, as has been implicated in Fz signaling in some other contexts (Luchtenborg, 2014).

A recent study proposed a role for Gαo downstream of the octopamine receptor Octβ1R. This signaling was proposed to regulate the acute behavioral response to starvation both on type II NMJs (octapaminergic) and on the type I NMJs (glutamatergic) analyzed in this study. In contrast to the current observations, downregulation of Gαo in these NMJs was proposed to increase, rather than decrease, type I bouton numbers. It is suspected that the main reason for the discrepancy lies in the Gal4 lines used. The BG439-Gal4 and C380-Gal4 lines of Koon are poorly characterized and, unlike the well-analyzed pan-neuronal elav-Gal4 and motoneuron-specific OK371-Gal4 and D42-Gal4 driver lines used in the current study, might mediate a more acute expression. In this case, this study reflects the positive role of Gαo in the developmental formation of glutamatergic boutons, as opposed to a role in acute fine-tuning in response to environmental factors as studied by Koon (Luchtenborg, 2014).

Postsynaptic expression of fz2 was found to fully rescue fz2 null NMJs. This study found that presynaptic knockdown of Fz2 (and other components of Wg-Fz2-Gαo signaling) recapitulates fz2 null phenotypes, whereas presynaptic overactivation of this pathway increases bouton numbers; furthermore, presynaptic overexpression of fz2 or Gαo rescues the fz2 nulls, just as postsynaptic overexpression of fz2 does. The current data thus support a crucial role for presynaptic Wg-Fz2-Gαo signaling in NMJ formation. Interestingly, both pre- and postsynaptic re-introduction of Arrow, an Fz2 co-receptor that is normally present both pre- and postsynaptically, as is Fz2 itself, can rescue arrow mutant NMJs. Thus, it appears that the pre- and postsynaptic branches of Fz2 signaling are both involved in NMJ development. A certain degree of redundancy between these branches must exist. Indeed, wild-type levels of Fz2 in the muscle are not sufficient to rescue the bouton defects induced by presynaptic expression of RNAi-fz2, yet overexpression of fz2 in the muscle can restore the bouton integrity of fz2 nulls. One might hypothesize that postsynaptic Fz2 overexpression activates a compensatory pathway - such as that mediated by reduction in laminin A signaling - that leads to restoration in bouton numbers in fz2 mutants. The current data showing that the targeted downregulation of Fz2 in the presynapse is sufficient to recapitulate the fz2 null phenotype underpin the crucial function of presynaptic Fz2 signaling in NMJ formation (Luchtenborg, 2014).

This study found that downregulation of Ank2 produces NMJ defects similar to those of wg, fz2 or Gαo silencing. However, Ank2 mutant phenotypes appear more pronounced, indicating that Wg-Fz2-Gαo signaling might control a subset of Ank2-mediated activities in the NMJ. Ank2 was proposed to play a structural role in NMJ formation, binding to microtubules through its C-terminal region. However, since the C-terminal region was insufficient to rescue Ank2L mutant phenotypes, additional domains are likely to mediate Ank2 function through binding to other proteins. This study demonstrates in the yeast two-hybrid system and in pull-down experiments that the ankyrin repeat region of Ank2 physically binds Gαo, suggesting that the function of Ank2 in NMJ formation might be regulated by Wg-Fz2-Gαo signaling. Indeed, epistasis experiments place Ank2 downstream of Gαo in NMJ formation (Luchtenborg, 2014).

Upon dissociation of the heterotrimeric G_o protein by activated GPCRs such as Fz2, the liberated Gαo subunit can signal to its downstream targets both in the GTP- and GDP-bound state (the latter after hydrolysis of GTP and before re-association with Gβγ). The free signaling Gαo-GDP form is predicted to be relatively long lived, and a number of Gαo target proteins have been identified that interact equally well with both of the nucleotide forms of this G protein. In the context of NMJ formation, this study found that Gαo-GTP and -GDP are efficient in the activation of downstream signaling, and identifies Ank2 as a binding partner of Gαo that interacts with both nucleotide forms. The importance of signaling by Gα-GDP released from a heterotrimeric complex by the action of GPCRs has also been demonstrated in recent studies of mammalian chemotaxis, planar cell polarity and cancer (Luchtenborg, 2014).

Gαo[G203T], which largely resides in the GDP-binding state owing to its reduced affinity for GTP, might be expected to act as a dominant-negative. However, in canonical Wnt signaling, regulation of asymmetric cell division as well as in planar cell polarity (PCP) signaling in the wing, Gαo[G203T] displays no dominant-negative activity but is simply silent, whereas in eye PCP signaling this form acts positively but is weaker than other Gαo forms. Biochemical characterization of the mammalian Gαi2[G203T] mutant revealed that it can still bind Gβγ and GTP, but upon nucleotide exchange Gαi2[G203T] fails to adopt the activated confirmation and can further lose GTP. The current biochemical characterization confirms that Gαo[G203T] still binds GTP. Interestingly, Gαi2[G203T] inhibited only a fraction of Gαi2-mediated signaling, suggesting that the dominant-negative effects of the mutant are effector specific. Thus, it is inferred that a portion of Gαo[G203T] can form a competent Fz2-transducing complex, and a portion of overexpressed Gαo[G203T] resides in a free GDP-loaded form that is also competent to activate downstream targets - Ank2 in the context of NMJ formation (Luchtenborg, 2014).

These experiments place Ank2 downstream of Gαo and also of Sgg (GSK3β). It remains to be investigated whether Ank2 can directly interact with and/or be phosphorylated by Sgg. Meanwhile, it is proposed that the microtubule-binding protein Futsch might be a linker between Sgg and Ank2. Futsch is involved in NMJ formation and is placed downstream of Wg-Sgg signaling, being the target of phosphorylation and negative regulation by Sgg as the alternative target to β-catenin, which is dispensable in Wg NMJ signaling. Abnormal Futsch localization has been observed in Ank2 mutants. In Drosophila wing and mammalian cells in culture, Gαo acts upstream of Sgg/GSK3β. Cumulatively, these data might suggest that the Wg-Fz2-Gαo cascade sends a signal to Futsch through Sgg, parallel to that mediated by Ank2 (Luchtenborg, 2014).

The importance of the Gαo-Ank2 interaction for Drosophila NMJ development is corroborated by findings in mammalian neuronal cells, where it was demonstrated that the ability of Gαo to induce neurite outgrowth is critically dependent on AnkB and AnkG. Knockdown of either or both ankyrin reduces neurite production. Remarkably, upon AnkB/G downregulation, Gαo switches its activity from the induction of microtubule-dependent processes (neurites) to actin-dependent protrusions (lamellopodia). Furthermore, Gαo recruits AnkB to the growing neurite tips. These data demonstrate that the Gαo-ankyrin mechanistic interactions are conserved from insects to mammals and are important for control over the neuronal tubulin cytoskeleton in the context of neurite growth and synapse formation. The novel signaling mechanism that were uncovered might thus be of general applicability in animal nervous system development and function (Luchtenborg, 2014).

Gamma-secretase promotes Drosophila postsynaptic development through the cleavage of a Wnt receptor

Developing synapses mature through the recruitment of specific proteins that stabilize presynaptic and postsynaptic structure and function. Wnt ligands signaling via Frizzled (Fz) receptors play many crucial roles in neuronal and synaptic development, but whether and how Wnt and Fz influence synaptic maturation is incompletely understood. This study showed that Fz2 receptor cleavage via the γ-secretase complex is required for postsynaptic development and maturation. In the absence of γ-secretase, Drosophila neuromuscular synapses fail to recruit postsynaptic scaffolding and cytoskeletal proteins, leading to behavioral deficits. Introducing presenilin mutations linked to familial early-onset Alzheimer's disease into flies leads to synaptic maturation phenotypes that are identical to those seen in null alleles. This conserved role for γ-secretase in synaptic maturation and postsynaptic development highlights the importance of Fz2 cleavage and suggests that receptor processing by proteins linked to neurodegeneration may be a shared mechanism with aspects of synaptic development (Restrepo, 2022).

Postsynaptic development and maturation enable the essential transition from nascent, unreliable synapse to robust connection capable of high-fidelity neurotransmission. In Drosophila, the Wg ligand promotes neurodevelopment by activating postsynaptic Fz2 receptors, but the precise downstream events utilized by Fz2 to influence events like synaptic maturation remained controversial and unclear. This study found an unexpected developmental role for postsynaptic γ-secretase in enabling Fz2-mediated postsynaptic development via receptor cleavage. This role is also perturbed by AD patient-derived PSEN1 alleles in Drosophila, suggesting a link between AD and Wnts. These data first solve longstanding mysteries as to the significance of, and factors required for, Fz2 cleavage in postsynaptic maturation. Second, it suggests that proteolytic receptor cleavage may be a shared mechanism between developmental and degenerative processes, raising the possibility of a prior unappreciated neurodevelopmental component to AD (Restrepo, 2022).

In the last 20 years, work at the Drosophila NMJ revealed the importance of Wnts in regulating synaptic function and development through Fz2 and multiple downstream pathways. The roles of each downstream pathway, however, remained unclear. In postsynaptic muscles, Fz2 signals via a Frizzled nuclear import (FNI) pathway resulting in nuclear import of cleaved Fz2 receptor. Three major questions about FNI remained unanswered: (1) what is its physiological significance, (2) how does the Fz2 cleavage site lend to its physiological role, and (3) what protease is necessary for Fz2 cleavage to promotes this physiological role? This work addresses each question, highlighting a role for FNI in postsynaptic development, the function of the cleavage site in synaptic maturation, and identifying γ-secretase as being required for Fz2 cleavage (Restrepo, 2022).

First, it was shown that postsynaptic Fz2 cleavage (and not canonical Wnt signaling) specifically promotes postsynaptic maturation. This does not preclude a postsynaptic Fz2 function in presynaptic growth and active zone establishment. Instead, postsynaptic Fz2 may promote growth via canonical pathways; this will be an important area for future study. Second, identification of γ-secretase in promoting Fz2 cleavage is critical to understand downstream Fz2 pathways and begins to answer longstanding questions in synaptic neurodevelopment and postsynaptic maturation. As the developmental defects in γ-secretase mutants are suppressed by activating the postsynaptic FNI pathway, this indicates that the relevant cleavage target in maturation is Fz2. As maturation defects underlie neurodevelopmental disorders, further understanding the relevant machinery that promotes maturation can inform a grasp of neurodevelopment disease progression (Restrepo, 2022).

Maturation of Drosophila central and NMJ synapses and mammalian dendritic spines all require γ-secretase. γ-secretase promotes axon guidance and early synaptic development, but its role in synaptic maturation is largely unknown. THe data suggest γ-secretase-dependent cleavage and translocation of signaling proteins may be a fundamental feature of postsynaptic development. Indeed, blocking Presenilin::Syt I interaction modestly reduces spine density and mature spines (Zoltowska, 2017). This is consistent with the current data, although the more general blockade of γ-secretase function has a larger effect on spine maturation. The mechanism remains unclear as γ-secretase modulates multiple downstream pathways, including Wnts. More work will be needed to determine if Wnt signaling is conserved in downstream signaling for synaptic maturation across evolution. In vertebrates, Fz5 and Fz8 are homologous to Drosophila Fz2 and contain the same consensus cleavage site. Fz8 is expressed in the nervous system , but its neuronal function and cleavage status remain unknown. Intriguingly, recent work showed that Fz5 can be cleaved in motor neuron-like NSC-34 cells. As Fz5 promotes activity-dependent synaptogenesis and neuronal survival, these data raise a tantalizing prospect that the γ-secretase / Wnt / Fz pathway is conserved in mammalian synaptic development (Restrepo, 2022).

Identifying a role for γ-secretase and its mechanistic basis in neurodevelopment offers insight into the potential function of γ-secretase in disease. PSEN1 mutations are the most widely known genetic cause of Early-Onset Alzheimer Disease (EOAD), but thorough understanding of this genetic link is lacking. The data indicate that neurodevelopmental and neurodegenerative mechanisms may be united by a shared requirement for γ-secretase-dependent receptor processing. In AD, this adds a layer of connection between neurodevelopment and neurodegeneration via synapse-to-nucleus communication. In a model, γ-secretase cleavage enables Fz2-C generation and transition from synapse to nucleus. In AD, the synapse-to-nucleus signals AIDA-1, ATF4, and CRTC1 (that are protein partners of cleaved receptor products) are also altered and blocking the synapse-to-nucleus translocation of Jacob, a protein that couples activity to CREB signaling, suppresses pathogenic Aβ-induced impairments. Neurodevelopmental defects were observed in EOAD mutations indistinguishable from loss-of-function mutants that could be suppressed by restoring nuclear Fz2-C . It will be important for future study to determine the intersection of proteolytic processing, synapse-to-nucleus signaling, and Wnts on AD. The data raise a tantalizing possibility of connecting AD to a previously unappreciated neurodevelopmental process, which can potentially provide earlier hallmarks to assess disease progression and inform therapeutic strategies to ameliorate disease states (Restrepo, 2022).

A prevailing mystery surrounding neurodegenerative diseases involves its onset. Although patients carry gene mutations all their lives, why do these disorders manifest later in life? A further challenge is to identify the earliest changes resulting from neurodegenerative disease gene mutations to better understand if symptoms are causative or correlative. The presence of synaptic maturation defects presents a hypothesis: if neurodegenerative disease risk genes are involved in synaptic maturation, the first reflection of mutations in those genes may be immature synapses that still function but lack the longevity of mature synapses. In advanced age, synapses constructed incorrectly during development may be the first to fail, leading to degeneration. AD models show reductions in postsynaptic proteins that precede neurodegeneration. Further, growing evidence suggests that neurodegenerative diseases have synaptopathic origins and early developmental defects. It is further intriguing that molecules which influence synaptogenesis, like Ephs, are modified by γ-secretase, potentially underlying further connection. Postsynaptic development and maturation may provide a unique angle to consider neurodegeneration, leading to earlier detection. With γ-secretase, maturation defects are suppressed by activating the downstream pathway; the data suggests analogous approaches may have clinical relevance. Whether synaptic NMJ phenotypes indicate a preclinical phenotype in disease models is an open question but examining postsynaptic maturation provides an opportunity to study shared mechanisms of neurodegenerative diseases, inform strategies to combat disease, and better understand neurodevelopment (Restrepo, 2022).

The data indicates that γ-secretase activity is required for Fz2 cleavage. First, γ-secretase and Fz2 localize close enough for interaction. Second, Fz2 and γ-secretase colocalize in the same subcellular compartments and traffic similarly. Third, a structural model predicts that the cleavage site embeds in the membrane, keeping it within the 'lid' distance to be recognized as a cleavage site. Moving the cleavage site so it no longer abuts the membrane prevents the cleavage-dependent functions of Fz2. The most parsimonious interpretation supports Fz2 cleavage by γ-secretase. However, it is important to note that the data do not conclusively imply a direct cleavage event. Fz2 is not a canonical γ-secretase substrate for several reasons. First, γ-secretase typically cleaves single pass transmembrane proteins; Fz2 is a 7-pass transmembrane domain atypical G-protein coupled receptor (GPCR). This is not a requisite, however, as multipass transmembrane proteins like neuregulin-1 can be cleaved by γ-secretase. Second, γ-secretase typically cleaves in a processive fashion: the substrate ectodomain is first shed via an ADAM a disintegrin and metallprotease) enzyme or β-secretase, after which γ-secretase cleavage releases the substrate intracellular domain. There is no evidence that Fz2 undergoes ectodomain shedding and the receptor lacks consensus N-terminal cleavage sites. Third, γ-secretase typically cleaves in the plane of the membrane. The Fz2 cleavage site is cytosolic, although predicted to juxtapose the membrane. Recent years, however, identified noncanonical substrates that serve as exceptions to this rule (Restrepo, 2022).

The data suggest that GPCRs may be atypical γ-secretase cleavage targets. This broadens the potential repertoire of γ-secretase, underscoring its importance in diverse processes. However, alternate interpretations cannot be ruled out, where Fz2 cleavage requires ectodomain shedding or occurs via successive events. In the latter, Fz2 would still be a direct target of γ-secretase that follows a preceding event. Also, γ-secretase may cleave a secondary target that allows Fz2 to be cleaved. In all cases, the activity of γ-secretase is still required for Fz2 cleavage. Future study in a cell-free system will be required for formal proof. Regardless, the complete suppression of the γ-secretase developmental phenotypes by nuclear Fz2-C expression indicates the most relevant cellular substrate in promoting postsynaptic development and maturation via γ-secretase is cleaved Fz2-C (Restrepo, 2022).

PROTEIN STRUCTURE

Amino Acids - 694

Structural Domains

FZ2 resembles all other members of the frizzled family in having the following structural motifs (beginning at the N terminus): a signal sequence, a domain of 120 amino acids with an invariant pattern of ten cysteine residues, a highly divergent region of 40-100 largely hydrophilic amino acids that is predicted to be flexible, and seven putative transmembrane segments. The C terminus resembles that of most mammalian Frizzled proteins, ending with the sequence S/T-X-V. FZ2 most closely resembles human fz5 and mouse fz8, with which it shares 49% and 45% amino acid identity, respectively. FZ and FZ2 share 33% amino-acid identity (Bhanot, 1996).

date revised: 22 November 2022  

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