InteractiveFly: GeneBrief

wallenda: Biological Overview | References

Long-distance organelle transport toward axon terminals, critical for neuron development and function, is driven along microtubules by kinesins. The biophysics of force production by various kinesins is known in detail. However, the mechanisms of in vivo transport processes are poorly understood because little is known about how motor-cargo linkages are controlled. A c-Jun N-terminal kinase (JNK)-interacting protein (JIP1) has been identified previously as a linker between kinesin-1 and certain vesicle membrane proteins, such as Alzheimer's APP protein and a reelin receptor ApoER2. JIPs are also known to be scaffolding proteins for JNK pathway kinases. Evidence is presented that a Drosophila ubiquitin-specific hydrolase (Fat facets) and a JNK signaling pathway that it modulates can regulate a JIP1-kinesin linkage. The JNK pathway includes a MAPKKK (Wallenda/DLK), a MAPKK (Hemipterous/MKK7), and the Drosophila JNK homolog Basket. Genetic tests indicate that those kinases are required for normal axonal transport. Biochemical tests show that activation of Wallenda (DLK) and Hemipterous (MKK7) disrupts binding between kinesin-1 and APLIP1, which is the Drosophila JIP1 homolog. This suggests a control mechanism in which an activated JNK pathway influences axonal transport by functioning as a kinesin-cargo dissociation factor (Horiuchi, 2007).

Maintaining proper distributions of protein complexes, RNAs, vesicles, and other organelles in axons is critical for the development, function, and survival of neurons. The primary distribution mechanism relies on long-distance transport driven by microtubule motor proteins. Components newly synthesized in the cell body, but needed in the axon, bind kinesin motors that carry them toward microtubule plus ends and the axon terminal (anterograde transport). Neurotrophic signals and endosomes, examples of axonal components that require transport to the cell body, bind dynein motors that carry them toward minus ends (retrograde transport). The importance of these processes is highlighted by the observation that mutation of motors and other transport machinery components can cause neurodegenerative diseases in humans and analogous phenotypes in model organisms (Horiuchi, 2007).

Two key questions are (1) how do cargoes link to particular motors, and (2) how are such linkages regulated to ensure appropriate pickup and dropoff dynamics? For kinesin-vesicle linkages, scaffolding proteins have emerged as key connectors. For example, the cargo-binding kinesin light chain (Klc) subunit of kinesin-1 binds not only the kinesin-1 heavy chain (Khc) but also JNK-interacting proteins (JIPs). Vertebrate JIPs can bind multiple components of the JNK signaling pathway, e.g., JNK itself, upstream activating kinases (MAPKKs), and regulatory kinases (MAPKKKs). JIPs can also bind vesicle-associated membrane proteins, such as ApoER2, which is a reelin receptor, and APP, a key factor in Alzheimer's disease. Therefore, JIP scaffolding proteins are likely to link JNK pathway kinases and kinesin-1 to vesicles carrying these membrane proteins. This raises an interesting question: Are the JNK pathway kinases simply passive hitchhikers on the kinesin-1/JIP/vesicle complex, or can they actively regulate its transport (Horiuchi, 2007)?

A genetic screen was conducted for factors that control kinesin-JIP linkage during axonal transport. The screen was based on the previous observation that neuron-specific overexpression of Aplip1, which encodes the Drosophila JIP1, causes synaptic protein accumulation in axons, larval paralysis, and larval-pupal lethality, the classic axonal-transport-disruption phenotypes caused by Khc and Klc mutations. Why might overexpression of the JIP1 cargo linker for kinesin-1 disrupt axonal transport? The disruptive effect requires APLIP1 (JIP1)-Klc binding. It may be that excess APLIP1 (JIP1) competes with other Klc-binding proteins, for example, different linkers that may attach kinesin-1 to other cargoes. In search of factors that can disrupt or antagonize APLIP1 (JIP1)-Klc binding, a screen was performed for genes that can suppress the axonal-transport phenotypes when co-overexpressed with Aplip1. An 'EP' collection of fly strains capable of the targeted overexpression of endogenous Drosophila genes was screened and P{EP}faf^EP381, a line that overexpresses fat facets (faf), was identified as a strong suppressor of the APLIP1 (JIP1)-induced lethality and other neuronal overexpression phenotypes (Horiuchi, 2007).

Faf protein antagonizes ubiquitination and proteasome-mediated degradation of its target proteins. Interestingly, Faf was recently reported to stimulate a Drosophila neuronal JNK signaling pathway that is regulated by the MAPKKK Wallenda (Wnd), a homolog of dual leucine zipper-bearing kinase (DLK) that is known to bind JIP1. Overexpression of faf leads to increased levels of Wnd (MAPKKK) protein and thereby causes excessive synaptic sprouting through a pathway that requires the Drosophila JNK homolog Basket. It was found that mutating just one copy of wnd blocked the suppression of Aplip1 overexpression by P{EP}faf^EP381. This suggests that faf overexpression suppresses APLIP1 (JIP1)-Klc interaction by elevating the level of Wnd (MAPKKK). Consistent with this, direct overexpression of wnd in neurons with a wild-type transgene (UAS-wnd) was as effective as P{EP}faf^EP381 in suppressing UAS-Aplip1-induced axonal accumulation of synaptic proteins. Equivalent expression of a 'kinase-dead' mutant transgene (UAS-wnd^KD) did not suppress the defects. Thus, Wnd (MAPKKK) and its downstream phosphorylation targets may actively regulate APLIP1 (JIP1)-Klc binding in neurons (Horiuchi, 2007).

If Wnd (MAPKKK) signaling plays a role in normal axonal transport, disrupting its function should cause axonal-transport phenotypes. Consistent with this, wnd loss-of-function mutations (wnd¹/wnd²) in an otherwise wild-type background caused accumulation of synaptic proteins in axons. The accumulation phenotype was rescued by motoneuron expression of the wild-type wnd transgene but not by equivalent expression of the kinase-dead mutant transgene. The likely target of Wnd (MAPKKK) kinase activity is the Drosophila homolog of MKK7, Hemipterous (Hep), a MAPKK that activates Bsk (JNK). Mutation of hep also causes axonal accumulations, as does neuronal expression of a dominant-negative mutant bsk transgene. The results of these genetic-inhibition tests combined with those of the Aplip1-overexpression-suppression tests suggest that a Wnd (MAPKKK)-activated JNK pathway influences fast axonal transport by regulating APLIP1 (JIP1)-Klc binding (Horiuchi, 2007).

Is a Wnd (MAPKKK)-Hep (MAPKK)-Bsk (JNK) signaling module bound by APLIP1 (JIP1)? Although all three components of the homologous vertebrate module (DLK-MKK7-JNK) bind JIP1, APLIP1 (JIP1) lacks a conserved JNK-binding domain, and it does not bind directly to Bsk (JNK). However, APLIP1 (JIP1) does bind Hep (MAPKK), Klc, and the Drosophila APP homolog APPL. To determine whether Wnd (MAPKKK) associates with Hep (MAPKK) and APLIP1 (JIP1), coexpression and immunoprecipitation tests were performed in Drosophila S2 cultured cells. Wnd (MAPKKK) did not coprecipitate with APLIP1 (JIP1). However, Hep (MAPKK) did coprecipitate with APLIP1 (JIP1), and Wnd (MAPKKK) coprecipitated with Hep (MAPKK). Thus, Wnd (MAPKKK) may bind and influence the APLIP1 (JIP1)-kinesin complex via Hep (MAPKK) (Horiuchi, 2007).

Can Wnd (MAPKKK) and Hep (MAPKK) control the binding of APLIP1 (JIP1) to Klc? When expressed in S2 cells, APLIP1 (JIP1) and Klc exhibit strong binding, as assessed by coimmunoprecipitation. Coexpression of wild-type Wnd (MAPKKK) partially inhibited APLIP1 (JIP1) binding to Klc, but coexpression of a kinase-dead mutant Wnd (MAPKKK) did not. Wild-type Hep (MAPKK) also caused a partial inhibition of APLIP1 (JIP1)-Klc binding, and a constitutively active mutant Hep (MAPKK) caused nearly complete inhibition. Finally, coexpression of wild-type Wnd (MAPKKK) and Hep (MAPKK) together caused an almost complete inhibition of APLIP1 (JIP1)-Klc binding. In addition to inhibiting APLIP1 (JIP1)-Klc binding, Wnd-Hep activation in S2 cells increased the level of Bsk (JNK) activation. Hence, there is a correlation between decreased levels of APLIP1 (JIP1)-Klc binding and elevated levels of Bsk (JNK) activation. This suggests that, despite the lack of a known JNK-binding site on APLIP1 (JIP1), Bsk (JNK) may be the kinase that disrupts the APLIP1 (JIP1)-Klc complex. These results suggest that Wnd (MAPKKK) activation of Hep (MAPKK), and perhaps also Hep (MAPKK) activation of Bsk (JNK), can regulate the linkage between kinesin-1 and a cargo complex via the JIP1-like scaffolding protein, APLIP1 (Horiuchi, 2007).

Hep (MAPKK) may regulate the APLIP1 (JIP1) complex either by activating JNK or by a mechanism independent of JNK. Observations that motoneuron-specific inhibition of Bsk (JNK) caused transport defects similar to those caused by mutations in wnd and hep and that decreased APLIP1 (JIP1)-Klc binding in S2-cell lysates coincided with increased phosphorylated Bsk (JNK) support pathway 1, i.e., Hep (MAPKK) activation of Bsk (JNK), which then directly or indirectly inhibits APLIP1 (JIP1)-Klc binding. A second pathway, Pathway 2, employs an alternative mechanism in which activated Hep (MAPKK) does not need Bsk (JNK) to inhibit APLIP1 (JIP1)-Klc binding. There is little current evidence that Hep or its vertebrate MAPKK homolog MKK7 have phosphorylation targets other than Bsk (JNK). However, that does not exclude the possibility that activated Hep induces in APLIP1 (JIP1) a direct conformational change that causes Klc dissociation. Regardless of how Hep (MAPKK) disrupts binding, when kinesin-1 is not attached to cargo via JIP1, it can fold into a compact form that does not interact with microtubules. Hence, the activated Wnd (MAPKKK) pathway could both inhibit APLIP1 (JIP1)-Klc binding and cause dissociation of kinesin-1 from microtubules. Consistent with this, recent studies report that stimulation of JNK pathways in cultured cells or axoplasm can disrupt the association of kinesin-1 with microtubules (Horiuchi, 2007 and references therein).

From a broader perspective on axonal-transport regulation, it is interesting to consider that there are multiple types of kinesin-1 cargoes, that there are various JIPs that could be specific for different cargoes, and that different MAPKKKs can associate with different JIPs. By sitting at the top of a classic signaling cascade, MAPKKKs such as Wnd are in a good position to differentially control the transport of specific subsets of anterograde kinesin-1 cargoes in response to specific cellular signals. It is known in mammals that other MAPKKKs such as MLK, ASK1, and MEKK1 can bind JIP scaffolding proteins. It will be interesting to determine whether they too influence kinesin-cargo interactions (Horiuchi, 2007).

This work provides the first demonstration that a kinesin and its transport functions can be influenced by a MAPKKK. More specifically, the MAPKKK Wnd and its downstream MAPKK Hep can regulate attachment of a JIP1 cargo linker to kinesin-1. These results also provide the first indication that ubiquitination pathways, by way of MAPKKKs, could be important for proper regulation of axonal transport. Finally, these results suggest that JNK pathway kinases are not just hitchhikers on the axonal kinesin-1/JIP/cargo complex; rather, they can actively regulate its transport dynamics (Horiuchi, 2007).

Highwire restrains synaptic growth by attenuating a MAP kinase signal

Highwire is an extremely large, evolutionarily conserved E3 ubiquitin ligase that negatively regulates synaptic growth at the Drosophila NMJ. Highwire has been proposed to restrain synaptic growth by downregulating a synaptogenic signal. This study identifies such a downstream signaling pathway. A screen for suppressors of the highwire synaptic overgrowth phenotype yielded mutations in wallenda, a MAP kinase kinase kinase (MAPKKK) homologous to vertebrate DLK and LZK. wallenda is both necessary for highwire synaptic overgrowth and sufficient to promote synaptic overgrowth, and synaptic levels of Wallenda protein are controlled by Highwire and ubiquitin hydrolases. highwire synaptic overgrowth requires the MAP kinase JNK and the transcription factor Fos. These results suggest that Highwire controls structural plasticity of the synapse by regulating gene expression through a MAP kinase signaling pathway. In addition to controlling synaptic growth, Highwire promotes synaptic function through a separate pathway that does not require Wallenda (Collins, 2006).

JNK signaling affects many cellular processes, often by regulating transcription factor activity that leads to changes in gene expression. A common downstream effector of JNK-mediated changes in gene expression is the AP-1 complex of Fos and Jun transcription factors, which can regulate synaptic growth at the Drosophila NMJ. To investigate whether Drosophila Fos or Jun (known as D-fos and D-jun, respectively) are required for highwire-dependent synaptic overgrowth, each was inhibited by expressing dominant-negative transgenes that contain the DNA binding and dimerization domains of Fos and Jun but lack the transcriptional activation domains. Expression of these dominant-negative transgenes in postmitotic neurons allowed circumvention of early embryonic requirements for D-fos and D-jun (Collins, 2006).

When Fos^DN and Jun^DN are neuronally expressed in a wild-type background, there is a modest trend toward inhibition of synaptic growth. When expressed in a highwire mutant background, the Fos^DN transgene confers dramatic suppression of the highwire synaptic phenotype, reducing bouton number and branching (42%) and increasing the intensity of staining for synaptic vesicle markers at the synapse. The reduction in highwire-dependent synaptic overgrowth is much greater than the reduction of growth in a wild-type background. In contrast, Jun^DN does not suppress the highwire phenotype. This suggests the existence of a pathway that is separate from AP-1, consistent with results in Drosophila demonstrating that D-Fos can act independently of D-Jun. The requirement for D-Fos in highwire synaptic overgrowth suggests that the highwire phenotype involves changes in gene expression rather than exclusively local changes to the synapse (Collins, 2006).

If Fos^DN acts downstream of Wallenda to inhibit synaptic overgrowth, it should also suppress the synaptic overgrowth caused by overexpressing wallenda. Indeed, when Fos^DN was coexpressed with UAS-wnd in neurons, Fos^DN could suppress the wallenda gain-of-function phenotype, leading to a 38% reduction in synaptic bouton number, a 52% reduction in synaptic branching, a 54% increase in bouton size, and a 3.8-fold increase in the intensity of staining of synaptic vesicle markers. This is consistent with D-Fos acting downstream of Wallenda to promote synaptic growth. Therefore, the synaptic overgrowth phenotypes caused by loss of highwire and by overexpression of wallenda are similar in their requirements for the transcription factor D-Fos (Collins, 2006).

Current models suggest that Highwire functions as an E3 ubiquitin ligase to downregulate a signaling pathway that promotes synaptic growth. This study identified a MAPKKK, Wallenda, whose protein levels are controlled by Highwire and the activity of ubiquitin hydrolases. Wallenda is both necessary for highwire-dependent synaptic overgrowth and sufficient to promote synaptic growth. Downstream of Wallenda, the MAP kinase JNK and transcription factor Fos are required for highwire-dependent synaptic overgrowth. It is proposed that Highwire restrains synaptic growth by downregulating the MAPKKK Wallenda, thereby inhibiting signaling through the JNK MAP kinase and the Fos transcription factor. In the absence of highwire, this signaling pathway is overactive, leading to changes in gene expression that result in excessive synaptic growth (Collins, 2006).

The regulation of the MAPKKK Wallenda is conserved in Drosophila and C. elegans (Nakata, 2005). In both organisms, the synaptic phenotype of highwire/rpm-1 requires the Wallenda/DLK-1 MAPKKK and downstream MAPK signaling. However, the downstream MAPK pathways diverge: in C. elegans, the rpm-1 phenotype requires a p38 MAP kinase (Nakata, 2005), while the highwire phenotype requires JNK signaling. This suggests that regulation of the specific MAPKKK Wallenda/DLK-1, rather than a particular downstream MAP kinase pathway, is a fundamental activity of Highwire and its orthologs (Collins, 2006).

Since Highwire functions as an E3 ubiquitin ligase to restrain synaptic growth, Wallenda is a compelling candidate target for the following reasons: (1) wallenda functions downstream of highwire and is essential for the synaptic overgrowth in highwire mutants; (2) increasing the levels of Wallenda by overexpression is sufficient to confer synaptic overgrowth; (3) Highwire regulates Wallenda protein levels through a posttranscriptional and most likely posttranslational mechanism. Each of the points above is conserved in C. elegans (Nakata, 2005). (4) Wallenda protein levels are regulated by ubiquitination in vivo, since inhibiting ubiquitination by overexpressing ubiquitin hydrolases increases the levels of Wallenda protein. (5) The RING domain of the C. elegans homolog rpm-1 can interact with the Wallenda homolog DLK-1 (Nakata, 2005) and stimulate its ubiquitination when both are overexpressed in 293T cells (Collins, 2006).

Targeting a MAPKKK, which sits at the top of a MAP kinase signaling pathway, is an attractive mechanism for spatially and temporally controlling a synaptogenic signal without affecting downstream components shared by multiple MAPK signaling cascades. Restraining MAP kinase signaling is essential for controlling diverse cellular processes, including cell proliferation, differentiation, and apoptosis. The targeting of MAPKKKs by specific ubiquitin ligases may be a powerful and general mechanism for regulating MAP kinase signals (Collins, 2006).

While Wallenda is an essential mediator of the highwire mutant phenotypes in both Drosophila and C. elegans, an endogenous synaptic function for Wallenda has not yet been identified in either organism: the wallenda mutants have surprisingly normal synapse morphology and function. This may be due to another pathway that compensates for the loss of wallenda function. Such redundancy would obscure the role of wallenda. A second possibility is that wallenda functions in an aspect of synaptic growth that is not detected or required under laboratory culture conditions. For instance, wallenda could promote synaptic growth as part of a structural plasticity program that responds to unknown experience-dependent stimuli. A third possibility is that Wallenda does not normally function at synapses, but its upregulation in highwire mutants causes a neomorphic phenotype. In this scenario, the regulation of Wallenda by Highwire is required for normal synaptic development, but endogenous Wallenda would not itself regulate the synapse. The neuropil and synaptic localization of Wallenda and the vertebrate homolog DLK (Hirai, 2005) is, however, consistent with a synaptic function (Collins, 2006).

As an activator of MAP kinase signaling, Wallenda and its homologs might also control other processes beyond the synapse. Functional studies in vertebrates suggest that DLK and JNK signaling regulate neuronal migration and axon outgrowth in the developing cortex (Hirai, 2002). Outside of the nervous system, DLK influences keratinocyte differentiation, and LZK is highly expressed in the pancreas, liver, and placenta. In Drosophila, wallenda mutants are female sterile. It is predicted that the regulation of DLK and LZK is conserved from worms and flies to vertebrates. Therefore, the vertebrate homologs of Highwire might regulate some of these neuronal and/or extraneuronal developmental processes (Collins, 2006 and references therein).

Highwire is a large, multidomain protein that, in addition to acting as an E3 ubiquitin ligase, has been shown to inhibit adenylate cyclase, influence TSC signaling and pteridine biosynthesis, and interact with the myc oncogene and the co-SMAD Medea. It is remarkable that throughout millions of years of evolution, members of the Highwire family have retained an exceptionally large size and complex domain structure. An attractive explanation for this conservation is that this molecule could serve as an intersection point for multiple signaling pathways, integrating MAP kinase and other signals during neural development (Collins, 2006).

The ubiquitin ligase activity alone could be responsible for regulating more than one downstream target. Interactions with components of TSC (tuberin/hamartin) and TGF-β signaling pathways suggest that Highwire might target either or both of these pathways. The model that Highwire regulates TGF-β signaling through interaction with the co-SMAD Medea has received considerable attention. Since the TGF-β pathway regulates synaptic growth at the NMJ, it has been proposed that synaptic overgrowth of highwire mutants is caused by overactivity of this pathway. Null alleles of wit, which completely disrupt TGF-β signaling at the NMJ, can partially suppress the highwire phenotypes: they partially suppress the increase in bouton number, but show little or no suppression of the reduced bouton size and the reduced intensity for synaptic vesicle markers. This partial suppression of highwire by wit is consistent with the model that overactive TGF-β signaling contributes to the highwire phenotype. However, the data are also consistent with the alternate model that TGF-β signaling and Highwire act in parallel pathways. An assay for the activity of TGF-β signaling is to stain for phosphorylated-MAD (phospho-MAD), the major transducer of BMP signals in Drosophila, in motoneuron nuclei. No change was detected in the levels of phospho-MAD staining in highwire mutants compared to wild-type. This assay is sensitive to changes in pathway activity—neuronal expression of the constitutively active type I receptor thick veins leads to a 40% increase in phospho-MAD staining. Interestingly, this increase in TGF-β signaling does not lead to excess synaptic growth. Combining a highwire mutant with expression of constitutively active thick veins does cause excess growth, but it does not lead to any further increase in phospho-MAD staining. These data are consistent with highwire and TGF-β signaling acting in parallel pathways (Collins, 2006).

Whether or not Highwire regulates TGF-β signaling, it is likely to target an additional pathway. Highwire not only restrains synaptic growth, but also promotes synaptic function. Synaptic function requires the ubiquitin ligase activity of Highwire and is sensitive to the levels of the ubiquitin hydrolase fat facets. This study demonstrates that this regulation of neurotransmitter release does not require Wallenda. Therefore, Highwire must regulate at least two distinct molecular pathways. If Wallenda is a substrate whose downregulation is essential for restraining synaptic growth, there is likely another substrate for Highwire whose downregulation promotes neurotransmitter release (Collins, 2006).

Downstream of Wallenda, the JNK MAP kinase and Fos transcription factor are required for the highwire synaptic morphology phenotype. Therefore, Highwire attenuates a JNK signaling pathway that presumably controls gene expression to regulate synaptic growth. Previous studies have implicated JNK-dependent transcriptional control in activity-dependent growth of the Drosophila NMJ. However, this previously described pathway is probably distinct from the JNK signal that is controlled by Highwire and activated by Wallenda. The previously described role for JNK requires AP-1, a heterodimer of Fos and Jun transcription factors; inhibiting either D-Fos or D-Jun disrupts this pathway. In contrast, highwire-induced overgrowth requires D-Fos, but not D-Jun. The Wallenda pathway could therefore involve a homodimer of D-Fos or another transcription factor that interacts with Fos. Such D-Jun-independent functions of D-Fos have been described previously in Drosophila. The differential requirement for transcription factors suggests that the output of Wallenda signaling cannot simply be activation of JNK, but instead activation of JNK in a particular spatial or temporal context, such as in the presence of cofactors that influence downstream signaling (Collins, 2006).

In addition to transcription factors, substrates for activated JNK include components of the cytoskeleton. Because the NMJ is distant from the motoneuron nucleus, and because vertebrate DLK colocalizes with tubulin in axonal regions of the brain, it was initially expected that the Highwire/Wallenda/JNK pathway would influence synaptic morphology through local action upon the synaptic cytoskeleton. Instead, a requirement was identified for a transcription factor and presumably changes in gene expression. However, this does not exclude an interaction with the cytoskeleton or local changes at the synapse. It is possible that Highwire regulates the Wallenda signal in the cell body. However, the observation that Wallenda accumulates in the synapse-rich neuropil and at the NMJ when Highwire is absent suggests that Wallenda could become activated at the synapse. This would imply the need for a mechanism to transport the activated JNK signal back to the nucleus. In addition, cell-wide changes in gene expression must then be translated into localized growth at the synapse. Activated Wallenda at the synapse is an attractive candidate to integrate changes in gene expression with regulation of the synaptic cytoskeleton to control synaptic growth (Collins, 2006).

DFsn collaborates with Highwire to down-regulate the Wallenda/DLK kinase and restrain synaptic terminal growth

The growth of new synapses shapes the initial formation and subsequent rearrangement of neural circuitry. Genetic studies have demonstrated that the ubiquitin ligase Highwire restrains synaptic terminal growth by down-regulating the MAP kinase kinase kinase Wallenda/dual leucine zipper kinase (DLK). To investigate the mechanism of Highwire action, DFsn has been identified as a binding partner of Highwire and characterized the roles of DFsn (CG4643) in synapse development, synaptic transmission, and the regulation of Wallenda/DLK kinase abundance. This study identified DFsn as an F-box protein that binds to the RING-domain ubiquitin ligase Highwire and that can localize to the Drosophila neuromuscular junction. Loss-of-function mutants for DFsn have a phenotype that is very similar to highwiremutants -- there is a dramatic overgrowth of synaptic termini, with a large increase in the number of synaptic boutons and branches. In addition, synaptic transmission is impaired in DFsn mutants. Genetic interactions between DFsn and highwire mutants indicate that DFsn and Highwire collaborate to restrain synaptic terminal growth. Finally, DFsn regulates the levels of the Wallenda/DLK kinase, and wallendais necessary for DFsn-dependent synaptic terminal overgrowth. In conclusion, the F-box protein DFsn binds the ubiquitin ligase Highwire and is required to down-regulate the levels of the Wallenda/DLK kinase and restrain synaptic terminal growth. It is proposed that DFsn and Highwire participate in an evolutionarily conserved ubiquitin ligase complex whose substrates regulate the structure and function of synapses (Wu, 2007; full text of article).

Highwire and the C. elegans homolog, RPM-1, act as ubiquitin ligases to regulate synaptic development. Liao (2004) proposed that, in C. elegans, RPM-1 participates in an atypical SCF ubiquitin ligase complex with the F-box protein FSN-1. Consistent with this hypothesis, RPM-1 binds to FSN-1 as well as to Skp-1 and Cullin-1, core components of SCF complexes. In addition, FSN-1 null mutants have very similar phenotypes to rpm-1 mutants at GABAergic synapses, but weaker phenotypes in DD motoneurons and sensory neurons. The difference in phenotypes suggests that RPM-1 interacts with other F-box proteins in addition to FSN-1, acts as a ubiquitin ligase without an F-box partner, or has ubiquitin-independent functions. The target of the RPM-1/FSN-1 complex in C. elegans is not clear. Biochemical and genetic data indicate that the receptor tyrosine kinase ALK is the functionally relevant target for FSN-1, while the MAPKKK DLK is the functionally relevant target for RPM-1. The data in Drosophila support the model from worms that RPM-1 and FSN-1 form a functional ubiquitin ligase complex, but simplify the model by demonstrating that in Drosophila both components target the same substrate (Wu, 2007).

This study demonstrates that Highwire binds the Drosophila homolog of FSN-1, DFsn. Therefore, the physical association of Highwire/RPM-1 and DFsn/FSN-1 is evolutionarily conserved. While eukaryotic genomes can encode hundreds of F-box proteins like DFsn, in worms, flies, mice, and humans there is only a single F-box protein that also contains an SPRY domain, and each is more closely related by sequence to each other than to other F-box proteins. Since the binding of DFsn/FSN-1 to Highwire/RPM-1 is conserved, it is speculated that the mouse and human homologs of DFsn, F-box protein 45 and hCG1734196, will bind to and function with Phr and PAM, the mouse and human homologs of Highwire, respectively. Indeed, expression analysis demonstrates that both the F-box protein 45 and Phr are expressed in a very similar pattern in the mouse brain (Wu, 2007).

These results suggest that the interaction of Highwire with DFsn is required for Highwire activity. Loss-of-function mutants for highwire and DFsn have qualitatively and quantitatively similar phenotypes - both are required to restrain synaptic terminal growth and promote synaptic release. Both Highwire and DFsn are necessary to down-regulate the levels of the MAPKKK Wallenda/DLK, and wallenda mutants suppress the morphological but not physiological phenotypes of both highwire and DFsn. Finally, genetic data support the model that Highwire and DFsn function together during synaptic development - DFsn mutants enhance the phenotype of a highwire hypomorph but not of a highwire null. All of these data are consistent with the model that Highwire and DFsn act together to form a functional ubiquitin ligase complex. In this model, the ligase complex targets Wallenda/DLK to restrain synaptic terminal growth, and an unknown substrate to promote synaptic function. It is speculated that the targeting of the Wallenda/DLK MAPKKK by the Highwire/DFsn complex will be conserved from worms to mammals. While Highwire and DFsn collaborate for synaptic development, the male sterility of DFsn but not highwire mutants suggests that DFsn has Highwire-independent functions in other developmental processes (Wu, 2007).

The Drosophila epsin 1 is required for ubiquitin-dependent synaptic growth and function but not for synaptic vesicle recycling

The ubiquitin-proteasome system plays an important role in synaptic development and function. However, many components of this system, and how they act to affect synapses, are still not well understood. This study used the Drosophila neuromuscular junction to study the in vivo function of Liquid facets (Lqf), a homolog of mammalian epsin 1. The data show that Lqf plays a novel role in synapse development and function. Contrary to prior models, Lqf is not required for clathrin-mediated endocytosis of synaptic vesicles. Lqf is required to maintain bouton size and shape and to sustain synapse growth by acting as a specific substrate of the deubiquitinating enzyme Fat facets. However, Lqf is not a substrate of the Highwire (Hiw) E3 ubiquitin ligase; neither is it required for synapse overgrowth in hiw mutants. Interestingly, Lqf converges on the Hiw pathway by negatively regulating transmitter release in the hiw mutant. These observations demonstrate that Lqf plays distinct roles in two ubiquitin pathways to regulate structural and functional plasticity of the synapse (Bao, 2008).

One important finding from this study is that Lqf does not play a detectable role in SV endocytosis. Multiple lines of evidence obtained from electrophysiological, ultrastructural and optical imaging studies support this conclusion. This is the first in vivo study of Lqf or epsin 1 on SV recycling. The finding is also clearly surprising given that epsin 1 has been highly implicated to play a key role in the initiation of clathrin-coated vesicle formation and endocytosis. Does the observation reflect the special property of the fly NMJ? Lqf lacking either the ENTH domain or the clathrin-interacting C-terminus has been shown to rescue the mutant phenotype in the developing eye. These rescue results are intriguing, but they do not readily support a specific role for Lqf in CME. In particular, there are no clear mechanisms on how these truncated fragments could fulfill Lqf's clathrin-dependent functions. Interestingly, RNA interference and small interfering RNA-induced knockdown of epsin 1 fails to block the internalization of EGF receptors in HeLa cells. There is also evidence that epsin 1 functions only in clathrin-independent endocytosis. Furthermore, Lqf has been shown to be required for endocytosis of select receptors but not of all receptors. More importantly, Lqf itself is not required for receptor-mediated endocytosis. Rather, Lqf appears to signal select ligands (such as Delta/Serrate/Lag2) for internalization or recycling. Hence, these studies lend strong support to observations that Lqf does not play a significant role in CME of SVs (Bao, 2008).

It should be noted that recent studies reveal that the epsin 1-interacting protein Eps15 is required for SV recycling in both C. elegans and Drosophila. However, Eps15 is required to maintain the level of endocytotic proteins in nerve terminals. Strikingly, key endocytotic proteins such as Dynamin and Dap160 are reduced in synaptic boutons by ∼90% and ∼80%, respectively, in eps15 mutants. These observations make it difficult, if not impossible, to assign a direct role for Eps15 in CME (Bao, 2008).

Synapse development is a highly regulated process involving a large number of molecules. The first suggestion that Lqf could have a potential role in synapse development came from studies of its deubiquitinating enzyme Faf. This notion was further supported by a direct biochemical demonstration that Lqf is a specific substrate of Faf. The current studies provide the first experimental test of this hypothesis by showing that Lqf acts downstream of Faf in promoting synaptic overgrowth. This effect on NMJ growth appears to be Faf dependent as lqf mutations alone do not dramatically affect bouton numbers. It is interesting to note that neuronal overexpression of Lqf promotes bouton budding but does not mimic the exuberant synaptic overgrowth induced by overexpression of Faf. Hence, it is suggested that Lqf is necessary but insufficient for synaptic overgrowth, raising the possibility that Lqf is not the only substrate of Faf in motoneurons (Bao, 2008).

Another important finding emerging from this study is that two distinct UPS pathways may be employed at the Drosophila larval NMJ to regulate synapse growth. The Hiw/RPM-1/Phr1 proteins have a conserved role in inhibiting presynaptic development in Drosophila, C. elegans and mammals. In C. elegans and Drosophila, the substrates of RPM-1/Hiw have been shown to be MAP kinases and MAPKKK. The current study indicates that Lqf is unlikely a substrate of Hiw in conditioning synaptic growth. In contrast, this study shows that the Faf pathway is a positive regulator of synaptic growth at the NMJ in which Lqf is an essential substrate. Hence, it is suggested that Hiw and Faf/Lqf are two distinct UPS pathways that regulate synapse development in Drosophila (Bao, 2008).

However, the relationship between the Faf and Hiw pathways in synapse development is rather complex. Intriguingly, the MAPKKK Wnd is required for synaptic overgrowth mediated by both Hiw and Faf pathways. One possibility is that Wnd acts downstream of Lqf to fulfill the function of both the Hiw and the Faf pathways. However, this idea is inconsistent with the observation that unlike lqf mutants, the wnd null mutant itself has no morphological or electrophysiological defect. More importantly, wnd mutations do not suppress the transmitter release defect seen in the hiw mutant, whereas the lqf mutant does. Alternatively, it is suggested that Hiw and Faf act through two parallel pathways and that the suppression of Faf-induced overgrowth by the wnd mutation may be mediated by Fos/Jun kinase signaling. Based on the observation that overexpression of Ubp2A increases neuronal Wnd levels, it is possible that Faf may also use Wnd as a substrate for synaptic overgrowth. However, this has yet to be tested experimentally (Bao, 2008).

Recent genetic studies have revealed an interesting feature of synapse growth and function that closely depends on protein turnover by specific UPS pathways. In Drosophila, faf or lqf mutations are capable of partially suppressing the defect in transmitter release in hiw mutants. This partial suppression is specific and should not be viewed simply as a reduction of transmitter release in faf or lqf mutant backgrounds by hiw mutations. If there were no partial suppression by faf or lqf mutations, the amplitude of EJPs would be similar to that in hiw single mutants. Because faf null mutations reduce Lqf levels, it is reasonable to suggest that Lqf acts downstream of Faf to inhibit synaptic transmission in hiw mutants. Unlike the functional interactions with hiw, however, faf or lqf mutations do not affect synaptic overgrowth in hiw mutants. Differing from lqf and faf mutations, wnd mutations fully suppress synaptic overgrowth but do not affect synaptic physiology in the hiw mutant. Hence, different ubiquitin pathways can specifically dissociate synapse growth from function (Bao, 2008).

The physiological stimuli involved in such selective modulation of synapse growth and function remain to be identified. Given the conserved role of the ubiquitin-proteasome system in synaptic plasticity across animal species, the findings reported in this study may have general neurobiological implications. In particular, it is noted that the Faf homolog in mouse, Usp9x or Fam is differentially expressed in different regions of the brain. Such spatial distribution patterns may provide a means for Usp9x to locally regulate synaptic function. Importantly, Usp9x is localized at synapses, where calcium influx rapidly regulates its enzymatic activity and deubiquitination of epsin 1. Hence, Faf and Lqf/epsin 1 are good candidate mediators of activity-dependent synaptic plasticity (Bao, 2008).

Autophagy promotes synapse development in Drosophila

Autophagy, a lysosome-dependent degradation mechanism, mediates many biological processes, including cellular stress responses and neuroprotection. This study demonstrates that autophagy positively regulates development of the Drosophila larval neuromuscular junction (NMJ). Autophagy induces an NMJ overgrowth phenotype closely resembling that of highwire (hiw), an E3 ubiquitin ligase mutant. Moreover, like hiw, autophagy-induced NMJ overgrowth is suppressed by wallenda (wnd) and by a dominant-negative c-Jun NH2-terminal kinase (bsk^DN). Autophagy promotes NMJ growth by reducing Hiw levels. Thus, autophagy and the ubiquitin-proteasome system converge in regulating synaptic development. Because autophagy is triggered in response to many environmental cues, these findings suggest that it is perfectly positioned to link environmental conditions with synaptic growth and plasticity (Shen, 2009).

Autophagy involves multiple steps, including induction, autophagosome formation, fusion of autophagosomes with lysosomes, and recycling of autophagy components. Disrupting any of these steps impairs autophagy. Several highly conserved ATG genes encoding core components of the autophagy machinery have been identified in yeast. Mutations in genes, including atg1, -2, -6, and -18, have also been isolated and characterized in Drosophila (Scott, 2004; Berry, 2007). To assess the role of autophagy in NMJ development, the effects were examined of mutations in atg genes, whose normal functions span the entire process: atg1 is defective in autophagy induction, atg6 is defective in autophagosome formation, and atg2 and -18 are defective in retrieval of other ATG proteins from autophagosomes (Levine, 2008). Regardless of the step impaired, all of these atg mutants exhibited significant reduction in NMJ size. These results demonstrate that a basal level of autophagy is required to promote NMJ development (Shen, 2009).

Overexpression of atg1⁺ is sufficient to induce high levels of autophagy in larval fat bodies and salivary glands (Berry, 2007; Scott, 2007). If autophagy is a positive regulator of NMJ development, an increase in autophagy might enhance synaptic growth. Consistent with previous studies, panneuronal overexpression of UAS-atg1⁺ under the control of C155-Gal4 or elav-Gal4 drivers induced high levels of autophagy in the nervous system, as indicated by increased staining with LysoTracker, an acidophilic dye which has been used to assess autophagy by labeling acidic structures, including lysosomes. Under these conditions, bouton number increased more than twofold. To further verify that this NMJ overgrowth was caused by elevated autophagy rather than to some other effect of atg1⁺ overexpression, whether mutations in other atg genes suppress this phenotype was examined. For this purpose, a null allele of atg18 (atg18^δ) was generated. Removal of one copy of atg18⁺ had no affect on NMJ growth in an otherwise wild-type background but significantly suppressed NMJ overgrowth caused by atg1⁺ overexpression. Removal of both copies of atg18⁺ conferred almost complete suppression. Therefore, NMJ overgrowth caused by atg1⁺ overexpression is primarily caused by elevated levels of autophagy (Shen, 2009).

As a further test, NMJ morphology was examined after feeding larvae with rapamycin, which induces autophagy by inhibiting TOR (target of rapamycin), the key negative regulator of autophagy (Rubinsztein, 2007). Wild-type larvae fed rapamycin exhibited striking NMJ overgrowth similar to that caused by overexpressing atg1⁺, consistent with the results of Knox (2007). Rapamycin-induced NMJ overgrowth was completely suppressed by mutations in atg18. Collectively, these results demonstrate that autophagy is a key positive regulator of NMJ growth (Shen, 2009).

Wairkar (2009) observed NMJ undergrowth in atg1 mutants but did not see overgrowth with atg1⁺ overexpression. This discrepancy likely results from the use of different UAS-atg1⁺ transgenes. For example, Wairkar was able to obtain only partial (~50%) rescue of NMJ undergrowth in atg1 mutants by overexpression of their UAS-atg1^rescue construct, whereas this study obtained complete rescue of this phenotype (Shen, 2009).

Atg1 has several functions unrelated to autophagy. It was found that axonal transport is disrupted in atg1-null mutants, which is a result also recently reported by Toda (2008) and Wairkar (2009). In addition, Atg1 suppresses translation by inhibiting the S6K kinase (Lee, 2007; Scott, 2007) and controls active zone density by inhibiting extracellular signal-regulated kinase (ERK) signaling (Wairkar, 2009). However, several lines of evidence indicate that these functions of Atg1 are not responsible for the NMJ phenotypes observed when Atg1 activity was altered. (1) atg2 or -18 mutants exhibited similar NMJ undergrowth but did not have defects in axonal transport. Thus, in agreement with Toda (2008), it is concluded that Atg1's role in axonal transport is distinct from its function in autophagy and NMJ growth. (2) Blocking or activating translation by overexpressing a dominant-negative S6K transgene or constitutively activated S6K transgenes by elav-Gal4 driver had little affect on NMJ growth. Moreover, coexpression of any of the three constitutively activated S6K transgenes failed to suppress NMJ overgrowth caused by atg1⁺ overexpression. Thus, the role of Atg1 in S6K-dependent translation does not contribute to the NMJ phenotypes associated with manipulations of Atg1. (3) An ERK mutation does not affect NMJ growth. Although this ERK mutation suppresses the deficit in active zone density in atg1 mutants, it does not suppress atg1's NMJ undergrowth phenotype (Wairkar, 2009), indicating that it is not mediated by the ERK pathway. Collectively, these results demonstrate that altered levels of autophagy are primarily responsible for the effects of Atg1 on NMJ development (Shen, 2009).

NMJ overgrowth induced by autophagy is distinctive and offers potential clues about pathways that may be involved. Formation of multiple long synaptic branches containing many small diameter boutons without any hyperbudding or satellite boutons most closely resembles the hiw phenotype, suggesting that autophagy and Hiw may function through the same pathway. Recent evidence indicates that Hiw inhibits NMJ growth by down-regulating Wnd, which in turn activates a Jun kinase encoded by bsk (basket). NMJ overgrowth in hiw is suppressed by mutations of wnd and by a dominant-negative mutation of bsk (bsk^DN; Collins, 2006). If the phenotypic similarity between hiw and increased autophagy reflects convergence on a common pathway, autophagy-induced NMJ overgrowth should also be suppressed by wnd and bsk^DN. Indeed, this is what was observed in this study. These results strongly support the idea that autophagy and Hiw converge on a Wnd-dependent MAPK signaling pathway to regulate NMJ development (Shen, 2009).

If autophagy and Hiw act via a common pathway, where do they converge? As a positive regulator of NMJ growth, autophagy could promote degradation of a negative regulator. An intriguing possibility is that Hiw is the negative regulator affected by autophagy. If a decrease in Hiw levels is responsible for NMJ overgrowth when autophagy is elevated, restoration of Hiw should suppress overgrowth. This possibility was tested by coexpressing wild-type Hiw with Atg1; Atg1-mediated NMJ overgrowth was found to be significantly suppressed. This suppression is not simply an indirect consequence of the dilution of GAL4 caused by addition of a second UAS element because coexpression of UAS-nwk⁺ did not suppress such NMJ overgrowth. This result also shows that Nwk (Nervous wreck), another negative regulator of NMJ growth, is not an apparent target of autophagy, as predicted by differences in phenotypes. Thus, autophagy appears to regulate NMJ growth through its effects on particular presynaptic proteins, and Hiw represents a key downstream effector (Shen, 2009).

To further test whether autophagy promotes NMJ growth by limiting Hiw, one copy of hiw⁺ was eliminated to determine whether this further decrease in Hiw levels enhanced the effects of atg1⁺ overexpression. In an otherwise wild-type background, loss of one copy of hiw⁺ had no affect, but it significantly enhanced atg1⁺-induced NMJ overgrowth. The phenotype of hiw homozygotes overexpressing atg1⁺ was no more extreme than hiw homozygote alone. The absence of any additive or synergistic effects further supports the hypothesis that autophagy promotes NMJ development by down-regulating Hiw (Shen, 2009).

Because Hiw antibodies do not work for immunohistochemistry, Hiw was visualized using a fully functional GFP-tagged construct to test directly whether abundance of Hiw is affected by autophagy. In an otherwise wild-type background, Hiw-GFP was strongly expressed in neurons throughout the ventral ganglion and brain lobes driven by C155-Gal4, as detected by anti-GFP staining. However, in larvae co-overexpressing atg1⁺, the GFP signal was reduced by ~60% relative to anti-HRP staining. This result was confirmed by Western blot analysis. Reduction of Hiw-GFP is not caused by the dilution of GAL4 by the presence of a second UAS element because coexpression of UAS-myr-RFP did not affect abundance of Hiw-GFP. These results further indicate that autophagy promotes NMJ growth by down-regulating Hiw (Shen, 2009).

These results indicate that NMJ overgrowth caused by elevated autophagy is primarily caused by reduction in Hiw. Is the converse also true? Is NMJ undergrowth in atg mutants caused by elevated levels of Hiw? To address these questions, Hiw-GFP was expressed in neurons using C155-Gal4 in various backgrounds. Hiw-GFP levels were significantly elevated in atg1 and -6 mutants compared with the controls, consistent with the idea that Hiw is down-regulated by autophagy. If this increase in Hiw is a primary cause of NMJ undergrowth in atg loss-of-function mutants, eliminating Hiw should prevent this undergrowth; i.e., mutations in hiw should be epistatic to atg mutations. Thus, NMJ morphology was examined in hiw; atg2 and hiw; atg18 double mutants, and it was found that hiw was completely epistatic, demonstrating the role of elevated levels of Hiw in NMJ undergrowth of atg mutants (Shen, 2009).

A more direct test is to determine whether overexpression of Hiw can reduce NMJ size. However, this experiment is complicated because overexpression of Hiw by a relatively weak neuronal driver (elav-Gal4) does not affect NMJ size, whereas overexpression of Hiw by a strong neuronal driver (Elav-GeneSwitch) has a modest dominant-negative effect. To determine whether increased levels of Hiw can limit NMJ growth, it appears necessary to overexpress Hiw at an intermediate level. Therefore, NMJs were examined in larvae overexpressing UAS-hiw⁺ via C155-Gal4. C155-Gal4/+; UAS-hiw⁺/+ female larvae exhibited very mild NMJ undergrowth. Stronger undergrowth was observed in C155-Gal4/Y; UAS-hiw⁺/+ male larvae. This difference is consistent with higher levels of C155-Gal4 expression in males than in females, owing to dosage compensation. No differences were observed in NMJ growth between C155-Gal4/+ female and C155-Gal4/Y male larvae, indicating that the undergrowth phenotypes are dependent on the levels of Hiw overexpression and not on differences in gender or expression of GAL4 alone. Thus, moderate increases in Hiw levels result in NMJ undergrowth. Furthermore, the modest NMJ undergrowth in C155-Gal4/+; UAS-hiw⁺/+ larvae was enhanced when one copy of atg1⁺, -2⁺, or -6⁺ was removed. Together, these results indicate that elevated levels of Hiw account for most of the NMJ undergrowth in atg mutants. However, excess Hiw cannot fully explain NMJ undergrowth in atg mutants because NMJ undergrowth caused by Hiw overexpression is less severe than that of atg1 and -18 mutants. Thus, when autophagy is impaired, additional negative regulators may accumulate to depress NMJ growth. It is also likely that elevated levels of Hiw target proteins other than Wnd to limit synaptic growth because loss-of-function mutations of wnd do not affect NMJ development (Shen, 2009).

Because autophagy is generally thought of as a nonselective bulk degradation process, the idea that autophagy regulates NMJ growth primarily through its effects on Hiw levels seems difficult to understand at first. However, recent studies demonstrate that autophagy can also operate in a substrate-selective mode in regulating specific developmental events (Rowland, 2006; Zhang, 2009). For example, in Caenorhabditis elegans, when postsynaptic cells fail to receive presynaptic contact, GABA_A receptors selectively traffic to autophagosomes (Rowland, 2006). However, the detailed mechanism of such selectivity is unknown. Zhang identified SEPA-1 as a bridge that mediates the specific recognition and degradation of P granules by autophagy in C. elegans. Thus, one possibility is that Hiw is specifically targeted to autophagosomes via a mechanism that remains to be elucidated. It is also possible that many presynaptic proteins besides Hiw are degraded by autophagy, but it is the reduction in Hiw that primarily affects NMJ size. Moreover, although the idea that autophagy regulates Hiw directly is favored, the possibility cannot be ruled out that autophagy promotes degradation of Hiw through an indirect mechanism involving the proteasome or other pathway (Shen, 2009).

In principle, autophagy could be acting on either side of the NMJ to regulate its development. Because atg1⁺ overexpression in muscle results in lethality at the first larval instar, it was not possible to assess whether this affects NMJ growth. Although a postsynaptic role of autophagy in NMJ development cannot be ruled out, several results suggest that the effects of autophagy are primarily presynaptic: neuronal expression of UAS-atg1⁺ is sufficient to completely rescue the NMJ undergrowth in atg1 mutants, the Hiw-Wnd pathway functions presynaptically (Wu, 2005; Collins, 2006), and hiw is completely epistatic to autophagy for NMJ growth (Shen, 2009).

Autophagy is of particular interest as a regulator of synaptic growth because it is triggered in response to many environmental cues. These results demonstrate that decreasing or increasing autophagy from basal levels results in corresponding effects on synaptic size. Thus, autophagy is perfectly positioned to link environmental conditions with synaptic growth and plasticity. As such, it is intriguing to speculate on a role for autophagy in learning and memory (Shen, 2009).

A dual leucine kinase-dependent axon self-destruction program promotes Wallerian degeneration

Axon degeneration underlies many common neurological disorders, but the signaling pathways that orchestrate axon degeneration are unknown. This study found that dual leucine kinase (DLK) promotes degeneration of severed axons in Drosophila and mice, and that its target, c-Jun N-terminal kinase, promotes degeneration locally in axons as they committed to degenerate. This pathway also promotes degeneration after chemotherapy exposure and may be a component of a general axon self-destruction program (Miller, 2009).

Candidate components of axon breakdown pathways should be present in axons and activated by diverse cellular insults. One such candidate is DLK, a mitogen-activated protein kinase kinase kinase (MAP3K). One of DLK's downstream targets, the mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK), is activated following axonal injury. The hypothesis that DLK promotes axon degeneration using Drosophila olfactory receptor neuron (ORN) axotomy model. Freen fluorescent protein (GFP) was expresssed in ORNs to visualize their axons, which extend from cell bodies in the antennae into the antennal lobes of the brain and across a midline commissure. To severe ORN axons and induce degeneration, the antennae was removedfrom wildtype flies and mutants lacking the Drosophila ortholog of DLK, wallenda (wnd). Most wildtype axons degenerated within twenty-four hours, while wnd mutant axons were significantly preserved. Wnd is therefore required for normal axon degeneration in Drosophila (Miller, 2009).

Wnd could act within neurons to promote breakdown after injury or within surrounding cells to promote axon clearance. To distinguish between these possibilities, Wnd was expressed in the GFP-expressing subpopulation of ORNs in wnd mutant flies. Such Wnd expression was not sufficient to induce degeneration in the absence of injury. However, it was found that the Wnd expressing axons of these otherwise wnd mutant flies were not preserved twenty-four hours after axotomy. Thus, Wnd functions in an internal neuronal pathway that promotes injury-induced axon degeneration. Wnd may selectively promote injury induced axon degeneration, since no defects in the developmental pruning of mushroom body gamma-lobe axons (Miller, 2009).

To determine if DLK promotes Wallerian degeneration in mammals, dorsal root ganglion (DRG) cultures were used from littermate wildtype and DLK-deficient embryos. DRG axons were severed to induce degeneration and degeneration of the distal axon segment was evaluated. Twenty-four hours after severing, wildtype axons distal to the transection deteriorated into axon fragments, while DLK-deficient axons remained continuous. To quantify the extent of axon fragmentation, the fraction of total axonal area occupied by axon fragments (degeneration index, DI) was measured, and it was found that DLK-deficient axons were significantly preserved. This delay in fragmentation persisted for forty-eight hours. Since non-neuronal cells are eliminated in this DRG culture system, DLK must operate within mammalian neurons to promote axon breakdown. Neuronal DLK therefore promotes axon fragmentation after injury in flies and mice (Miller, 2009).

To determine if DLK promotes degeneration in response to multiple insults, the response was determined of DLK-deficient DRG axons to vincristine, a chemotherapeutic drug that induces axon degeneration in vitro, and whose dose-limiting side effects in patients include neuropathy. It was found that DLK-deficient axons were significantly protected from vincristine-induced fragmentation, suggesting that DLK operates in a general axon breakdown program (Miller, 2009).

To determine if disrupting DLK protects injured axons in vivo, the sciatic nerves of littermate wildtype and DLK-deficient adult mice were transected. Fifty-two hours post-transection, wildtype axons degenerated, whereas DLK-deficient axons were significantly preserved. Electron microscopy revealed that preserved axon profiles contain mitochondria and a cytoskeleton. Thus, normal Wallerian degeneration in vivo in adult mice requires DLK (Miller, 2009).

DLK is a MAP3K that can activate JNK and p38 via intermediary MAP2Ks. To determine whether either downstream kinase promotes axon degeneration, JNK and p38 were inhibited in the DRG axotomy model using wildtype cultures. Inhibition of JNK, but not p38, protected transected axons from fragmentation, and a significant delay in fragmentation persisted for over forty-eight hours. Thus JNK, like DLK, acts within neurons to promote axon degeneration (Miller, 2009).

Axon degeneration is hypothesized to comprise at least three distinct phases; (1) competence to degenerate, much of which is determined transcriptionally before axotomy, (2) commitment to degenerate, which occurs in the substantial delay period between injury and axon fragmentation, and (3) the execution phase, when axons fragment. If JNK';s primary role were to promote competence to degenerate, then JNK activity should be required prior to axotomy. This is not the case: applying the JNK inhibitor twenty-four hours prior to axotomy and then removing it just before axotomy was not protective. In contrast, JNK inhibition started concurrently with axotomy was protective. Thus, JNK promotes axon fragmentation after the competence period and acts within the severed distal axon segment (Miller, 2009).

JNK could commit axons to degenerate during the delay between injury and breakdown, or it could operate during the subsequent execution phase of axon breakdown. To test whether JNK activity is required during the execution phase, the JNK inhibitor was added three hours after axotomy, which is approximately nine hours before the onset of fragmentation. This treatment schedule spans the transition from the proposed commitment phase to the execution phase and the entire execution phase itself. Continuous JNK inhibition beginning three hours post-axotomy did not delay axon fragmentation. Therefore, JNK activity is not required during the execution phase of axon fragmentation. Rather, JNK activity is required during the early response to injury that commits the axon to breakdown hours later (Miller, 2009).

Converging lines of evidence suggest that there is a general internal axon self-destruction program, but its molecular components are unknown. This study shows that the MAP3K DLK and its downstream MAPK JNK are important elements of such a program. Disrupting this pathway delays axon fragmentation in response to both axotomy and the neurotoxic chemotherapeutic agent vincristine. Thus, a common self-destruction program may promote axon breakdown in response to diverse insults, and so may be targetable in multiple clinical settings (Miller, 2009).

The DLK-1 kinase promotes mRNA stability and local translation in C. elegans synapses and axon regeneration

Growth cone guidance and synaptic plasticity involve dynamic local changes in proteins at axons and dendrites. The Dual-Leucine zipper Kinase MAPKKK (DLK) has been implicated in synaptogenesis and axon outgrowth in C. elegans and other animals. This study shows that in C. elegans DLK-1 regulates not only proper synapse formation and axon morphology but also axon regeneration by influencing mRNA stability. DLK-1 kinase signals via a MAPKAP kinase, MAK-2, to stabilize the mRNA encoding CEBP-1, a bZip protein related to CCAAT/enhancer-binding proteins, via its 3'UTR. Inappropriate upregulation of cebp-1 in adult neurons disrupts synapses and axon morphology. CEBP-1 and the DLK-1 pathway are essential for axon regeneration after laser axotomy in adult neurons, and axotomy induces translation of CEBP-1 in axons. These findings identify the DLK-1 pathway as a regulator of mRNA stability in synapse formation and maintenance and also in adult axon regeneration (Yan, 2009).

Neurons respond to environmental stimuli and insults in a compartmentalized manner. Local protein synthesis in dendrites and axonal growth cones has emerged as a major mechanism allowing compartmentalized responses in growth cone guidance and neuronal plasticity. Several mRNAs are known to be transported and localized to growth cones or axons; transport of such mRNAs often is mediated by their 3' untranslated regions (3'UTRs) (Yan, 2009).

However, it remains controversial whether mature neurons employ axonal regulation of mRNA and local protein synthesis. mRNA-binding proteins, such as Zip-code binding proteins (ZBPs) and cytoplasmic polyadenylation element-binding proteins (CPEBs), are abundant in growing neurites but are mostly undetectable in axons and synapses of mature neurons. Polyribosomes are rarely seen in axons. Nonetheless, several reports suggest that axonal mRNA regulation must occur in mature neurons. In Aplysia neurons, mRNA for the peptide neurotransmitter Sensorin is concentrated at synapses upon contact with target motor neurons, and local translation and secretion of Sensorin promote synapse maturation and plasticity. A number of mRNAs are upregulated in axons of injured adult dorsal root ganglion (DRG) neurons. Regulation of Ran GTPase via local translation of RanBP1 is implicated in retrograde signaling of axon injury. Local translation thus can transmit injury signals and initiate local repair processes (Yan, 2009 and references therein).

CCAAT/enhancer-binding proteins (C/EBP) are widely expressed basic-leucine-zipper (bZip) domain transcription factors with long-studied roles in cell proliferation, differentiation, and stress. In neurons the transcriptional roles of C/EBP proteins have been linked to learning and memory. Learning and memory tasks trigger activation of Erk or p38 kinases, leading to phosphorylation of specific C/EBP isoforms. mRNAs of murine and leech C/EBP are also upregulated following axonal injury, and murine C/EBPβ can activate the transcription of an α-tubulin gene associated with injury responses. The pathways that induce C/EBP after injury are largely unknown (Yan, 2009 and references therein).

The function of the conserved ubiquitin E3 ligase RPM-1 in synaptogenesis and axon formation has been studied in C. elegans. C. elegans neurons have simple unipolar or bipolar axon trajectories and form synapses en passant. For example, the ALM and PLM mechanosensory neurons have a long axon that bifurcates into a branch exclusively forming synapses and another branch transducing mechanoreception. RPM-1 regulates the organization and stabilization of presynaptic terminals and axon termination in both mechanosensory and motor neurons. A major target of RPM-1 ubiquitination is the Dual-Leucine zipper Kinase DLK-1 MAPKKK, which acts in a MAPK cascade consisting of the MAPKK MKK-4 and the p38 kinase PMK-3 (Nakata, 2005). By controlling the level of DLK-1, RPM-1 keeps the activity of the DLK-1 cascade at optimal levels. This negative regulation of the DLK pathway by ubiquitin-mediated protein degradation is conserved in Drosophila and mammalian neurons (Collins, 2006; Jin, 2008; Lewcock, 2007; Yan, 2009 and references therein).

The targets of the DLK-1/p38 cascade have not previously been identified. This study reports the identification of MAK-2, a member of the MAPKAP kinase family, and CEBP-1, a member of the C/EBP class of bZip factors, as effectors of the C. elegans DLK-1 cascade. MAPKAPKs are conserved Ser/Thr kinases that are direct targets of p38 and Erk kinases. cebp-1 mRNA is destabilized by RPM-1 via the DLK-1/MAK-2 cascade, acting on the cebp-1 3.UTR. The DLK-1/MAK-2/CEBP-1 pathway is essential for regenerative regrowth of mature axons following laser axotomy, in part by regulating axonal cebp-1 mRNA stability and translation (Yan, 2009).

Search PubMed for articles about Drosophila Wallenda

Bao, H., Reist, N. E. and Zhang, B. (2008). The Drosophila epsin 1 is required for ubiquitin-dependent synaptic growth and function but not for synaptic vesicle recycling. Traffic 9(12): 2190-205. PubMed Citation: 18796008

Berry, D. L. and Baehrecke, E. H. (2007). Growth arrest and autophagy are required for salivary gland cell degradation in Drosophila. Cell 131: 1137-1148. PubMed Citation: 18083103

Collins, C. A., Wairkar, Y. P., Johnson, S. L. and DiAntonio, A. (2006). Highwire restrains synaptic growth by attenuating a MAP kinase signal. Neuron 51(1): 57-69. PubMed Citation: 16815332

Hirai, S., et al. (2002). MAPK-upstream protein kinase (MUK) regulates the radial migration of immature neurons in telencephalon of mouse embryo. Development 129(19): 4483-95. PubMed Citation: 12223406

Hirai, S., et al. (2005). Expression of MUK/DLK/ZPK, an activator of the JNK pathway, in the nervous systems of the developing mouse embryo. Gene Expr. Patterns 5(4): 517-23. PubMed Citation: 15749080

Horiuchi, D., Collins, C. A., Bhat, P., Barkus, R. V., Diantonio, A. and Saxton, W. M. (2007). Control of a kinesin-cargo linkage mechanism by JNK pathway kinases. Curr. Biol. 17(15): 1313-7. PubMed Citation: 17658258

Jin, Y. and Garner, C. C. (2008). Molecular mechanisms of presynaptic differentiation. Annu. Rev. Cell Dev. Biol. 24: 237-262. PubMed Citation: 18588488

Knox, S., et al. (2007). Mechanisms of TSC-mediated control of synapse assembly and axon guidance. PLoS One. 2: e375. PubMed Citation: 17440611

Lee, S. B., et al. (2007). ATG1, an autophagy regulator, inhibits cell growth by negatively regulating S6 kinase. EMBO Rep. 8: 360-365. PubMed Citation: 17347671

Levine, B., and Kroemer, G. (2008). Autophagy in the pathogenesis of disease. Cell 132:27-42. PubMed Citation: 18191218

Lewcock, J. W., Genoud, N., Lettieri, K. and Pfaff, S. L. (2007). The ubiquitin ligase Phr1 regulates axon outgrowth through modulation of microtubule dynamics. Neuron 56: 604-620. PubMed Citation: 18031680

Liao, E. H, Hung, W., Abrams, B. and Zhen, M. (2004) An SCF-like ubiquitin ligase complex that controls presynaptic differentiation. Nature 430: 345-350. 15208641

Miller, B. R., et al. (2009). A dual leucine kinase-dependent axon self-destruction program promotes Wallerian degeneration. Nat. Neurosci. 12(4): 387-9. PubMed Citation: 19287387

Nakata, K., et al. (2005). Regulation of a DLK-1 and p38 MAP kinase pathway by the ubiquitin ligase RPM-1 is required for presynaptic development. Cell 120: 407-420. PubMed Citation: 15707898

Rowland, A. M., et al. (2006). Presynaptic terminals independently regulate synaptic clustering and autophagy of GABAA receptors in Caenorhabditis elegans. J. Neurosci. 26: 1711-1720. PubMed Citation: 16467519

Rubinsztein, D. C., et al. (2007). Potential therapeutic applications of autophagy. Nat. Rev. Drug Discov. 6: 304-312. PubMed Citation: 17396135

Scott, R.C., Schuldiner, O. and Neufeld, T. P. (2004). Role and regulation of starvation-induced autophagy in the Drosophila fat body. Dev. Cell. 7: 167-178. PubMed Citation: 15296714

Scott, R. C., Juhász, G. and Neufeld, T. P. (2007). Direct induction of autophagy by Atg1 inhibits cell growth and induces apoptotic cell death. Curr. Biol. 17: 1-11. PubMed Citation: 17208179

Shen, W. and Ganetzky, B. (2009). Autophagy promotes synapse development in Drosophila. J Cell Biol. 187(1): 71-9. PubMed Citation: 19786572

Toda, H., et al. (2008). UNC-51/ATG1 kinase regulates axonal transport by mediating motor-cargo assembly. Genes Dev. 22: 3292-3307. PubMed Citation: 19056884

Wairkar, Y. P., et al. (2009). Unc-51 controls active zone density and protein composition by downregulating ERK signaling. J. Neurosci. 29: 517-528. PubMed Citation: 19144852

Wu, C., Wairkar, Y. P., Collins, C. A. and DiAntonio, A. (2005). Highwire function at the Drosophila neuromuscular junction: spatial, structural, and temporal requirements. J. Neurosci. 25(42): 9557-66. 16237161

Wu, C., Daniels, R. W. and Diantonio, A. (2007). DFsn collaborates with Highwire to down-regulate the Wallenda/DLK kinase and restrain synaptic terminal growth. Neural Develop. 2: 16. PubMed Citation: 17697379

Yan, D., Wu, Z., Chisholm, A. D. and Jin, Y. (2009). The DLK-1 kinase promotes mRNA stability and local translation in C. elegans synapses and axon regeneration. Cell 138(5): 1005-18. PubMed Citation: 19737525

Zhang, Y., et al. (2009). SEPA-1 mediates the specific recognition and degradation of P granule components by autophagy in C. elegans. Cell 136: 308-321. PubMed Citation: 19167332

date revised: 30 October 2009

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