InteractiveFly: GeneBrief

wallenda: Biological Overview | References


Gene name - wallenda

Synonyms -

Cytological map position - 76B9-76B9

Function - signaling

Keywords - MAPKKK in the JNK pathway, axonal transport, autophagy, synaptic terminal growth, promotion of Wallerian degeneration

Symbol - wnd

FlyBase ID: FBgn0036896

Genetic map position - 3L: 19,617,505..19,628,696 [-]

Classification - protein kinase

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | EntrezGene

Recent literature
Ma, X., Chen, Y., Zhang, S., Xu, W., Shao, Y., Yang, Y., Li, W., Li, M. and Xue, L. (2015). Rho1-Wnd signaling regulates loss-of-cell polarity-induced cell invasion in Drosophila. Oncogene [Epub ahead of print]. PubMed ID: 25961917
Summary:
Both cell polarity and c-Jun N-terminal kinase (JNK) activity are essential to the maintenance of tissue homeostasis, and disruption of either is commonly seen in cancer progression. Despite the established connection between loss-of-cell polarity and JNK activation, much less is known about the molecular mechanism by which aberrant cell polarity induces JNK-mediated cell migration and tumor invasion. This study presents results from a genetic screen using an in vivo invasion model via knocking down cell polarity gene in Drosophila wing discs, and identifies Rho1-Wnd signaling as an important molecular link that mediates loss-of-cell polarity-triggered JNK activation and cell invasion. Wallenda (Wnd), a protein kinase of the mitogen-activated protein kinase kinase kinase family, by forming a complex with the GTPase Rho1, is both necessary and sufficient for Rho1-induced JNK-dependent cell invasion, MMP1 activation and epithelial-mesenchymal transition. Furthermore, Wnd promotes cell proliferation and tissue growth through Wingless production when apoptosis is inhibited by p35. Finally, Wnd shows oncogenic cooperation with RasV12 to trigger tumor growth in eye discs and causes invasion into the ventral nerve cord. Together, these data not only provides a novel mechanistic insight on how cell polarity loss contributes to cell invasion, but also highlights the value of the Drosophila model system to explore human cancer biology.

Ma, X., Xu, W., Zhang, D., Yang, Y., Li, W. and Xue, L. (2015). Wallenda regulates JNK-mediated cell death in Drosophila. Cell Death Dis 6: e1737. PubMed ID: 25950467
Summary:
The c-Jun N-terminal kinase (JNK) pathway plays essential roles in regulating a variety of cellular processes including proliferation, migration and survival. Previous genetic studies in Drosophila have identified numerous cell death regulating genes, providing new insights into the mechanisms for related diseases. Despite the known role of the small GTPase Rac1 in regulating cell death, the downstream components and underlying mechanism remain largely elusive. This study shows that Rac1 promotes JNK-dependent cell death through Wallenda (Wnd). In addition, Wnd triggers JNK activation and cell death via its kinase domain. Moreover, both MKK4 and Hep are critical for Wnd-induced cell death. Furthermore, Wnd is essential for ectopic Egr- or Rho1-induced JNK activation and cell death. Finally, Wnd is physiologically required for loss of Scribble-induced JNK-dependent cell death. Thus, these data suggest that wnd encodes a novel essential cell death regulator in Drosophila.

Hao, Y., Frey, E., Yoon, C., Wong, H., Nestorovski, D., Holzman, L. B., Giger, R. J., DiAntonio, A. and Collins, C. (2016). An evolutionarily conserved mechanism for cAMP elicited axonal regeneration involves direct activation of the dual leucine zipper kinase DLK. Elife 5 [Epub ahead of print]. PubMed ID: 27268300
Summary:
A broadly known method to stimulate the growth potential of axons is to elevate intracellular levels of cAMP, however the cellular pathway(s) that mediate this are not known. This study identifies the Dual Leucine-zipper Kinase (DLK, Wallenda in Drosophila) as a critical target and effector of cAMP in injured axons. DLK/Wnd is thought to function as an injury 'sensor', as it becomes activated after axonal damage. These findings in both Drosophila and mammalian neurons indicate that the cAMP effector kinase PKA is a conserved and direct upstream activator of Wnd/DLK. PKA is required for the induction of Wnd signaling in injured axons, and DLK is essential for the regenerative effects of cAMP in mammalian DRG neurons. These findings link two important mediators of responses to axonal injury, DLK/Wnd and cAMP/PKA, into a unified and evolutionarily conserved molecular pathway for stimulating the regenerative potential of injured axons.
Chen, L., Nye, D.M., Stone, M.C., Weiner, A.T., Gheres, K.W., Xiong, X., Collins, C.A. and Rolls, M.M. (2016). Mitochondria and caspases tune Nmnat-mediated stabilization to promote axon regeneration. PLoS Genet 12: e1006503. PubMed ID: 27923046
Summary:
Axon injury can lead to several cell survival responses including increased stability and axon regeneration. Using an accessible Drosophila model system, this study investigated the regulation of injury responses and their relationship. Axon injury stabilizes the rest of the cell, including the entire dendrite arbor. After axon injury, it was found that mitochondrial fission in dendrites is upregulated, and reducing fission increases stabilization or neuroprotection (NP). Thus axon injury seems to both turn on NP, but also dampen it by activating mitochondrial fission. Caspases were identified to be negative regulators of axon injury-mediated NP, so mitochondrial fission could control NP through caspase activation. In addition to negative regulators of NP, it was found that nicotinamide mononucleotide adenylyltransferase (Nmnat) is absolutely required for this type of NP. Increased microtubule dynamics, which has previously been associated with NP, requires Nmnat. Indeed Nmnat overexpression is sufficient to induce NP and increase microtubule dynamics in the absence of axon injury. DLK, JNK and fos are also required for NP. Because NP occurs before axon regeneration, and NP seems to be actively downregulated, it was tested whether excessive NP might inhibit regeneration. Indeed both Nmnat overexpression and caspase reduction reduce regeneration. In addition, overexpression of fos or JNK extend the timecourse of NP and dampen regeneration in a Nmnat-dependent manner. These data suggest that NP and regeneration are conflicting responses to axon injury, and that therapeutic strategies that boost NP may reduce regeneration.

Li, J., Zhang, Y. V., Asghari Adib, E., Stanchev, D. T., Xiong, X., Klinedinst, S., Soppina, P., Jahn, T. R., Hume, R. I., Rasse, T. M. and Collins, C. A. (2017). Restraint of presynaptic protein levels by Wnd/DLK signaling mediates synaptic defects associated with the kinesin-3 motor Unc-104. Elife 6. PubMed ID: 28925357
Summary:
The kinesin-3 family member Unc-104/KIF1A is required for axonal transport of many presynaptic components to synapses, and mutation of this gene results in synaptic dysfunction in mice, flies and worms. Studies at the Drosophila neuromuscular junction indicate that many synaptic defects in unc-104-null mutants are mediated independently of Unc-104's transport function, via the Wallenda (Wnd)/DLK MAP kinase axonal damage signaling pathway. Wnd signaling becomes activated when Unc-104's function is disrupted, and leads to impairment of synaptic structure and function by restraining the expression level of active zone (AZ) and synaptic vesicle (SV) components. This action concomitantly suppresses the buildup of synaptic proteins in neuronal cell bodies, hence may play an adaptive role to stresses that impair axonal transport. Wnd signaling also becomes activated when pre-synaptic proteins are over-expressed, suggesting the existence of a feedback circuit to match synaptic protein levels to the transport capacity of the axon.

BIOLOGICAL OVERVIEW

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}fafEP381, 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}fafEP381. 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}fafEP381 in suppressing UAS-Aplip1-induced axonal accumulation of synaptic proteins. Equivalent expression of a 'kinase-dead' mutant transgene (UAS-wndKD) 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 FosDN and JunDN 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 FosDN 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, JunDN 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 FosDN acts downstream of Wallenda to inhibit synaptic overgrowth, it should also suppress the synaptic overgrowth caused by overexpressing wallenda. Indeed, when FosDN was coexpressed with UAS-wnd in neurons, FosDN 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).

Protein turnover of the Wallenda/DLK kinase regulates a retrograde response to axonal injury

Regenerative responses to axonal injury involve changes in gene expression; however, little is known about how such changes can be induced from a distant site of injury. This study describes a nerve crush assay in Drosophila to study injury signaling and regeneration mechanisms. Wallenda (Wnd), a conserved mitogen-activated protein kinase (MAPK) kinase kinase homologous to dual leucine zipper kinase, was found to function as an upstream mediator of a cell-autonomous injury signaling cascade that involves the c-Jun NH(2)-terminal kinase MAPK and Fos transcription factor. Wnd is physically transported in axons, and axonal transport is required for the injury signaling mechanism. Wnd is regulated by a conserved E3 ubiquitin ligase, named Highwire (Hiw) in Drosophila. Injury induces a rapid increase in Wnd protein concomitantly with a decrease in Hiw protein. In hiw mutants, injury signaling is constitutively active, and neurons initiate a faster regenerative response. These data suggest that the regulation of Wnd protein turnover by Hiw can function as a damage surveillance mechanism for responding to axonal injury (Xiong, 2010).

A conditioning lesion protects axons from degeneration via the Wallenda/DLK MAP kinase signaling cascade

Axons are vulnerable components of neuronal circuitry, and neurons are equipped with mechanisms for responding to axonal injury. A highly studied example of this is the conditioning lesion, in which neurons that have been previously injured have an increased ability to initiate new axonal growth. This study investigated the effect of a conditioning lesion on axonal degeneration, which occurs in the distal stump after injury, and also occurs in neuropathies and neurodegenerative disorders. It was found that Drosophila motoneuron axons that had been previously injured had an increased resiliency to degeneration. This requires the function of a conserved axonal kinase, Wallenda (Wnd)/DLK, and a downstream transcription factor. Because axonal injury leads to acute activation of Wnd, and overexpression studies indicate that increased Wnd function is sufficient to promote protection from degeneration, it is proposed that Wnd regulates an adaptive response to injury that allows neurons to cope with axonal stress (Xiong, 2012a).

The findings suggest that the Wnd kinase plays a protective role in Drosophila motoneuron axons. Wnd protein is normally kept at a low level in these axons, but becomes rapidly induced by axonal injury. This leads to activation of a nuclear signaling cascade that promotes an increased resiliency of axons to degeneration after a second injury. The requirement for Wnd in the protective effect of a conditioning lesion leads to a proposal that Wnd regulates a stress response pathway that allows neurons to adapt to axonal injury (Xiong, 2012a).

A pathway that inhibits axonal degeneration would be very advantageous in instances where the integrity of the axon is compromised but not completely lost, such as anoxia, loss of myelination, or defects in axonal transport. Such problems are a concern not only for neuronal injuries, but also neurodegenerative disorders, whose onset may be influenced by neuronal stress. Along these lines, mutations that lead to activation of Wnd/DLK signaling can suppress a synaptic retraction phenotype in Drosophila (Xiong, 2012a).

The conditioning lesion is classically studied for its role in facilitating axonal regeneration, although it has been noted that a conditioning lesion can also attenuate degeneration of motoneurons in a rat model of ALS. In vertebrates, a conditioning injury of axons in the PNS induces widespread cellular changes, including chromatolysis and changes in gene expression, translation, trafficking, cytoskeleton, and physiology. Studies in multiple model organisms have already implicated Wnd/DLK in nuclear signaling, translation, axonal transport, and cytoskeletal dynamics; hence, this kinase may function as an upstream regulator of multiple downstream responses to axonal injury. The exact cellular events that lead to protection from degeneration and their relationship to the regenerative response remain to be determined (Xiong, 2012a).

Importantly, the action of Wnd/DLK is not always beneficial. In contrast to motoneuron axons, Wnd is unable to protect olfactory neuron axons in the adult. Instead, Wnd plays a modest role in promoting degeneration in these neurons. Also in contrast, the vertebrate homolog DLK plays a pro-degenerative role in DRG neurons, both after injury and after nerve growth factor withdrawal ). The distinct downstream outcomes may depend on distinct features of downstream signaling events in different cell types, developmental contexts, and cellular location. For instance, the protective role of Wnd requires time, the neuronal cell body, and downstream gene expression, while the distal stump of injured axon would be unable to receive the benefits of a nuclear signaling cascade induced by injury. However, even the nuclear signaling cascade in the proximal stump is not always beneficial: in cultured DRG neurons, DLK promotes apoptosis after nerve growth factor withdrawal. This negative outcome of DLK action shares mechanistic similarities with the beneficial outcome of Wnd for regeneration in that both depend on retrograde signaling to the nucleus after an axonal stimulus (Xiong, 2012a).

Similar to previous studies, the findings of this study suggest that JNK plays a pro-degenerative role in Drosophila motoneuron axons; however, this action may occur independently of Wnd regulation. Indeed, a Wnd-independent pool of phosphorylated JNK has been described in Drosophila axons. While JNK plays an important role in the regenerative response to injury, it is not clear whether JNK functions in the injury-induced protection from degeneration. An equally likely candidate mediator, based on studies in C. elegans, is the other stress-activated MAPK, p38. Future characterization of the mechanism of Wnd/DLK signaling, including the cofactors and effectors for its different functions in different contexts, will be important for delineating the molecular differences between positive and negative responses to axonal damage (Xiong, 2012a).

The Highwire ubiquitin ligase promotes axonal degeneration by tuning levels of Nmnat protein

Axonal degeneration is a hallmark of many neuropathies, neurodegenerative diseases, and injuries. Using a Drosophila injury model this study has identified a highly conserved E3 ubiquitin ligase, Highwire (Hiw), as an important regulator of axonal and synaptic degeneration. Mutations in hiw strongly inhibit Wallerian degeneration in multiple neuron types and developmental stages. This new phenotype is mediated by a new downstream target of Hiw, the NAD+ biosynthetic enzyme nicotinamide mononucleotide adenyltransferase (Nmnat), which acts in parallel to a previously known target of Hiw, the Wallenda dileucine zipper kinase (Wnd/DLK) MAPKKK. Hiw promotes a rapid disappearance of Nmnat protein in the distal stump after injury. An increased level of Nmnat protein in hiw mutants is both required and sufficient to inhibit degeneration. Ectopically expressed mouse Nmnat2 is also subject to regulation by Hiw in distal axons and synapses. These findings implicate an important role for endogenous Nmnat and its regulation, via a conserved mechanism, in the initiation of axonal degeneration. Through independent regulation of Wnd/DLK, whose function is required for proximal axons to regenerate, Hiw plays a central role in coordinating both regenerative and degenerative responses to axonal injury (Xiong, 2012b).

Since the discovery of the dramatic inhibition of degeneration by the WldS mutation, many studies have focused upon the action of the NAD+ biosynthetic enzyme isoforms, Nmnat1, Nmnat2, and Nmnat3, which in some circumstances can confer protection against axonal degeneration (reviewed in Coleman, 2010). Most of these studies involve gain-of-function overexpression experiments; it has been difficult to address the role of endogenous Nmnat enzymes in this process. Recent observations indicate that endogenous Nmnat activity plays an essential role in neuronal survival, and its depletion leads to neurodegeneration. In addition, recent studies in vertebrate neurons suggest that the cytoplasmic isoform, Nmnat2, has a short half-life in neurons. An attractive model proposes that Nmnat2 is rapidly turned over in axons, and that its loss in the distal stump of an axon, which has become disconnected from its cell body, leads to the initiation of Wallerian degeneration (Xiong, 2012b).

Some aspects of this model are supported by current in vivo characterization in Drosophila. This study identfies Hiw, a highly conserved protein with features of an E3 ubiquitin ligase, as an important regulator of Wallerian degeneration. Hiw's role in this process involves the Nmnat protein, whose levels in axons and synapses are regulated post-transcriptionally by Hiw function. In hiw mutants, Wallerian degeneration is strongly inhibited, and the increased level of Nmnat protein in hiw mutants is both required and sufficient to inhibit degeneration (Xiong, 2012b).

While the localization of endogenous Hiw in Drosophila is not known, homologues in mice and Caenorhabditis elegans have been detected in axons and at synapses, so it is in the appropriate location to target the destruction of Nmnat in distal axons. However, it remains to be determined whether the down-regulation of Nmnat in the distal stump per se is the trigger for Wallerian degeneration. When HA-Nmnat was overexpressed, axons were protected from degeneration long after the rapid disappearance of detectable protein in the distal stump. It is possible that even very low levels of Nmnat protein are sufficient to protect from degeneration. It is also formally possible that the basal levels of Nmnat before injury, rather than the disappearance of Nmnat after injury, is an important determinant of degeneration. It is also acknowledged that axonal degeneration likely involves additional steps downstream or in parallel to the regulation of Nmnat by Hiw. While overexpression of Hiw can induce a reduction in HA-Nmnat levels, it was not possible to observe an enhanced rate of degeneration when Hiw was overexpressed (Xiong, 2012b).

Studies almost a decade ago suggested a role for the ubiquitin protease system (UPS) in the initiation of Wallerian degeneration (Zhai, 2003). It is tempting to propose that this role is manifested by the regulation of Nmnat by Hiw. However the current observations caution against a simple interpretation that Hiw regulates Nmnat via the UPS, since Hiw can promote disappearance of Nmnat protein in cells in a manner unaffected by proteasome inhibitors. Moreover, in vivo, inhibition of the proteasome had only a minor effect upon Nmnat levels in a wild-type background. However in hiw mutants, Nmnat levels were very sensitive to the function of the proteasome. It is interpreted that additional ubiquitin ligases and the UPS may regulate Nmnat independently of Hiw (Xiong, 2012b).

Regardless of the role of the proteasome, the current observations suggest that ubiquitin plays an important role in Nmnat regulation. Overexpression of the yeast de-ubiquitinating protease UBP2 leads to increased levels of Nmnat protein and inhibition of Wallerian degeneration, in a manner that requires endogenous Nmnat. Future studies of the mechanism by which Hiw regulates Nmnat will therefore consider potential proteasome-independent roles of ubiquitination. Of note, in yeast UBP2 has been shown to preferentially disassemble polyubiquitin chains linked at Lys63, which have been found to perform non-proteolytic functions in DNA repair pathways, kinase activation, and receptor endocytosis. The possibility should also be considered that Hiw regulates Nmnat indirectly: since thus far it has not been possible to detect any ubiquitinated Nmnat species, it is possible that an intermediate, yet unknown, regulator of Nmnat may be the actual substrate of ubiquitination. Nevertheless, co-immunoprecipitation studies from S2R+ cells indicate that Hiw and Nmnat have the capacity to interact (Xiong, 2012b).

The mechanism and cellular location of Nmnat's protective action is a highly debated subject. Observations in the literature point to both NAD+-dependent and NAD+-independent models for the strong protection by the WldS mutation (Coleman, 2010). The location of its protective action may be the mitochondria, since mitochondrially localized Nmnat can protect axons from degeneration. However golgi/endosomal localized Nmnat2 can also be protective. The findings suggest that mutation of hiw leads to an increase in the pool of endogenous Nmnat that functionally impacts degeneration (Xiong, 2012b).

While the site of endogenous Nmnat function during axonal degeneration remains to be identified, this study found that the levels of ectopically expressed mouse Nmnat2 were specifically increased in the hiw mutant background. In contrast, the levels of nuclearly localized mNmnat1 or mitochondrially localized mNmnat3 were unaffected by Hiw. Since Nmnat2 has a short half-life in vertebrate neurons, it is intriguing to propose that it is regulated by Hiw orthologs via an analogous mechanism (Xiong, 2012b).

Since Nmnat2 does not appear to localize to mitochondria, does this favor a non-mitochondrial activity, such as function as a chaperone, for the protective action? It remains challenging to determine the exact location of protection, since the most apparent changes in Nmnat protein may not necessarily be the functionally relevant changes (Xiong, 2012b).

A previously characterized target of Hiw regulation is the Wallenda (Wnd) MAP kinase kinase kinase. This axonal kinase is also capable of inhibiting Wallerian degeneration in motoneurons. The protective action of Wnd requires a downstream signaling cascade and changes in gene expression mediated by the Fos transcription factor. Loss of nmnat does not affect this signaling cascade nor does it affect the protective action of Wnd. Conversely, loss of wnd does not affect the protection caused by overexpressing nmnat. Importantly, the regulation of Nmnat by Hiw does not appear to require Wnd function, and Wnd and Nmnat can protect axons independently of each other. These findings favor the model that Wnd and Nmnat are both regulated by Hiw and influence axonal degeneration through independent mechanisms (Xiong, 2012b).

The Wnd kinase plays additional roles in neurons, which can be genetically separated from Nmnat function. These include regulation of synaptic growth: a dramatic synaptic overgrowth phenotype in hiw mutants is fully suppressed by mutation of wnd, but is not at all affected by knockdown of nmnat. Wnd/DLK also promotes axonal sprouting in response to axonal injury, which is also unaffected by nmnat knockdown. It is therefore clear that by regulating both Wnd and Nmnat, Hiw regulates multiple independent pathways in neurons (Xiong, 2012b).

It is intriguing that the actions of both Wnd and Nmnat promote cellular responses to axonal injury. Axonal regeneration requires an initiation of a growth program within the axon, which depends upon the function of Wnd and its homologues. Equally important is a clearance of the distal stump to make room for the regenerating axon. Since both Wnd and Nmnat are transported in axons a model is proposed in which Hiw function in the distal axon terminal could simultaneously promote destruction of Nmnat in the distal stump, and accumulation of Wnd in the proximal stump. The latter is observed after injur, and is required to promote new axonal growth. The actual location in which Hiw regulates Nmnat remains to be determined. As an upstream regulator of both sprouting in the proximal stump and degeneration of the distal stump, Hiw may play a central role in regulating the ability of a neuron to regenerate its connection after injury (Xiong, 2012b).

Importantly, the protective action of Nmnat may not be limited to Wallerian degeneration. The WldS mutation can protect neurons from degeneration in a wide variety of paradigms, from models of neurodegenerative disease, diabetic neuropathy, excitotoxity, and loss of myelination. These findings suggest that action and regulation of Nmnat function is broadly important for neuronal function and maintenance. As a critical regulator of Nmnat, the Hiw ubiquitin ligase and its vertebrate homologues deserve further scrutiny for potential roles in human health and disease (Xiong, 2012b).

Loss of the spectraplakin Short stop activates the DLK injury response pathway in Drosophila

The MAPKKK dual leucine zipper-containing kinase (DLK, Wallenda in Drosophila) is an evolutionarily conserved component of the axonal injury response pathway. After nerve injury, DLK promotes degeneration of distal axons and regeneration of proximal axons. This dual role in coordinating degeneration and regeneration suggests that DLK may be a sensor of axon injury, and so understanding how DLK is activated is important. Two mechanisms are known to activate DLK. First, increasing the levels of DLK via overexpression or loss of the PHR ubiquitin ligases that target DLK activate DLK signaling. Second, in Caenorhabditis elegans, a calcium-dependent mechanism, can activate DLK. This study describe as new mechanism that activates DLK in Drosophila: loss of the spectraplakin short stop (shot). In a genetic screen for mutants with defective neuromuscular junction development, this study identified a hypomorphic allele of shot that displays synaptic terminal overgrowth and a precocious regenerative response to nerve injury. Both phenotypes are the result of overactivation of the DLK signaling pathway. It was further shown that, unlike mutations in the PHR ligase Highwire, loss of function of shot activates DLK without a concomitant increase in the levels of DLK. As a spectraplakin, Shot binds to both actin and microtubules and promotes cytoskeletal stability. The DLK pathway is also activated by downregulation of the TCP1 chaperonin complex, whose normal function is to promote cytoskeletal stability. These findings support the model that DLK is activated by cytoskeletal instability, which is a shared feature of both spectraplakin mutants and injured axons (Valakh, 2013).

Spectraplakins are huge, multidomain proteins that bind to both actin and microtubules to regulate cytoskeletal dynamics. The family of spectraplakins consists of mammalian bpag1/dystonin and ACF7/MAC1, Drosophila short stop (shot)/kakapo, and C. elegans vab-10. Spectraplakins function in many cellular processes, including regulating ER-Golgi transport in mammalian sensory neurons (Ryan, 2012a; Ryan, 2012b) and mediating polarized locomotion of skin stem cells upon injury (Wu, 2011). They also play an essential role in axons, as mutations in mammalian spectraplakins lead to peripheral neuropathy in both mice and humans. In Drosophila, the spectraplakin short stop has been extensively studied for its role in embryonic axon outgrowth and its regulation of microtubule dynamics. All prior loss-of-function mutants in shot are embryonic lethal, and these strong alleles have severe impairments of their cytoskeleton and poor axon outgrowth, leading axons to 'stop short' of their targets. This embryonic phenotype is in apparent contradiction to the larval phenotype described for the shotVV allele, in which the synaptic terminal is overgrown with additional synaptic boutons. However the shotVV allele is a hypomorph and motor axons successfully navigate to their targets, so this mutant must retain sufficient function to allow for axonal outgrowth. Although shotVV behaves as a hypomorph, the molecular lesion was not identified, and so it is plausible that this allele is a neomorph and that its regulation of DLK may not reflect the normal function of the protein. However, RNAi knockdown of shot was found to generate the same phentoypes as shotVV and leads to the same activation of Wallenda/DLK. Hence, both the shotVV allele and shot RNAi provide evidence for a new function for shot, namely, as a negative regulator of Wallenda/DLK signaling. It will be interesting to determine whether mammalian spectraplakins also restrain DLK signaling and whether dysregulated MAP kinase pathways may mediate some of the phenotypes of spectraplakin mutants, such as peripheral neuropathy (Valakh, 2013).

A series of recent studies highlights the central role of DLK in the developing and injured mammalian nervous system. DLK is required for normal developmental cell death in motor and sensory neurons, for Wallerian degeneration of injured peripheral axons, for cell death and axon degeneration of retinal ganglion cells in models of glaucoma, and for the proregenerative preconditioning response in injured DRG axons. Because DLK appears to be central to the neuronal injury response, there is great interest in understanding its mechanism of activation. In particular, it is important to understand how axon injury leads to the activation of DLK (Valakh, 2013).

To date, methods that increase the levels of DLK are the best understood mechanism for increasing DLK activity. In worms, flies, and mice, loss of the PHR ubiquitin ligase leads to an increase in the levels of DLK; and in worms and flies, there are extensive data demonstrating that this activates the kinase. Similarly, in both worms and flies, the overexpression of DLK is sufficient to activate the DLK signaling pathway. In Drosophila, injury leads to a loss of the PHR ubiquitin ligase Highwire, potentially via autophagosomal degradation, which in turn leads to an increase in DLK and may be a method of injury-induced activation. In mammals, a positive feedback loop between DLK and JNK inhibits Phr1-dependent degradation of DLK, increasing the levels of DLK and activating the pathway (Huntwork-Rodriguez, 2013). In addition, a calcium-dependent activation mechanism was recently demonstrated in C. elegans, which is very exciting because calcium influx is an early step after axon injury. However, the key hexapeptide sequence that mediates this calcium-dependent regulation is absent from both Drosophila and mouse DLK, suggesting that additional activation mechanisms for DLK may exist. Indeed, the current data show that loss of the spectraplakin shot can also activate DLK and leads to the hypothesis that cytoskeletal disruptions may activate DLK. These findings demonstrate that loss of function of shot leads to activation of Wallenda/DLK without a concomitant increase in the levels of Wallenda/DLK. Hence, loss of shot is not acting upstream or in concert with Highwire because loss of Highwire or components of the Highwire ubiquitin ligase complex lead to increased levels of Wallenda/DLK. The shot mutant is the first manipulation that activates Wallenda/DLK signaling in Drosophila without altering the levels of Wallenda/DLK. Hence, loss of shot must activate Wallenda/DLK via a novel mechanism. It is hypothesized that this mechanism is related to the biochemical function of Shot, which is to stabilize the cytoskeleton by simultaneously binding both actin and microtubules. Prior studies demonstrate that shot null mutants have a destabilized microtubule network, and ehis study demonstrates that the microtubule network is more dynamic in shot RNAi knockdown larvae. It is proposed that a destabilized cytoskeleton activates Wallenda/DLK. Consistent with this model, mutations in either of two subunits of the TCP-1 complex, which like Shot regulates both the actin and microtubule cytoskeleton, leads to activation of DLK signaling. By demonstrating this activation of DLK signaling, the current results support and extend the work of Bounoutas (2011) who demonstrated that microtubule disruption leads to DLK-dependent changes in protein levels in C. elegans. Moreover, the findings are consistent with studies in mammalian cell culture demonstrating that drugs that destabilize microtubules can activate MAP kinase signaling, as well as studies in Drosophila showing that genetic disruption of the cytoskeleton can activate JNK signaling (Valakh, 2013).

Axonal injury as a result of trauma or neurotoxic insults, such as chemotherapy drug treatment, is accompanied by a change in microtubule network stability. A model is proposed in which DLK functions as a sensor of microtubule network stability. When the cytoskeleton is destabilized as a result of injury, DLK will be activated. The consequence of that activation will depend on downstream signaling pathways and may differ by cellular compartment. For example, DLK in the distal axon will promote axonal degeneration, whereas DLK activation in proximal axons will facilitate the retrograde transport of injury signals that can activate regenerative and/or apoptotic gene expression programs (Valakh, 2013).

In the mammalian PNS, DLK is required for the preconditioning response that boosts the efficacy of peripheral DRG axon regeneration after a prior nerve injury (Shin, 2012). In both worms and flies, activation of DLK by increasing its abundance improves the regenerative response in the absence of a prior nerve injury. Hence, it is attractive to speculate that activation of DLK in the absence of injury may also improve regeneration in mammalian axons. The findings with shot suggest that relatively mild disruptions to the axonal cytoskeleton can activate DLK and accelerate the regenerative response in Drosophila in the absence of a prior trauma. Future studies will test whether pharmacological agents that disrupt the cytoskeleton can activate DLK in mammalian neurons and whether such activation promotes axon regeneration (Valakh, 2013).

In conclusion, this study demonstrates that, in the absence of Shot, Wallenda/DLK signaling is activated resulting in synaptic terminal overgrowth and more rapid regenerative axonal sprouting. The role of Shot as an actin-microtubule cross-linker suggests that Wallenda/DLK is activated by cytoskeletal disruption and suggests novel approaches for controlling DLK activity in the injured or diseased nervous system (Valakh, 2013).

A permissive role of mushroom body α/β core neurons in long-term memory consolidation in Drosophila

Memories are not created equally strong or persistent for different experiences. In Drosophila, induction of long-term memory (LTM) for aversive olfactory conditioning requires ten spaced repetitive training trials, whereas a single trial is sufficient for LTM generation in appetitive olfactory conditioning. Although, with the ease of genetic manipulation, many genes and brain structures have been related to LTM formation, it is still an important task to identify new components and reveal the mechanisms underlying LTM regulation. This study shows that single-trial induction of LTM can also be achieved for aversive olfactory conditioning through inhibition of highwire (hiw)-encoded E3 ubiquitin ligase activity or activation of its targeted proteins in a cluster of neurons, localized within the α/β core region of the mushroom body. Moreover, the synaptic output of these neurons is critical within a limited posttraining interval for permitting consolidation of both aversive and appetitive LTM. It is proposed that these α/β core neurons serve as a 'gate' to keep LTM from being formed, whereas any experience capable of 'opening' the gate is given permit to be consolidated into LTM (Huang, 2012).

The current study began with the finding that 24 hr memory resulting from single session was enhanced in two hiw mutant alleles. This enhanced memory component was identified as facilitated LTM, given that it was sensitive to protein synthesis inhibition. The behavioral effect of hiwδRING and the presence of a hiw-GAL4 line allowed mapping the neural circuitry to a cluster of MB α/β core neurons, within which Hiw and its downstream targets regulate LTM. Furthermore, it was shown that the MB α/β core neurons are involved in the consolidation of both aversive and appetitive LTM. In conclusion, the observations that the MB α/β core neurons are capable of both facilitating and limiting LTM suggests a working model in which the ability to form LTM is gated through these neurons. The significance of these results is further elaborated below (Huang, 2012).

Not only synthesis but also degradation of proteins plays a critical role in the remodeling of synapses, learning, and memory. Altered memory formation in ubiquitin ligase mutants has been reported in mice and Drosophila. This study reports that Hiw, an evolutionarily conserved E3 ubiquitin ligase, negatively regulated LTM formation through restraining its downstream target Wallenda (Wnd). The results indicated Hiw function as an inhibitory constraint, as the memory suppressor gene, on LTM formation. Removal of this suppressor or direct activation of its downstream signals could lead to the facilitated LTM induction without the repetitive training that is normally required. So far, the physiological consequence of Hiw or Wnd in core neurons still remains an open question. Because of the extensively shared components with hiw’s function in synaptic growth and transmission, the attenuated Hiw activity may elevate Wnd level and then lead to the excessive synaptogenesis or abnormal synaptic activity in core neurons. It will also be interesting to test whether the hiw-mediated LTM facilitation shares some common molecular components with the corkscrew-regulated spacing effect in LTM induction, in which the MB α/β lobes also play an important role (Huang, 2012).

In a recent report, Hiw was shown to regulate the axon guidance in MB (Shin, 2011). The morphological defect was also observed in MB α/β lobes in a portion of hiw mutant flies. It is striking that hiw mutants with MB defect can form LTM even more efficiently, given the observation of LTM impairment in another MB structural mutant, ala. However, comparing to the total loss of vertical lobes (including α and α') in ala mutant, most hiw mutants had the abnormal thickness of α/β lobes caused by the unequal distribution of the MB axonal projections between the α and β lobes, and about 39% of hiwDN mutant had the shortened α lobe. One of the possible explanations is that the remaining function of α/β lobe in hiw mutants is sufficient to support the LTM. Moreover, expression of Hiw dominant-negative protein or acutely increasing Wnd protein level in MB was sufficient to promote LTM but did not give rise to any observable gross morphological change in MB. Thus, it is suggested that Hiw mediates memory phenotype through a different mechanism from the one that led to the structure change in the MB (Huang, 2012).

The involvement of MB in the hiw-mediated LTM facilitation led an examination of the function of this structure in LTM regulation. It has been well documented that the MB, a bilateral brain structure that consists of approximately 2,500 neurons in each hemisphere, plays the central role in olfactory memories, both aversive and appetitive. Intrinsic MB neurons are organized into physically distinct α, β, α', β' and γ lobes. All three lobes exhibit different functions in memory processing, such that the output of α/β lobes is required for retrieval of memory, α' β' lobes are transiently required to stabilize memory or to retrieve immediate memory, and γ lobe mediate a rutabaga-dependent mechanism and dopaminergic signal to support short-term memory (STM) and LTM formation. Moreover, memory traces mapped to different lobes exhibit different temporal features (Huang, 2012).

Through gene expression patterns and enhancer trap lines, each lobe of MB can be classified into more specific subgroups such as the posterior, surface, and core regions in the α/β lobes. The current work shows that α/β core neurons play a distinct role in LTM induction. The synaptic outputs of these neurons are critical during consolidation of LTM for both aversive and appetitive conditioning, but these neurons are not involved in LTM cellular consolidation per se because a landmark of LTM cellular consolidation, CREB-mediated protein synthesis, occurs in non-MB neurons. Thus, one of the roles for this cluster of neurons can be viewed as simply providing connections to channel learning information to the downstream neurons in which LTM is formed. However, the remarkable feature of enabling single-trial induction of aversive LTM through targeted genetic manipulation of this cluster of neurons suggests that they play a unique permissive role in determining whether an experience should be consolidated (Huang, 2012).

This newly identified function for permitting an experience to be consolidated leads to proposal of a gating theory. This theory proposes that the α/β core neurons serve as a 'gate,' and activation of this gating mechanism functions as a checkpoint that keeps LTM from being formed for general experiences, whereas only specific experiences, capable of 'opening' this gate, can and are bound to trigger LTM consolidation and to form LTM ultimately. There is little survival advantage in committing never-to-be-repeated episodes to memory, particularly because the very act of LTM formation may be deleterious to the fly. In contrast, repetitively occurring experience, such as spaced repetitive aversive conditioning, and events critical for survival, such as finding food or single-trial appetitive conditioning, would be able to 'open' the gate, and therefore, LTM is formed for such experiences (Huang, 2012).

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 (bskDN). 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 (atg18δ) 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-atg1rescue 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 (bskDN; 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 bskDN. 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, GABAA 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).

Highwire regulates guidance of sister axons in the Drosophila mushroom body

Axons often form synaptic contacts with multiple targets by extending branches along different paths. PHR (Pam/Highwire/RPM-1) family ubiquitin ligases are important regulators of axon development, with roles in axon outgrowth, target selection, and synapse formation. This study reports the function of Highwire, the Drosophila member of the PHR family, in promoting the segregation of sister axons during mushroom body (MB) formation. Loss of highwire results in abnormal development of the axonal lobes in the MB, leading to thinned and shortened lobes. The highwire defect is attributable to guidance errors after axon branching, in which sister axons that should target different lobes instead extend together into the same lobe. The highwire mutant MB displays elevation in the level of the MAPKKK Wallenda/DLK (dual leucine zipper kinase), a previously identified substrate of Highwire, and genetic suppression studies show that Wallenda/DLK is required for the highwire MB phenotype. The highwire lobe defect is limited to α/β lobe axons, but transgenic expression of highwire in the pioneering α'/β' neurons rescues the phenotype. Mosaic analysis further shows that α/β axons of highwire mutant clones develop normally, demonstrating a non-cell-autonomous role of Highwire for axon guidance. Genetic interaction studies suggest that Highwire and Plexin A signals may interact to regulate normal morphogenesis of α/β axons (Shin, 2011).

In Drosophila, highwire is best studied for its role in restraining synaptic terminal growth at the NMJ. Studies in the fly neuromuscular system did not, however, find a role for highwire in motoneuron axon guidance. The current study demonstrates that highwire does regulate axon guidance of MB neurons in Drosophila. The gross morphological defects, such as the short α lobe and thinning of either the α or β lobe, present in the highwire MB lobes of the adult could be attributable to defects in either the development or maintenance of axons. However, similar defects were observed in both the developing and adult MB, so the phenotype is not attributable to degeneration of previously formed axons. The defect is also inconsistent with a gross alteration in axon outgrowth or guidance. The α/β axons form, path-find appropriately through the peduncle, and branch at the appropriate location. Instead, the data suggest a selective deficit in responding to guidance cues at this choice point. After bifurcation of the axon, sister branches do not segregate into distinct lobes as in WT but rather travel together into the same lobe. This phenotype is consistent with loss of homotypic repulsion of sister branches and/or the inability to respond to selective guidance cues targeting the axons to particular lobes (Shin, 2011).

In both fly and worm, PHR proteins sculpt synaptic terminals by restraining Wnd/DLK MAPKKK activity. In Drosophila, highwire acts as an ubiquitin ligase to limit the abundance of Wnd/DLK. Excess Wnd/DLK protein overactivates a MAP kinase signaling pathway that promotes synaptic terminal overgrowth. This study demonstrates that highwire-dependent downregulation of Wnd/DLK is also required for segregation of sister branches of α/β axons and, hence, proper MB development. In the absence of highwire, levels of Wnd/DLK are elevated in the axons of the developing MB. Furthermore, genetic deletion of Wnd/DLK suppresses the highwire-dependent phenotypes, demonstrating that Wnd/DLK is required for the aberrant behavior of α/β axons in the highwire mutant. Attempted were made to test whether overexpression of Wnd/DLK phenocopies highwire mutant MB by driving Wnd/DLK transgenic expression with OK107-Gal4 or MB subset Gal4 lines, including α'/β'-specific NP2748-Gal4. However, the strong overexpression of Wnd/DLK resulted in either lethality or massive cell death in the MB, probably because of the excess activation of downstream JNK MAPK signaling (Shin, 2011).

Although the relationship between the PHR ubiquitin ligase and DLK kinase is clear in flies and worms, studies in vertebrate systems paint a murkier picture. Analysis of PHR mutants in mice and zebrafish consistently demonstrate an important role in various aspects of axon development. However, the molecular mechanism of PHR action and the potential involvement of DLK in vertebrate axons is less clear. In cultured sensory axons from the Phr1 mutant magellan, axon morphology is disrupted and DLK protein is mislocalized. In addition, pharmacological inhibition of p38, a MAP kinase that can be downstream of DLK, reduced the size of the abnormally large growth cones present in these mutant axons. These findings are consistent with a role for DLK activity in generating the Phr1-dependent phenotypes. However, in an independently generated Phr1 mutant, no gross change was observed in DLK levels and it was found that genetic deletion of DLK failed to suppress either corticothalamic axon guidance defects or motoneuron sprouting defects. In zebrafish, mutations in the PHR ortholog esrom disrupt axon guidance and lead to an increase in JNK activation, consistent with a role for DLK, but inhibition of neither JNK nor p38 can suppress the esrom phenotypes, arguing against a functional role for DLK (Hendricks, 2009). The finding that Wnd/DLK is essential for axonal phenotypes in Drosophila while it is dispensable for at least some axonal phenotypes in mice and fish suggests that there is no simple relationship between PHR targets and the cellular function of PHR proteins. PHR proteins do interact with a number of other proteins besides DLK, and so the mechanism of PHR-dependent axonal phenotypes is likely context dependent (Shin, 2011).

During MB development, the α/β neurons are the last to be born and the last to extend their axons into the MB lobes. These α/β axons follow the path established by the earlier-born γ and α'/β' axons. In the highwire mutant, the α/β axons form short, thin, or absent α/β lobes, whereas the γ and α'/β' axons form morphologically normal lobes. Although such results would be consistent with a unique requirement for highwire in α/β neurons, a series of findings instead indicate that highwire is required in α'/β' neurons and indirectly affects the development of α/β axons via a non-cell-autonomous mechanism. First, in the highwire mutant, Wnd/DLK levels are elevated in γ, α'/β', and α/β axons, demonstrating that the Highwire ligase is likely targeting Wnd/DLK in all three cell types. Second, in the highwire mutant, the sister branches of the α/β axons fail to segregate but instead travel into the same lobe. However, within a brain hemisphere, most of the sister branches choose the same lobe, resulting in either a thickened α or β lobe. Hence, the decision as to which lobe to enter is apparently not determined independently by each axon. Third, expression of highwire in the earlier-born α'/β' neurons is sufficient to rescue the defects in the α/β lobes. Fourth, in single-cell highwire α/β clones, sister axons segregate normally in an otherwise heterozygous background. Together, these data demonstrate a non-cell-autonomous requirement for highwire (Shin, 2011).

How might α'/β' axons affect the guidance decision of α/β axons? Misexpression of the cell adhesion molecule Fas II in the α'/β' neurons leads to the loss of either α or β projections, demonstrating that inter-axonal interactions can affect α/β axon development and suggesting that α'/β' axons act as “pioneering axons” for the later-arriving α/β axons. Because the α'/β' axons form morphologically normal lobes in the highwire mutant, the defect is likely at the molecular level, potentially involving a change in either membrane-associated or secreted guidance cues. In the vertebrate CNS, the highwire ortholog Phr1 is also required for a non-cell-autonomous mechanism that guides cortical axons. In the absence of Phr1, cortical axons stall at the corticostriatal border and do not contribute to the internal capsule. In contrast, after conditional excision of Phr1 exclusively in cortical neurons, these same cortical axons can now cross this choice point and path-find to the thalamus. Hence, the requirement for PHR proteins for non-cell-autonomous axon guidance mechanisms is evolutionarily conserved, although there is no evidence that the molecular mechanism is conserved (Shin, 2011).

To investigate the molecular mechanism of the non-cell-autonomous requirement for highwire, genetic interactions between highwire and candidate guidance molecules were tested. The data suggest that Highwire promotes a Plexin A signaling mechanism that is required for proper α/β lobe development. Loss of a single copy of the plexin A gene has no effect on MB development in an otherwise WT background but enhances the phenotype of a weak highwire allele. Furthermore, RNAi-mediated knockdown of plexin A in the MB has a very similar phenotype to loss of highwire, with abnormal thickness of α/β lobes and shortened α lobes. Hence, Plexin A is required for normal MB development. Plexins are receptors for semaphorins, and both Sema-1a and Sema-5c are required for normal MB development. The genetic studies did not uncover a genetic interaction between either of these semaphorins and highwire, but the absence of such an interaction does not rule out the involvement of these or other semaphorins. Two potential models are consistent with these genetic interaction studies. First, Plexin A may function to downregulate Wnd/DLK, potentially via inhibition of Rac GTPase signaling. In the absence of either plexin A or highwire, Wnd/DLK activity would be upregulated, disrupting axonal interactions between α'/β' axons and α/β axons via unknown mechanisms. Alternatively, excess Wnd/DLK activity in the highwire mutant could disrupt the Plexin A signaling pathway that is necessary for α/β lobe development. The mechanisms by which Highwire and Plexin A signaling converge will be the subject of future studies (Shin, 2011).

Diminished MTORC1-dependent JNK activation underlies the neurodevelopmental defects associated with lysosomal dysfunction

This study evaluated the mechanisms underlying the neurodevelopmental deficits in Drosophila and mouse models of lysosomal storage diseases (LSDs). Lysosomes promote the growth of neuromuscular junctions (NMJs) via Rag GTPases and mechanistic target of rapamycin complex 1 (MTORC1). However, rather than employing S6K/4E-BP1, MTORC1 stimulates NMJ growth via JNK, a determinant of axonal growth in Drosophila and mammals. This role of lysosomal function in regulating JNK phosphorylation is conserved in mammals. Despite requiring the amino-acid-responsive kinase MTORC1, NMJ development is insensitive to dietary protein. This paradox is attributed to anaplastic lymphoma kinase (ALK), which restricts neuronal amino acid uptake, and the administration of an ALK inhibitor couples NMJ development to dietary protein. These findings provide an explanation for the neurodevelopmental deficits in LSDs and suggest an actionable target for treatment (Wong, 2015).

Mucolipidosis type IV (MLIV) and Batten disease are untreatable lysosomal storage diseases (LSDs) that cause childhood neurodegeneration. MLIV arises from loss-of-function mutations in the gene encoding TRPML1, an endolysosomal cation channel belonging to the TRP superfamily. The absence of TRPML1 leads to defective lysosomal storage and autophagy, mitochondrial damage, and macromolecular aggregation, which together initiate the protracted neurodegeneration observed in MLIV). Batten disease arises from the absence of a lysosomal protein, CLN3), and results in psychomotor retardation. Both diseases cause early alterations in neuronal function. For instance, brain imaging studies revealed that MLIV and Batten patients display diminished axonal development in the cortex and corpus callosum, the causes of which remain unknown (Wong, 2015).

To better understand the etiology of MLIV in a genetically tractable model, flies were generated lacking the TRPML1 ortholog. The trpml-deficient (trpml1) flies have led to insight into the mechanisms of neurodegeneration and lysosomal storage (Wong, 2015).

This study reports that trpml1 larvae exhibit diminished synaptic growth at the NMJ, a well-studied model synapse. Lysosomal function supports Rag GTPases and MTORC1 activation, and this is essential for JNK phosphorylation and synapse development (Wong, 2015).

Drosophila larvae and mice lacking CLN3 also exhibit diminished Rag/ MTORC1 and JNK activation, suggesting that alterations in neuronal signaling are similar in different LSDs and are evolution- arily conserved. Interestingly, the NMJ defects in the two fly LSD models were suppressed by the administration of a high-protein diet and a drug that is currently in clinical trials to treat certain forms of cancer. These findings inform a pharmacotherapeutic strategy that may suppress the neurodevelopmental defects observed in LSD patients (Wong, 2015).

This study shows that lysosomal dysfunction in Drosophila MNs results in diminished bouton numbers at the larval NMJ. Evidence is presented that lysosomal dysfunction results in decreased activation of the amino-acid-responsive cascade involving Rag/MTORC1, which are critical for normal NMJ development (Wong, 2015).

Despite the requirement for MTORC1 in NMJ synapse development, previous studies and the current findings show that bouton numbers are independent of S6K and 4E-BP1. Rather, MTORC1 promotes NMJ growth via a MAP kinase cascade culminating in JNK activation. Therefore, decreasing lysosomal function or Rag/MTORC1 activation in hiwND8 suppressed the associated synaptic overgrowth. However, the 'small-bouton' phenotype of hiwND8 was independent of MTORC1. Thus, MTORC1 is required for JNK-dependent regulation of bouton numbers, whereas bouton morphology is independent of MTORC1. Furthermore, although both rheb expression and hiw loss result in Wnd-dependent elevation in bouton numbers, the supernumerary boutons in each case show distinct morphological features. Additional studies are needed for deciphering the complex interplay between MTORC1-JNK in regulating the NMJ morphology (Wong, 2015).

Biochemical analyses revealed that both JNK phosphorylation and its transcriptional output correlated with the activity of MTORC1, which are consistent with prior observations that cln3 overexpression promotes JNK activation and that tsc1/tsc2 deletion in flies result in increased JNK-dependent transcription. These findings point to the remarkable versatility of MTORC1 in controlling both protein trans lation and gene transcription (Wong, 2015).

Using an in vitro kinase assay, this study demonstrates that Wnd is a target of MTORC1. Because axonal injury activates both MTORC1 and DLK/JNK, these findings imply a functional connection between these two pathways. Interestingly, the data also suggest that MTORC1 contains additional kinases besides MTOR that can phosphorylate Wnd. One possibility is that ULK1/Atg1, which associates with MTORC1, could be the kinase that phosphorylates Wnd. Consistent with this notion, overexpression of Atg1 in the Drosophila neurons has been shown to promote JNK signaling and NMJ synapse overgrowth via Wnd) (Wong, 2015).

This study also found that developmental JNK activation in axonal tracts of the CC and pJNK levels in cortical neurons were compromised in a mouse model of Batten disease. Thus, the signaling deficits identified in Drosophila are also conserved in mammals. The activity of DLK (the mouse homolog of Wnd) and JNK signaling are critical for axonal development in the mouse CNS. Therefore, decreased neuronal JNK activation during development might underlie the thinning of the axonal tracts observed in many LSDs (Wong, 2015).

Although the findings of this study demonstrate a role for an amino-acid- responsive cascade in the synaptic defects associated with lysosomal dysfunction, simply elevating the dietary protein content was not sufficient to rescue these defects. These findings were reminiscent of an elegant study that showed that the growth of Drosophila neuroblasts is uncoupled from dietary amino acids owing to the function of ALK, which suppresses the uptake of amino acids into the neuroblasts (Cheng, 2011). Indeed, simultaneous administration of an ALK inhibitor and a high-protein diet partially rescued the synaptic growth defects associated with the lysosomal dysfunction, and improved the rescue of pupal lethality associated with trpml1. Although these studies do not causally link the defects in synapse development with pupal lethality, they do raise the intriguing possibility that multiple phenotypes associated with LSDs could be targeted using ALK inhibitors along with a protein-rich diet (Wong, 2015).

Although LSDs result in lysosomal dysfunction throughout the body, neurons are exceptionally sensitive to these alterations. The cause for this sensitivity remains incompletely understood. Given the findings of this study that mature neurons do not efficiently take up amino acids from the extracellular medium, lysosomal degradation of proteins serves as a major source of free amino acids in these cells. Therefore, disruption of lysosomal degradation leads to severe shortage of free amino acids in neurons, regardless of the quantity of dietary proteins, thus explaining the exquisite sensitivity of neurons to lysosomal dysfunction (Wong, 2015).

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).

Insulin/IGF1 signaling inhibits age-dependent axon regeneration

The ability of injured axons to regenerate declines with age, yet the mechanisms that regulate axon regeneration in response to age are not known. This study shows that axon regeneration in aging C. elegans motor neurons is inhibited by the conserved insulin/IGF1 receptor DAF-2. DAF-2's function in regeneration is mediated by intrinsic neuronal activity of the forkhead transcription factor DAF-16/FOXO. DAF-16 regulates regeneration independently of lifespan, indicating that neuronal aging is an intrinsic, neuron-specific, and genetically regulated process. In addition, DAF-18/PTEN was found to inhibit regeneration independently of age and FOXO signaling via the TOR pathway. Finally, DLK-1, a conserved regulator of regeneration, is downregulated by insulin/IGF1 signaling, bound by DAF-16 in neurons, and required for both DAF-16- and DAF-18-mediated regeneration. Together, these data establish that insulin signaling specifically inhibits regeneration in aging adult neurons and that this mechanism is independent of PTEN and TOR (Byrne, 2004).

Cytoskeletal disruption activates the DLK/JNK pathway, which promotes axonal regeneration and mimics a preconditioning injury

Nerve injury can lead to axonal regeneration, axonal degeneration, and/or neuronal cell death. Remarkably, the MAP3K dual leucine zipper kinase, DLK or Wallenda, promotes each of these responses, suggesting that DLK is a sensor of axon injury. In Drosophila, mutations in proteins that stabilize the actin and microtubule cytoskeletons activate the DLK pathway, suggesting that DLK may be activated by cytoskeletal disruption. This model was tested in mammalian sensory neurons. Pharmacological agents designed to disrupt either the actin or microtubule cytoskeleton were found to activate the DLK pathway, and that activation is independent of calcium influx or induction of the axon degeneration program. Moreover, activation of the DLK pathway by targeting the cytoskeleton induces a pro-regenerative state, enhancing axon regeneration in response to a subsequent injury in a process akin to preconditioning. This highlights the potential utility of activating the DLK pathway as a method to improve axon regeneration. Moreover, DLK is required for these responses to cytoskeletal perturbations, suggesting that DLK functions as a key neuronal sensor of cytoskeletal damage (Valakh, 2015).


REFERENCES

Search PubMed for articles about Drosophila Wallenda

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date revised: 12 November 2017

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