A neuropeptide signaling pathway regulates synaptic growth in Drosophila

Neuropeptide signaling is integral to many aspects of neural communication, particularly modulation of membrane excitability and synaptic transmission. However, neuropeptides have not been clearly implicated in synaptic growth and development. Here, we demonstrate that cholecystokinin-like receptor (CCKLR) and drosulfakinin (DSK), its predicted ligand, are strong positive growth regulators of the Drosophila melanogaster larval neuromuscular junction (NMJ). Mutations of CCKLR or dsk produced severe NMJ undergrowth, whereas overexpression of CCKLR caused overgrowth. Presynaptic expression of CCKLR was necessary and sufficient for regulating NMJ growth. CCKLR and dsk mutants also reduced synaptic function in parallel with decreased NMJ size. Analysis of double mutants revealed that DSK/CCKLR regulation of NMJ growth occurs through the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA)-cAMP response element binding protein (CREB) pathway. Our results demonstrate a novel role for neuropeptide signaling in synaptic development. Moreover, because the cAMP-PKA-CREB pathway is required for structural synaptic plasticity in learning and memory, DSK/CCKLR signaling may also contribute to these mechanisms.     

Neuron-Type Specific Functions of DNT1, DNT2 and Spz at the Drosophila Neuromuscular Junction

Retrograde growth factors regulating synaptic plasticity at the neuromuscular junction (NMJ) in Drosophila have long been predicted but their discovery has been scarce. In vertebrates, such retrograde factors produced by the muscle include GDNF and the neurotrophins (NT: NGF, BDNF, NT3 and NT4). NT superfamily members have been identified throughout the invertebrates, but so far no functional in vivo analysis has been carried out at the NMJ in invertebrates. The NT family of proteins in Drosophila is formed of DNT1, DNT2 and Spätzle (Spz), with sequence, structural and functional conservation relative to mammalian NTs. Here, we investigate the functions of Drosophila NTs (DNTs) at the larval NMJ. All three DNTs are expressed in larval body wall muscles, targets for motor-neurons. Over-expression of DNTs in neurons, or the activated form of the Spz receptor, Toll ^10b, in neurons only, rescued the semi-lethality of spz ² and DNT1 ⁴¹ , DNT2 ^e03444 double mutants, indicating retrograde functions in neurons. In spz ² mutants, DNT1 ⁴¹ , DNT2 ^e03444 double mutants, and upon over-expression of the DNTs, NMJ size and bouton number increased. Boutons were morphologically abnormal. Mutations in spz and DNT1,DNT2 resulted in decreased number of active zones per bouton and decreased active zone density per terminal. Alterations in DNT function induced ghost boutons and synaptic debris. Evoked junction potentials were normal in spz ² mutants and DNT1 ⁴¹ , DNT2 ^e03444 double mutants, but frequency and amplitude of spontaneous events were reduced in spz ² mutants suggesting defective neurotransmission. Our data indicate that DNTs are produced in muscle and are required in neurons for synaptogenesis. Most likely alterations in DNT function and synapse formation induce NMJ plasticity leading to homeostatic adjustments that increase terminal size restoring overall synaptic transmission. Data suggest that Spz functions with neuron-type specificity at the muscle 4 NMJ, and DNT1 and DNT2 function together at the muscles 6,7 NMJ.     

JNK Pathway Activation Is Controlled by Tao/TAOK3 to Modulate Ethanol Sensitivity

Neuronal signal transduction by the JNK MAP kinase pathway is altered by a broad array of stimuli including exposure to the widely abused drug ethanol, but the behavioral relevance and the regulation of JNK signaling is unclear. Here we demonstrate that JNK signaling functions downstream of the Sterile20 kinase family gene tao/Taok3 to regulate the behavioral effects of acute ethanol exposure in both the fruit fly Drosophila and mice. In flies tao is required in neurons to promote sensitivity to the locomotor stimulant effects of acute ethanol exposure and to establish specific brain structures. Reduced expression of key JNK pathway genes substantially rescued the structural and behavioral phenotypes of tao mutants. Decreasing and increasing JNK pathway activity resulted in increased and decreased sensitivity to the locomotor stimulant properties of acute ethanol exposure, respectively. Further, JNK expression in a limited pattern of neurons that included brain regions implicated in ethanol responses was sufficient to restore normal behavior. Mice heterozygous for a disrupted allele of the homologous Taok3 gene (Taok3Gt) were resistant to the acute sedative effects of ethanol. JNK activity was constitutively increased in brains of Taok3Gt/+ mice, and acute induction of phospho-JNK in brain tissue by ethanol was occluded in Taok3Gt/+ mice. Finally, acute administration of a JNK inhibitor conferred resistance to the sedative effects of ethanol in wild-type but not Taok3Gt/+ mice. Taken together, these data support a role of a TAO/TAOK3-JNK neuronal signaling pathway in regulating sensitivity to acute ethanol exposure in flies and in mice.     

The genome sequence of the protostome Daphnia pulex encodes respective orthologues of a neurotrophin, a Trk and a p75NTR: Evolution of neurotrophin signaling components and related proteins in the bilateria

Background

Neurotrophins and their Trk and p75NTR receptors play an important role in the nervous system. To date, neurotrophins, Trk and p75NTR have only been found concomitantly in deuterostomes. In protostomes, homologues to either neurotrophin, Trk or p75NTR are reported but their phylogenetic relationship to deuterostome neurotrophin signaling components is unclear. Drosophila has neurotrophin homologues called Spätzles (Spz), some of which were recently renamed neurotrophins, but direct proof that these are deuterostome neurotrophin orthologues is lacking. Trks belong to the receptor tyrosine kinase (RTK) family and among RTKs, Trks and RORs are closest related. Flies lack Trks but have ROR and ROR-related proteins called NRKs playing a neurotrophic role. Mollusks have so far the most similar proteins to Trks (Lymnaea Trk and Aplysia Trkl) but the exact phylogenetic relationship of mollusk Trks to each other and to vertebrate Trks is unknown. p75NTR belongs to the tumor necrosis factor receptor (TNFR) superfamily. The divergence of the TNFR families in vertebrates has been suggested to parallel the emergence of the adaptive immune system. Only one TNFR representative, the Drosophila Wengen, has been found in protostomes. To clarify the evolution of neurotrophin signaling components in bilateria, this work analyzes the genome of the crustacean Daphnia pulex as well as new genetic data from protostomes.

Results

The Daphnia genome encodes a neurotrophin, p75NTR and Trk orthologue together with Trkl, ROR, and NRK-RTKs. Drosophila Spz1, 2, 3, 5, 6 orthologues as well as two new groups of Spz proteins (Spz7 and 8) are also found in the Daphnia genome. Searching genbank and the genomes of Capitella, Helobdella and Lottia reveals neurotrophin signaling components in other protostomes.

Conclusion

It appears that a neurotrophin, Trk and p75NTR existed at the protostome/deuterostome split. In protostomes, a "neurotrophin superfamily" includes Spzs and neurotrophins which respectively form two paralogous families. Trks and Trkl proteins also form closely related paralogous families within the protostomian RTKs, whereby Trkls are absent in deuterostomes. The finding of p75NTR in several protostomes suggests that death domain TNFR superfamily proteins appeared early in evolution.     

A Toll-Spätzle pathway in the tobacco hornworm, Manduca sexta

Insects synthesize a battery of antimicrobial peptides (AMPs) and expression of AMP genes is regulated by the Toll and Imd (immune deficiency) pathways in Drosophila melanogaster. Drosophila Toll pathway is activated after Spätzle (Spz) is cleaved by Spätzle processing enzyme (SPE) to release the active C-terminal C106 domain (DmSpz-C106), which then binds to the Toll receptor to initiate the signaling pathway and regulate expression of AMP genes such as drosomycin. Toll and Spz genes have been identified in other insects, but interaction between Toll and Spz and direct evidence for a Toll-Spz pathway in other insect species have not been demonstrated. Our aim is to investigate a Toll-Spz pathway in Manduca sexta, and compare M. sexta and D. melanogaster Toll-Spz pathways. Co-immunoprecipitation (Co-IP) assays showed that MsToll^ecto (the ecto-domain of M. sexta Toll) could interact with MsSpz-C108 (the active C-terminal C108 domain of M. sexta Spz) but not with full-length MsSpz, and DmToll^ecto could interact with DmSpz-C106 but not DmSpz, suggesting that Toll receptor only binds to the active C-terminal domain of Spz. Co-expression of MsToll-MsSpz-C108, but not MsToll-MsSpz, could up-regulate expression of drosomycin gene in Drosophila S2 cells, indicating that MsToll-MsSpz-C108 complex can activate the Toll signaling pathway. In vivo assays showed that activation of AMP genes, including cecropin, attacin, moricin and lebocin, in M. sexta larvae by purified recombinant MsSpz-C108 could be blocked by pre-injection of antibody to MsToll, further confirming a Toll-Spz pathway in M. sexta, a lepidopteran insect.     

Autophagy promotes synapse development in Drosophila

Autophagy, a lysosome-dependent degradation mechanism, mediates many biological processes, including cellular stress responses and neuroprotection. In this study, we demonstrate that autophagy positively regulates development of the Drosophila melanogaster larval neuromuscular junction (NMJ). Autophagy induces an NMJ overgrowth phenotype closely resembling that of highwire (hiw), an E3 ubiquitin ligase mutant. Moreover, like hiw, autophagy-induced NMJ overgrowth is suppressed by wallenda (wnd) and by a dominant-negative c-Jun NH₂-terminal kinase (bsk^DN). We show that 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, our findings suggest that it is perfectly positioned to link environmental conditions with synaptic growth and plasticity.     

Neurotrophin receptor interacting factor (NRIF) is an essential mediator of apoptotic signaling by the p75 neurotrophin receptor

Activation of the p75 neurotrophin receptor leads to a variety of effects within the nervous system, including neuronal apoptosis. Both c-Jun N-terminal kinase (JNK) and the tumor suppressor p53 have been reported to be critical for this receptor to induce cell death; however, the mechanisms by which p75 activates these pathways is undetermined. Here we report that the neurotrophin receptor interacting factor (NRIF) is necessary for p75-dependent JNK activation and apoptosis. Upon nerve growth factor withdrawal, nrif-/- sympathetic neurons underwent apoptosis, whereas p75-mediated death was completely abrogated. The lack of cell death correlated with a lack of JNK activation in the nrif-/- neurons, suggesting that NRIF is a selective mediator for p75-dependent JNK activation and apoptosis. Moreover, we document that NRIF expression is sufficient to induce cell death through a mechanism that requires p53. Taken together, these results establish NRIF as an essential component of the p75 apoptotic pathway.     

The PDZ-GEF Gef26 regulates synapse development and function via FasII and Rap1 at the Drosophila neuromuscular junction

Guanine nucleotide exchange factors (GEFs) are essential for small G proteins to activate their downstream signaling pathways, which are involved in morphogenesis, cell adhesion, and migration. Mutants of Gef26, a PDZ-GEF (PDZ domain-containing guanine nucleotide exchange factor) in Drosophila, exhibit strong defects in wings, eyes, and the reproductive and nervous systems. However, the precise roles of Gef26 in development remain unclear. In the present study, we analyzed the role of Gef26 in synaptic development and function. We identified significant decreases in bouton number and branch length at larval neuromuscular junctions (NMJs) in Gef26 mutants, and these defects were fully rescued by restoring Gef26 expression, indicating that Gef26 plays an important role in NMJ morphogenesis. In addition to the observed defects in NMJ morphology, electrophysiological analyses revealed functional defects at NMJs, and locomotor deficiency appeared in Gef26 mutant larvae. Furthermore, Gef26 regulated NMJ morphogenesis by regulating the level of synaptic Fasciclin II (FasII), a well-studied cell adhesion molecule that functions in NMJ development and remodeling. Finally, our data demonstrate that Gef26-specific small G protein Rap1 worked downstream of Gef26 to regulate the level of FasII at NMJs, possibly through a βPS integrin-mediated signaling pathway. Taken together, our findings define a novel role of Gef26 in regulating NMJ development and function.     

JNK functions in the non-canonical Wnt pathway to regulate convergent extension movements in vertebrates

Recent genetic studies in Drosophila identified a novel non-canonical Wnt pathway, the planar cell polarity (PCP) pathway, that signals via JNK to control epithelial cell polarity in Drosophila. Most recently, a pathway regulating convergent extension movements during gastrulation in vertebrate embryos has been shown to be a vertebrate equivalent of the PCP pathway. However, it is not known whether the JNK pathway functions in this non-canonical Wnt pathway to regulate convergent extension movements in vertebrates. In addition, it is not known whether JNK is in fact activated by Wnt stimulation. Here we show that Wnt5a is capable of activating JNK in cultured cells, and present evidence that the JNK pathway mediates the action of Wnt5a to regulate convergent extension movements in Xenopus. Our results thus demonstrate that the non-canonical Wnt/JNK pathway is conserved in both vertebrate and invertebrate and define that JNK has an activity to regulate morphogenetic cell movements.     

Activation of neutral-sphingomyelinase,MAPKs, and p75 NTR-mediating caffeic acid phenethyl ester–induced apoptosis in C6 glioma cells

Background

Caffeic acid phenethyl ester (CAPE), a component of propolis, is reported to possess anti-inflammatory, anti-bacterial, anti-viral, and anti-tumor activities. Previously, our laboratory demonstrated the in vitro and in vivo bioactivity of CAPE and addressed the role of p53 and the p38 mitogen-activated protein kinase (MAPK) pathway in regulating CAPE-induced apoptosis in C6 glioma cells.

Results

C6 cancer cell lines were exposed to doses of CAPE; DNA fragmentation and MAPKs and NGF/P75NTR levels were then determined. SMase activity and ceramide content measurement as well as western blotting analyses were performed to clarify molecular changes. The present study showed that CAPE activated neutral sphingomyelinase (N-SMase), which led to the ceramide-mediated activation of MAPKs, including extracellular signal-regulated kinase (ERK), Jun N-terminus kinase (JNK), and p38 MAPK. In addition, CAPE increased the expression of nerve growth factor (NGF) and p75 neurotrophin receptor (p75NTR). The addition of an N-SMase inhibitor, GW4869, established that NGF/p75NTR was the downstream target of N-SMase/ceramide. Pretreatment with MAPK inhibitors demonstrated that MEK/ERK and JNK acted upstream and downstream, respectively, of NGF/p75NTR. Additionally, CAPE-induced caspase 3 activation and poly [ADP-ribose] polymerase cleavage were reduced by pretreatment with MAPK inhibitors, a p75NTR peptide antagonist, or GW4869.

Conclusions

Taken together, N-SMase activation played a pivotal role in CAPE-induced apoptosis by activation of the p38 MAPK pathway and NGF/p75NTR may explain a new role of CAPE induced apoptosis in C6 glioma.     

Anterograde Activin Signaling Regulates Postsynaptic Membrane Potential and GluRIIA/B Abundance at the Drosophila Neuromuscular Junction

Members of the TGF-β superfamily play numerous roles in nervous system development and function. In Drosophila, retrograde BMP signaling at the neuromuscular junction (NMJ) is required presynaptically for proper synapse growth and neurotransmitter release. In this study, we analyzed whether the Activin branch of the TGF-β superfamily also contributes to NMJ development and function. We find that elimination of the Activin/TGF-β type I receptor babo, or its downstream signal transducer smox, does not affect presynaptic NMJ growth or evoked excitatory junctional potentials (EJPs), but instead results in a number of postsynaptic defects including depolarized membrane potential, small size and frequency of miniature excitatory junction potentials (mEJPs), and decreased synaptic densities of the glutamate receptors GluRIIA and B. The majority of the defective smox synaptic phenotypes were rescued by muscle-specific expression of a smox transgene. Furthermore, a mutation in actβ, an Activin-like ligand that is strongly expressed in motor neurons, phenocopies babo and smox loss-of-function alleles. Our results demonstrate that anterograde Activin/TGF-β signaling at the Drosophila NMJ is crucial for achieving normal abundance and localization of several important postsynaptic signaling molecules and for regulating postsynaptic membrane physiology. Together with the well-established presynaptic role of the retrograde BMP signaling, our findings indicate that the two branches of the TGF-β superfamily are differentially deployed on each side of the Drosophila NMJ synapse to regulate distinct aspects of its development and function.     

Nibbling away at synaptic development

Beyond its role as a response to starvation, autophagy has been increasingly implicated as part of the normal mechanisms regulating growth and remodeling of various cells and tissues during development. In recently published work we demonstrate that autophagy promotes synaptic development of the Drosophila larval neuromuscular junction (NMJ). We find that autophagy acts by downregulating an E3 ubiquitin ligase, Highwire (Hiw), which limits NMJ growth via a MAPKKK pathway. A similar role for autophagy in the synaptic remodeling that occurs during learning and memory remains an intriguing possibility.     

CARD4/Nod1 mediates NF-κB and JNK activation by invasive Shigella flexneri

Epithelial cells are refractory to extracellular lipopolysaccharide (LPS), yet when presented inside the cell, it is capable of initiating an inflammatory response. Using invasive Shigella flexneri to deliver LPS into the cytosol, we examined how this factor, once intracellular, activates both NF-κB and c-Jun N-terminal kinase (JNK). Surprisingly, the mode of activation is distinct from that induced by toll-like receptors (TLRs), which mediate LPS responsiveness from the outside-in. Instead, our findings demonstrate that this response is mediated by a cytosolic, plant disease resistance-like protein called CARD4/Nod1. Biochemical studies reveal enhanced oligomerization of CARD4 upon S. flexneri infection, an event necessary for NF-κB induction. Dominant-negative versions of CARD4 block activation of NF-κB and JNK by S. flexneri as well as microinjected LPS. Finally, we showed that invasive S. flexneri triggers the formation of a transient complex involving CARD4, RICK and the IKK complex. This study demonstrates that in addition to the extracellular LPS sensing system mediated by TLRs, mammalian cells also possess a cytoplasmic means of LPS detection via a molecule that is related to plant disease-resistance proteins.     

The Anopheles gambiae Oxidation Resistance 1 (OXR1) Gene Regulates Expression of Enzymes That Detoxify Reactive Oxygen Species

Background

OXR1 is an ancient gene, present in all eukaryotes examined so far that confers protection from oxidative stress by an unknown mechanism. The most highly conserved region of the gene is the carboxyl-terminal TLDc domain, which has been shown to be sufficient to prevent oxidative damage.

Methodology/Principal Findings

OXR1 has a complex genomic structure in the mosquito A. gambiae, and we confirm that multiple splice forms are expressed in adult females. Our studies revealed that OXR1 regulates the basal levels of catalase (CAT) and glutathione peroxidase (Gpx) expression, two enzymes involved in detoxification of hydrogen peroxide, giving new insight into the mechanism of action of OXR1. Gene silencing experiments indicate that the Jun Kinase (JNK) gene acts upstream of OXR1 and also regulates expression of CAT and GPx. Both OXR1 and JNK genes are required for adult female mosquitoes to survive chronic oxidative stress. OXR1 silencing decreases P. berghei oocyst formation. Unexpectedly, JNK silencing has the opposite effect and enhances Plasmodium infection in the mosquito, suggesting that JNK may also mediate some, yet to be defined, antiparasitic response.

Conclusion

The JNK pathway regulates OXR1 expression and OXR1, in turn, regulates expression of enzymes that detoxify reactive oxygen species (ROS) in Anopheles gambiae. OXR1 silencing decreases Plasmodium infection in the mosquito, while JNK silencing has the opposite effect and enhances infection.