InteractiveFly: GeneBrief

Neurotrophin 1: Biological Overview | References

BIOLOGICAL OVERVIEW

Neurotrophic interactions occur in Drosophila, but to date, no neurotrophic factor had been found. Neurotrophins are the main vertebrate secreted signalling molecules that link nervous system structure and function: they regulate neuronal survival, targeting, synaptic plasticity, memory and cognition. This study has identified a neurotrophic factor in flies, Drosophila Neurotrophin (DNT1), structurally related to all known neurotrophins and highly conserved in insects. By investigating with genetics the consequences of removing DNT1 or adding it in excess, it was shown that DNT1 maintains neuronal survival, as more neurons die in DNT1 mutants and expression of DNT1 rescues naturally occurring cell death, and it enables targeting by motor neurons. Spätzle and a further fly neurotrophin superfamily member, DNT2, also have neurotrophic functions in flies. These findings imply that most likely a neurotrophin was present in the common ancestor of all bilateral organisms, giving rise to invertebrate and vertebrate neurotrophins through gene or whole-genome duplications. This work provides a missing link between aspects of neuronal function in flies and vertebrates, and it opens the opportunity to use Drosophila to investigate further aspects of neurotrophin function and to model related diseases (Zhu, 2008).

In vertebrate brain development, neurons are produced in excess, and surplus neurons are eliminated through apoptosis (cell death), adjusting innervation, targeting, and connectivity to target size. Neurotrophins (NTs) are the major class of molecules promoting neuronal survival in vertebrates. They also control cell proliferation and neuronal differentiation, and they are required for axonal and dendritic elaborations, synaptic plasticity, excitability, and long-term potentiation (LTP, the basis of memory and learning). NTs underlie most aspects of vertebrate nervous system development and function, and abnormal NT function is linked to psychiatric disorders. NTs are the key molecules linking nervous system structure and function in vertebrates. Despite such fundamental roles, NTs have been missing from invertebrates (Zhu, 2008).

There is compelling evidence that neurotrophic factors exist in Drosophila. As in vertebrates, about half the neurons die in the fruit fly central nervous system (CNS) during embryogenesis. Apoptosis occurs in most neuroblast lineages, and there is dramatic hyperplasia in mutant embryos lacking programmed cell death. In multiple CNS contexts, the survival of subsets of neurons and glia requires long-range, nonautonomous support. For instance, there are no glial cells of retinal origin; glia enter the retina through the optic stalk, and if they are defective, such as in repo mutants, retinal neurons die in excess (Xiong, 1995 ). In disconnected mutants, the optic lobes (where the retinal photoreceptor neurons project to in the brain) degenerate. When mosaic clones of disconnected mutant cells are generated in the brain optic lobes in otherwise normal flies, retinal neurons die. Lack of connectivity at the optic lobe also results in massive optic lobe neuronal death due to abnormal function originating from the retina rather than the brain. A trophic factor for retinal neurons is predicted to emanate from the brain optic lobe glia (Dearborn, 2004; Fishbach, 1984). In the embryonic CNS, upon glial ablation or mutations in glial cells missing, there is excess neuronal apoptosis. Glia are also produced in excess: most dramatically, in the embryo, 75% midline glia and a small subset of longitudinal glia die during axon guidance (prior to homeostatic functions of glia). Identified gliatrophic factors include the neuregulin homolog Vein and the TGFα homolog Spitz, both ligands of EGFR, and the ligands of the PDGR homolog PVR. Other properties commonly assigned to complex brains and to NT function, such as synaptic plasticity, LTP, and complex behaviour, all occur in flies. However, no neurotrophic factor has been identified in Drosophila (Zhu, 2008).

The NTs comprise brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), NT3, and NT4/5 (plus NT6/7 in fish) and bind the Receptor Tyrosine Kinases TrkA, -B, -C, the atypical TNFR superfamily member p75, and Integrin α9β. Pro-NTs bind p75 to promote cell death, and mature NTs bind Trk and p75 receptors, or p75 alone, to promote cell survival. Vertebrate NTs bind Trks to activate the MAPKinase/ERK and AKT pathways (promoting cell survival), PLC-γ (regulating calcium levels), and NFkappaB (promoting cell survival). Binding of NTs to p75 independently of Trks results in cell death or cell survival, through JNK and NFkappaB, respectively. In an evolutionary context, p75 is more ancient than the Trks. The most conserved NT among vertebrates is BDNF, and BDNF mutations correlate with epilepsy, anxiety, depression, attention deficit disorder, autism, and other cognitive and psychiatric disorders. NTs underlie an endogenous mechanism of CNS repair, and disregulation of NGF underlies chronic pain (e.g., in cancer). Drosophila is a very powerful model organism used to understand gene networks and model disease; however, a surprising void has been the lack of NT studies in flies (Zhu, 2008 and references therein).

NT ligands and receptors have been identified throughout the invertebrate deuterostomes. There are functional Trk receptors in the lancelet Amphioxus, and p75 and Trk orthologs have been identified in sea urchin and acorn worm. Searches of sequenced genomes have revealed NTs in all deuterostome groups, represented by Amphioxus NT (Bf-NT), acorn worm NT (Sk-NT), and sea urchin NT (Sp-NT). In protostome invertebrates, a bona fide Trk (in the snail Lymnea) and an atypical Trk (in the snail Aplysia), have also been identified in molluscs. The function of these ancient NTs and receptors is unknown. These findings indicate that NTs are more ancient in evolution than previously thought, although no NT has been identified in protostomes (Zhu, 2008 and references therein).

The presence of NTs in flies has been controversial. Structural and biochemical features of the Drosophila protein Spätzle (Spz) revealed an NGF domain. However, a parallel similarity to horseshoe crab coagulogen, involved in the blood-clotting cascade, overshadowed that earlier finding. An initial computational analysis of the sequenced genomes based on BLAST searches declared lack of NTs in flies. However, this simple BLAST search missed 30% of Drosophila genes and would have missed any proteins with structural conservation despite sequence divergence. Structural predictions have confirmed that Spz belongs to the NT superfamily (Weber, 2007). There are to date no functional studies of Spz in the CNS, so whether it plays neurotrophic roles is unknown (Zhu, 2008).

To investigate whether a NT may underlie some of the structural and functional aspects of the insect nervous system, we searched the sequenced Drosophila genome for NT sequences. This study shows that Drosophila Neurotrophin 1 (DNT1) is a NT superfamily member that promotes neuronal survival and targeting, and that there is a NT family in Drosophila formed by DNT1, DNT2, and Spz (Zhu, 2008).

Twenty-eight known full-length and Cystine-knot domain (Cysknot, characteristic of NTs) vertebrate NT sequences were used to query the Drosophila genome with TBLASTN and PSI-BLAST, which is specific to detect distantly related sequences. When using carp BDNF as query, both searches identified CG18318. In turn, CG18318 identified BDNF from multiple species, from fish to human. After isolating the full-length cDNA3 from CG18318, the protein sequence was used to carry out a structure-based search using FUGUE. FUGUE identifies distantly related proteins, the sequence of which may have diverged through evolution while retaining structural conservation. FUGUE compares the query protein sequence with the HOMSTRAD database of proteins of known structure, and it assigns amino-acid substitutions a score depending on how this affects protein structure. FUGUE identified the human NTs with over 99% certainty as probable homologs of cDNA3 from CG18318, above similarity to coagulogen. Search of the ENSEMBL human database using cDNA3 protein sequence as query also identified human BDNF. Thus, the protein encoded by cDNA3 was named Drosophila Neurotrophin1 (DNT1). PSI-BLAST searches using Spz as query to the Drosophila genome had identified distant spz paralogs: DNT1 is spätzle 2 (spz2) (Zhu, 2008).

To verify the structural features of DNT1, a structural alignment of DNT1 was carried out to known NT sequences from human, Xenopus, and the ancient NTs from lamprey (Lf-NT), Amphioxus (Bf-NT), sea urchin (Sp-NT), and acorn worm (Sk-NT). All the essential residues that form the NT Cysknot (positions 499-601 in DNT1) are conserved in all these sequences, i.e., the six cysteines, the glutamine (position 539), and conservative substitutions of all the residues of the hydrophobic core. Interestingly, DNT1 shares more conserved residues with acorn worm Sk-NT than with other NTs. The DNT1 Cysknot is highly conserved in all sequenced insects, such as fruit fly (Drosophila), mosquito (Anopheles), and bee (Apis), and conservation outside the Cysknot is also high among all Drosophila species (Zhu, 2008).

Neurotrophic factors had been anticipated in Drosophila but not previously found. DNT1 satisfies the criteria to be a NT superfamily member. First, DNT1 was identified by sequence homology to NTs through sequence-based bioinformatic searches. Sequence identity to NTs is not high and is restricted to the Cysknot domain. However, this conservation is sufficient to ensure the structural features of a NT Cysknot. Second, DNT1 is structurally a NT superfamily member. DNT1 is predicted to be secreted, it is cleaved and forms a NT-Cysknot, which dimerises to become functional. A structure-based alignment shows conservation of all the residues relevant to forming the Cysknot, not only between DNT1, vertebrate, and human NTs, but also including the ancient NTs from Amphioxus, sea urchin, and acorn worm. Third, DNT1 functions like a canonical NT: loss of DNT1 function results in increased neuronal apoptosis, gain of DNT1 function rescues naturally occurring cell death (NOCD), and interfering with DNT1 function affects targeting by embryonic motor axons. In the CNS, neuronal survival depends on DNT1 produced in limiting amounts from the midline intermediate target. Targeting by the motor axons requires DNT1 at the muscle. The high conservation of DNT1 in insects supports its functional relevance. Adult flies mutant for spz or double mutant for DNT1 and DNT2 have distinct locomotion deficits. DNT1 is expressed in the brain, in the centres for learning and memory, suggesting possible higher neuronal functions (Zhu, 2008).

Previous reports had revealed an NGF domain in Spz and biochemical evidence supports a similar mechanism of activation for Spz and the vertebrate NTs (Weber, 2003). A theoretical structural analysis of Spz had shown that Spz forms a NT Cysknot. These are features also found in DNT1. However, when bioinformatic searches were carried out for Spz, a relationship between Spz and the NTs could not be established. Sequence identity between Spz and NGF is lower than for DNT1 and BDNF. Spz is also less conserved in insects than DNT1 is. The sequence of Spz is more diverged from the vertebrate NTs than DNT1 is. Nevertheless, Spz, together with Toll, also plays neurotrophic functions (Zhu, 2008).

Structural analysis of the spz paralogs indicates that DNT1, Spz, and Spz5 are more closely related to each other and to the NTs, whereas Spz3, Spz4, and Spz6 are highly diverged. The possibility cannot at this stage be ruled out that Spz3, Spz4, and Spz6 may also play functions in the nervous system. Spz5 is structurally close to the NTs and very highly conserved in insects. This study has shown that Spz5/DNT2 has neurotrophic functions, as it rescues NOCD, and loss of Spz5/DNT2 function results in increased CNS apoptosis and axon targeting errors. spz5 has been renamed as DNT2. Thus, there is a NT family in Drosophila formed of at least DNT1/Spz2, DNT2/Spz5, and Spz (Zhu, 2008).

Orthologs are genes related by ancestry. The identification of DNT1 by sequence homology to BDNF does not mean that DNT1 is a BDNF ortholog. BDNF resulted from the duplication of an ancestral vertebrate NT, thus a relationship between DNT1 and vertebrate NTs goes back to an ancestral NT. Consistently, DNT1 and Spz are more closely related to Sk-NT from acorn worm. The sequence relatedness between DNT1 and the NTs is unlikely to be due to convergence since it was found using three independent types of searches, including a structure-based search, and confirmed with two types of reverse searches, and biochemical features and function are also conserved. Direct proof that DNT1, DNT2, and spz are general NT orthologs cannot be obtained. High sequence divergence among all invertebrate NTs precludes the phylogenies to resolve. The same conclusion had been reached for the analysis of ancient deuterostomian NTs (Hallböök, 2006). The phylogenetic analyses of DNT1 and spz compared to all known NTs, carried out in this study, revealed interesting features: first, the invertebrate deuterostomian NTs are closer to DNT1 and Spz than the vertebrate NT. Second, among those, acorn worm NT (Sk-NT) is the closest to DNT1 and Spz. Third, two other protein families contain Cysknots, TGFβ and PDGF, but these Cysknots differ from that of NTs. The Cysknot in DNT1 and Spz is unequivocally closer to the NT Cysknot. The most parsimonious explanation is that an ancestral NT gene present in Urbilateria (the presumed common ancestor of all bilateral organisms) gave rise to the NTs in deuterostomes and in protostomes. The deuterostome NT duplicated twice to give rise to BDNF, NGF, NT3, and NT4 in vertebrates, and the protostome ancestor duplicated more than once to generate at least DNT1, DNT2, and spz, while sequences diverged, retaining the structural features of the NT Cysknot that enabled function (Zhu, 2008).

A similar scenario is encountered in the tumour necrosis factor (TNF) superfamily, in which sequence similarity and identity between TNF members is restricted to the TNF homology domain where it is also low (19%-30%), but they are nevertheless considered members of a protein superfamily based on structural and functional conservation. Thus, deuterostomian invertebrate NTs (Bf-NT, Sp-NT, and Sk-NT) belong to the NT superfamily based on sequence similarity in the Cysknot, and this study shows that DNT1, DNT2, and Spz belong to the NT superfamily based on sequence, structural, and functional criteria (Zhu, 2008).

It had long been thought that NTs were missing from the Drosophila genome. A similarity between Spz and NGF had been previously proposed but remained controversial. First, structural considerations had also revealed a similarity between Spz and horseshoe crab coagulogen, involved in the blood-clotting cascade. This phylogenetic analysis does not resolve coagulogen as sufficiently distinct from DNT1, Spz, or the NTs. The Toll signalling cassette is conserved in horseshoe crab, including a Toll receptor and the downstream target NFkappaB. Although it is unknown whether coagulogen may also have NT function in the horseshoe crab CNS, it is an intriguing possibility. This study shows that that FUGUE analysis comparing DNT1 to all proteins of known structure reveals a closer relationship of DNT1 to vertebrate NTs than to coagulogen (Zhu, 2008).

Second, an initial comparison of the sequenced human and Drosophila genomes with BLAST reported that there were no NTs in Drosophila. However, this simple BLAST missed 30% of the Drosophila genes and would have missed any proteins with structural conservation despite sequence divergence. In fact, a recent report has reiterated the relationship of Spz to the NT superfamily (Weber, 2007). This study identified DNT1 using searches optimised for distantly related sequences, PSI-BLAST and FUGUE. In PSI-BLAST sequence searches, carp BDNF reveals sequence relatedness of DNT1 to NTs. Reverse BLAST and PSI-BLAST reveal similarity of DNT1 to BDNF from multiple fish species and humans. Structure-based searches with FUGUE demonstrate that DNT1 is structurally related to human BDNF, NGF, NT3, and NT4. Thus, DNT1 retains the features of all four human NTs. Thus, there is high sequence divergence among the NTs that nevertheless retain the functional Cysknot (Zhu, 2008).

The neurotrophic theory originally proposed that NTs promote neuronal survival in a target-dependent manner, although NTs can also promote neuronal survival prior to innervation and in autocrine and paracrine manners. Important evidence that vertebrate NTs promote neuronal (and glial) survival was the finding that exogenous application of NTs rescues neurons (and glia) from NOCD, both in cell culture and in vivo. This study finds that expressing DNT1 either in all CNS neurons or at the midline can rescue NOCD in vivo. Expressing DNT2 or activated Toll in all CNS neurons also rescues NOCD. These findings indicate that, like in vertebrates, the DNTs can promote cell survival. The prosurvival functions of the DNTs are nonautonomous as the three DNTs are expressed virtually only at the CNS midline, but in the mutants, apoptosis is induced throughout the VNC; DNT1-RNAi targeted to the midline induces apoptosis throughout the VNC, and overexpression of DNT1 only at the midline rescues NOCD throughout the VNC (Zhu, 2008).

Loss of vertebrate NTs in individual mouse NT knockouts or their receptors affect the CNS very weakly, and do not generally cause an increase in CNS apoptosis. Loss of DNT1, spz, Toll, or DNT2 function does not cause massive CNS neuronal death either. Nevertheless, apoptosis increases significantly in the embryonic CNS in all DNT mutants. The dying cells are at least partly HB9 and Eve neurons. No significant apoptosis were found phenotypes in DNT1 mutants or upon gain of function in the developing retina (Zhu, 2008).

Vertebrate NTs play partially redundant functions: some can substitute for one another to rescue apoptosis in mutants, and in multiple knock-out combinations, e.g., BDNF^-/-NT3^-/-NT4^-/- or TrkB^-/-TrkC^-/-, a 20% reduction in motor neurons and a dramatic increase in brain apoptosis, respectively, were observed compared to single mutants. The DNTs play redundant roles in the embryonic CNS in some, but not all, contexts. Expression of activated spz in DNT1⁴¹ mutant embryos is not sufficient to fully rescue apoptosis (however, we have not tested the reciprocal experiment), but apoptosis increases in DNT1^-/- DNT2^-/- double mutants, indicating redundancy between DNT1 and DNT2 for cell survival (Zhu, 2008).

Vertebrate NT function depends on neuronal modality: different neurons require different NTs for survival, and increases in apoptosis in the brain were observed when looking at specific neuronal types (e.g., parvalbumin-positive neurons in BDNF knock-out mice). In DNT1 mutants, an increase in apoptosis of HB9- and Eve-positive neurons was observed, and loss of Eve neurons. Neuronal modality differences are revealed in the targeting by motor axons. Alterations in DNT1 function affect primarily ISNb/d motor axons, whereas loss of Spz function affects SNa motor axons, correlating with complementary domains of spz and DNT1 expression in different subsets of muscles (Zhu, 2008).

Locomotion deficits and/or lethality are a further feature of NT knock-out mice. In fruit flies, some double-mutant combinations of the DNTs and triple mutants die during embryogenesis. DNT1 DNT2 double-mutant and spz² mutant viable adult flies have distinct locomotion and/or behavioural deficits. Locomotion defects can reflect proprioception or muscle or synaptic problems. NTs play roles in synaptic plasticity, LTP, and behaviour, and altered NT function causes psychiatric and cognitive disorders in humans. At least DNT1 is expressed in the adult central brain in the centres controlling learning and memory. Perhaps the DNTs are involved in higher neuronal functions (Zhu, 2008).

DNT1 produces two types of transcripts: the longer contain the Cysknot domain (cDNA3), and shorter ones (cDNA 1, cDNA2, and cDNA4) comprise only most of the pro-domain. Expression of the shorter isoform does not rescue apoptosis, rather it (and the full-length protein) may increase it. This is reminiscent of the opposite functions of the mature and full-length vertebrate NTs in the control of neuronal survival and death, respectively, and of the fact that in transgenic flies, full-length Spz is not functional in immunity, whereas the cleaved Cysknot is. It is not known whether the shorter DNT1 isoforms play other roles, but conceivably they may modulate the function of mature DNT1, as the pro-domain of spz can inhibit signalling by the Spz-Cysknot (Zhu, 2008).

Loss of vertebrate NTs severely affects the PNS, and rather weakly affects the motor neurons. Virtually all vertebrate PNS neurons require NTs for survival. In Drosophila, the effect of DNT1 mutations in the embryonic PNS is milder than in the CNS (unpublished data). Exogenous application of NTs can rescue vertebrate motor neuron survival, but loss of individual vertebrate NTs does not induce motor neuron apoptosis. Only 20%-30% of motor neurons die in triple knock-out mice lacking multiple NTs or all Trk receptors . In fact, the main trophic factor maintaining vertebrate motor neuron survival is GDNF, which does not belong to the NT superfamily. Motor neurons are not produced in vast excess in Drosophila, but there is motor neuron apoptosis in normal embryos, as detected with the motor neuron markers HB9 and Eve, although the underlying cause is not known. A significant increase was observed in HB9 neuronal apoptosis in DNT1 mutant embryos compared to wild type (although HB9 also labels interneurons). Loss of Eve motor neurons is also observed in DNT1 mutants, as well as loss of all the FasII-positive ISNb/d axons in triple-mutant embryos. It has previously been reported that RP motor neurons can be missing in Toll mutant embryos, although this could reflect an autocrine function. It was not possible to conclusively determine whether motor neuron death in DNT1 and triple mutants is due to the target-derived function of DNTs in the muscle, or an autocrine/paracrine requirement in the motor neurons. Expression of DNTs at the midline could influence the motor neurons within the CNS. Abundant evidence indicates that motor neurons live and function well in the absence of the muscle target in Drosophila. For instance, upon genetic elimination or surgical ablation of the muscle and in the absence of muscle-derived signals, motor neurons grow towards the muscle but fail to target or target to ectopic sites. In normal embryos and larvae, the projection patterns of motor neurons is very stereotypic. Accordingly, it would appear that motor neuron survival may not depend on the target muscle in Drosophila embryos and larvae (Zhu, 2008).

Vertebrate NTs influence muscle innervation by motor neurons. In Drosophila, the existence of a muscle-derived sprout-promoting factor to which Toll-expressing motor neurons would respond had been anticipated (Halfon, 1995). This study has shown that a target-derived function of DNTs in the muscle is required for guidance and targeting by motor axons. Loss of function for all three DNTs, as well as gain of DNT1 function, disrupts axon guidance and targeting by motor axons. The domains of expression of DNT1 and spz in the muscles are complementary, and both overlap that of DNT2. Consistently, DNT1 and spz, together with DNT2, affect targeting by complementary sets of motor axons, and the triple mutants have dramatic defects in all motor neuron projections (Zhu, 2008). The larval neuromuscular junction (NMJ) offers the most amenable synapse in Drosophila. There is abundant evidence of synaptic plasticity at the NMJ. However, so far, the identification of the responsible retrograde signals has been rather scarce. The identification of the muscle-derived secreted DNTs is promising in this context (Zhu, 2008).

All three DNTs are expressed at the CNS midline and in the muscles. At least the Spz receptor Toll is expressed transiently in the muscle; Toll and spz mutants have muscle defects, and Toll is involved in motor neuron synaptogenesis, although some of the Toll mutant muscle defects may be due to nonautonomous effects. Muscle defects have also been observedin spz mutants and most severely in the triple mutants. However, targeting errors were also observed in the presence of normal muscle patterns, indicating that targeting and putative muscle functions can be dissociated. The possibility cannot be ruled out that DNTs may play roles in midline-derived glia or neurons, including motor neurons, or in the muscles. Interestingly, vertebrate NTs also have functions in the muscle (Zhu, 2008).

Signalling by DNT1 and DNT2 may not necessarily proceed by binding canonical vertebrate-like Trk and p75 receptors. Ligand and receptor pairs do not necessarily coevolve. For instance, Toll-like receptors are highly conserved, but bind very different ligand types in flies and vertebrates. DNT1 and DNT2 may bind yet-unidentified Trk and p75 homologs in Drosophila or other receptors that activate equivalent signalling pathways and result in equivalent cellular, neurotrophic responses. Trk homologs were originally reported in Drosophila and subsequently showed not to belong to the Trk family. However, a Trk homolog has been found in the protostome mollusc Lymnea, suggesting that either Trks may have been lost in Drosophila or not found. Trk receptors are modular, thus exon shuffling during evolution could have led to the separation of domains into different proteins while retaining function. Consistently, an intracellular Trk-like tyrosine kinase domain has been found in Aplysia in a receptor, ApTrk, with an extracellular domain unrelated to the Trks. The converse situation is conceivable (Zhu, 2008).

DNT1 may bind a receptor tyrosine kinase, or a TNFR-like receptor (as p75 is), or resembling Spz, a Toll-like receptor, or, as with vertebrate NTs, DNT1 may be a promiscuous ligand binding multiple receptor types. As with vertebrate NT receptors, binding to one receptor type may result also in interactions with other receptors that alter cellular outcomes depending on context. There is a TNF receptor and multiple Toll-like receptors in Drosophila. Signalling by Toll and mammalian Toll-like receptors underlies innate immunity, and it is an ancient pathway present also in the cnidarian Nematostella and in C. elegans. Vertebrate NTs are also involved in immunity. Perhaps Toll signalling is an ancient mechanism underlying the functions of both the nervous and immune systems. Interestingly, the extracellular domain of Toll resembles that of Trk receptors (with the unusual combination of Leu-rich repeats and cysteine repeats), and intracellularly, Toll activates a downstream signalling pathway very similar to that of p75, resulting in the activation of NFkappaB. The current data indicate that the evolutionary trajectory of neurotrophin signalling in arthropods travelled through (although may not be restricted to) Toll. DNTs may also bind other receptor types (Zhu, 2008).

Toll, p75 and the TNFR family are more ancient than the Trks. Drosophila Spz/Toll, and vertebrate Toll-related, p75 and TNFR receptors signal through NFkappaB (promoting cell survival) and c-Jun (promoting cell death). Vertebrate Toll-like-related receptors also activate MAPKinases, and p75 also activates AKT. These pathways are compatible with the neurotrophic functions of DNT1, DNT2, and Spz. NFkappaB is also involved in synapse formation, synaptic plasticity, learning, and memory, and alterations in NFkappaB function also lead to psychiatric conditions. Inhibition of NFkappaB signalling in crabs (protostome arthropods like flies) leads to deficits in learning and memory, functions traditionally assigned to NTs. Conceivably, also higher functions of DNTs may be controlled by NFkappaB (Zhu, 2008).

The findings and those of others suggest that the evolution of neurotrophin signalling may have resulted in diversification of receptors and/or downstream signalling pathways (Zhu, 2008).

DNT1 sequences were not found in the snail Aplysia. This could mean that NTs appeared independently in deuterostomes and insects, and their similarity is due to convergence. However, it is equally possible that structure and function were conserved despite high sequence divergence, that the sequences have not been found yet, or that NT were lost from some or many animals. A Trk-like tyrosine kinase domain has been found in Aplysia, ApTrk, and a bona fide Trk ortholog in another snail, Lymnea, suggesting that the NT signalling pathway is present in molluscs. The unsuccessful search in Aplysia is likely due to incomplete genome sequence and expressed sequence tag (EST) collection (Zhu, 2008).

If a NT was present in Urbilateria, then NTs may be important in the nervous system development and function of all animals with a centralised nervous system or brain. What about simpler animals such as anemones and corals, which do not have a centralised nervous system, but a diffuse, nerve net? To ask this, NTs were sought in a cnidarian, Nematostella, but no DNT1 homolog was found. Sequence divergence and/or incomplete EST database may have also prevented the identification of NT sequences in Nematostella. Orthologs of Toll and downstream targets of Toll, p75, and Trk receptors, such as NFkappaB, MAPKinase, and ERK, are all present in Nematostella. Alternatively, NTs may have originated in Urbilateria and are absent from simpler animals, or perhaps a preexisting NT may have been lost in Nematostella and other cnidarians (just as NTs were lost in the deuterostome Ciona, as extensive gene loss is known to have occurred in cnidarians. Consistently with the view that elaborations of neurotrophin signalling underlie brain complexity, perhaps the diffuse net structure of the cnidarian nervous system does not require neurotrophin signalling, resulting in their loss. However, the acorn worm also has a diffuse, nerve net nervous system, and it has a NT and p75 receptor. This suggests that NTs may also be present in other animals with a nerve net, where they may have a subset of functions (e.g., axon guidance, connectivity, or synaptic functions) (Zhu, 2008).

These data suggest that a NT was most likely present in Urbilateria, the common ancestors of all bilateral organisms, protostomes and deuterostomes. This NT duplicated independently in vertebrates and invertebrates, and NTs were retained in organisms with a centralised nervous system and/or brain. NTs may be more ancient and have been either retained or lost in animals with diffuse neuronal nets. The current findings imply that the control of cell survival and targeting by the NT superfamily is an ancient mechanism of nervous system development. Further functions of the DNTs could also include synaptic and neuronal activity, learning, and memory. The current findings support the notion of a common origin for nervous system centralisation in evolution. They suggest that in the course of evolution 'elaborations of what went before' — an available molecular mechanism involving the ancestral NTs—and 'tinkering' with NT signalling accompanied the diversification of nervous systems and behaviours (Zhu, 2008).

The identification of DNTs bridges a void in neuronal studies using Drosophila as a model for understanding the brain. In flies, conserved molecular mechanisms involving the NT superfamily may underlie aspects of retrograde transport, dendrite formation, axonal remodelling, synaptic plasticity, LTP, and learning and memory -- these are all functions for which NTs are responsible in vertebrates. This work opens a wide range of opportunities to further the understanding of brain formation and evolution and to model human brain diseases using Drosophila (Zhu, 2008).

Toll-6 and Toll-7 function as neurotrophin receptors in the Drosophila melanogaster CNS

Neurotrophin receptors corresponding to vertebrate Trk, p75^NTR or Sortilin have not been identified in Drosophila, thus it is unknown how neurotrophism may be implemented in insects. Two Drosophila neurotrophins, DNT1 and DNT2, have nervous system functions, but their receptors are unknown. The Toll receptor superfamily has ancient evolutionary origins and a universal function in innate immunity. This study shows that Toll paralogs unrelated to the mammalian neurotrophin receptors function as neurotrophin receptors in fruit flies. Toll-6 and Toll-7 are expressed in the CNS throughout development and regulate locomotion, motor axon targeting and neuronal survival. DNT1 (also known as NT1 and spz2) and DNT2 (also known as NT2 and spz5) interact genetically with Toll-6 and Toll-7, and DNT1 and DNT2 bind to Toll-6 and Toll-7 promiscuously and are distributed in vivo in domains complementary to or overlapping with those of Toll-6 and Toll-7. It is concluded that in fruit flies, Tolls are not only involved in development and immunity but also in neurotrophism, revealing an unforeseen relationship between the neurotrophin and Toll protein families (McIlroy, 2013).

The Toll receptor superfamily, comprising Toll and Toll-like receptors (TLRs), has ancient evolutionary origins, arising over 700 million years ago, and is present throughout metazoans. Toll and TLRs have a universal function in innate immunity, and they initiate adaptive responses in vertebrates. In humans the ten TLRs are pattern recognition receptors that directly bind to microbial antigens and activate proinflammatory and co-stimulatory responses. Mammalian TLRs were identified by homology to Drosophila Toll (Toll-1). The Drosophila genome contains nine Toll receptor genes (Toll-1 to Toll-9), which, except for Toll-9, are phylogenetically distinct from the vertebrate TLRs. Thus, Drosophila Toll-1 to Toll-8 form one clade and Toll-9 together with vertebrate TLRs form another. Toll-1 functions in developmental processes, including the establishment of the embryonic dorso-ventral axis, in axon targeting and degeneration, and in innate immunity, but the roles of the remaining Tolls are largely unresolved. Reports have indicated that Toll-7 to Toll-9 have developmental functions but no antibacterial immunity functions, although Toll-7 is involved in antiviral responses, and Toll-6 and Toll-7 are expressed in the CNS. Unlike the TLRs, Toll-1 does not bind microbial products directly. Instead, detection of bacterial molecules by the soluble recognition proteins PGRP and GNBP triggers a serine protease cascade. This leads to the cleavage and activation of Spätzle (Spz), an endogenous protein ligand for Toll-1 (McIlroy, 2013).

Spz belongs to the neurotrophin family of growth factors, which in vertebrates comprises nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3) and neurotrophin-4 (NT4). Spz comprises a signal peptide, an unstructured pro-domain and an active cystine knot domain of 13 kDa (also known C-106), which dimerizes, binding Toll with 2:2 stoichiometry. Spz is secreted as a pro-protein and is cleaved extracellularly by the serine proteases Easter, acting in development, and Spätzle Processing Enzyme (SPE), acting in immunity, to release the active cystine knot. This mechanism resembles the extracellular cleavage of BDNF at the synaptic cleft by the serine protease plasmin (which is also involved in the blood-clotting cascade) and which is activated by the presynaptic release of plasminogen activating factor (tPA) upon high frequency stimulation. The characteristic neurotrophin cystine knot, formed by antiparallel β-sheets held together by three intersecting disulfide bonds, can be precisely aligned between the crystal structures of Spz and NGF (McIlroy, 2013).

DNT1 was identified independently as related to BDNF, using vertebrate neurotrophin sequences as query to search the Drosophila sequenced genome with bioinformatics tools. DNT1 was found to be spz2, a paralog of spz. Structural prediction analysis showed that, of the spz paralogs, DNT1 and DNT2 (spz5) (Parker, 2001) are closest to the neurotrophin superfamily, followed by spz (McIlroy, 2013).

There is also functional conservation between DNT1, DNT2 and Spz and the mammalian neurotrophins in the nervous system (Zhu, 2008). The vertebrate neurotrophins have essential functions during development in neuronal survival, axon targeting and connectivity and during adult life in learning, memory and cognition. During development, DNT1, DNT2 and spz are expressed in target cells for CNS neurons, such as the embryonic en-passant midline target of interneurons and the muscles, the target of motor neurons DNT1 and DNT2 are required for neuronal survival, as neuronal apoptosis decreases upon their overexpression in the CNS and increases in the loss-of-function mutants, leading to neuronal loss, with apoptotic neurons comprising those identified as bearing the Even-skipped (Eve) or Homeobox 9 (HB9) neuronal markers. DNTs are required for motor-axon targeting, as interfering with the function of DNT1, DNT2 and Spz causes misrouting, mistargeting and sprouting defects in motor axon terminals. Thus, DNT1 and DNT2, as well as Spz, are Drosophila neurotrophins on the basis of sequence, structural and functional homology to the vertebrate neurotrophins (McIlroy, 2013).

There is further cellular and molecular evidence that neurotrophism operates in the Drosophila nervous system. During normal Drosophila development, many neurons and glial cells die, and ablation or mutation in glial cells results in neuronal death in several contexts. Identified Drosophila neurotrophic factors include the homolog of mesencephalic astrocyte-derived neurotrophic factor (MANF), which promotes dopaminergic neuron survival in fruit flies using a noncanonical pathway (Palgi, 2009), and Netrin, which promotes interneuron survival from the en-passant midline target. Gliotrophic factors of the transforming growth factor (TGF)-α, neuregulin and PVF/platelet-derived growth factor (PGDF) protein families have also been shown to maintain glial survival in Drosophila (McIlroy, 2013).

The mammalian neurotrophins signal through three distinct receptor types (p75^NTR, Trk and Sortilin) and share a downstream target, the activation of NF-κB. In Drosophila there are no canonical homologs of these receptors. The receptors for DNT1 and DNT2 are unknown, although one hypothesis is that orphan Tolls fulfill this function in insects. Toll receptors are generally thought to function by activating NF-κB signaling, which regulates the production of antimicrobial peptides in immunity. Neurotrophins also function in immunity, but these roles have been largely unexplored. TLRs are also present in the CNS, primarily in microglia, where they have immunity-related functions (Rivest, 2009). Thus, potential relationships between the Toll and neurotrophin families may have been overlooked. This study asked whether Toll-6 and Toll-7 can function as receptors for DNT1 and DNT2 during CNS development (McIlroy, 2013).

This study found that neurotrophic functions in the fruit fly are carried out by Toll-7 and Toll-6 binding DNT1 and DNT2, respectively. Toll-6 and Toll-7 are expressed in the locomotor circuit, including motor neurons and interneurons of the embryonic CNS central pattern generator and locomotion centers of the adult central brain. By removing Toll-6 and Toll-7 function in mutants or adding them in excess, it was shown that Toll-6 and Toll-7 are required for normal locomotion and motor axon targeting, and to maintain neuronal survival. In the absence of Toll-6 and Toll-7 function, at least some of the dying cells are HB9⁺ and Eve⁺ EL interneurons that normally express the receptors. Using genetic interaction analysis, it was shown that Toll-6 and Toll7 function together with DNT1 and DNT2 in vivo. Using biochemical approaches in vitro, in cell culture and in vivo, it was shown that Toll-6 and Toll-7 directly bind DNT2 and DNT1, respectively. Finally, the relative in vivo protein distribution patterns of the ligands and the receptors are consistent with their shared functions. Most importantly, it was shown that Toll receptors underlie neurotrophism in fruit flies, which is therefore implemented using a different molecular mechanism from the canonical vertebrate mechanism involving p75^NTR, Trks and Sortilin (McIlroy, 2013).

The data show that Toll-6 and Toll-7 have neurotrophic functions in the Drosophila CNS matching those of DNT1 and DNT2. As in the mammalian neurotrophin system, these functions are pleiotropic. Mammalian neurotrophin ligands and receptors have functions ranging from maintaining neuronal survival to axon targeting, dendritic arborization and synaptic transmission, which vary with context, cell type and time. For instance, whereas vertebrate neurotrophins and Trk receptors maintain neuronal survival in the peripheral nervous system, they do not have a prominent role in maintaining motor neuron survival, instead functioning at the neuromuscular junction in synaptogenesis and synaptic plasticity. The data show that Toll-6 and Toll-7 also have pleiotropic functions, maintaining predominantly interneuron survival and regulating motor-axon targeting (McIlroy, 2013).

The data indicate that Toll-7/DNT1 and Toll-6/DNT2 are the most likely ligand-receptor pairs, but there appears to be promiscuity in ligand binding, as at least DNT2 can bind both receptors. This may also be the case for DNT1, but pure mature DNT1 protein could not be obtained using the baculovirus system, restricting the tests that could be performed. Such promiscuity may account for the redundancy between Toll-6 and Toll-7 observed in genetic and functional tests (for example, compromised locomotion and viability in the double mutants only). It may indicate that in vivo the binding partners might be determined by the relative temporal and spatial distribution patterns of the proteins. Alternatively, it is also conceivable that DNT1 and DNT2 have distinct functions and may bind each receptor according to functional requirements. DNT1 and DNT2 have distinct biochemical properties: whereas DNT2 is consistently secreted from S2 cells as a mature, cleaved form consisting of the cystine knot domain, DNT1 is secreted both as full-length and mature forms, as well as products of cleavage in the disordered pro-domain. The protease that might cleave DNT1 in vivo is unknown, but these properties are akin for DNT2 to the intracellular cleavage of NGF and for DNT1 the extracellular cleavage of BDNF. In either case, the observed promiscuity is reminiscent of the binding of all mammalian neurotrophins to a common p75^NTR receptor (McIlroy, 2013).

Although vertebrate neurotrophin receptors are structurally and functionally distinct from the Tolls, both regulate NF-κB. NF-κB is also one of the transcription factors that activates the innate immune response downstream of the TLRs, and it also has extensive and highly conserved functions in neurons. Neuronal NF-κB controls gene expression as a potent prosurvival factor; it controls neurite extension; it also has non-nuclear synaptic functions, including the clustering of glutamate receptors; and it underlies synaptic plasticity during learning and memory, from crustaceans to mammals. In humans, alterations in NF-κB function lead to psychiatric disorders. Previous reports have shown that Toll-6 and Toll-7 do not activate Drosomycin upon immune challenge, indicating that Toll-6 and Toll-7 do not have innate immunity functions and do not activate NF-κB&-Dif in cell types involved in immunity. Future work will focus on elucidating the signaling mechanism downstream of Toll-6 and Toll-7 in the CNS and, in particular, to determine whether it uses downstream signal transducers such as MyD88 that are required for the immune and developmental functions of Toll-1. The mammalian TLR-8 is required for neurite extension in the neonatal brain, but this activity is not MyD88 dependent. Thus, although the current data do not confirm or refute whether Toll-6 and Toll-7 can signal through the canonical Toll signaling pathway, they do show that Toll-6 and Toll-7 function upstream of NF-κB (McIlroy, 2013).

This conclusion is supported by several observations reported in this study. First, in cell culture, activated forms of Toll-6 and Toll-7 and stimulation with DNT ligands were able to induce NF-κB signaling via Dorsal and Dif. Second, in vivo, overexpression of activated Toll-6^CY and Toll-7^CY in retinal photoreceptor neurons resulted in the elevation of Dorsal, Dif and Cactus proteins, as was previously reported for Toll-1. Third, in vivo, overexpression in neurons of activated Toll-6^CY and Toll-7^CY, like activated Toll^10b, rescued the semi-lethality of the spz² mutation; and conversely, overexpression of activated Toll^10b in neurons rescued the semi-lethality of the DNT1 DNT2 double mutation. The data also show that signaling by Toll-6 and Toll-7 differs in at least some respects from that mediated by Spz-Toll-1. For example, in cell culture the activation of NF-κB signaling by Toll-6 and Toll-7 was not as strong as that reported by others to be induced by Toll-1; and in vivo genetic rescues revealed a specific and stronger relationship between Toll-6 and Toll-7 and DNT1 and DNT2, compared to Toll-1. Understanding the molecular mechanisms of Toll-6 and Toll-7 signaling that underlie the developmental programs that they promote is a key objective of future research (McIlroy, 2013).

Notably, NF-κB, p75^NTR and Toll receptors are all evolutionarily very ancient molecules, present in cnidarians (for example, Nematostella); thus, they evolved long before the common ancestor of flies and humans and since the origin of the nervous and immune systems. Of note, the Toll homolog in the worm Caenorhabditis elegans is expressed in neurons and can implement an immune function by means of a behavioral response of pathogen avoidance. p75^NTR is a member of the tumor necrosis factor receptor superfamily, which is closer to the Tolls than to the Trks. Toll receptors resemble p75^NTR intracellularly, through their ability to activate a downstream signaling pathway resulting in the activation of NF-κB, and Trk receptors in the extracellular ligand-binding module, with a combination of leucine-rich repeats and cysteine repeats. Trk receptors, with an intracellular tyrosine kinase domain, emerged later in evolution. Although Toll receptors are evolutionarily conserved, they are not, at least in the innate immunity context, activated by the same ligands in flies and humans. This raises questions: if in Drosophila the Trk receptors were lost and Tolls are the only neurotrophic receptors, is this a key difference that underlies the distinct brain types and behaviors in flies and humans? In the course of evolution, did the Tolls become specialized for immunity functions in vertebrates? Or is the relationship uncovered in this study between the neurotrophin-ligand and Toll-receptor superfamilies an ancient mechanism of nervous system formation? In mammals TLRs also have nervous system functions, including ones in neurogenesis, neurite growth, plasticity and behavior, but the endogenous ligands in the mammalian CNS are unknown. A key objective of future research will be to investigate whether the neurotrophin and TLR protein families interact in the mammalian brain, particularly in the context of learning, memory, and neurodegenerative and neuroinflammatory diseases (McIlroy, 2013).

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). 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. This study investigated 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, in neurons only, rescued the semi-lethality of spz and DNT1;DNT2 double mutants, indicating retrograde functions in neurons. In spz mutants, DNT1;DNT2 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. Frequency and amplitude of spontaneous events were reduced in spz² mutants suggesting defective neurotransmission. These 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 (Sutcliffe, 2013).

These data show that the Drosophila NTs DNT1, DNT2 and Spz are produced in muscles, required at the larval NMJ synapse with neuron type specificity, that alterations in their function affect synaptic structure and, at least in the case of Spz, also physiology. The spz² allele is a mutation in the pro-domain that interferes with the secretion of Spz in cell culture. These data show that the semi-lethality of the spz² allele can be rescued with the over-expression of activated Toll^10b in neurons implying that the spz² mutation causes a reduction in normal spz function. The mechanism by which the spz² allele affects function is not currently known, and this is a question that should be solved in the future. The DNT1⁴¹ and DNT1⁵⁵ mutant alleles are null and produce no protein. DNT2^e03444 is a Piggy-Bac insertion allele that is hypomorphic. It has not been possible to generate a DNT2 null allele, thus it is conceivable that a null might have revealed more dramatic phenotypes. It was shown that DNT1, DNT2 and spz are expressed in the larval body wall muscle, and the temperature sensitive semi-lethality of the mutants was used to address the question of whether the DNTs might be functional in neurons. The data showed that over-expression of cleaved DNT1CK’+, DNT2CK and spzCK restricted to neurons can rescue the semi-lethality of the mutants. No link was hypothesized between the larval muscle expression and adult survival, and it does not necessarily exist. The data show importantly that DNT1, DNT2 and Spz can function in neurons and that these neuronal functions are essential for viability (Sutcliffe, 2013).

Reduced Spz, DNT1 and DNT2 function increased terminal size and bouton number, caused abnormal post-synaptic bouton morphology, reduced number of active zones per bouton, reduced active zone density per NMJ terminal and increased shedding of synaptic material. The deficit in active zones in DNT1 DNT2 double mutants could be rescued by the over-expression of either DNT1CK3’+ or DNT2CK in neurons, demonstrating that this phenotype was the direct result of loss of DNT1 and DNT2 function. The experiments did not reveal rescue of the spz² phenotype, which could be due to technical reasons or to the nature of spz² allele (Sutcliffe, 2013).

To ease the analysis, an automatic method was developed to quantify anti-Brp (nc82) staining at the NMJ that was named DeadEasy Synapse. Data was acquired both using conventional manual counting and DeadEasy Synapse. Both methods revealed the same results, thus validating DeadEasy Synapse, which will be of great use to the Drosophila NMJ community. The plug-in works with ImageJ and will be made publicly available through the lab webpage (Sutcliffe, 2013).

Aberrant Spz function in the spz² allele resulted in reduced frequency and amplitude of spontaneous mEJPs. However, evoked EJPs were normal for all mutants examined (Sutcliffe, 2013).

Altogether, the data strongly suggest that DNT1, DNT2 and Spz are required for synaptogenesis. They also suggest that the increase in NMJ terminal size and bouton number in the mutants corresponds to a homeostatic structural adjustment that compensates for the reduced number of active zones, thus restoring the overall number of functional release sites and synaptic transmission. Remarkably, Spz functions at the muscle 4 NMJ, whereas the combined functions of DNT1 and DNT2 are required for the muscle 6,7 NMJ (Sutcliffe, 2013).

It has long been known that homeostatic compensatory mechanisms adjust the NMJ terminal to muscle size as the larva grows, to maintain synaptic efficacy within an appropriate physiological range. Increased synaptic growth is accompanied by a decrease in transmitter release per bouton resulting in normal muscle excitation. Conversely, mutants with fewer boutons have normal physiology, as each bouton has more active zones maintaining a constant overall number per NMJ. An analogous scenario is seen in mammals: in NT4 knockout mice, a reduction in post-synaptic AChR density induces a compensatory increase in NMJ terminal area. In Drosophila, it has long been anticipated that retrograde factors produced in the muscle regulate pre-synaptic neurotransmitter release, perhaps by regulating the number of presynaptic active zones in each bouton or some aspect of the presynaptic release mechanism, but their discovery has been scarce. One retrograde growth factor at the Drosophila NMJ is Gbb (Sutcliffe, 2013).

The data are consistent with the DNTs functioning as retrograde growth factors. Firstly, they are expressed at the muscle, and muscle over-expression of spz and DNT2 can rescue the semi-lethality of spz² mutants. Secondly, activated Toll rescues the semi-lethality of spz² mutants when expressed in neurons but not in muscle. Thirdly, DNT2 transcripts are localised at the boutons and over-expression of DNT2CK in neurons rescues the semi-lethality of DNT1⁴¹, DNT2^e0344 double mutants. However, further evidence that over-expressed cleaved spzCK, DNT1CK3’+ and DNT2CK using the GAL4 system can be secreted and taken up normally by receiving cells would be desirable. There is robust evidence that spzCK, DNT1CK3’+ and DNT2CK are functional cell-autonomously in the cells in which they are over-expressed using the GAL4/UAS system. However, inappropriate secretion upon over-expression of the cleaved forms could explain why over-expression from muscle is not as effective as over-expression from neurons at rescuing the semi-lethality of the mutants (Sutcliffe, 2013).

Autocrine and anterograde functions of the DNTs in neurons and/or muscle are also likely. Toll is expressed in muscle where it functions as an inhibitor of synaptogenesis. Targeting by motoraxons coincides with a downregulation of Toll at the target muscle, and over-expression of Toll in muscle reduces bouton number at the muscle 6,7 NMJ. The DNTs may also have bidirectional functions. DNT2 transcripts are localised post-synaptically, reminiscent of the post-synaptic expression of BDNF and NT4. Their transcripts are translated in response to neuronal activity and the combination of anterograde and retrograde functions results in synaptic potentiation. DNTs, perhaps particularly DNT2, may also have bidirectional functions at the synapse. The full characterisation of retrograde and/or anterograde functions of the DNTs must await the discovery of the receptors functioning at the NMJ, and the production of good antibodies that enable visualisation of the endogenous ligands (Sutcliffe, 2013).

The data indicate that the DNTs are required for synaptogenesis. DNT loss of function mutants display increased bouton number and increased terminal size but reduced number of active zones per bouton and normal EJPs. Most likely, the increase in terminal size and bouton number is a homeostatic compensation for the deficient formation and function of active zones. In DNT1⁴¹, DNT2^e0344 mutants, muscles are smaller compared to wild-type and active zone density is reduced, but the NMJ is larger. This suggests that since in DNT mutants boutons have fewer synapses, the NMJ expands making more boutons, thus compensating for the synaptic deficits and maintaining overall normal function. Over-expression of DNTs in neurons increases muscle size, ghost boutons and shed synaptic debris, phenotypes that are consistent with a function of the DNTs in promoting axonal and muscle growth and/or in synaptic transmission. On the other hand, the observation that loss and gain of DNT function results in increased production of synaptic debris and ghost boutons, could also reflect defective clearance of synaptic material (e.g. by muscle or glia) upon interference with DNT function (Sutcliffe, 2013).

In spz² mutants in physiological calcium levels no changes in synaptic transmission were observed; lowering the extracellular calcium concentration reveals reduced frequency and amplitude of mEJPs, correlating with a reduction in the number of active zones per bouton. Despite the aberrant frequency and amplitude of spontaneous mEJPs in spz² mutants in low calcium, evoked EJPs were normal under all conditions for all mutants examined and loss of DNTs did not affect synaptic transmission (Sutcliffe, 2013).

These phenotypes are reminiscent of those found upon manipulation of another invertebrate neurotrophin superfamily member in mollusks, Aplysia neurotrophin (ApNT). Alterations in ApNT levels influence synaptic structure and the formation of synaptic varicosities. Expression of a dominant negative form of the receptor Ap-Trk-DN in cultured neurons had no effect in synaptic transmission following a single pulse of 5-HT, and effects were only seen in Long Term Facilitation after 5 consecutive pulses. Similarly, expression of ApNT or bathing cells in ApNT also led to more pronounced increases in evoked potential in Long Term Facilitation. Conceivably, high frequency stimulation might reveal enhanced effects in synaptic transmission upon manipulation of DNT function, and this is something worth testing in the future (Sutcliffe, 2013).

To conclude, in the case of Drosophila, it is most likely that the increase in NMJ terminal size and bouton number in the DNT mutants corresponds to a homeostatic structural adjustment that compensates for the reduced number of active zones, restoring the overall number of functional release sites and subsequent synaptic transmission (Sutcliffe, 2013).

The DNTs are the first growth factors to be identified to have neuron-type specificity at the Drosophila NMJ: the spz² mutation affected the muscle 4 NMJ, and DNT1⁴¹, DNT2^e0344 double mutants affected the muscle 6,7 NMJ. This observed neuron-type specificity is reminiscent of the neuronal modality of mammalian NT and Trk receptor function in the central and peripheral nervous systems. However, this study analysed the muscles 4 and 6,7 NMJs, not all muscles in the larva, thus the possibility cannot be ruled out that the DNTs may have more redundant and less specific functions in other muscles. How the observed specificity comes about is not yet understood, since the current evidence indicates that the three ligands are expressed throughout the muscles. In future work, antibodies to the DNTs may provide higher resolution and reveal distinct distribution patterns. For now, the distribution of DNT2 transcripts in synaptic boutons is a significant difference. In any case, the data also show that there is some functional redundancy between Spz, DNT1 and DNT2. DNTs may be promiscuous ligands that in some circumstances (e.g. upon over-expression) can bind multiple receptors, and thus neurons may be able to respond to the excess of any of the DNTs. This is reminiscent of the redundancy between vertebrate NT ligands, and the fact that different NTs can bind the same Trk receptor. In the normal larva, the distinct NMJ-specific effects may reflect the distribution of the DNT receptors in distinct neuronal types, and testing this hypothesis will await the identification of the receptors for DNT1 and DNT2. In any case, the NMJ specificity precisely reflects the neuron type specificity for the DNTs that also takes place during motoraxon targeting in the embryo: DNT1 is required for targeting of ISNb/d and Spz for targeting of SNa motoraxons. Intriguingly, since specific DNTs appear to be required for both motor-axon targeting and synapse formation at the NMJ, this would suggest that such neuron-type specific functions serve to shape motor-neuron-muscle connectivity required for the organisation of locomotor behaviour. Future progress will tackle the identification of the DNT receptors, the basis of this neuron-type specificity and the relevance for behaviour (Sutcliffe, 2013).

Three-tier regulation of cell number plasticity by neurotrophins and Tolls in Drosophila

Cell number plasticity is coupled to circuitry in the nervous system, adjusting cell mass to functional requirements. In mammals, this is achieved by neurotrophin (NT) ligands, which promote cell survival via their Trk and p75NTR receptors and cell death via p75NTR and Sortilin. Drosophila NTs (DNTs; see NT1) bind Toll receptors (see Toll-6 & Toll-7) instead to promote neuronal survival, but whether they can also regulate cell death is unknown. This study show that DNTs and Tolls can switch from promoting cell survival to death in the central nervous system (CNS) via a three-tier mechanism. First, DNT cleavage patterns result in alternative signaling outcomes. Second, different Tolls can preferentially promote cell survival or death. Third, distinct adaptors downstream of Tolls can drive either apoptosis or cell survival. Toll-6 promotes cell survival via MyD88-NF-κB and cell death via Wek-Sarm-JNK. The distribution of adaptors changes in space and time and may segregate to distinct neural circuits. This novel mechanism for CNS cell plasticity may operate in wider contexts (Foldi, 2017).

Balancing cell death and cell survival enables structural plasticity and homeostasis, regeneration, and repair and fails in cancer and neurodegeneration. In the nervous system, cell number plasticity is linked to neural circuit formation, adjusting neuronal number to functional requirements. In mammals, the neurotrophin (NT) protein family [NGF, brain-derived neurotrophic factor (BDNF), NT3, and NT4] regulates neuronal number through two mechanisms. First, full-length pro-NTs, comprised of a disordered prodomain and a cystine-knot (CK) domain, induce cell death; in contrast, mature NTs formed of CK dimers promote cell survival. Second, pro-NTs bind p75NTR and Sortilin receptors, inducing apoptosis via JNK signaling, whereas mature NTs bind p75NTR, promoting cell survival via NF-κB and TrkA, B, and C, promoting cell survival via PI3K/AKT and MAPK/ERK. As the NTs also regulate connectivity and synaptic transmission, they couple the regulation of cell number to neural circuitry and function, enabling structural brain plasticity. There is abundant evidence that cell number plasticity occurs in Drosophila melanogaster central nervous system (CNS) development, with neurotrophic factors including NTs and mesencephalic astrocyte-derived neurotrophic factor (MANF), but fruit flies lack p75NTR and Trk receptors, raising the question of how this is achieved in the fly. Finding this out is important, as it could lead to novel mechanisms of structural plasticity for both flies and humans (Foldi, 2017).

The Drosophila NTs (DNTs) Spätzle (Spz), DNT1, and DNT2 share with mammalian NTs the characteristic structure of a prodomain and a conserved CK of 13-15 kD, which forms a disulfide-linked dimer. Spz resembles NGF biochemically and structurally, and the binding of its Toll-1 receptor resembles that of NGF to p75NTR. DNT1 (also known as spz2) was discovered by homology to BDNF, and DNT2 (also known as spz5) as a paralogue of spz and DNT1. DNT1 and 2 promote neuronal survival, and DNT1 and 2, Spz, and Spz3 are required for connectivity and synaptogenesis. Spz, DNT1, and DNT2 are ligands for Toll-1, -7, and -6, respectively, which function as NT receptors and promote neuronal survival, circuit connectivity, and structural synaptic plasticity. Tolls belong to the Toll receptor superfamily, which underlies innate immunity. There are nine Toll paralogues in flies, of which only Toll-1, -5, -7, and -9 are involved in immunity. Tolls are also involved in morphogenesis, cell competition, and epidermal repair. Whether DNTs and Tolls can balance cell number plasticity is unknown (Foldi, 2017).

Like the p75NTR receptor, Toll-1 activates NF-κB (a potent neuronal prosurvival factor with evolutionarily conserved functions also in structural and synaptic plasticity) signaling downstream. Toll-1 signaling involves the downstream adaptor MyD88, which forms a complex with Tube and Pelle. Activation of Toll-1 triggers the degradation of the NF-κB inhibitor Cactus, enabling the nuclear translocation of the NF-κB homologues Dorsal and Dorsal-related immunity factor (Dif), which function as transcription factors. Other Tolls have also been suggested to activate NF-κB. However, only Toll-1 has been shown to bind MyD88, raising the question of how the other Tolls signal in flies (Foldi, 2017).

Whether Tolls regulate cell death is also obscure. Toll-1 activates JNK, causing apoptosis, but its expression can also be activated by JNK to induce nonapoptotic cell death. Toll-2, -3, -8, and -9 can induce apoptosis via NF-κB and dSarm independently of MyD88 and JNK. However, in the CNS, dSarm induces axonal degeneration, but there is no evidence that it can promote apoptosis in flies. In other animals, Sarm orthologues are inhibitors of Toll signaling and MyD88, but there is no evidence that dSarm is an inhibitor of MyD88 in Drosophila. Thus, whether or how Tolls may regulate apoptosis in flies is unclear (Foldi, 2017).

In the mammalian brain, Toll-like receptors (TLRs) are expressed in neurons, where they regulate neurogenesis, apoptosis, and neurite growth and collapse in the absence of any insult. However, their neuronal functions have been little explored, and their endogenous ligands in neurons remain unknown (Foldi, 2017).

Because Toll-1 and p75NTR share common downstream signaling pathways and p75NTR can activate NF-κB to promote cell survival and JNK to promote cell death, this study asked whether the DNTs and their Toll receptors could have dual roles controlling cell survival and death in the Drosophila CNS (Foldi, 2017).

In the first regulatory tier, each DNT has unique features conducive to distinctive functions. Spz, DNT1, and DNT2 share with the mammalian NTs the unequivocal structure of the CK domain unique to this protein family. However, DNT1, DNT2, and Spz have distinct prodomain features and are processed differently, leading to distinct cellular outcomes. Spz is only secreted full length and cleaved by serine proteases. DNT1 and 2 are cleaved intracellularly by conserved furins. In cell culture, DNT1 was predominantly secreted with a truncated prodomain (pro-DNT1), whereas DNT2 was secreted mature. In vivo, both pro- and mature DNTs were produced from neurons. Interestingly, DNT1 also has an isoform lacking the CK domain, and Spz has multiple isoforms with truncated prodomains. Thus, in vivo, whether DNT1 and 2 are secreted full length or cleaved and whether Spz is activated will depend on the proteases that each cell type may express. Pro-DNT1 activates apoptotic JNK signaling, whereas mature DNT1 and 2 activate the prosurvival NF-κB (Dorsal and Dif) and ERK signaling pathways. Mature Spz does not activate ERK. This first tier is evolutionarily conserved, as mammalian pro-NTs can promote cell death, whereas furin-cleaved mature NTs promote cell survival. NF-κB, JNK, and ERK are downstream targets shared with the mammalian NTs, downstream of p75NTR (NF-κB and JNK) and Trks (ERK), to regulate neuronal survival and death. Thus, whether a cell lives or dies will depend on the available proteases, the ligand type, and the ligand cleavage product it receives (Foldi, 2017).

In a second regulatory tier, this study showed that the specific Toll family receptor activated by a DNT matters. Toll-6 and -7 could maintain neuronal survival, whereas Toll-1 had a predominant proapoptotic effect. Because there are nine Tolls in Drosophila, some Tolls could have prosurvival functions, whereas others could have proapoptotic functions. Different Tolls also lead to different cellular outcomes in immunity and development. Thus, the life or death of a neuron will depend on the Toll or combination of Tolls it expresses. Binding of Spz to Toll-1 is most likely unique, but DNT1 and 2 bind Toll-6 and -7 promiscuously, and, additionally, DNT1 and 2 with Toll-6 and -7 activate NF-κB and ERK, whereas pro-DNT1 activates JNK. This suggests that ligand prodomains might alter the affinity for Toll receptors and/or facilitate the formation of heterodimers between different Tolls and/or with other coreceptors to induce cell death. A 'DNT-Toll code' may regulate neuronal numbers (Foldi, 2017).

In a third tier, available downstream adaptors determine the outcome between cell survival and death. Toll-6 and -7 activate cell survival by binding MyD88 and activating NF-κB and ERK (whether ERK activation depends on MyD88 is not known), and Toll-6 can activate cell death via Wek, dSarm, and JNK signaling. Toll-6 was shown to bind MyD88 and Wek, which binds dSarm, and dSarm binds MyD88 and promotes apoptosis by inhibiting MyD88 and activating JNK. Wek also binds MyD88 and Toll-1. So, evidence suggests that Wek recruits MyD88 and dSarm downstream of Tolls. Because Toll-6 binds both MyD88 and Wek and Wek binds both MyD88 and dSarm, Wek functions like a hinge downstream of Toll-6 to facilitate signaling via MyD88 or dSarm, resulting in alternative outcomes. Remarkably, adaptor expression profiles change over time, switching the response to Toll-6 from cell survival to cell death. In the embryo, when both MyD88 and dSarm are abundant, there is virtually no Wek, and Toll-6 can only bind MyD88 to promote cell survival. As Wek levels rise, Toll-6 signaling can also induce cell death. If the Wek-Sarm-JNK route prevails, Toll-6 induces apoptosis; if the Wek-MyD88-NF-κB route prevails, Toll-6 signaling induces cell survival (Foldi, 2017).

Thus, the cellular outcome downstream of DNTs and Tolls is context and time dependent. Whether a cell survives or dies downstream of DNTs and Tolls will depend on which proteases are expressed nearby, which ligand it receives and in which form, which Toll or combination of Tolls it expresses, and which adaptors are available for signaling (Foldi, 2017).

How adaptor profiles come about or change is not understood. A neuronal type may be born with a specific adaptor gene expression profile, or Toll receptor activation may influence their expression. In fact, MyD88 reinforces its own signaling pathway, as Toll-6 and -7 up-regulate Dorsal, Dif, and Cactus protein levels and TLR activation increases Sarm levels. This study showed that apoptosis caused by MyD88 excess depends on JNK signaling. Because JNK functions downstream of Wek and dSarm, this suggests that MyD88, presumably via NF-κB, can activate the expression of JNK, wek, or dsarm. By positively regulating wek expression, MyD88 and dSarm could establish positive feedback loops reinforcing their alternative pathways. Because dSarm inhibits MyD88, mutual regulation between them could drive negative feedback. Positive and negative feedback loops underlie pattern formation and structural homeostasis and could regulate neuronal number in the CNS as well. Whether cell-autonomous or -nonautonomous mechanisms result in the diversification of adaptor profiles, either in time or cell type, remains to be investigated (Foldi, 2017).

Either way, over time the Toll adaptors segregate to distinct neural circuits, where they exert further functions in the CNS. Toll-1, -6, and -8 regulate synaptogenesis and structural synaptic plasticity. Sarm regulates neurite degeneration, and in the worm, it functions at the synapse to determine neuronal identity. The reporters used in this study revealed a potential segregation of MyD88 to the motor circuit and dSarm to the sensory circuit, but this is unlikely to reflect the endogenous complexity of Toll-signaling circuitry, as dsarmMIMIC- has a GFP insertion into one of eight potential isoforms, and dsarm also functions in the motor system (McLaughlin, 2016). Importantly, cell death in the normal CNS occurs mostly in late embryogenesis and in pupae, coinciding with neural circuit formation and remodeling, when neuronal number is actively regulated. Thus, the link by DNTs and Tolls from cell number to circuitry offers a complex matrix of possible ways to regulate structural plasticity in the CNS (Foldi, 2017).

This study has uncovered remarkable similarities between Drosophila Toll-6 and mammalian TLR signaling involving MyD88 and Sarm. All TLRs except TLR3 signal via MyD88 and activate NF-κB . Neuronal apoptosis downstream of TLRs is independent of NF-κB and instead depends on TRIF and Sarm1. Sarm1 is a negative regulator of TLR signaling, an inhibitor of MyD88 and TRIF. sarm1 is expressed in neurons, where it activates JNK and promotes apoptosis. However, the endogenous ligands for TLRs in the normal undamaged brains are not known. Preliminary analysis has revealed the intriguing possibility that NTs either can bind TLRs or induce interactions between Trks, p75NTR, and TLRs. It is compelling to find out whether TLRs regulate structural plasticity in the mammalian brain in concert with NTs (Foldi, 2017).

To conclude, DNTs with Tolls constitute a novel molecular system for structural plasticity in the Drosophila CNS. This could be a general mechanism to be found also in the mammalian brain and in other contexts as well, such as epithelial cell competition and regeneration, and altered in cancer and neurodegeneration (Foldi, 2017).

Toll-8/Tollo negatively regulates antimicrobial response in the Drosophila respiratory epithelium

Barrier epithelia that are persistently exposed to microbes have evolved potent immune tools to eliminate such pathogens. If mechanisms that control Drosophila systemic responses are well-characterized, the epithelial immune responses remain poorly understood. This study consisted of a genetic dissection of the cascades activated during the immune response of the Drosophila airway epithelium i.e. trachea. Evidence is presented that bacteria induced-antimicrobial peptide (AMP) production in the trachea is controlled by two signalling cascades. AMP gene transcription is activated by the inducible IMD pathway that acts non-cell autonomously in trachea. This IMD-dependent AMP activation is antagonized by a constitutively active signalling module involving the receptor Toll-8/Tollo, the ligand Spätzle2/DNT1 (Neurotrophin 1) and Ect-4, the Drosophila ortholog of the human Sterile alpha and HEAT/ARMadillo motif (SARM). The data show that, in addition to Toll-1 whose function is essential during the systemic immune response, Drosophila relies on another Toll family member to control the immune response in the respiratory epithelium (Akhouayri, 2011).

Epithelial responses are local responses to prevent the epithelium from unnecessary immune reactions. Since the recognition steps in Drosophila respiratory epithelia involve the transmembrane receptor PGRP-LC and occur within the extracellular space, it is expected that molecular mechanisms must be at work to prevent constitutive or excessive immune response in this tissue, particularly essential for animal growth and viability. This report presents data demonstrating that the transmembrane receptor Tollo is part of a signalling network, whose function is to specifically down-regulate AMP production in the trachea. Tollo antagonizes IMD pathway activation in the respiratory epithelium, and DNT1/Spz2 and Ect4/SARM are putative Tollo ligand and transducer, respectively, in this process. These data demonstrate that, in addition to the family founder Toll-1, another member of the Leucine-Rich-Repeats family of Toll proteins, is regulating the Drosophila innate immune response. Although it has been abundantly documented that every single mammalian TLR has an immune function, the putative implication of Toll family members, other than Toll-1 itself, in the Drosophila immune response has been a subject of controversy. Data showing that Drosophila Toll-9 over-expression was sufficient to induce AMPs expression in vivo has prompted the idea that Toll-9 could maintain significant levels of anti-microbial molecules, thus providing basal protection against microbes. However, a recent analysis of a complete Toll-9 loss-of-function allele has shown that this receptor is neither implicated in basal anti-microbial response nor required to mount an immune response to bacterial infection (Narbonne-Reveau, 2011. The present data are also fully consistent with a recent report showing that Toll-6, Toll-7 and Toll-8 are not implicated in systemic AMP production in flies, and demonstrate that a Toll family member, Tollo, is a negative regulator of local airway epithelial immune response upon bacterial infection. In contrast to Toll-1, whose activation is inducible in the fat body, Tollo pathway activation seems to be constitutive in the trachea. Despite these differences, both receptors use a member of the Spz family as ligand. Interestingly, sequence similarities, intron's size and conservation of key structural residues, indicate that Spz2/DNT1 is phylogenetically the closest family member to the Toll ligand Spz. Furthermore, both Spz and Spz2/DNT1 have been shown to have neurotrophic functions in flies. It would be of great interest to test whether Tollo also mediates Spz2 function in the nervous system (Akhouayri, 2011).

Both during embryonic development and immune response, Spz is activated by proteolytic cleavage. This step depends upon the Easter protease that is implicated in D/V axis specification and on SPE for Toll pathway activation by microbes. Since Spz orthologs are also produced as longer precursors, they are likely to be activated by proteolysis. The fact that Tollo and Spz2 loss-of-function phenotypes correspond to excessive AMP production, suggests that in wild-type conditions, the Tollo pathway is constitutively activated by an active form of the Spz2 ligand. This situation is reminiscent to that observed in the embryonic ventral follicle cells, in which a Pipe-mediated signal induces a constitutive activation of the Easter cascade leading to Spz cleavage, Toll activation and, in turn, ventral fate acquisition. It should be noted that Easter and one Pipe isoform are very strongly expressed in the trachea cells, and are candidate proteins in mediating Tollo activity in the respiratory epithelia (Akhouayri, 2011).

The fact that Ect4, but not dMyd88 mutant, loss-of-function mutant phenocopies Tollo mutant suggest that Ect4 could be the TIR domain adaptor transducing Tollo signal in the tracheal cells. Alternatively, Ect4/SARM could mediate Tollo function by interfering with IMD pathway signalling. In mammals, SARM is under the transcriptional control of TLR and negatively regulates TLR3 signalling by directly interfering with the association between the RHIM domain-containing proteins TRIF and RIP (Carty, 2006). Since PGRP-LC contains a RHIM domain as TRIF, and IMD is the Drosophila counterpart of RIP, one can envisage that Drosophila SARM could act by interfering with the PGRP-LC/IMD association required for IMD pathway signalling. Similarly to its function as a negative regulator in fly immunity, SARM is the only TIR domain-containing adaptor that acts as a suppressor of TLR signalling (Akhouayri, 2011).

One obvious question relates to the mode of action of Tollo on IMD pathway downregulation. Two mechanisms have been recently described that result in the down-regulation of the IMD pathway. The first one regulates PGRP-LC membrane localization, and is dependent on the PIRK protein (Lhocine, 2008). Upon infection, the intracellular PIRK protein is up-regulated and, in turn, represses PGRP-LC plasma membrane localization leading to the shutdown of the IMD signalling (Lhocine, 2008). In infected pirk mutants, IMD-dependent AMPs are overproduced in both the gut and the fat body. In the conditions used in this study, however, inactivation of PIRK specifically in the trachea did not influence Drosomycin activation in trachea. To verify whether Tollo is acting via a mechanism similar to PIRK, PGRP-LC membrane localization was examined using a UAS-PGRP-LC::GFP construct. PGRP-LC membrane localization was identical in wild-type and Tollo mutant tracheal cells. The second mechanism that modulates IMD activation, acts directly on the promoters of IMD target genes. Caudal transcription factor has been shown to sit on some of the IMD target promoters preventing their activation by Relish. The putative implication of Caudal in Tollo signalling was tested by using Drs-GFP reporter transgenes containing either wild-type Caudal Responsive Elements (CDREs) or mutated versions unresponsive to Caudal activity. Upon infection, Drs-GFP with mutated CDREs was activated in fat body but not in gut or trachea. In conclusion, Caudal acts as a transcriptional activator, rather than a repressor, for the Drs-GFP reporter in trachea. These results indicate that Tollo does not regulate the IMD pathway via PGRP-LC membrane localization or through promoter targeting of Caudal. One challenging task for the future will be to identify the mechanism used by Tollo to counter-balance tracheal PGRP-LC activation. It has been reported that the loss of Tollo function in ectodermal cells during embryogenesis alters glycosylation in nearby differentiating neurons. Since the pattern of oligosaccharides expressed in a cell can influence its interactions with others and with pathogens, Tollo could function by modifying glycosylation pattern in response to microbes. It could be envisaged that Tollo mediates PGRP-LC glycosylation, and thereby reduces its ability to respond to bacterial elicitors. Further work will be required to address the above hypothesis, whereby Tollo activity and glycosylation modification could be linked in order to regulate the IMD pathway activation in trachea (Akhouayri, 2011).

Search PubMed for articles about Drosophila Neurotrophin 1

Akhouayri, I., Turc, C., Royet, J. and Charroux, B. (2011). Toll-8/Tollo negatively regulates antimicrobial response in the Drosophila respiratory epithelium. PLoS Pathog. 7(10): e1002319. PubMed ID: 22022271

Carty, M., et al. (2006). The human adaptor SARM negatively regulates adaptor protein TRIF-dependent Toll-like receptor signaling. Nat. Immunol. 7: 1074-1081. PubMed ID: 16964262

Dearborn, R. and Kunes, S. (2004). An axon scaffold induced by retinal axons directs glia to destinations in the Drosophila optic lobe. Development 131: 2291-2303. PubMed ID: 15102705

Fischbach, K. -F. and Technau, G. M. (1984). Cell degeneration in the developing optic lobes of the sine oculis and small-optic-lobes mutants of Drosophila melanogaster. Dev. Biol. 104: 219-239. PubMed ID: 6428950

Foldi, I., Anthoney, N., Harrison, N., Gangloff, M., Verstak, B., Nallasivan, M. P., AlAhmed, S., Zhu, B., Phizacklea, M., Losada-Perez, M., Moreira, M., Gay, N. J. and Hidalgo, A. (2017). Three-tier regulation of cell number plasticity by neurotrophins and Tolls in Drosophila. J Cell Biol 216(5):1421-1438. PubMed ID: 28373203

Halfon, M. S., Hashimoto, C. and Keishishian, H. (1995). The Drosophila Toll gene functions zygotically and is necessary for proper motorneuron and muscle development. Dev. Biol. 169: 151-167. PubMed ID: 7750635

Hallböök, F., Wilson, K., Thorndyke, M. and Olinski, R. (2006). Formation and evolution of the chordate neurotorphin and Trk receptor genes. Brain Behav. Evol. 68: 133-144. PubMed ID: 16912467

Lhocine, N., et al. (2008). PIMS modulates immune tolerance by negatively regulating Drosophila innate immune signaling. Cell Host Microbe 4: 147-158. PubMed ID: 18692774

McIlroy, G., Foldi, I., Aurikko, J., Wentzell, J. S., Lim, M. A., Fenton, J. C., Gay, N. J. and Hidalgo, A. (2013). Toll-6 and Toll-7 function as neurotrophin receptors in the Drosophila melanogaster CNS. Nat Neurosci 16: 1248-1256. PubMed ID: 23892553

McLaughlin, C. N., Nechipurenko, I. V., Liu, N. and Broihier, H. T. (2016). A Toll receptor-FoxO pathway represses Pavarotti/MKLP1 to promote microtubule dynamics in motoneurons. J Cell Biol 214(4): 459-474. PubMed ID: 27502486

Narbonne-Reveau, K., Charroux, B. and Royet, J. (2011). Lack of an antibacterial response defect in Drosophila Toll-9 mutant. PLoS One 6: e17470. PubMed ID: 21386906

Sutcliffe, B., Forero, M. G., Zhu, B., Robinson, I. M. and Hidalgo, A. (2013). Neuron-type specific functions of DNT1, DNT2 and Spz at the Drosophila neuromuscular junction. PLoS One 8: e75902. PubMed ID: 24124519

Weber, A. N. R., et al. (2003). Binding of Drosophila cytokine Spatzle to Toll is direct and establishes signaling. Nat Immunol 4: 794-800. PubMed ID: 12872120

Weber, N. R., et al. (2007). Role of the Spätzle pro-domain in the generation of an active Toll receptor ligand. J. Biol. Chem. 282: 13522-13531. PubMed ID: 17324925

Xiong, W.-C. and Montell, C. (1995). Defective glia induce neuronal apoptosis in the repo visual system of Drosophila. Neuron 14: 581-590. PubMed ID: 7695904

Zhu, B., et al. (2008). Drosophila neurotrophins reveal a common mechanism for nervous system formation. PLoS Biol. 6(11): e284. PubMed ID: 19018662

date revised: 23 August 2017

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