InteractiveFly: GeneBrief

Neurotrophin 1: Biological Overview | References

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 α9?β. 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-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 Citation: 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 Citation: 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 Citation: 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 Citation: 6428950

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 Citation: 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 Citation: 16912467

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

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 Citation: 21386906

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 Citation: 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 Citation: 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 Citation: 7695904

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

date revised: 20 April 2012

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