InteractiveFly: GeneBrief

Toll-6 & Toll-7: Biological Overview | References


Gene name - Toll-6 & Toll-7

Synonyms -

Cytological map positions - 71C2-71C2 & 56E4-56E4

Functions - transmembrane receptor

Keywords - CNS, motor axon targeting, Neurotrophin receptor, Toll-like receptor, Innate immunity

Symbols - Toll-6 & Toll-7

FlyBase IDs: FBgn0036494 & FBgn0034476

Genetic map positions - chr3L:15329792-15337417 & chr2R:15714410-15718750

Classification - Toll-interleukin 1-resistance & Leucine rich repeat

Cellular location - surface transmembrane



NCBI links for Toll-6: Precomputed BLAST | EntrezGene

NCBI links for Toll-7: EntrezGene
Recent literature
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: 459-474. PubMed ID: 27502486
Summary:
FoxO proteins are evolutionarily conserved regulators of neuronal structure and function, yet the neuron-specific pathways within which they act are poorly understood. To elucidate neuronal FoxO function in Drosophila melanogaster, a screen was performed for FoxO's upstream regulators and downstream effectors. On the upstream side, genetic and molecular pathway analyses is presented indicating that the Toll-6 receptor, the Toll/interleukin-1 receptor domain adaptor dSARM, and FoxO function in a linear pathway. On the downstream side, it was found that Toll-6-FoxO signaling represses the mitotic kinesin Pavarotti/MKLP1 (Pav-KLP), which itself attenuates microtubule (MT) dynamics. In vivo functions were probed for this novel pathway, and it was found to be essential for axon transport and structural plasticity in motoneurons. Elevated expression of Pav-KLP underlies transport and plasticity phenotypes in pathway mutants, indicating that Toll-6-FoxO signaling promotes MT dynamics by limiting Pav-KLP expression. In addition to uncovering a novel molecular pathway, this work reveals an unexpected function for dynamic MTs in enabling rapid activity-dependent structural plasticity.
BIOLOGICAL OVERVIEW

Neurotrophin receptors corresponding to vertebrate Trk, p75NTR 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 (Yagi, 2010; Nakamoto, 2012; Tauszig, 2000; Ooi, 2002; Seppo, 2003), and Toll-6 and Toll-7 are expressed in the CNS (Kambris, 2002). 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 (Lu, 2005). 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 (Zhu, 2008). 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 (Zhu, 2008). 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 (Zhu, 2008). 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 (Newquist, 2013). 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 (p75NTR, 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 p75NTR, 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 p75NTR 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-6CY and Toll-7CY 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-6CY and Toll-7CY, like activated Toll10b, rescued the semi-lethality of the spz2 mutation; and conversely, overexpression of activated Toll10b 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, p75NTR 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 (Pujol, 2001). p75NTR is a member of the tumor necrosis factor receptor superfamily, which is closer to the Tolls than to the Trks (Bothwell, 2006). Toll receptors resemble p75NTR 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 (Bothwell, 2006). Trk receptors, with an intracellular tyrosine kinase domain, emerged later in evolution (Sossin, 2006). 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 (Okun, 2011). 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).

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).

Virus recognition by Toll-7 activates antiviral autophagy in Drosophila

Innate immunity is highly conserved and relies on pattern recognition receptors (PRRs) such as Toll-like receptors (identified through their homology to Drosophila Toll) for pathogen recognition. Although Drosophila Toll is vital for immune recognition and defense, roles for the other eight Drosophila Tolls in immunity have remained elusive. This study shows that Toll-7 is a PRR both in vitro and in adult flies; loss of Toll-7 led to increased vesicular stomatitis virus (VSV) replication and mortality. Toll-7, along with additional uncharacterized Drosophila Tolls, was transcriptionally induced by VSV infection. Furthermore, Toll-7 interact with VSV at the plasma membrane and induces antiviral autophagy independently of the canonical Toll signaling pathway. These data uncover an evolutionarily conserved role for a second Drosophila Toll receptor that links viral recognition to autophagy and defense and suggest that other Drosophila Tolls may restrict specific as yet untested pathogens, perhaps via noncanonical signaling pathways (Nakamoto, 2012).

Multiple innate immune pathways in Drosophila rely on the activation of the transcription factor NF-κB; however, the Toll-7 dependent autophagy response is likely elicited via an NF-κB-independent mechanism. Unlike Toll-7 deficient flies, flies lacking core Toll pathway components did not demonstrate increased susceptibility to VSV. Moreover, the IMD pathway was not activated by viral infection. In agreement with these data, MyD88 was also not required for the induction of antiviral autophagy. This NF-κB independence is consistent with previous studies that found that the NFκB-dependent AMPs Diptericin and Drosomycin are not induced in Drosophila cells when stimulated with a hyperactive form of Toll-7 (Tauszig, 2000) and that Toll-7 is dispensable for immunity to NF-κB-dependent bacterial challenges (Yagi, 2010). Hence, while Toll-7 likely activates non-canonical signaling pathways, the exact pathways downstream of Toll-7 remain to be determined (Nakamoto, 2012).

Recent studies in mammals found that TLR activation can lead to the induction of autophagy in a variety of cultured cells. However, the mechanism by which TLR stimulation converges on autophagy is unclear. Moreover, the dependence on specific signaling molecules is controversial and whether TLR-induced autophagy is important in restricting infection in vivo is unknown. The current data, together with the findings that Listeria recognition via a peptidoglycan recognition protein induces autophagy, suggest that multiple classes of PRRs are involved in the induction of antimicrobial autophagy, which plays an important role in the control of a diverse set of pathogens (Nakamoto, 2012).

While the discovery of Toll as an innate immune receptor led to the identification of TLRs as a large family of PRRs, studies demonstrating a role for the additional eight Toll receptors in immunity have lagged behind. This discrepancy may be in part due to the lack of studies probing the role of the additional eight Toll receptors in antiviral defense. Perhaps the lack of classical cytoplasmic sensors (RIG-I and MDA5) has required Drosophila to be more heavily dependent on the Tolls for viral recognition, opening up the possibility that additional Drosophila Toll receptors play roles in antiviral immunity. This hypothesis is further supported by the finding that a number of uncharacterized Tolls are induced by viral infection similar to the two major antiviral TLRs, TLR3 and TLR7, which are transcriptionally induced by viral infection in mammalian systems. Importantly, Toll-7 is conserved in vector mosquitoes, suggesting that Toll-7 and other Toll receptors may be involved in the recognition and restriction of human arboviruses (Nakamoto, 2012).

TLRs are generally thought to directly bind their pathogen-associated molecular patterns (PAMPS), whereas Drosophila Toll functions indirectly by recognizing a host cytokine. The findings that Toll-7 interacts with VSV virions suggest that Toll-7 might act directly as a pattern recognition receptor more similar to mammalian TLRs, a previously unknown mechanism for an insect Toll receptor. Although VSV is an arbovirus, the natural vectors have been proposed to be biting insects such as sand flies and blackflies; nevertheless, for several reasons it is believed that VSV is a bona fide ligand for Drosophila Toll-7. First, Toll-7 is highly conserved between insect species that have been sequenced (66% identity and 77% homology to Aedes aegypti Toll-7), indeed, more so than many other Toll receptors. Second, while nucleic acids have been well-characterized as viral PAMPs, emerging evidence suggests that viral proteins including glycoproteins can also activate TLRs. Importantly, there are several examples of murine TLRs that recognize PAMPs from viruses that naturally do not infect mice. Humans are the natural host of measles virus, yet the viral hemagglutinin still activates mouse TLR2. Likewise, Tlr2−/− murine macrophages have reduced cytokine responses to hepatitis C virus core and NS3, as well as to human cytomegalovirus, despite the fact that both viruses are human viruses. Moreover, in mouse macrophages and myeloid dendritic cells, VSV-G activates an antiviral response dependent on TLR4, even though VSV does not normally infect mice in the wild. These results are consistent with the idea that PAMPs are molecular signatures often conserved across wide groups of pathogens and not necessarily restricted to a single microbe. It is therefore not unexpected that TLRs (as well as Tolls) can recognize these structures even if they have not yet encountered that particular pathogen. Third, while the Rhabdovirus VSV does not normally infect fruit flies, the closely related Rhabdovirus sigma virus is a natural Drosophila pathogen. The Drosophila sigmaviruses phylogenetically cluster more closely to the vesiculoviruses than other groups of Rhabdoviruses. Furthermore, while autophagy has not formally been shown to restrict sigma virus, flies deficient in Drosophila p62 (ref(2)p), which serves as an autophagy cargo receptor implicated in the clearance of Sindbis virus capsids and other pathogens, are more susceptible to infection. Given the relatedness of sigma virus to VSV, it is posited that the Toll-7 ligand on VSV may be similar to that of a natural Drosophila pathogen (Nakamoto, 2012).

Intriguingly, the interaction between Toll-7 and VSV suggests that other Toll receptors may recognize presently undefined ligands, including pathogen-derived molecules. Taken together with studies on Toll in microbial defense, the data suggest that Toll receptors likely evolved to recognize foreign microbes and elicit antimicrobial effector mechanisms, therefore uncovering an evolutionarily conserved intrinsic antiviral program that links pathogen recognition to autophagy, which may be amenable to therapeutic intervention (Nakamoto, 2012).

A positional Toll receptor code directs convergent extension in Drosophila

Elongation of the head-to-tail body axis by convergent extension is a conserved developmental process throughout metazoans. In Drosophila, patterns of transcription factor expression provide spatial cues that induce systematically oriented cell movements and promote tissue elongation. However, the mechanisms by which patterned transcriptional inputs control cell polarity and behaviour have long been elusive. This study demonstrates that three Toll family receptors, Toll-2 (18 wheeler), Toll-6 and Toll-8, are expressed in overlapping transverse stripes along the anterior-posterior axis and act in combination to direct planar polarity and polarized cell rearrangements during convergent extension. Simultaneous disruption of all three receptors strongly reduces actomyosin-driven junctional remodelling and axis elongation, and an ectopic stripe of Toll receptor expression is sufficient to induce planar polarized actomyosin contractility. These results demonstrate that tissue-level patterns of Toll receptor expression provide spatial signals that link positional information from the anterior-posterior patterning system to the essential cell behaviours that drive convergent extension (Pare, 2014).

Together, these results demonstrate that the spatial signals that establish planar polarity and direct polarized cell behaviour during convergent extension in Drosophila are encoded at the cell surface by three Toll family receptors expressed in overlapping stripes along the AP axis of the embryo. Simultaneous disruption of Toll-2, Toll-6 and Toll-8 significantly impairs planar polarity, cell intercalation, and convergent extension, and removing one or two receptors disrupts planar polarity in distinct subsets of cells, indicating that these proteins serve non-redundant and highly localized functions. These findings support a model in which planar polarity is induced by interactions between neighbouring cells with different levels of Toll receptor activity. Therefore, Drosophila Toll receptors provide the basis of a spatial code that translates patterned Eve and Runt transcriptional activity into planar polarized actomyosin contractility, linking positional information provided by the embryonic AP patterning system to the essential cell behaviours that drive convergent extension. The Toll receptor code is incomplete in certain regions, such as the parasegmental boundaries, suggesting the existence of additional polarity cues at these interfaces. Toll-2,6,8 mutants are similar to runt mutants with respect to all measures of cell rearrangement and planar polarity, but are not as severe as eve mutants. Thus, although Toll-2,6,8 mutants recapitulate much of the eve mutant phenotype, Eve likely has additional targets important for planar polarity (Pare, 2014).

Toll family receptors have a highly conserved structure in vertebrates and invertebrates, including extracellular LRR motifs that are often present in proteins involved in cell adhesion and cell-cell recognition. Although individual receptors are not orthologous between flies and humans, mammalian Toll-like receptors are required for epithelial regeneration and wound healing, processes that involve dynamic and spatially regulated changes in cell adhesion. In the innate immune system, pathogen detection by Toll family receptors activates transcriptional pathways mediated by NF-κB and MAP kinase signalling. However, the spatial information provided by patterned Toll receptor expression in Drosophila, as well as the rapid timescale of cell rearrangements during convergent extension, suggest a more direct connection between Toll receptor signalling and the cellular contractile machinery. Consistent with this possibility, activation of mammalian Toll-like receptors in dendritic cells induces a rapid remodelling of the actin cytoskeleton and mammalian Toll-like receptors can inhibit neurite outgrowth and trigger rapid growth cone collapse in neurons, reminiscent of Toll receptor functions in the Drosophila nervous system. Elucidating the mechanisms that link Toll family receptors to dynamic changes in cell polarity and behaviour may provide insight into conserved and relatively unexplored aspects of Toll receptor signalling (Pare, 2014).

Functional analysis of Toll-related genes in Drosophila

The Drosophila genome encodes a total of nine Toll and related proteins. The immune and developmental functions of Toll and 18Wheeler (18W) have been analyzed extensively, while the in vivo functions of the other Toll-related proteins require further investigation. This study performed transgenic experiments and found that overexpression of Toll-related genes caused different extents of lethality and developmental defects. Moreover, 18w, Toll-6, Toll-7 and Toll-8 often caused related phenotypic changes, consistent with the idea that these four genes have more conserved molecular structure and thus may regulate similar processes in vivo. Deletion alleles of Toll-6, Toll-7 and Toll-8 were generated by targeted homologous recombination or P element excision. These mutant alleles were viable, fertile, and had no detectable defect in the inducible expression of antimicrobial peptide genes except for the Toll-8 mutant had some defects in leg development. The expression of 18w, Toll-7 and Toll-8 mRNA showed wide and overlapping patterns in imaginal discs and the 18w, Toll-8 double and Toll-7, Toll-8 double mutants showed substantially increased lethality. Overall these results suggest that some of the Toll-related proteins, such as 18W, Toll-7 and Toll-8, may have redundant functions in regulating developmental processes (Yagi, 2010).

Fain-of-function studies suggest that 18w together with Toll-6, Toll-7 and Toll-8 to form a subgroup which has similar functions, and loss-of-function analyses provide some support for this model. In particular there is increased lethality of the double mutant combinations of 18w, Toll-8 and Toll-7, Toll-8. However, the Toll-6 mutant did not show significant phenotype, and the 18w, Toll-6 and Toll-7, Toll-6 double mutants did not show more severe phenotype or lethality. Other analyses may help to unveil the Toll-6 functions. Furthermore, it was not possible to examine other double mutant combinations, as the short distance between 18w and Toll-7 (290 kb) and between Toll-6 and Toll-8 (100 kb) makes it difficult to generate the double mutants by meiotic recombination. RNAi should be useful method to overcome such difficulty but RNAi lines that were tested did not efficiently knockdown the expression of these genes. Further analysis of the reason for the lethality of the double mutants may shed light on the mechanism and developmental function of Toll-related proteins (Yagi, 2010).

Polarized expression of p75NTR specifies axons during development and adult neurogenesis

Newly generated neurons initiate polarizing signals that specify a single axon and multiple dendrites, a process critical for patterning neuronal circuits in vivo. This study reports that the pan-neurotrophin receptor p75NTR is a polarity regulator that localizes asymmetrically in differentiating neurons in response to neurotrophins and is required for specification of the future axon. In cultured hippocampal neurons, local exposure to neurotrophins causes early accumulation of p75NTR) into one undifferentiated neurite to specify axon fate. Moreover, knockout or knockdown of p75NTRb results in failure to initiate an axon in newborn neurons upon cell-cycle exit in vitro and in the developing cortex, as well as during adult hippocampal neurogenesis in vivo. Hence, p75NTR governs neuronal polarity, determining pattern and assembly of neuronal circuits in adult hippocampus and cortical development (Zuccaro, 2014).


REFERENCES

Search PubMed for articles about Drosophila Toll-6 & Toll-7

Bothwell, M. (2006). Evolution of the neurotrophin signaling system in invertebrates. Brain Behav Evol 68: 124-132. PubMed ID: 16912466

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

Kambris, Z., Hoffmann, J. A., Imler, J. L. and Capovilla, M. (2002). Tissue and stage-specific expression of the Tolls in Drosophila embryos. Gene Expr Patterns 2: 311-317. PubMed ID: 12617819

Lu, B., Pang, P. T. and Woo, N. H. (2005). The yin and yang of neurotrophin action. Nat Rev Neurosci 6: 603-614. PubMed ID: 16062169

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

Nakamoto, M., Moy, R. H., Xu, J., Bambina, S., Yasunaga, A., Shelly, S. S., Gold, B. and Cherry, S. (2012). Virus recognition by Toll-7 activates antiviral autophagy in Drosophila. Immunity 36: 658-667. PubMed ID: 22464169

Newquist, G., Drennan, J. M., Lamanuzzi, M., Walker, K., Clemens, J. C. and Kidd, T. (2013). Blocking apoptotic signaling rescues axon guidance in Netrin mutants. Cell Rep 3: 595-606. PubMed ID: 23499445

Okun, E., Griffioen, K. J. and Mattson, M. P. (2011). Toll-like receptor signaling in neural plasticity and disease. Trends Neurosci 34: 269-281. PubMed ID: 21419501

Ooi, J. Y., Yagi, Y., Hu, X. and Ip, Y. T. (2002). The Drosophila Toll-9 activates a constitutive antimicrobial defense. EMBO Rep 3: 82-87. PubMed ID: 11751574

Palgi, M., Lindstrom, R., Peranen, J., Piepponen, T. P., Saarma, M. and Heino, T. I. (2009). Evidence that DmMANF is an invertebrate neurotrophic factor supporting dopaminergic neurons. Proc Natl Acad Sci U S A 106: 2429-2434. PubMed ID: 19164766

Pare, A. C., Vichas, A., Fincher, C. T., Mirman, Z., Farrell, D. L., Mainieri, A. and Zallen, J. A. (2014). A positional Toll receptor code directs convergent extension in Drosophila. Nature 515(7528):523-7. PubMed ID: 25363762

Parker, J. S., Mizuguchi, K. and Gay, N. J. (2001). A family of proteins related to Spatzle, the toll receptor ligand, are encoded in the Drosophila genome. Proteins 45: 71-80. PubMed ID: 11536362

Pujol, N., Link, E. M., Liu, L. X., Kurz, C. L., Alloing, G., Tan, M. W., Ray, K. P., Solari, R., Johnson, C. D. and Ewbank, J. J. (2001). A reverse genetic analysis of components of the Toll signaling pathway in Caenorhabditis elegans. Curr Biol 11: 809-821. PubMed ID: 11516642

Rivest, S. (2009). Regulation of innate immune responses in the brain. Nat Rev Immunol 9: 429-439. PubMed ID: 19461673

Seppo, A., Matani, P., Sharrow, M. and Tiemeyer, M. (2003). Induction of neuron-specific glycosylation by Tollo/Toll-8, a Drosophila Toll-like receptor expressed in non-neural cells. Development 130: 1439-1448. PubMed ID: 12588858

Sossin, W. S. (2006). Tracing the evolution and function of the Trk superfamily of receptor tyrosine kinases. Brain Behav Evol 68: 145-156. PubMed ID: 16912468

Tauszig, S., Jouanguy, E., Hoffmann, J. A. and Imler, J. L. (2000). Toll-related receptors and the control of antimicrobial peptide expression in Drosophila. Proc Natl Acad Sci U S A 97: 10520-10525. PubMed ID: 10973475

Yagi, Y., Nishida, Y. and Ip, Y. T. (2010). Functional analysis of Toll-related genes in Drosophila. Dev Growth Differ 52: 771-783. PubMed ID: 21158756

Zhu, B., Pennack, J. A., McQuilton, P., Forero, M. G., Mizuguchi, K., Sutcliffe, B., Gu, C. J., Fenton, J. C. and Hidalgo, A. (2008). Drosophila neurotrophins reveal a common mechanism for nervous system formation. PLoS Biol 6: e284. PubMed ID: 19018662

Zuccaro, E., Bergami, M., Vignoli, B., Bony, G., Pierchala, B. A., Santi, S., Cancedda, L. and Canossa, M. (2014). Polarized expression of p75NTR specifies axons during development and adult neurogenesis. Cell Rep 7: 138-152. PubMed ID: 24685135


Biological Overview

date revised: 16 December 2014

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