InteractiveFly: GeneBrief

spatzle 5: Biological Overview | References

Tumor-suppressive cell competition is an evolutionarily conserved process that selectively removes precancerous cells to maintain tissue homeostasis. Using the polarity-deficiency-induced cell competition model in Drosophila, this study identify Toll-6, a Toll-like receptor family member, as a driver of tension-mediated cell competition through α-Spectrin (α-Spec)-Yorkie (Yki) cascade. Toll-6 aggregates along the boundary between wild-type and polarity-deficient clones, where Toll-6 physically interacts with the cytoskeleton network protein α-Spec to increase mechanical tension, resulting in actomyosin-dependent Hippo pathway activation and the elimination of scrib mutant cells. Furthermore, this study show that Spz5 secreted from fat body, the key innate organ in fly, facilitates the elimination of scrib clones by binding to Toll-6. These findings uncover mechanisms by which fat bodies remotely regulate tumor-suppressive cell competition of polarity-deficient tumors through inter-organ crosstalk and identified the Toll-6-α-Spec axis as an essential guardian that prevents tumorigenesis via tension-mediated cell elimination (Kong, 2022).

Epithelial cells possess intrinsic mechanisms to outcompete and eliminate early precancerous cells to maintain homeostasis. For instance, in a mouse model of esophageal carcinogenesis, the majority of newly developed tumor clones are eliminated through cell competition by adjacent normal epithelium. Similarly, surveillance mechanisms also exist in Drosophila epithelium to actively remove oncogenic clones composed of polarity-deficient cells. Genetic studies in flies have uncovered numerous mechanisms that regulate tumor-suppressive cell competition, including c-Jun-N-terminal kinase (JNK) signaling activation-mediated cell elimination, direct cell-cell interaction, secreted factors from epithelial cells, and inter-organ crosstalk between insulin-producing cells and precancerous cell-bearing discs (Kong, 2022).

Initially identified in Drosophila, the Hippo pathway is an evolutionarily conserved signaling cascade that plays crucial roles in various physiological and pathological contexts, ranging from tumor progression and embryogenesis to stem cell renewal and immune surveillance. Apart from its well-established roles in controlling cell proliferation and cell death, numerous studies have proved that the Hippo pathway also functions as a key mechanotransducer to sense mechanical changes in the microenvironment. Despite the identification of multiple essential mechanosensitive signaling molecules including RAP2, MAP4K, Agrin, and Spectrin, it remains poorly understood how mechanical stimuli are transmitted from plasma membrane localized receptors to activate Hippo signaling cascade-mediated cellular responses, especially in intact tissues. This study, through a genetic screen in Drosophila, uncovered a regulatory mechanism whereby mechanical tension drives tumor-suppressive cell competition though the Hippo pathway. The genetic and biochemistry data uncovered Toll-6 as an essential regulator of Hippo signaling and further identified α-Spec as an essential downstream component that regulates cell competition via tension-mediated actomyosin activation. Moreover, this study further demonstrated that inter-organ communication is critical for the removal of precancerous cells at a systemic level and discovered fat body (FB)-derived Spz5 as a crucial ligand (Kong, 2022).

This study demonstrated that polarity-deficient oncogenic clones are eliminated through tension-dependent cell competition and has identified Toll-6 as a key membrane receptor that physically interacts and acts through α-Spec to activate the Hippo pathway. It has long been recognized that both extrinsic cues such as ligands and intrinsic factors such as stiffness and cell-cell contact-mediated mechanical cues can determine cell fate and affect cell proliferation, yet relatively little is known about how the cytoskeleton system contributes to the elimination of precancerous cells during cell competition in vivo. The data show that both &alpha-Spec and Rho1, two essential cytoskeleton regulators, accumulate and facilitate the elimination of scrib clones. In addition, α-Spec as a crucial linker that bridges Toll-6 activation-induced tensional changes to cytosolic Hippo pathway activation. Interestingly, studies in the mammalian system showed that RhoA (human Rho1 ortholog) is responsible for mechanical force-induced cell extrusion. Thus, a similar tension-mediated cell-elimination mechanism might exist in the mammalian system to actively remove unfitted precancerous cells (Kong, 2022).

TLRs play critical roles in the innate immune response. The Drosophila genome encodes nine TLRs, of which only Toll (Tl/Toll-1) has a clear function in innate immunity. Interestingly, a paradoxical role of Tl in regulating cell competition has been reported. Activation of Tl in polarity-deficient clones suppresses the elimination of losers, whereas in the Myc-induced cell competition model, increased Tl activity accelerates the elimination of losers. Apart from Tl, Toll-2, Toll-3, Toll-7, Toll-8, and Toll-9 have been implicated in regulating cell competition in different contexts, while Toll-4 and Toll-5 have little effect. It is noteworthy that none of above studies has investigated the role of Toll-6 in cell competition. The current data not only reveal Toll-6 as a crucial regulator of tumor-suppressive cell competition but also show how the mechanical tension-mediated Hippo cascade is initiated from the cell membrane through the Toll-6-&alpha-Spec axis. Notably, this study found that Toll-6 was not required for Myc-induced cell competition. Given that TLRs are highly conserved in vertebrates and the elimination of scrib-depleted cells also exists in the mammalian system, further experiments are necessary to determine whether analogous mechanisms exist in mammals and humans to regulate mechanical tension-induced, Hippo pathway-mediated tumor-suppressive cell competition (Kong, 2022).

Inter-organ communication is essential for proper development and homeostasis maintenance of multicellular organisms under both physiological and pathological conditions. The tumor progression process is also shaped by the interactions between tumor and other organs, including the immune system. Recent studies in Drosophila have provided insightful understanding of the complex crosstalk between organs during tumorigenesis. The FB is the major immune organ of Drosophila, and it has been shown that intestinal tumor progression or abdominal tumor transplantation promotes the wasting behavior of FBs. The current findings that the transcription of spz5 is increased in the FB from scrib clone bearing larvae to facilitate tumor-suppressive cell competition may provide an in vivo mechanistic understanding of the inter-organ communications between FBs and remotely colonized precancerous clones. Together, the ism) around the boundary between losers and winners, which recruits α-Spec and provokes Hippo pathway-dependent elimination of scrib^-/- clones. Meanwhile, the presence of scrib^-/- loser cells in the eye disc will trigger a systemic effect on the distal organs, including FBs, which results in the transcription upregulation and secretion of Spz5, in turn forming a feedforward loop to reinforce the tumor-suppressive cell competition by binding to Toll-6 (Kong, 2022).

Although biochemical and genetic data demonstrate that Toll-6 physically interacts with α-Spec and that α-Spec is required for the elimination of scrib clones, these experiments were unable to explain the molecular mechanisms by which Toll-6 recruits α-Spec and initiates the downstream signaling transduction. Another limitation is that because the substantial analysis relies heavily on genetics to infer mechanism, enough rigorous biochemistry data was not included to prove how the binding of Toll-6 with α-Spec triggers Hippo signaling activation. Additionally, this study found that FB-derived Spz5 is essential for the elimination of scrib clones through inter-organ communications, but it is not understood completely how the spz5 mRNA level is upregulated systematically in the FBs of larvae that bear scrib mutant clones. Future work will be required to dissect the transcriptome changes of FBs upon precancerous clone induction in distal organs. Finally, this study showed that Spz5 acts through Toll-6 to regulate cell competition, and it is known that Spz5 can bind to other TLRs to regulate both cell death and survival through a three-tier mechanism (Foldi, 2017), suggesting that Spz5 can trigger intracellular signal transduction through ligand receptor binding. Nonetheless, a question that remains unsolved is why a signaling network that relays cell mechanical properties (Toll-6-α-Spec axis) should be regulated by a chemical ligand/receptor interaction; it would be interesting to further explore the underlying mechanisms (Kong, 2022).

Neuron-type specific functions of DNT1, DNT2 and Spz at the Drosophila neuromuscular junction

Retrograde growth factors regulating synaptic plasticity at the neuromuscular junction (NMJ) in Drosophila have long been predicted but their discovery has been scarce. In vertebrates, such retrograde factors produced by the muscle include GDNF and the neurotrophins (NT: NGF, BDNF, NT3 and NT4). NT superfamily members have been identified throughout the invertebrates, but so far no functional in vivo analysis has been carried out at the NMJ in invertebrates. The NT family of proteins in Drosophila is formed of DNT1, DNT2 and Spatzle (Spz), with sequence, structural and functional conservation relative to mammalian NTs. This study investigated the functions of Drosophila NTs (DNTs) at the larval NMJ. All three DNTs are expressed in larval body wall muscles, targets for motor-neurons. Over-expression of DNTs in neurons, or the activated form of the Spz receptor, Toll^10b, in neurons only, rescued the semi-lethality of spz² and DNT1⁴¹, DNT2^e03444 double mutants, indicating retrograde functions in neurons. In spz² mutants, DNT1⁴¹, DNT2^e03444 double mutants, and upon over-expression of the DNTs, NMJ size and bouton number increased. Boutons were morphologically abnormal. Mutations in spz and DNT1,DNT2 resulted in decreased number of active zones per bouton and decreased active zone density per terminal. Alterations in DNT function induced ghost boutons and synaptic debris. Evoked junction potentials were normal in spz² mutants and DNT1⁴¹, DNT2^e03444 double mutants, but frequency and amplitude of spontaneous events were reduced in spz² mutants suggesting defective neurotransmission. The data indicate that DNTs are produced in muscle and are required in neurons for synaptogenesis. Most likely alterations in DNT function and synapse formation induce NMJ plasticity leading to homeostatic adjustments that increase terminal size restoring overall synaptic transmission. Data suggest that Spz functions with neuron-type specificity at the muscle 4 NMJ, and DNT1 and DNT2 function together at the muscles 6,7 NMJ (Sutcliffe, 2013).

Characterization of Spz5 as a novel ligand for Drosophila Toll-1 receptor

The Drosophila Toll-1 receptor is involved in embryonic development, innate immunity, and tissue homeostasis. Currently, as a ligand for the Toll-1 receptor, only Spatzle (Spz) has been identified and characterized. It has been previously reported that Drosophila larva-derived tissue extract contains ligand activity for the Toll-1 receptor, which differs from Spz based on the observation that larval extract prepared from spz mutants possessed full ligand activity. This study demonstrates that Spz5, a member of the Spz family of proteins, functions as a ligand for the Toll-1 receptor. Processing of Spz5 by Furin protease, which is known to be important for ligand activity of Spz5 to Toll-6, is not required for its function to the Toll-1 receptor. By generating a spz5 null mutant, it was further shown that the Toll-1 ligand activity of larva-derived extract is mainly derived from Spz5. Finally, a genetic interaction was found between spz and spz5 in terms of developmental processes. This study identified a novel ligand for the Drosophila Toll-1 receptor, providing evidence that Toll-1 is a multi-ligand receptor, similar to the mammalian Toll-like receptor (Nonaka, 2018).

This study has demonstrated that Spz5 is most likely the protein in charge of the Toll pathway-stimulating activity in larva-derived tissue extract, which was reported in a previous paper (Kanoh, 2015). Since it was not possible to examine a direct effect of Spz5, i.e., spz5-expressing cell extracts in the Drs reporter assay with DL1 cells because of the technical difficulty in purifying Spz5, it can be at most concluded that Spz5 was required for novel Toll-1 ligand activity in the larval extract based on the mutant experiment (Nonaka, 2018).

The Spz family of proteins is known to become active via protease cleavage. Spz is cleaved by Easter or SPE, Spz3 by Easter, and Spz5 by Furin proteases. This study, however, showed that Spz5 activity in terms of the Toll-1 ligand does not require processing by Furin, and Spz5 is likely to function in a full-length form, which was suggested by the result that Furin cleavage site-lacking Spz5 (Spz5R284G) retained the Toll-1 ligand activity. This implied that Spz5 might work as a DAMP that is released by cell damage or necrotic death, because proteolytic processing is required for secretion of Spz5 from cells. Because this study demonstrated that Spz5 is not involved in pinching-induced activation of innate immunity in larvae, Spz5 might function in a different physiological or pathological context, which should be pursued in future studies (Nonaka, 2018).

It is widely appreciated that homozygous mutant flies from loss-of-function Toll-1 alleles are scarce but those from Spz can be substantially obtained. Because spzΔ8 must be a null allele, it would be possible that the Toll-1 receptor might have other ligands during developmental processes (though this could be simply accounted for by the amount of their maternal mRNAs). In the current study, it was observed that spz5 single mutant flies were homozygous viable, but the double mutants for spz and spz5 produced fewer homozygous progenies. This implied that Spz5 might have a role in development with Spz. Elucidating when and how Spz5 functions during development will be of interest (Nonaka, 2018).

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

Neurotrophin receptors corresponding to vertebrate Trk, p75^NTR or Sortilin have not been identified in Drosophila, thus it is unknown how neurotrophism may be implemented in insects. Two Drosophila neurotrophins, DNT1 and DNT2a (Spz5), have nervous system functions, but their receptors are unknown. The Toll receptor superfamily has ancient evolutionary origins and a universal function in innate immunity. This study shows that Toll paralogs unrelated to the mammalian neurotrophin receptors function as neurotrophin receptors in fruit flies. Toll-6 and Toll-7 are expressed in the CNS throughout development and regulate locomotion, motor axon targeting and neuronal survival. DNT1 (also known as NT1 and spz2) and DNT2 (also known as NT2 and spz5) interact genetically with Toll-6 and Toll-7, and DNT1 and DNT2 bind to Toll-6 and Toll-7 promiscuously and are distributed in vivo in domains complementary to or overlapping with those of Toll-6 and Toll-7. It is concluded that in fruit flies, Tolls are not only involved in development and immunity but also in neurotrophism, revealing an unforeseen relationship between the neurotrophin and Toll protein families (McIlroy, 2013).

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

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

DNT1 was identified independently as related to BDNF, using vertebrate neurotrophin sequences as query to search the Drosophila sequenced genome with bioinformatics tools. DNT1 was found to be spz2, a paralog of spz. Structural prediction analysis showed that, of the spz paralogs, DNT1 and DNT2 (spz5) 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. The vertebrate neurotrophins have essential functions during development in neuronal survival, axon targeting and connectivity and during adult life in learning, memory and cognition. During development, DNT1, DNT2 and spz are expressed in target cells for CNS neurons, such as the embryonic en-passant midline target of interneurons and the muscles, the target of motor neurons. DNT1 and DNT2 are required for neuronal survival, as neuronal apoptosis decreases upon their overexpression in the CNS and increases in the loss-of-function mutants, leading to neuronal loss, with apoptotic neurons comprising those identified as bearing the Even-skipped (Eve) or Homeobox 9 (HB9) neuronal markers. DNTs are required for motor-axon targeting, as interfering with the function of DNT1, DNT2 and Spz causes misrouting, mistargeting and sprouting defects in motor axon terminals. Thus, DNT1 and DNT2, as well as Spz, are Drosophila neurotrophins on the basis of sequence, structural and functional homology to the vertebrate neurotrophins (McIlroy, 2013).

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

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

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

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

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

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

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

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

Drosophila neurotrophins reveal a common mechanism for nervous system formation

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. Spatzle 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).

Search PubMed for articles about Drosophila Spz5

Kanoh, H., Kuraishi, T., Tong, L. L., Watanabe, R., Nagata, S. and Kurata, S. (2015). Ex vivo genome-wide RNAi screening of the Drosophila Toll signaling pathway elicited by a larva-derived tissue extract. Biochem Biophys Res Commun 467(2): 400-406. PubMed ID: 26427875

Kong, D., Zhao, S., Xu, W., Dong, J. and Ma, X. (2022). Fat body-derived Spz5 remotely facilitates tumor-suppressive cell competition through Toll-6-α-Spectrin axis-mediated Hippo activation. Cell Rep 39(12): 110980. PubMed ID: 35732124

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

Nonaka, S., Kawamura, K., Hori, A., Salim, E., Fukushima, K., Nakanishi, Y. and Kuraishi, T. (2018). Characterization of Spz5 as a novel ligand for Drosophila Toll-1 receptor. Biochem Biophys Res Commun. PubMed ID: 30361090

Sutcliffe, B., Forero, M. G., Zhu, B., Robinson, I. M. and Hidalgo, A. (2013). Neuron-type specific functions of DNT1, DNT2 and Spz at the Drosophila neuromuscular junction. PLoS One 8(10): e75902. PubMed ID: 24124519

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(11): e284. PubMed ID: 19018662

date revised: 10 October 2022

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