Myd99 overexpression functions upstream of Drosomycin (Drs). S2 cells were cotransfected with a Myd88 expression vector and reporter plasmids. Overexpression of Myd88 is sufficient to trigger a marked (nearly 40-fold) induction of the Drs promoter. In contrast, the promoter of AttA, the gene encoding the antibacterial peptide Attacin A, was increased only threefold. Overexpression of the isolated TIR domain is sufficient to trigger induction, whereas the NH2-terminal death domain is not active. Double-stranded RNA interference (RNAi) technique was used to abolish expression of Myd88 in S2 cells. RNAi-mediated inhibition of Myd88 expression results in a marked and specific reduction of the Toll-mediated induction of the Drs promoter but does not affect the LPS-mediated induction of the AttA promoter. These results indicated that Myd88 participates in the Toll pathway in S2 cells and is not required for the response to LPS treatment (Tauszig-Delamasure, 2002).
Micro-RNAs are a class of small non-coding regulatory RNAs that impair translation by imperfect base pairing to mRNAs. For analysis of their cellular function, different miRNA-specific DNA antisense oligonucleotides were injected into Drosophila embryos. In four cases severe interference with normal development was observed; in another case, this had a moderate impact and six other oligonucleotides did not cause detectable phenotypes. The miR-13a DNA antisense oligonucleotide was used as a PCR primer on a cDNA library template. In this experimental way nine Drosophila genes were identifed, each characterized by 3' untranslated region motifs that allow imperfect duplex formation with miR-13 or related miRNAs. These genes, which include Sos and Myd88, represent putative targets for miRNA regulation. Mutagenesis of the target motif of two genes followed by transfection in Drosophila Schneider 2 (S2) cells and subsequent reporter gene analysis confirms the hypothesis that the binding potential of miR-13 is inversely correlated with gene expression (Boutla, 2003).
Drosophila Myd88 is an adapter in the Toll signaling pathway that associates with both the Toll receptor and the downstream kinase Pelle. Expression of Myd88 in S2 cells strongly induces activity of a Drosomycin reporter gene, whereas a dominant-negative version of Myd88 potently inhibits Toll-mediated signaling. Myd88 associates with the death domain-containing adapter Drosophila Fas-associated death domain-containing protein (FADD), which in turn interacts with the apical caspase Dredd. This pathway links a cell surface receptor to an apical caspase in invertebrate cells and therefore suggests that the Toll-mediated pathway of caspase activation may be the evolutionary ancestor of the death receptor-mediated pathway for apoptosis induction in mammals (Horng, 2001).
A BLAST search of the Drosophila genome identified the sequence encoding Myd88, a Drosophila homolog of human MyD88. Similar to its human homolog, Drosophila Myd88 contains an N-terminal death domain, an intermediate domain, and a TIR domain. However, unlike human MyD88, Drosophila Myd88 contains an additional 81 amino acids preceding the death domain and a 162-aa-long C-terminal region following the TIR domain (Horng, 2001).
Transfection of Myd88 into Drosophila S2 cells potently induces a Drosomycin reporter gene but not an Attacin reporter gene. This preferential ability to induce an antifungal gene is similar to that of Toll 10b, a constitutively active form of Toll, and suggests that Myd88 may be a component of the Toll-Tube-Pelle-Cactus-Dif signaling pathway. Previous studies have demonstrated that Toll-mediated Drosomycin induction requires the nuclear translocation of Dif. Dif is normally retained in the cytoplasm by the IkappaB inhibitor Cactus and is released only in response to signal-dependent degradation of Cactus. To test whether Myd88-mediated Drosomycin induction also depends on Cactus degradation, a Cactus mutant was constructed that contains mutations of the conserved serine residues that, in mammalian IkappaB, are the targets of signal-dependent phosphorylation. A Cactus mutant inhibits Drosomycin induction by Myd88 and, as expected, by Toll. This result indicates that, similar to Toll, Myd88 regulates Drosomycin induction through the Cactus-dependent pathway (Horng, 2001).
For further analyses, various deletion mutants of Myd88 were generated. Two of the deletion mutants, one containing the TIR domain and the C-terminal domain (amino acids 237-537) and another containing the intermediate, TIR, and C-terminal domains (amino acids 176-537), activate the Drosomycin reporter weakly (10-fold) in comparison to full length Myd88, indicating that the intact protein is required for optimal activity. However, the fact that these truncation mutants can still induce signaling is surprising, since they lack the death domain that mediates interactions with downstream signaling components. Moreover, similar analyses of human MyD88 have shown that a combination of the death domain and the intermediate domain is sufficient to induce signaling activity comparable to that of the wild-type protein. An equivalent truncation of Myd88 (amino acids 1-237) retains no residual activity despite being well expressed, suggesting that there are some differences in domain function between human and Drosophila Myd88 proteins (Horng, 2001).
To determine whether Myd88 is a component of the Toll signaling pathway, attempts were made to identify a deletion mutant that would have dominant-negative activity. Therefore, three Myd88 deletion mutants that do not activate the Drosomycin reporter were tested for their ability to inhibit Toll-mediated Drosomycin induction. The strongest inhibitor was the death domain- and middle domain-containing construct (amino acids 1-237), which at low concentrations potently inhibits Toll-mediated Drosomycin induction in a dose-dependent manner (Horng, 2001).
To order Myd88 in the pathway with respect to Pelle, Myd88 was tested for its ability to be inhibited by PelleN, a dominant-negative form of Pelle that consists of the N-terminal death domain-containing region of Pelle. Myd88, like Toll, is strongly inhibited by PelleN. Myd88, however, does not inhibit Pelle, demonstrating that, similar to the mammalian pathway, Myd88 functions upstream of Pelle (Horng, 2001).
To further establish Myd88 as a component of the Toll pathway, whether Myd88 interacts with Toll was tested by coimmunoprecipitation assays. The TIR domain-containing Myd88 construct is detected in anti-Toll immunoprecipitates. Interestingly, when cotransfected with Toll 10b, Myd88 reproducibly appears as two distinct bands -- a slower migrating upper band that may correspond to phosphorylated Myd88 construct and a faster migrating lower band. The predominant form of Myd88 detected in immunoprecipitates is the faster migrating species. Myd88 therefore associates with Toll, presumably through TIR domains, and is a component of the active receptor complex (Horng, 2001).
Because human MyD88 associates with IRAK through death domains, a likely immediate downstream target of MyD88 is the IRAK homolog Pelle. Interaction between the death domain-containing Drosophila Myd88 construct (amino acids 1-237) and Pelle was examined. Myd88 is detected in Pelle immunoprecipitates, indicating that Myd88 interacts with Pelle, presumably through their death domains (Horng, 2001).
These results therefore demonstrate that Myd88 is an adaptor in the Toll signaling pathway downstream of the receptor and upstream of Pelle. From genetic analyses, the adaptor protein Tube has also been implicated to be downstream of Toll and upstream of Pelle in the Toll signaling pathway. The death domain of Tube also interacts with Pelle. Because Tube and Myd88 also contain death domains that could potentially mediate their interaction, tests were performed for association between these two proteins in immunoprecipitation assays; Tube and Myd88 do indeed interact. Therefore, Myd88 and Tube both function as adaptors downstream of Toll, exist in the same active complex along with Pelle, and are probably both involved in the recruitment and/or activation of Pelle. Understanding functional differences between these two adapters will require further analysis (Horng, 2001).
To identify other potential downstream targets of Myd88, a search of the Drosophila genome was performed for other sequences that encode death domain-containing proteins that may interact with Myd88. One such sequence encodes a protein with a death domain as well as a death effector domain and appears to be a homolog of mammalian FADD. This cDNA has been identified and named FADD (Hu, 2000). Whether FADD can interact with Myd88 was tested. Lysates from S2 cells transfected with Myd88 were incubated with anti-Flag beads to immunoprecipitate FADD, and immunoprecipitates were blotted with anti-V5 antibody to look for associated Myd88. A strong band corresponding to Myd88 was observed, indicating that Myd88 can interact with FADD through death domains. Overexpression of FADD in S2 cells, however, does not lead to activation of either the Drosomycin or Attacin reporters (Horng, 2001).
Mammalian FADD is recruited to the tumor necrosis factor receptor complex through homophilic death domain interactions with the adapter TNFR-associated death domain-containing protein (TRADD). In turn, FADD recruits procaspase-8 through homophilic death effector domain associations. It is speculated that Drosophila FADD may likewise recruit a Drosophila caspase to the Toll receptor complex. A potential candidate caspase is Dredd, an apical caspase with a long prodomain shown to be essential for induction of antibacterial genes. Indeed, analysis of immunoprecipitated lysates from cells cotransfected with Drosophila FADD, and either full length Dredd or the death effector domain of Dredd showed strong association of Dredd with FADD. A second study (Hu, 2000) has also shown interaction of dFADD with Dredd (Horng, 2001).
Thus Drosophila Myd88 is an adapter in the Toll signaling pathway. Myd88 associates with both Toll and Pelle and functions upstream of Pelle. Tube is known from genetic studies to be an adapter in the Toll pathway that functions upstream of Pelle. Why Toll should signal through Myd88 and Tube, two receptor-proximal adapters with seemingly similar functions, is not yet clear. Myd88 associates with the receptor Toll as well as the downstream adapter FADD, which in turn interacts with the apical caspase Dredd. Because caspases are essential executioners of the apoptotic machinery in organisms from nematodes to mammals, and because Dredd has been shown to be involved in apoptosis during Drosophila development, it is possible that Toll-1 or some of the other eight Tolls that exist in Drosophila may induce apoptosis (or another Dredd-dependent pathway) through the Myd88/dFADD/Dredd pathway in a cell-type specific and/or developmental stage-specific manner. The pathway comprised of Toll, Myd88, dFADD, and Dredd would be the first description of a pathway in invertebrates that links a cell surface receptor to an apical caspase. Such a pathway, if it exists, would enable extracellular stimuli, perhaps ligands secreted by other cells during development or pathogen-derived products during infection, to instruct invertebrate cells to undergo cell death. In addition, the Toll/Myd88/dFADD/Dredd pathway is remarkably similar to that activated by the receptors of the tumor necrosis factor receptor (TNFR) superfamily in mammals, in which FADD-mediated recruitment of caspase-8 leads to induction of apoptosis. Since the Drosophila genome does not encode any cell surface receptors homologous to TNFRs, it appears that the Toll/Myd88/dFADD/Dredd pathway is the evolutionary ancestor of the mammalian death receptor pathways. This possibility is further supported by the recent finding that human TLR2 can induce apoptosis through the Myd88/FADD/Caspase-8 pathway (Horng, 2001).
Myd88 specifically associates with Toll in S2 cells The interaction of tagged versions of Myd88 and Toll was examined; Myd88 associates with Toll in transfected S2 cells. Higher expression of the V5-tagged Myd88 protein is reproducibly seen in cells cotransfected with the Toll expression vector; this might reflect stabilization of Myd88 by interaction with Toll. Similar experiments with truncated versions of Myd88 indicate that the interaction with the intracytoplasmic domain of Toll is mediated by the TIR domain of Myd88. However, no association of Myd88 was detected with the related receptors 18-wheeler, Toll-5, Toll-6, Toll-7 or Toll-8, showing that the interaction with Toll is specific. Myd88 is associated with the IRAK-related kinase Pelle in transfected S2 cells (Tauszig-Delamasure, 2002).
Innate immunity mediated by Toll signalling has been extensively studied, but how Toll signalling is precisely controlled in balancing innate immune responses remains poorly understood. It has been reported that the plasma membrane localization of Drosophila MyD88 is necessary for the recruitment of cytosolic adaptor Tube to the cell surface, thus contributing to Toll signalling transduction. This study demonstrates that Drosophila Pellino functions as a negative regulator in Toll-mediated signalling. Pellino accumulates at the plasma membrane upon the activation of Toll signalling in a MyD88-dependent manner. Moreover, Pellino was found to be associated with MyD88 via its CTE domain, which is necessary and sufficient to promote Pellino accumulation at the plasma membrane where it targets MyD88 for ubiquitination and degradation. Collectively, this study study uncovers a mechanism by which a feedback regulatory loop involving MyD88 and Pellino controls Toll-mediated signalling, thereby maintaining homeostasis of host innate immunity (Ji, 2014).
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).
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