InteractiveFly: GeneBrief

Allatostatin C: Biological Overview | References

BIOLOGICAL OVERVIEW

The link between the biological clock and reproduction is evident in most metazoans. The fruit fly Drosophila melanogaster, a key model organism in the field of chronobiology because of its well-defined networks of molecular clock genes and pacemaker neurons in the brain, shows a pronounced diurnal rhythmicity in oogenesis. Still, it is unclear how the circadian clock generates this reproductive rhythm. A subset of the group of neurons designated 'posterior dorsal neuron 1' (DN1p), which are among the ~150 pacemaker neurons in the fly brain, produces the neuropeptide Allatostatin C (AstC-DN1p). This study reports that six pairs of AstC-DN1p neurons send inhibitory inputs to the brain's insulin-producing cells that express two AstC receptors: star1 and AICR2. Consistent with the roles of insulin/insulin-like signaling in oogenesis, activation of AstC-DN1p suppresses oogenesis through the insulin-producing cells. This study shows evidence that AstC-DN1p activity plays a role in generating an oogenesis rhythm by regulating juvenile hormone and vitellogenesis indirectly via insulin/insulin-like signaling. AstC is orthologous to the vertebrate neuropeptide somatostatin (SST). Like AstC, SST inhibits gonadotrophin secretion indirectly through gonadotropin-releasing hormone neurons in the hypothalamus. The functional and structural conservation linking the AstC and SST systems suggest an ancient origin for the neural substrates that generate reproductive rhythms (Zhang, 2021).

Six pairs of DN1p neurons were discovered that are part of the circadian pacemaker neuron network in the brain and make functional inhibitory connections to the brain IPCs. The IPCs are endocrine sensors that link the organism's nutritional status with anabolic processes, such as those associated with growth in developmental stages and with reproduction in adults. In juvenile stages, activation of insulin and insulin-like growth factor (IGF) signaling (IIS) through the InR results in larger flies, whereas inhibition of this pathway produces smaller flies. Consistent with this, it was also found that forced activation of the AstC-DN1p (i.e., CNMa-Gal4/UAS-NaChBac) during development resulted in 12% smaller adults, confirming their role as a negative regulator of the IPCs. In adults, the IPCs are associated with many physiological and behavioral processes, such as feeding, glycaemic homeostasis, sleep, lifespan, and stress resistance. As such, the IPCs receive a variety of modulatory inputs from both central and peripheral sources, such as sNPF, corazonin, tachykinins, limostatin, allatostatin A, adipokinetic hormone, GABA, serotonin, and octopamine. Regarding reproduction, IIS directed by the IPCs stimulates GSC proliferation and vitellogenesis. The results also indicate that AstC from AstC-DN1p suppresses the secretory activity of the IPCs and JH (juvenile hormone)-dependent oocyte development (i.e., vitellogenesis). Indeed, it was found that the JH mimic, methoprene, can rescue the suppression of oogenesis induced by AstC-DN1p activation. From these results it is concluded that IPCs are inhibited by AstC, released by AstC-DN1p. A similar link between IIS and the circadian clock has also been reported in mammals, but the mechanism remains unclear (Zhang, 2021).

Although the genetic evidence supporting the inhibitory action of AstC-DN1p on IPCs is compelling, it is also puzzling because a previous study found forced activation of 8 to 10 pairs of DN1p neurons (i.e., Clk4.1-LexA+ neurons) induced Ca2+ transients in IPCs. This earlier study also found that, under LD 12:12 conditions, the IPCs showed electrical activity early in the morning when DN1p neurons are also active. However, the same study reported that under DD conditions the IPCs showed no bursting activity in the morning (i.e., CT0 to -4). Instead, they showed bursting activity in the late afternoon (i.e., CT8 to -12) when DN1 activity falls. Furthermore, DN1p activation evokes varying levels of Ca2+ transients from individual IPCs, some of which produce no detectable Ca2+ transient. Thus, like mammalian pancreatic β-cells, the IPCs in Drosophila seem to comprise a heterogeneous cell population. It was noted that individual IPCs show highly variable AstC-R1 expression, which would also lead to individual IPCs showing variable responses to AstC (Zhang, 2021).

In D. melanogaster, the LD cycle generates an egg-laying rhythm by influencing oogenesis and oviposition. Oviposition depends on light cues, whereas oogenesis cycles with the circadian rhythm that itself continues to run in DD conditions. In live-brain Ca2+ imaging experiments, DN1 neurons show a circadian Ca2+ activity rhythm that peaks around CT19 and reaches its lowest point between CT6 and CT8. This DN1 activity rhythm correlates well with the rhythm of vitellogenesis initiation observed in this study. In this model, the lowest point in DN1 Ca2+ activity between CT6 and CT8 leads to a significant attenuation of AstC secretion. This leads to a derepression of IPC activity, which eventually induces JH biosynthesis and vitellogenesis initiation. The 6-h delay required for previtellogenic stage 7 follicles to develop into vitellogenic stage 8 follicles would result in a peak in the number of stage 8 follicles between CT12 and CT14. Notably, the ovaries of the AstC-deficient mutant showed similar numbers of stage 8 oocytes at all examined circadian time points, indicating that any other JH- or vitellogenesis-regulating factors play only minor roles in producing the circadian vitellogenesis rhythm (Zhang, 2021).

Like the IPCs, the DN1p cluster is also heterogeneous. A subset of the DN1p neurons is most active at dawn and promotes wakefulness. Another subset of the DN1p cluster (also known as spl-gDN1) promotes sleep. The DN1p cluster comprises two morphologically distinct subpopulations: a-DN1p and vc-DN1p. The a-DN1p subcluster promotes wakefulness by inhibiting sleep promoting neurons, whereas the vc-DN1p subcluster resembles the sleep-promoting spl-gDN1. These results indicate AstC-DN1p are a-DN1p neurons that project to the anterior optic tubercle (AOTU. Although the possibility cannot be ruled out that AstC-DN1p is also heterogeneous and includes some vc-DN1p neurons, the wake-promoting role of a-DN1p aligns well with the circadian vitellogenesis rhythm that requires the secretory activity of AstC-DN1p to be lowest in the afternoon and highest at dawn. Furthermore, AstC-DN1p neurons express Dh31. Dh31-expressing DN1 clock neurons are intrinsically wake-promoting and Dh31-DN1p activity in the late night or early morning suppresses sleep. Again, this is consistent with the observation that AstC-DN1p are also wake-promoting a-DN1p. It is speculated that Dh31 plays a limited role in oogenesis regulation, because unlike AstC, RNAi-mediated knockdown of Dh31 had a negligible impact on female fecundity (Zhang, 2021).

Besides AstC-DN1p, the female brain has many additional AstC neurons. However, it seems unlikely that other AstC neurons contribute to the circadian vitellogenesis rhythm. This is because restoring AstC expression specifically in AstC-DN1p almost completely restored the vitellogenesis rhythm in AstC-deficient mutants. It is feasible, however, that other AstC neurons contribute to different aspects of female reproduction. Indeed, a sizable difference was noted in the final oogenesis outcome between AstC-Gal4 neuron activation and brain-specific AstC-Gal4 neuron activation. This suggests that AstC cells outside of the brain also regulate oogenesis, probably in other physiological contexts, such as the postmating responses (Zhang, 2021).

AstC receptors are orthologous to mammalian SST receptors (sstr1-5). SST is a brain neuropeptide that was originally identified as an inhibitor of growth hormone (GH) secretion in the anterior pituitary. Thus, the observation that AstC inhibits IIS from IPCs (a major endocrine signal that promotes growth in Drosophila) suggests remarkable structural and functional conservation between the invertebrate AstC and vertebrate SST systems. In addition, SST inhibits the hypothalamic neuropeptide GnRH, which stimulates the anterior pituitary's production of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). FSH stimulates clutches of immature follicles to initiate follicular development, while LH stimulates ovulation. Thus, both AstC and SST regulate the secretion of gonadotropins (JH in insects, FSH and LH in mammals) indirectly through the IPCs in insects and through the hypothalamic GnRH neurons in mammals. This functional conservation between AstC and SST is also evident in the immune system. AstC inhibits the innate immune system in insects, while SST inhibits inflammation in mammals (Zhang, 2021).

In many seasonal breeders, the changing photoperiod as the seasons progress acts as an environmental cue for the biological clock system, which would then direct any necessary physiological changes. During the winter, Drosophila females enter a form of reproductive dormancy characterized by a pronounced suppression of vitellogenesis. A winter-like condition (i.e., short-day length, low temperature, and food shortage) down-regulates neural activity in the IPCs. But the IPCs are not equipped with a cell-autonomous clock, so they must receive seasonal information from the brain clock neuronal network. Indeed, two clock related neuropeptides (pigment dispersing factor and short neuropeptide F) from circadian morning pacemaker or M-cells have been implicated in regulating reproductive dormancy. Intriguingly, AstC-DN1p neurons are the DN1p subset that receives pigment dispersing factor signals from these M-cells. Furthermore, DN1p can process light and temperature information for the circadian regulation of behavior. Finally, the finding that AstC-DN1p generates the circadian vitellogenesis rhythm via the IPCs makes AstC-DN1p neurons the prime candidates for integrating the seasonal cues that control the entrance, maintenance, or exit from reproductive dormancy. Considering the functional and structural conservation between the AstC and SST systems, the SST system may also link the brain clock, GnRH, and/or its downstream reproductive pathways in controlling seasonal reproductive patterns in vertebrates (Zhang, 2021).

Alleviation of thermal nociception depends on heat-sensitive neurons and a TRP channel in the brain

Acute avoidance of dangerous temperatures is critical for animals to prevent or minimize injury. Therefore, surface receptors have evolved to endow neurons with the capacity to detect noxious heat so that animals can initiate escape behaviors. Animals including humans have evolved intrinsic pain-suppressing systems to attenuate nociception under some circumstances. Using Drosophila melanogaster, this study uncovered a new mechanism through which thermal nociception is suppressed. A single descending neuron was identified in each brain hemisphere, which is the center for suppression of thermal nociception. These Epi neurons, for Epione-the goddess of soothing of pain-express a nociception-suppressing neuropeptide Allatostatin C (AstC), which is related to a mammalian anti-nociceptive peptide, somatostatin. Epi neurons are direct sensors for noxious heat, and when activated they release AstC, which diminishes nociception. Epi neurons also express the heat-activated TRP channel, Painless (Pain), and thermal activation of Epi neurons and the subsequent suppression of thermal nociception depend on Pain. Thus, while TRP channels are well known to sense noxious temperatures to promote avoidance behavior, this work reveals the first role for a TRP channel for detecting noxious temperatures for the purpose of suppressing rather than enhancing nociception behavior in response to hot thermal stimuli (Liu, 2023).

Endogenous pain inhibitory systems can temporarily provide relief. Millions of people suffer from chronic and debilitating pain, some of which might be induced by abnormalities in the descending pain modulatory system. In mammals, neurotransmitters and neuromodulators, including endogenous opioids (β-endorphin, encephalin, and dynorphin) and endogenous cannabinoids, play important roles in nociception inhibition. Brain imaging and electrophysiological studies indicate that the pain-suppressing descending modulatory circuit receives input from multiple brain regions including the rostral anterior cingulate cortex, the periaqueductal gray region, and the rostral ventromedial medulla. However, the key neurons that are activated in the inhibitory pathway, and the target neurons that are silenced, have not been clearly delineated (Liu, 2023).

A pain inhibitory system has also been documented in worms. In C. elegans avoidance responses that are mediated through the polymodal ASH neurons are suppressed by complex signaling pathways initiated by octopamine and neuropeptides. Drosophila has also been employed to study the inhibition of nociception in addition to the far more extensive studies focusing on the mechanisms for detecting noxious stimuli, such as excessive heat, has been shown to to initiate escape responses. A Drosophila channel, Painless (Pain), which is related to the TRP channel in the fly's compound eye, is critical for sensing noxious heat. This work, which followed the seminal discovery of TRPV1 as a heat sensor in mammals and the finding that a related TRPV channel (Osm-9) contributes to several other sensory modalities in C. elegans, contributed significantly to the notion that TRP channels are evolutionarily conserved polymodal sensors (Liu, 2023).

In addition to Pain, two other Drosophila TRP channels also function in sensing high temperatures to promote escape behavior: Pyrexia (Pyx) and TRPA1. However, it is unclear whether any TRP channel serves to detect noxious heat for the purpose of alleviating thermally induced nociception (Liu, 2023).

This work used the fruit fly, Drosophila melanogaster, to investigate an intrinsic system for suppression of thermal nociception. A pair of bilaterally symmetrical neurons in the brain was identified that is required for decreasing the nociceptive response to hot temperatures. These Epi neurons respond directly to heat and release a neuropeptide, Allatostatin C (AstC), which is required for suppression of nociception. The ability of Epi neurons to sense noxious heat depends on Pain, demonstrating a role for a thermo-TRP in suppressing rather than enhancing the nociceptive response to high temperatures (Liu, 2023).

This study found that a single pair of bilaterally symmetrical Epi neurons in the fly brain is critical for suppressing thermal nociception. The importance of Epi neurons is underscored by the observation that artificial activation of these neurons is sufficient to suppress the aversive jump response to hot temperatures and that inhibition of signaling from these neurons increases the jump responses to moderate heat. The profound effect of a single pair of neurons in reducing thermal nociception is surprising given that multiple brain regions appear to function in pain suppression in mammals (Liu, 2023).

The dendrites of Epi neurons arborize to multiple regions of the brain, such as the optic lobes (OLs), the lateral horn (LH), and a region near the mushroom bodies, indicating that Epi neurons receive multiple signal inputs. The LH is a higher-order processing center that receives input from the antennal (olfactory) lobes and then sends relays to other brain regions such as the mushroom bodies. Therefore, it is intriguing to speculate that the Epi neurons may be activated by noxious odorants and aversive visual cues, which attenuate the avoidance behavioral responses to these stimuli. Epi neurons might also receive input from attractive olfactory and visual cues, which in turn diminish the escape responses to noxious stimuli such as high temperatures. In addition, the axons of Epi neurons project to the VNC, consistent with a role in descending control of motor output (Liu, 2023).

A key question is the mechanism through which Epi neurons respond to hot temperatures and alleviate thermal nociception. Epi neurons were found to be directly activated by hot temperatures and do so through activation of the thermo-TRP channel, Pain, which is expressed in Epi neurons. The Pain channel is critical for suppressing nociception since mutation of the pain gene causes an increase in thermal pain sensitivity (hyperalgesia). While Epi neurons respond directly to heat and are anti-nociceptors, other neurons in the fly brain, the so-called anterior cell neurons, respond directly to suboptimal warm temperatures. In contrast to the anti-nociceptive Epi neurons, the AC neurons function in thermal avoidance, which is mediated through thermal activation of TRPA1 (Liu, 2023).

The next question is the mechanism through which activation of Epi neurons suppresses thermal pain. Epi neurons express a neuropeptide, AstC, which binds to receptors that have sequence homology (39.0% identity for AstC-R1; 38.5% identity for AstC-R2) to human opioid receptors, which function in the suppression of nociception in mammals. Moreover, mutation of AstC or knockdown of AstC in Epi neurons causes thermal hyperalgesia, and mutation of AstC-R1 elicits a similar phenotype. Heat stimulation diminishes the level of AstC in Epi neurons, indicating that activation of these neurons promotes release of AstC. It is concluded that Epi neurons alleviate thermal nociception through a mechanism that depends on heat sensing by the Pain channel, leading to release of AstC (Liu, 2023).

Surprisingly, mutation of pain also reduced expression of AstC in Epi neurons below the level of detection. This effect was not due to elimination of Epi neurons since pain mutant brains express UAS-GCaMP6f under control of the Epi-Gal4. Expression of neuropeptides has been linked to neuronal activity. Moreover, there is an example in which a thermosensory TRPV channel affects expression of a neuropeptide receptor. Pain is activated by thermal heat, with the most pronounced activation in the noxious heat range. However, even at temperatures significantly below the flex point in which a given temperature rapidly opens the gate of a thermosensory TRP, such as Pain, there is some channel activity. It is suggested that low levels of Pain and Epi neuron activities are necessary for expression of AstC, while high levels of activities that are induced by noxious heat are required for release of the AstC (Liu, 2023).

A feature of activation of Epi neurons is that the pain suppression due to an acute 30-second activation of Epi neurons is sustained for several minutes. It is suggested that the slow termination of the pain suppression following stimulation of these neurons is mediated by release of the neuromodulator AstC, which persists for several minutes. Epi neurons appear to be non-adapting, as chronic activation of these neurons with the NaChBac channel leads to similar levels of pain suppression as acute stimulation with channelrhodopsin. This non-adapting feature of Epi neurons may be beneficial because it allows for pain suppression under conditions in which the aversive response to heat needs to be suppressed sufficiently long enough to allow activities that promote survival. Given that fruit flies are poikilothermic, and their body temperature equilibrates with the environment, direct activation of Epi neurons would allow the flies to suppress nociception and enter excessively warm environments to feed or avoid predators (Liu, 2023).

In conclusion, this study unveils a molecular and cellular basis for pain suppression in Drosophila. The observation that Pain is essential for suppressing nociception is surprising given that all other thermal-TRP channels function in avoidance of suboptimal or noxious temperatures. Mutation of pain in fly larvae eliminates the sensitivity to hot temperatures (hypoalgesia). Thus, it is remarkable that the same TRP channel has opposite functions in nociception and anti-nociception in larvae and adults (Liu, 2023).

Allatostatin-C/AstC-R2 is a novel pathway to modulate the circadian activity pattern in Drosophila

Seven neuropeptides are expressed within the Drosophila brain circadian network. Previous mRNA profiling suggested that Allatostatin-C (AstC) is an eighth neuropeptide and specifically expressed in dorsal clock neurons (DN1s). The results of this study show that AstC is, indeed, expressed in DN1s, where it oscillates. AstC is also expressed in two less well-characterized circadian neuronal clusters, the DN3s and lateral-posterior neurons (LPNs). Behavioral experiments indicate that clock-neuron-derived AstC is required to mediate evening locomotor activity under short (winter-like) and long (summer-like) photoperiods. The AstC-Receptor 2 (AstC-R2) is expressed in LNds, the clock neurons that drive evening locomotor activity, and AstC-R2 is required in these neurons to modulate the same short photoperiod evening phenotype. Ex vivo calcium imaging indicates that AstC directly inhibits a single LNd. The results suggest that a novel AstC/AstC-R2 signaling pathway, from dorsal circadian neurons to an LNd, regulates the evening phase in Drosophila (Diaz, 2019).

To learn more about how the ~150 clock neurons within the adult fly brain communicate, RNA-sequencing data were examined from the LNds, LNvs, and DN1s for neuropeptides not yet associated with this circuitry. AstC was a promising candidate, because mRNAs encoding both the peptide and one of its receptors (AstC-R2) were identified within the three clock neuron clusters; these data suggested a novel intra-clock circuitry signaling pathway. AstC transcripts as well as the neuropeptide are, indeed, well expressed in the DN1s, and the neuropeptide signal undergoes strong cycling in DD as well as LD conditions. Moreover, AstC is also expressed in two other circadian neuron subgroups, the DN3s and the LPNs, and it is the first neuropeptide identified in these circadian clusters. Behavioral data after RNAi knockdown indicate that the AstC binds to AstC-R2 expressed in E-cells to modulate the timing of evening locomotor activity. Ex vivo calcium imaging indicates that AstC directly inhibits a single LNd (Diaz, 2019).

AstC is required in the clock neurons to regulate the evening locomotor activity phase in short and long photoperiods, suggesting that 'masking' effects in 12:12 LD obscured a phenotypic effect. This shift in the timing of the E-peak occurs when AstC is reduced in all circadian neurons (tim-GAL4 driver) (Diaz, 2019).

AstC cycles in the DN1s, not only under standard 12:12 LD conditions but also under DD conditions and the short photoperiod condition of 6:18 LD. In all cases, AstC staining intensity in the DN1 soma is lowest during the early day. It was hypothesizefd that the AstC staining is reduced at this time, because the peptide is being transported from the soma to the dendritic arbors for secretion. Indeed, this early-day timing correlates with DN1 firing, as DN calcium and firing frequency are highest at this time and, thus, may be associated with activity-dependent peptide release. This temporal regulation could coincide with the timing of the AstC effect on the phase of the evening locomotor peak. If this is the case, however, residual AstC remaining after knockdown in the DN1s with the clk^4.1M-GAL4 driver must still be sufficient to promote a wild-type phenotype (Diaz, 2019).

In addition, the LPNs are probably not a key circadian source of AstC: their AstC levels were also dramatically reduced in the clk⁸⁵⁶-GAL4-mediated knockdown without any phenotypic effect. All AstC-expressing DN3s are targeted by the tim-GAL4 driver, yet most of these DN3s are not included in the clk⁸⁵⁶-GAL4 driver. Although a tim-GAL4 source from outside the circadian network cannot be excluded, these results suggest that the DN3s are the key source of AstC. This tentative conclusion is based on negative data, and the lack of a DN3-specific GAL4 driver makes it impossible to test this model directly. Therefore, three possible models are proposed: (1) the DN3s are the primary source of AstC within the circadian circuit; (2) the DN1s, DN3s, and LPNs, or some combination, are functionally redundant, or (3) a small amount of residual AstC within DN1s is sufficient for its behavioral role in the evening activity peak assay. Although neuron-specific deletion of AstC might contribute to distinguishing between these three possibilities, no accurate and efficient CRISPR-based strategy is available for achieving temporal and spatial specificity (Diaz, 2019).

Once released by dorsal circadian neurons, AstC signals to the LNds via binding to its receptor, AstC-R2. This is because AstC-R2 knockdown in the LNds and knockdown of AstC in the entire circadian circuit give rise to the identical delayed E-peak phenotype. Moreover, the DNs and the LNds both extend projections to the dorsal protocerebrum region (near the pars intercerebralis), where they come in close proximity. In summary, this study adds a new player to the neuronal circuitry governing E-peak modulation. It is suggested that the LNd neuronal activity is modulated not only by signaling from M-cells but also by AstC signaling from the dorsal region of the brain (Diaz, 2019).

The functional imaging strongly indicates that AstC binding to LNd-localized AstC-R2 leads to neuronal inhibition. The effect is consistent with several previous electrophysiology experiments and contributes to an emerging theme of LNd inhibition. It is also one of the first pieces of evidence indicating that the DNs can be a source of inhibition onto the LNds. Interestingly, only a single LNd is directly AstC sensitive, further attesting to LNd heterogeneity and suggesting that behavioral regulation of the evening phase arises from this signaling to a single LNd. It is suggested that this response is communicated directly to the rest of the LNds, for example via gap junctions, but a more indirect and circuitous route of communication cannot be excluded (Diaz, 2019).

It is noted that there are several caveats to the current model. The AstC and AstC-R2 knockdown experiments were conducted with only a single RNAi line each, due to the lack of additional functional RNAi lines. However, the two experiments show essentially identical phenotypes, suggesting that off-target effects are unlikely to pose a problem. It was not possible to rule out that the AstC knockdown phenotypes are not due to a developmental requirement. Experiments to address this point are challenging, because when the temperature is raised to reduce tubgal80^ts repression and allow for an adult-only knockdown, the heat itself dramatically changes the E-peak timing. Lastly, lack of LPN- and DN3-specific drivers precluded addressing whether DN3-derived AstC is required for this evening activity peak modulation and whether the DN3s can directly inhibit the LNds (Diaz, 2019).

Although the AstC peptide sequence is highly conserved among insect species, only the AstC-R2 receptor has a mammalian homolog: the somatostatin-galanin-opioid receptor family. Inhibitory somatostatin (SST) interneurons are present in the mammalian equivalent of a central core clock, the suprachiasmatic nucleus (SCN). SST interneurons are also known to affect sleep and circadian behaviors. Interestingly, SST is associated with proper adaptation under photoperiod conditions for both diurnal and nocturnal mammals, suggesting a highly conserved function with AstC/AstC-R2 for adaptation under different equinox environments. It will be interesting to see whether the SCN-resident SST interneurons are important for this adaptation, like the AstC-containing clock neurons described in this study (Diaz, 2019).

Allatostatin C modulates nociception and immunity in Drosophila

Bacterial induced inflammatory responses cause pain through direct activation of nociceptive neurons, and the ablation of these neurons leads to increased immune infiltration. This study investigated nociceptive-immune interactions in Drosophila and the role these interactions play during pathogenic bacterial infection. After bacterial infection, robust upregulation is found of ligand-gated ion channels and allatostatin receptors involved in nociception, which potentially leads to hyperalgesia. It was further found that Allatostatin-C Receptor 2 (AstC-R2) plays a crucial role in host survival during infection with the pathogenic bacterium Photorhabdus luminescens. Upon examination of immune signaling in AstC-R2 deficient mutants, it was demonstrated that Allatostatin-C Receptor 2 specifically inhibits the Immune deficiency pathway, and knockdown of AstC-R2 leads to overproduction of antimicrobial peptides related to this pathway and decreased host survival. This study provides mechanistic insights into the importance of microbe-nociceptor interactions during bacterial challenge. It is posited that Allatostatin C is an immunosuppressive substance released by nociceptors or Drosophila hemocytes that dampens IMD signaling in order to either prevent immunopathology or to reduce unnecessary metabolic cost after microbial stimulation. AstC-R2 also acts to dampen thermal nociception in the absence of infection, suggesting an intrinsic neuronal role in mediating these processes during homeostatic conditions. Further examination into the signaling mechanisms by which Allatostatin-C alters immunity and nociception in Drosophila may reveal conserved pathways which can be utilized towards therapeutically targeting inflammatory pain and chronic inflammation (Bachtel, 2018).

During bacterial challenge, the host immune response must be mounted in a tightly regulated and quantitatively precise manner. Overproduction of immune effectors results in immune-related pathophysiology, tissue damage, and metabolic cost whereas under-production of these effectors may permit bacterial expansion and subsequently bacterially derived damage. Recent studies have shown that bacteria can directly interact with nociceptive neurons, and that ablation of these neurons leads to increased lymph drainage during S. aureus infection most likely by suppressing immunomodulatory neuropeptide release. Thus, bacterial activation of nociceptive neurons may be a novel mechanism of immune control. This study represents the first attempt to characterize bacterially induced hyperalgesia and the effects of genes related to this process on host immunity in Drosophila melanogaster. This study provides support for a newly emerging idea that nociceptive neurons may be crucial to mounting an appropriate immune response during these infections (Bachtel, 2018).

This study investigated the gene kinetics, effect on noxious behavior, and immune consequences of nociceptive gene activation during microbial challenge. A robust upregulation was found of ligand gated ion channels (TRPA1 and ppk) and Allatostatin receptors (AstC-R1, AstC-R2, AstA-R1) upon microbial challenge, the homologs of these genes have been associated with hyperalgesia in mammalian systems. This study found that nociceptive gene activation differed temporally upon infection with E. coli as compared to pathogenic P. luminescens, and that bacterial load better correlated with nociceptive gene activation than immune activation (as measured by the IMD antimicrobial peptide encoding gene, Cecropin A1). Importantly, this correlation supports a recent paper demonstrating that S. aureus bacterial load better correlates with hyperalgesia than paw swelling (immune infiltration) in mice (Bachtel, 2018).

To determine whether the upregulation of these nociception-related genes contributed to hyperalgesia, immune and nociceptive knockdown fly mutants were generated for the genes upregulated, and changes to noxious heat sensitization were measured. Upon examining alterations to this behavior, it was found that AstC-R1 and AstC-R2 RNAi mutants displayed hyperalgesia whereas IMD and TRPA1 knockdown mutants showed robust hypoalgesia. These results are in agreement with previous studies demonstrating the importance of TRPA1 in noxious heat sensation. To determine whether it was possible to raise the noxious heat sensitivity of IMD mutants back to wild-type levels by infection with a bacterium, IMD knockdown flies were infected with a non-pathogenic strain of E. coli, and these mutants were found to display hyperalgesia, suggesting IMD activation contributes to, but is not necessary for hyperalgesia during bacterial infections. These results implicate NF-kappaB activation as a conserved mechanism of hyperalgesia in arthropod and mammalian lineages with the additional hyperalgesia seen upon infection of IMD knockdown mutants being attributed to Toll signaling or direct bacterial activation. Indeed, previous studies have found that a transcription factor downstream of IMD activation, Relish, alters thermal nociception as well (Bachtel, 2018).

Due to bacteria being able to potentially manipulate the expression of nociceptive genes in their favor, it was of interest to discover whether any of the nociception-related genes tested played a beneficial or detrimental role to the host during microbial challenge. To test this, each nociception-related gene was silenced ubiquitously in flies and their survival upon injection with the insect pathogen P. luminescens was measured. A trend towards decreased survival of AstC-R1 knockdown flies was found, and a significant decrease in survival was found upon knockdown of AstC-R2, suggesting a potential role for Allatostatin-C in modulating host immune processes during bacterial infection. However, when infecting AstC-R2 knockdown flies with the non-pathogen E. coli, no decreased survival was detected over 48 hours as compared to wild-type flies suggesting that this effect alone is not sufficient to cause death (Bachtel, 2018).

The mammalian homolog of Allatostatin is Somatostatin, which has documented effects in reducing systemic inflammation in mammalian systems, and thus whether knockdown of AstC-R2 leads to alterations in immune signaling that could contribute to the decreased survival was examined. A robust over-induction of IMD signaling was detected with a modest, but non-significant increase in Toll and decrease in Eiger as compared to wild-type flies, suggesting that AstC-R2 reduced IMD signaling independently of the Toll or Jak-Stat pathways respectively. Despite the robust upregulation of the IMD pathway, no changes were observed in bacterial load during P. luminescens infection of AstC-R2 knockdown flies as compared to wild-type controls. These results suggest that antimicrobial peptides related to this pathway are ineffective at controlling this pathogen. Indeed, recent reports have shown that an antimicrobial peptide-resistant sub-population of P. luminescens is responsible for the majority of the virulence during insect infection, and that P. luminescens is able to employ proteases that specifically degrade antimicrobial peptides, rendering them post-translationally ineffective (Bachtel, 2018).

By knocking down a receptor for Allatostatin C, which has dual role in inhibiting heat-driven nociception as well as inhibiting the IMD pathway during bacterial challenge, hyperactivation of this immune pathway, hyperalgesia, and reduced survival upon challenge with P. luminescens were observed. The hyperalgesia seen in AstC-R1 and AstC-R2 RNAi knockdown flies in the absence of bacterial challenge most likely is not due to dysregulation of the IMD pathway because similar basal transcript levels of Cecropin A1 were observed in AstC-R2 knockdown mutants as compared to wild-type flies. Indeed, AstC-R1 and AstC-R2 also share structural homology with mammalian opioid receptors. However, the reduced survival in AstC-R2 knockdown flies may be explained either directly or indirectly by over-activated IMD signaling and AstC-R2-IMD double knockdown mutants will be needed in order to confirm this hypothesis. Remarkably, the results recapitulate many of the findings found in a seminal study investigating the importance of somatostatin receptor 4 in the modulation of hyperalgesia and inflammation (Helyes, 2008). Therefore, Drosophila AstC-R2 may be more functionally similar to mammalian SSTR4 than previously perceived (Bachtel, 2018).

Due to the transcriptional upregulation of AstC-R1 and AstC-R2 during infection, it is likely that this upregulation reflects one mechanism of the host fine-tuning the immune response to prevent immune related damage from occurring as well as mediating avoidance behaviors while in a compromised state. Somatostatin regulatory circuits have been documented at sites of chronic inflammation where they have important roles in inhibiting pro-inflammatory cytokine production by macrophages and T-cells yet found processes have not been previously described in Drosophila. Interestingly, another neuropeptide that acts as a crucial component of this circuit by inhibiting somatostatin release is substance P, an additional molecule released from nociceptive neurons. Thus, immune manipulation during microbial challenge by nociceptive neurons is likely to be a well-orchestrated process that amplifies or suppresses pro-inflammatory cytokine production in a way to best ensure host survival (Bachtel, 2018).

The results imply that nociceptor-immune interactions during microbial infection in Drosophila may be more similar to mammalian systems than previously conceived (see Potential role for AstC-R2 in nociceptor-bacterial-immune interactions in Drosophila). This idea is supported by recent findings demonstrating that nociceptive neurons in flies are sensitive the proinflammatory cytokine Eiger, as well as bacterially derived lipopolysaccharides. Drosophila also possesses homologous genes for other immuno-modulatory substances released from nociceptors including substance P, CGRP and VIP (DTK, DH31, and Pdf respectively), yet their roles in pain sensation and immunity have not been characterized. Due to the wealth of transgenic lines available, quick developmental cycle and cheap cost of maintenance, Drosophila could prove to be a valuable tool in deciphering nociceptor-innate immune interactions in the future. Further studies into the interface of pain, immunity, and microbial challenge hold large promise for innovative treatments for inflammatory pain, auto-immune conditions, as well as potential explanations for host-tolerance of the gut microbiota (Bachtel, 2018).

Allatostatin C and its paralog allatostatin double C: the arthropod somatostatins

Arthropods have not one, but two genes encoding an allatostatin C-like peptide. The newly discovered paralog gene was called Ast-CC, and the peptide that it is predicted to make was called allatostatin double C (ASTCC). Genes for both allatostatin C (ASTC) and its paralog were found in the tick Ixodes scapularis as well as dipteran, lepidopteran, coleopteran, aphidoidean and phthirapteran insect species. In addition partial or complete cDNAs derived from Ast-CCs were found in a number of species, including Drosophila melanogaster, Bombyx mori and Rhodnius prolixus. The ASTCC precursors have a second conserved peptide sequence suggesting that they may produce two biologically active peptides. The predicted precursors encoded by the Ast-CCs have some unusual features, particularly in Drosophila, where they lack a signal peptide, and have instead a peptide anchor. These unusual structural features suggest that they are perhaps expressed by cells that are not specialized in neuropeptide synthesis and that in Drosophila ASTCC may be a juxtacrine. Data from the Fly Atlas project show that in Drosophila Ast-CC is little expressed. Nevertheless a P-element insertion in this gene is embryonic lethal, suggesting that it is an essential gene. Similarity between the precursors and receptors of ASTC/ASTCC and somatostatin suggests that ASTC/ASTCC and somatostatin have a common ancestor (Veenstra, 2009).

The sesquiterpenoid juvenile hormone is produced by the corpora allata and exercises essential functions in insect growth, metamorphosis and reproduction. The regulation of the biosynthetic activity of the corpora allata has been studied extensively and it is known that the activity of the corpora allata is at least in part regulated by neuropeptides, which may either stimulate (allatotropins) or inhibit (allatostatins) the corpora allata to synthesize juvenile hormone. The first allatostatins identified were isolated from the cockroach Diploptera punctata, but it became soon apparent that these peptides and their homologs are present in many insect species as well as other arthropods. Furthermore they are not only produced by the brain to inhibit the activity of the corpora allata, but they are also produced by other tissues, notably the midgut from where they could potentially be released in concentrations sufficient to inhibit the corpora allata. It thus appears, that like the somatostatins in vertebrates, the allatostatins are general inhibitory peptides used for different functions (Veenstra, 2009).

The identification of allatostatins from other insect species has been pursued by both immunoassay using antisera to the peptides identified from Diploptera as well as the synthetic activity of the corpus allatum to identify allatostatins. These bioassays yielded other neuropeptides with allatostatic activity. Unfortunately, though these peptides were clearly structurally different from the allatostatins from Diploptera, they were also given the name allatostatin. Thus from the cricket Gryllus bimaculatus a number of neuropeptides were identified having allatostatic activity; these are commonly called allatostatins B to distinguish them from the allatostatins identified from Diploptera, now called allatostatins A. From the tobacco hornworm moth Manduca sexta yet another allatostatin was identified, a peptide now known as allatostatin C (ASTC). As is the case for the allatostatins A, the allatostatins B and C are also present in other insect species, e.g., Drosophila melanogaster, Aedes aegypti and Tribolium castaneum and in different tissues, notably again in the midgut (Veenstra, 2009).

While the allatostatins A and B are produced from precursors producing from three to fourteen structurally related peptides, the known ASTC genes encode a single biologically peptide. ASTC seems very well conserved; from several lepidopteran species cDNAs have been isolated predicting the same peptide as the one initially isolated from Manduca. The ASTC predicted from the Drosophila genome differs from the Manduca peptide in a single amino acid residue; the ASTC isolated from A. aegypti adds a second substitution, as does the peptide predicted and tentatively identified by mass spectrometry from Tribolium. It was therefore somewhat of a surprise to see the honeybee peptide appear to be significantly different, so different in fact that it was not recognized by the authors as an ASTC. The peptide inhibits the synthesis of juvenile hormone in the moths M. sexta and Helicoverpa zea as well as the mosquito A. aegypti, but it is much less effective in other moth species, and does not inhibit the corpora allata in D. melanogaster. However, in the latter species it does inhibit the rate of heartbeat (Veenstra, 2009).

Although so far genomic analysis has only revealed a single allatostatin A, B or C gene per species, analysis of the various insect genomes which have been, or are in the progress of being sequenced, revealed that all these insect species have in fact not one but two genes encoding an ASTC-like peptide. These two genes were also found in the genome of the tick Ixodes scapularis and may thus well be generally present in arthropods. The predicted precursor of the second gene has some unusual features, particularly in Drosophila (Veenstra, 2009).

Search PubMed for articles about Drosophila

Bachtel, N. D., Hovsepian, G. A., Nixon, D. F. and Eleftherianos, I. (2018). Allatostatin C modulates nociception and immunity in Drosophila. Sci Rep 8(1): 7501. PubMed ID: 29760446

Diaz, M. M., Schlichting, M., Abruzzi, K. C., Long, X. and Rosbash, M. (2019). Allatostatin-C/AstC-R2 is a novel pathway to modulate the circadian activity pattern in Drosophila. Curr Biol 29(1): 13-22.e13. PubMed ID: 30554904

Helyes, Z., Pinter, E., Sandor, K., Elekes, K., Banvolgyi, A., Keszthelyi, D., Szoke, E., Toth, D. M., Sandor, Z., Kereskai, L., Pozsgai, G., Allen, J. P., Emson, P. C., Markovics, A. and Szolcsanyi, J. (2009). Impaired defense mechanism against inflammation, hyperalgesia, and airway hyperreactivity in somatostatin 4 receptor gene-deleted mice. Proc Natl Acad Sci U S A 106(31): 13088-13093. PubMed ID: 19622729

Liu, J., Liu, W., Thakur, D., Mack, J., Spina, A. and Montell, C. (2023). Alleviation of thermal nociception depends on heat-sensitive neurons and a TRP channel in the brain. Curr Biol 33(12): 2397-2406. PubMed ID: 37201520

Veenstra, J. A. (2009). Allatostatin C and its paralog allatostatin double C: the arthropod somatostatins. Insect Biochem Mol Biol 39(3): 161-170. PubMed ID: 19063967

Zhang, C., Daubnerova, I., Jang, Y. H., Kondo, S., Zitnan, D. and Kim, Y. J. (2021). The neuropeptide allatostatin C from clock-associated DN1p neurons generates the circadian rhythm for oogenesis. Proc Natl Acad Sci U S A 118(4). PubMed ID: 33479181

date revised: 15 September 2022

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