InteractiveFly: GeneBrief

short neuropeptide F precursor: Biological Overview | References

BIOLOGICAL OVERVIEW

Insulin and insulin growth factor have central roles in growth, metabolism and ageing of animals, including Drosophila melanogaster. In Drosophila, insulin-like peptides (Dilps) are produced by specialized neurons in the brain. This study shows that Drosophila short neuropeptide F (sNPF), an orthologue of mammalian neuropeptide Y (NPY), and sNPF receptor sNPFR1 regulate expression of Dilps. Body size was increased by overexpression of sNPF or sNPFR1. The fat body of sNPF mutant Drosophila had downregulated Akt, nuclear localized FOXO, upregulated translational inhibitor 4E-BP and reduced cell size. Circulating levels of glucose were elevated and lifespan was also extended in sNPF mutants. These effects are mediated through activation of extracellular signal-related kinase (ERK; Rolled in Drosophila) in insulin-producing cells of larvae and adults. Insulin expression was also increased in an ERK-dependent manner in cultured Drosophila central nervous system (CNS) cells and in rat pancreatic cells treated with sNPF or NPY peptide, respectively. Drosophila sNPF and the evolutionarily conserved mammalian NPY seem to regulate ERK-mediated insulin expression and thus to systemically modulate growth, metabolism and lifespan (Lee, 2008).

Neuropeptides regulate a wide range of animal behaviours related to nutrition. In particular, mammalian NPY produced in the hypothalamus of the brain controls food consumption. NPY injection in the hypothalamus of rats produces hyperphagia and obese phenotypes (Emeson, 2005; Wahlestedt, 1993). The Drosophila orthologue of NPY is sNPF. This peptide is expressed in the nervous system and it regulates food intake and body size; overexpression of sNPF produces bigger and heavier flies (Lee, 2004). Likewise, the G-protein-coupled receptor of sNPF (sNPFR1) is expressed in neurons and shows significant similarity with vertebrate NPY receptors (Feng, 2003). In mammals, however, little is known about how NPY and sNPF systemically modulate growth, metabolism and lifespan. This study shows that these neuropeptides control expression of insulin-like peptides and subsequently affect insulin signalling in target tissues (Lee, 2008).

Initially the effects of sNPF and sNPFR1 on body size were characterized by measuring the length of flies from head to abdomen. The body size of sNPF hypomorphic Drosophila mutants (sNPF^c00448) was 23% of that of the wild-type, whereas overexpressing two copies of the sNPF in the sensory neurons and sensory structures of the nervous systems by MJ94-Gal4 (MJ94>2XsNPF) increased body size by 24%. Similar changes were seen in the overall size of adult wings, which resulted from changes in both cell size and number. Effects on body size were associated with sNPF expression levels: relative to wild type, sNPF levels were 3.5-fold higher in MJ94>2XsNPF and less than half of the wild type in sNPF^c00448. In contrast to the effect of sNPF on body size, there was little effect on size from repression or overexpression of the sNPF receptor in MJ94-expressing cells (Lee, 2008).

Drosophila insulin-like peptides (Dilps) modulate growth and adult size; therefore, whether sNPF has a role in insulin-producing neurons was tested. For positive controls, Dilp2 was overexpressed in insulin-producing cells (IPCs) through Dilp2-Gal4, which increased body size, and the IPCs were ablated by expression of Dilp2>reaper to decrease body size. To investigate sNPF signalling, sNPFR1 was overexpressed in the IPCs and a 10% increase in body size was observed. Conversely, expression of the sNPFR1 dominant-negative mutant (Dilp2>sNPFR1-DN) reduced body size by 14%. Manipulation of the sNPF ligand with IPCs expressing Dilp2-Gal4, however, did not affect body size: flies overexpressing sNPF (Dilp2>2XsNPF) or in which sNPF was silenced by RNAi (Dilp2>sNPF-Ri) were of similar size to the wild type. Taken together, these results suggest that sNPF peptide may be secreted from MJ94-expressing sensory neurons and activate sNPFR1 of Dilp2-expressing IPCs (Lee, 2008).

To assess this model, the sNPF ligand and sNPFR1 receptor were visualized in the larval brain. Seven IPCs were detected in each brain hemisphere using the marker Dilp2-Gal4>nGFP. Neurons containing sNPF peptide in the axon terminal and cell body (sNPFnergic neurons) were stained adjacent to these IPCs. As expected, sNPFR1 receptors were localized in the plasma membrane of IPCs marked with Dilp2>DsRed. sNPFR1 was also localized in the neurons of the larval brain hemispheres, sub-oesophagus ganglion, ventral abdominal neurons and descending axons in the ventral ganglion (Lee, 2008).

To study genetic interactions between sNPFR1 and Dilps in IPCs, Dilp1 and Dilp2 interference mutants were generated in the sNPFR1 overexpression background. In contrast to the 10% body size increase by sNPFR1 overexpression in IPCs (Dilp2>sNPFR1), inhibition of Dilp1 and Dilp2 in IPCs (Dilp2>Dilp1-Ri and Dilp2>Dilp2-Ri) generated reduced body size by 10% and 15%, respectively. Inhibition of Dilp1 and Dilp2 with sNPFR1 overexpression in IPCs (Dilp2>sNPFR1+Dilp1-Ri and Dilp2> sNPFR1+Dilp2-Ri) also generated a reduction in body size of 8% and 13%, indicating that Dilp1 and Dilp2 are downstream genes of sNPFR1 in IPCs for regulating body size (Lee, 2008).

To test whether sNPF regulates Dilp expression in larval IPCs, expression of Dilp1, 2, 3 and 5 were assessed in sNPF mutants. Neuronal overexpression of sNPF (MJ94>2XsNPF) markedly increased expression of Dilp2 in IPCs; it also produced novel Dilp2 expression outside of these cells. As expected, reduction of sNPF by MJ94>sNPF-Ri inhibited expression of Dilp2. In common with Dilp2, the expression of Dilp1 was positively regulated by sNPF overexpression and reduced by sNPF hypomorphs (MJ94>sNPF-Ri and sNPF^c00448). Consistent with the model, expression of Dilp1 and Dilp2 was increased more than fourfold with overexpression of the receptor in IPCs (Dilp2>sNPFR1) and decreased by half with inhibition of the receptor gene in IPCs (Dilp2>sNPFR1-DN). Larval IPCs also express Dilp3 and Dilp5. Expression of Dilp3 was reduced by sNPF hypomorphs (MJ94>sNPF-Ri and sNPF^c00448) but expression of Dilp5 was not regulated by any sNPF mutants. There are few functions known to distinguish these various insulin-like peptides. Nutrition-dependent growth regulation is associated with expression of Dilp3 and Dilp5, but not with that of Dilp2. Recent reports show that Dilp2 is reduced in long-lived flies expressing dFOXO or Jun-N-terminal kinase (JNK), whereas Dilp5 is uniquely upregulated upon dietary restriction that increases lifespan (Lee, 2008).

To investigate how Drosophila sNPF regulates Dilp expression, the activation of Drosophila MAP kinase signalling, which includes the action of ERK (encoded by Rolled) and JNK, was measured. sNPF overexpression with MJ94-Gal4 increased phospho-activated pERK relative to basal ERK1/2. Expression of the receptor protein sNPFR1 in IPCs also increased pERK. There were no detectable changes in phospho-activated pJNK in these sNPF and sNPFR1 mutants. Next, whether ERK activation in IPCs was sufficient to induce Dilp expression was tested. Expression of a constitutively active ERK in IPCs (Dilp2>rolled^SEM) increased expression of Dilp1 and Dilp2 more than threefold, and both transcripts were repressed less than half by the expression of an ERK inhibitory phosphatase DMKP-3 in IPCs (Dilp2>DMKP-3). In addition, the inhibition of ERK with the sNPFR1 overexpression in IPCs (Dilp2>sNPFR1+DMKP-3) also repressed expression of Dilp1 and Dilp2 compared with that of sNPFR1 overexpression in IPCs (Dilp2>sNPFR1). These results indicate that sNPF and sNPFR1 signalling regulate ERK activation in IPCs, which in turn modulates expression of Dilp1 and Dilp2 (Lee, 2008).

To further examine the effect of sNPF on Dilp, Drosophila CNS-derived neural BG2-c6 cells, which endogenously express sNPFR1 were treated with a synthetic sNPF peptide. Dilp1 and Dilp2 were induced within 15 min, and the elevated transcript persisted for 1 h. Concomitant with this gene expression, sNPF-treated cells activated ERK. Importantly, sNPF did not induce Dilp expression significantly when cells were treated with ERK-specific kinase MEK inhibitor PD98059. To compare the functional conservation of sNPF and NPY in the regulation of insulin expression, similar tests were conduced with rat insulinoma INS-1 cells, which express NPY receptors NPYR1 and NPRY2. When treated with the human NPY peptide, expression of insulin1 and insulin2 and ERK was activated within 15 min. Furthermore, treatment with the MEK inhibitor PD98059 and NPY abolished the induction of insulin1 and insulin2. Together, these findings suggest that the regulation of insulin expression by sNPF or NPY through ERK is evolutionarily conserved in Drosophila and mammals (Lee, 2008).

To verify that sNPF induction of Dilp expression has a physiological consequence, insulin signals at a target tissue, the Drosophila fat body were examined. Fat body cells in flies with neuronal overexpression of sNPF (MJ94>2XsNP) were 42% larger than in the control, whereas inhibition of sNPF by MJ94>sNPF-Ri and sNPF^c00448 reduced cell size by 38% and 51% respectively. These differences in size correspond to changes in insulin signal transduction within the cells. Overexpression of sNPF (MJ94>sNPF and MJ94>2XsNPF) leads to phosphorylation and activation of Akt in the fat body, whereas the opposite effect was seen with neuronal inhibition of sNPF (MJ94>sNPF-Ri and sNPF^c00448). Activated Akt represses the transcription factor dFOXO by phosphorylation and subsequent cytoplasmic localization. In wild-type flies, dFOXO localized equally in the cytoplasm and nucleus. As predicted, neuronal induction of sNFP (MJ94>2XsNPF) increased the cytoplasmic localization of dFOXO, whereas inhibition of sNPF (MJ94>sNPF-Ri and sNPF^c00448) yielded fat body cells with dFOXO predominantly localized in the nucleus. Finally, dFOXO induces expression of the translational inhibitor d4E-BP, and, consistent with the current observations, expression of d4E-BP was elevated in animals where sNPF was inhibited (MJ94>sNPF-Ri and sNPF^c00448) and reduced in animals where sNPF was overexpressed (MJ94>2XsNPF) (Lee, 2008).

Besides cell growth, Drosophila insulin-like peptides modulate aspects of metabolism and ageing. For instance, ablation of the IPCs reduces animal size, elevates the level of haemolymph carbohydrates.Therefore trehalose and glucose were assessed in sNPF mutant flies. As predicted, both carbohydrates were reduced upon sNPF overexpression, and both were elevated in sNPF hypomorphs. Also the lifespan of sNPF mutants was measured. As expected, inhibition of sNPF by MJ94>sNPF-Ri increased median lifespan by 16-21%, whereas sNPF overexpression (MJ94>2XsNPF) did not affect lifespan in flies (Lee, 2008).

Overall, the effects on Dilp1 and Dilp2 expression in IPCs regulated by sNPF are associated with cellular, carbohydrate and lifespan responses that are predicted to be caused by changes in the actual level of available insulin peptides. It is concluded that sNPF ultimately regulates insulin secretion from the IPC to affect target tissue insulin/dFOXO signalling and thus modulate growth, metabolism and lifespan (Lee, 2008).

Regulation of food consumption by neuropeptides is a critical step for interventions for managing obesity and metabolic syndromes. Mammalian NPY is known to positively regulate appetite and has thus been thought to promote weight gain primarily by affecting food intake. Thus study revealed a novel physiological role for NPY that is conserved by sNPF of Drosophila. These neuropeptides can affect growth, metabolism and lifespan by modulating ERK-regulated transcription of insulin-like peptides. In Drosophila, sNPFnergic and IPC neurons are adjacent in the brain. This study found, however, that pancreatic β-cells are also responsive to NPY, which is of hypothalamic origin. Although the hypothalamic neurosecretory cells and responding pancreatic endocrine cells are spatially distinct in mammals, recent developmental analysis suggests a parallel developmental pathway for hypothalamic neurosecretory cells and the IPCs of Drosophila, raising the possibility of a common molecular mechanism for β-cell formation. This would suggest that β-cells are not only evolutionarily tied to the hypothalamic neurosecretory cells but also that they retain their functional relationship to their hypothalamic origin by regulating insulin in response to the neuropeptide NPY (Lee, 2008).

Neuronal energy-sensing pathway promotes energy balance by modulating disease tolerance

The starvation-inducible coactivator cAMP response element binding protein (CREB)-cAMP-regulated transcription coactivator (Crtc) has been shown to promote starvation resistance in Drosophila by up-regulating CREB target gene expression in neurons, although the underlying mechanism is unclear. This study found that Crtc and its binding partner CREB enhance energy homeostasis by stimulating the expression of short neuropeptide F (sNPF), an ortholog of mammalian neuropeptide Y, which was shown to be a direct target of CREB and Crtc. Neuronal sNPF was found to promote energy homeostasis via gut enterocyte sNPF receptors, which appear to maintain gut epithelial integrity. Loss of Crtc-sNPF signaling disrupts epithelial tight junctions, allowing resident gut flora to promote chronic increases in antimicrobial peptide (AMP) gene expression that compromised energy balance. Growth on germ-free food reduces AMP gene expression and rescues starvation sensitivity in Crtc mutant flies. Overexpression of Crtc or sNPF in neurons of wild-type flies dampens the gut immune response and enhances starvation resistance. These results reveal a previously unidentified tolerance defense strategy involving a brain-gut pathway that maintains homeostasis through its effects on epithelial integrity (Shen, 2016).

Disruptions in energy balance are a component of the collateral damage associated with mounting an immune response. In addition to regulating the magnitude of an immune response, energy allocation must be properly regulated to minimize physiological damage during infection. This study found that Drosophila sNPF, a mammalian NPY homolog, is regulated by CrebB/Crtc within the CNS, where it promotes energy balance by maintaining epithelial integrity and thereby attenuating overexuberant immune activation in the gut. The effects of sNPF were unexpected, given its role in food-seeking behavior. Indeed, food intake appears comparable between Crtc mutants and control flies (Shen, 2016).

The effects of sNPF are mediated by enterocyte sNPF-Rs, suggesting that the sNPF brain-gut signal is released by a subset of the sNPF+ neurons that directly innervate the gut. Although neuronal activity is known to contribute to energy homeostasis, the results suggest that the modulation of the gut immune system by CrebB/Crtc is a critical component in this process (Shen, 2016).

Epithelial tissues are typically colonized by both commensal and invasive microbes. sNPF appears to be actively expressed and released from the CNS in times of stress, providing nonautonomous control of gut immunity from the brain. Based on its widespread expression in the midgut, sNPF-R may provide ubiquitous attenuation of the innate immune response. Consistent with observations in Drosophila, activation of the NPY receptor ortholog (NPR-1) in Caenorhabditis elegans also down-regulates inflammatory gene expression. The current studies extend these findings by showing how a neuronal fasting-inducible pathway modulates energy balance via its effects on the gut immune system (Shen, 2016).

Following their activation, sNPF-Rs appear to promote energy balance by enhancing epithelial integrity. Although the mechanism underlying these effects is unclear, it is noted that disruption of the tight junction protein Bbg in flies also causes constitutive up-regulation of innate immunity genes. Future studies should reveal whether sNPF-R modulates the activity of Bbg or related proteins in enterocytes (Shen, 2016).

In mammals, inflammatory bowel diseases, such as ulcerative colitis, are often associated with profound weight loss, due, in part, to the chronic activation of the immune system. By reducing inflammatory gene expression and enhancing energy homeostasis, gut neuropeptides, such as NPY, may provide therapeutic benefit in this setting (Shen, 2016).

Starvation promotes concerted modulation of appetitive olfactory behavior via parallel neuromodulatory circuits

The internal state of an organism influences its perception of attractive or aversive stimuli and thus promotes adaptive behaviors that increase its likelihood of survival. The mechanisms underlying these perceptual shifts are critical to understanding of how neural circuits support animal cognition and behavior. Starved flies exhibit enhanced sensitivity to attractive odors and reduced sensitivity to aversive odors. This study shows that a functional remodeling of the olfactory map is mediated by two parallel neuromodulatory systems that act in opposing directions on olfactory attraction and aversion at the level of the first synapse. Short neuropeptide F sensitizes an antennal lobe glomerulus wired for attraction, while tachykinin (DTK) suppresses activity of a glomerulus wired for aversion. Thus this study shows parallel neuromodulatory systems functionally reconfigure early olfactory processing to optimize detection of nutrients at the risk of ignoring potentially toxic food resources (Ko, 2015).

This study demonstrates that shifts in the internal metabolic state of an animal lead to dramatic functional changes in its olfactory circuit and behaviors. Starved flies exhibit enhanced odor sensitivity in odorant receptor neurons (ORNs) that mediate behavioral attraction and decreased sensitivity in ORNs that mediate behavioral aversion. This functional remodeling of the olfactory map is mediated by parallel neuromodulatory systems that act in opposing directions on olfactory attraction and aversion. An earlier study showed that sNPFR signaling increases sensitivity in Or42b ORNs and thus enhances behavioral attraction (Root, 2011). The current study, however, shows that sNPFR signaling does not account for all changes induced by starvation in behavioral responses to a wider range of odor concentrations. Second, this study shows that starvation leads to a decreased sensitivity in the Or85a ORNs, an odorant channel that mediates behavioral aversion. Third, it was shown that DTKR signaling mediates the reduced sensitivity in the Or85a ORNs and partly accounts for enhanced behavioral attraction to high concentrations of vinegar. Fourth, eliminating DTKR and sNPFR signaling pathways together fully reverses the effect of starvation on behavioral attraction across all odor concentrations tested. Finally, evidence suggests that the same global insulin signal regulating sNPFR expression may also regulate DTKR expression (Ko, 2015).

In the wild, rotten fruits early in the fermentation process are more attractive to Drosophila than fresh or highly fermented fruits. In the laboratory, well fed flies display very little attraction to apple cider vinegar (Root, 2011). Low levels of vinegar are indicative of fresh fruit of limited nutritional value. Expanding odor sensitivity to lower concentrations of potential food odors may encourage flies to accept food sources of lower value. High odor concentrations typically accompany late stages of fermentation and are often aversive or uninteresting to flies. Starved flies are attracted to high concentrations of vinegar partly due to neuromodulatory mechanisms that enhance sensitivity in Or42b ORNs, an attractive odor channel, and partly through neuromodulatory mechanisms that reduce sensitivity in Or85a ORNs, an aversive odor channel. In the working model, behavioral attraction to higher odor concentrations of vinegar is the sum of the opposing effects of Or42b and Or85a. When flies face starvation, the balance of these inputs shifts to favor Or42b over Or85a inputs, as mediated by selective upregulation of sNPFR and DTKR in these ORNs, respectively. These processes could serve to encourage flies to risk ingestion of potentially toxic foods when under nutritional stress (Ko, 2015).

Given the broad array of glomeruli that can respond to odors such as vinegar, it may be surprising that the modulation of only two glomeruli is sufficient to significantly impact fly behavioral attraction. Whether these findings extend to a broad array of food associated odors and whether additional glomeruli are modulated by these neuromodulatory systems remain to be determined. In this context, it is noted that a recent correlational analysis predicts DM5 activity is highly correlated with behavioral attraction. However, this prediction has not been confirmed by direct testing of the DM5 glomerulus in behavioral experiments and is contradicted by more recent findings, as well as the data in this paper. Thus the current findings suggest that in starved flies the concentration range over which vinegar odor is attractive expands in both directions, with the acute need for caloric intake apparently outweighing considerations of food quality or risk (Ko, 2015).

This study highlights the importance of neuromodulators in shaping local neural circuit activity to accommodate the internal physiological state of an organism. The often unique expression patterns of specific GPCRs in sensory systems highlights the flexibility conferred by this evolutionarily ancient mechanism to translate neuroendocrine signals into local shifts in neuronal excitability and network properties that ultimately lead to adaptive behaviors. sNPF shares structural and functional similarities with its vertebrate homolog, NPY. Both neuropeptides show roles in controlling food intake and feeding behaviors in insects and vertebrates. Interestingly, NPY is also expressed in the vertebrate olfactory bulb and is thus positioned to shape olfactory processing during shifts in appetitive states as well. sNPF's broad expression pattern in the fly brain supports the possibility it is widely used to orchestrate changes across many different neuropils to shape appetitive behaviors. Indeed, sNPF and NPF, another NPY homolog in Drosophila, have been shown in the fly gustatory system to control sweet and bitter taste sensitivity, respectively, in parallel but opposing directions (Inagaki, 2014). The similar changes manifested by nutritional stress in both the olfactory and gustatory systems suggests complex networks of neuromodulators may shape sensory processing of aversive and attractive inputs differentially throughout the brain in a hunger state (Ko, 2015).

DTK and DTKR share homology with substance P and its receptor NK1, respectively. Interestingly, they seem to share roles in shaping the processing of stressful or negative sensory cues in both flies and mammals. For example, in rodents, emotional stressors cause long-lasting release of substance P to activate NK1 in the amygdala to generate anxiety-related behavior. In Drosophila, DTK signaling has also been shown to be critical for aggressive behaviors among male flies (Asahina, 2014). Previous work has shown Drosophila tachykinin mediates presynaptic inhibition in ORNs and detected expression in the LNs. This current study maps the locus of DTK's effects on behavioral responses to vinegar to the Or85a/DM5 ORNs using behavior and functional imaging. It was also confirmed that the source of the peptide is indeed the LNs as previous anatomical data had suggested. Thus, tachykinin's role in modulating stressful sensory inputs appears to extend to a glomerulus hardwired to behavioral aversion in the olfactory system (Ko, 2015).

The current results here resonate with discoveries in the gustatory system (Inagaki, 2014) and show that starvation changes the perception of both attractive and aversive sensory inputs beginning at the peripheral nervous system. Through the use of parallel neuromodulatory systems, the internal state of the organism functionally reconfigures early olfactory processing to optimize its detection of nutrients at the risk of ignoring potentially toxic food resources. It is certainly likely that neuromodulatory systems also impact and reconfigure central circuits in appetitive contexts. Thus, it will be of great interest to understand the contributions of peripheral and central circuits towards modifying appetitive behaviors (Ko, 2015).

Autophagy within the mushroom body protects from synapse aging in a non-cell autonomous manner

Macroautophagy is an evolutionarily conserved cellular maintenance program, meant to protect the brain from premature aging and neurodegeneration. How neuronal autophagy, usually loosing efficacy with age, intersects with neuronal processes mediating brain maintenance remains to be explored. This study shows that impairing autophagy in the Drosophila learning center (mushroom body, MB) but not in other brain regions triggered changes normally restricted to aged brains: impaired associative olfactory memory as well as a brain-wide ultrastructural increase of presynaptic active zones (metaplasticity), a state non-compatible with memory formation. Mechanistically, decreasing autophagy within the MBs reduced expression of an NPY-family neuropeptide, and interfering with autocrine NPY signaling of the MBs provoked similar brain-wide metaplastic changes. The results in an exemplary fashion show that autophagy-regulated signaling emanating from a higher brain integration center can execute high-level control over other brain regions to steer life-strategy decisions such as whether or not to form memories (Bhukel, 2019).

The maintenance of neuronal homeostasis is severely threatened by aging. The strictly postnatal character of deficits observed after KD of core autophagy machinery triggered the hope that autophagy might have a specific relation to the aging process. The last few years have indeed seen an accumulation of evidences that the efficiency of autophagic clearance in neurons declines with age on organismal level. Hence, rejuvenating autophagy in aging neurons is considered a promising strategy to restore cognitive performance. Successfully exploring this direction will, however, depend on deepening insights at the intersection of autophagy, the relevant neuronal sub-cellular compartments, importantly synaptic specializations, and relevant neuron populations/brain regions (Bhukel, 2019).

The endogenous polyamine spermidine has prominent cardio-protective and neuro-protective effects and recent work finds spermidine restoration to counteract otherwise deteriorating health in aging mice in an autophagy-dependent manner. In Drosophila, restoring spermidine specifically suppressed age-induced decay in their ability to form olfactory memories, again in an autophagy-dependent manner. Concomitantly, in the aged Drosophila brain, previous work found a brain-wide, age-induced upshift in the ultrastructural size (EM: larger T-bars; STED: increased diameter of BRP scaffold) of presynaptic AZs (metaplasticity). Two findings causally linked this upshift to decreased olfactory memory performance. First, when continuously fed with spermidine, flies of 30 days of age (normally suffering from a complete loss of age-sensitive component of memory) were largely protected from these changes. Secondly, genetically provoking this up-shift eliminated the normally age-sensitive memory component in young animals already. An upshift in the AZ size should increase synaptic strength, evident in increased SV release in response to natural odors observed in aged but not aged-spermidine-fed flies. Presynaptic plasticity is crucial for forming memory traces in Drosophila. Previous work thus suggests that this presynaptic metaplasticity shifts the operational range of synapses in a way that they become unable to execute the plastic changes faithfully in response to conditioning stimuli (Bhukel, 2019).

This study further addressed the relation between defective autophagy, presynaptic ultrastructure and plasticity and olfactory memory formation. Autophagosome biogenesis is very dominant close to presynaptic specializations in distal axons in compartmentalized fashion and efficient macro-autophagy is essential for neuronal homeostasis and survival. Retrograde transport of autophagosomes might play a role in broader neuronal signaling processes, promoting neuronal complexity and preventing neurodegeneration. Surprisingly, however, the data do not favor a direct substrate relationship between AZ proteins and autophagy. Instead, evidence was found for a seemingly non-cell autonomous relation between brain-wide synapse organization and the autophagic status of the mere MB. After genetic impairment of autophagy (via atg5 or atg9 KD) using two different MB-specific Gal4-driver lines, the presynaptic metaplasticity was observed across the Drosophila olfactory system and beyond. While the autophagic arrest (p62 staining) was largely limited to the expression domain of these drivers, the synapses were pushed towards a state of metaplasticity. Since the ultrastructural size of AZs and the per AZ BRP levels increased equally in aged and MB-autophagy-challenged animals, it is concluded that the autophagic status of the MB neuron population executes a signaling process, which can control the per AZ amounts of BRP and other AZ proteins. Further studies are warranted to dissect the nature of these signaling processes (Bhukel, 2019).

Notably, accumulating evidences support the important role of neuropeptide Y (NPY) in aging and lifespan determination. NPY levels decrease with age in mice and re-substituting NPY is able to counteract age-induced changes of the brain at several levels. A cross-talk between autophagy and NPY in regulating the feeding behavior has been demonstrated in mice (Bhukel, 2019).

This study found that transcript expression level of an NPY family member (sNPF) are controlled by autophagy within the MBs. snpf hypomorph allele mimicking the MB reduction of sNPF of the MB-specific autophagy KD situations as well as the sNPF expression in aged animals. In this hypomorph allele a similar up regulation was observed in BRP Nc82 signal. KD of the snpfr using an MB-specific driver drove the brain-wide metaplastic change even stronger than the sNPF hypomorph (obviously only partially affecting the sNPF-specific signaling). This scenario in ultrastructural detail resembled both the age-induced and MB-specific autophagy-KD-induced metaplasticity phenotypes. These results, therefore, support the essential role of MB in integrating the metabolic state of Drosophila in an autocrine fashion to modulate the presynaptic release scaffold state throughout the fly brain. The mechanistic basis of this exciting regulation warrants further investigation. Interestingly, elevated cAMP signaling is generally driving plasticity in Drosophila neurons, while sNPF signaling is meant to reduce cAMP and thus potentially might be able to reset plastic changes such as increased BRP levels. In apparent contradiction to sNPF signaling directly widely controlling metaplasticity is the finding that MB-specific KD of the sNPFR sufficed to increase BRP levels. At this moment, it can only be speculated as to why KD of sNPF-receptor also results in extended metaplastic changes. Potentially, sNPF-receptor signaling within the MB might be important to control sNPF secretion in a physiological manner via a quasi-autocrine mechanism (Bhukel, 2019).

Intriguingly, the metaplastic state characterized both aged and MB-specific autophagy KD animals, and in both cases provoked a specific loss of the ASM component of memory. Notably, olfactory MTM measured in this study, are considered to be the direct precursor of olfactory LTM, which in turn have been shown to be energetically costly. Notably, autophagy and NPY signaling are prime candidate mechanisms for the therapy of age-induced cognitive processes (Bhukel, 2019).

Recent research has uncovered several examples connecting autophagy and hormonal-type regulations interacting between organ systems in non-cell autonomous regimes. For instance, Atg18 acts non-cell autonomously both in neurons and in intestines to firstly, maintain the wild-type lifespan of C.elegans and secondly, to respond to the dietary restriction and DAF-2 longevity signals. Atg18 in chemosensory neurons and intestines acts in parallel and converges on unidentified neurons that secrete neuropeptides to mediate the influence of Daf-2 on C.elegans lifespan through the transcription factor DAF-16/FOXO in response to reduced IGF signaling. In Drosophila, neuronal up-regulation of AMPK induces autophagy, via up-regulation of Atg1 non-cell autonomously in intestines and slows intestinal aging and vice versa. Moreover, up-regulation of Atg1 in neurons extends lifespan and maintains intestinal homeostasis during aging and these inter-tissue effects of AMPK/Atg1 were linked to altered insulin-like signaling. On the contrary, this study found the insulin producing cells (IPCs) themselves to not mediate the observed metaplastic state, as neither the KD of atg9 nor the KD of snpfr in Pars intercerebralis had any impact on the synaptic status of these flies (Bhukel, 2019).

Autophagy regulation is tightly connected to cellular energetics, nutrient recycling, and the maintenance of cellular energy status. The fruit fly can evaluate its metabolic state by integrating hunger and satiety signals at the very KC-to-MBON synapses in MB under control of dopaminergic neurons to control hunger-driven food-seeking behavior. At the same time, long-term memory encoding necessitates an increase in MB energy flux with dopamine signaling mediating this energy switch in the MB. In line with these findings, this study now provides a modeling basis to study these delicate relations in an exemplary fashion. Taken together, these data suggest that MB integrates the metabolic state of the flies via cross talk between autophagy and sNPF signaling with the decision whether to form memories or not and a block in this cross talk with aging gives rise to synaptic metaplasticity which initiates the age-induced memory impairment in Drosophila. It is tempting to speculate that the MB executes hierarchically, a high-level control integrating the metabolic and caloric situation with a life-strategy decision of whether or not to form mid-term memories (Bhukel, 2019).

ER-Ca2+ sensor STIM regulates neuropeptides required for development under nutrient restriction in Drosophila

Neuroendocrine cells communicate via neuropeptides to regulate behaviour and physiology. This study examines how STIM (Stromal Interacting Molecule), an ER-Ca2+ sensor required for Store-operated Ca2+ entry, regulates neuropeptides required for Drosophila development under nutrient restriction (NR). Two STIM-regulated peptides, Corazonin and short Neuropeptide F, were found to be required for NR larvae to complete development. Further, a set of secretory DLP (Dorso lateral peptidergic) neurons which co-express both peptides was identified. Partial loss of dSTIM caused peptide accumulation in the DLPs, and reduced systemic Corazonin signalling. Upon NR, larval development correlated with increased peptide levels in the DLPs, which failed to occur when dSTIM was reduced. Comparison of systemic and cellular phenotypes associated with reduced dSTIM, with other cellular perturbations, along with genetic rescue experiments, suggested that dSTIM primarily compromises neuroendocrine function by interfering with neuropeptide release. Under chronic stimulation, dSTIM also appears to regulate neuropeptide synthesis (Megha, 2019).

Metazoan cells commonly use ionic Ca2+ as a second messenger in signal transduction pathways. To do so, levels of cytosolic Ca2+ are dynamically managed. In the resting state, cytosolic Ca2+ concentration is kept low and maintained thus by the active sequestration of Ca2+ into various organelles, the largest of which is the ER. Upon activation, ligand-activated Ca2+ channels on the ER, such as the ryanodine receptor or inositol 1,4,5-trisphosphate receptor (IP3R), release ER-store Ca2+ into the cytosol. Loss of ER-Ca2+ causes STromal Interacting Molecule (STIM), an ER-resident transmembrane protein, to dimerize and undergo structural rearrangements. This facilitates the binding of STIM to Orai, a Ca2+ channel on the plasma membrane, whose pore then opens to allow Ca2+ from the extracellular milieu to flow into the cytosol. This type of capacitative Ca2+ entry is called Store-operated Ca2+ entry (SOCE). Of note, key components of SOCE include the IP3R, STIM and Orai, that are ubiquitously expressed in the animal kingdom, underscoring the importance of SOCE to cellular functioning. Depending on cell type and context, SOCE can regulate an array of cellular processes (Megha, 2019).

Neuronal function in particular is fundamentally reliant on the elevation of cytosolic Ca2+. By tuning the frequency and amplitude of cytosolic Ca2+ signals that are generated, distinct stimuli can make the same neuron produce outcomes of different strengths. The source of the Ca2+ influx itself contributes to such modulation as it can either be from internal ER-stores or from the external milieu, through various activity-dependent voltage gated Ca2+ channels (VGCCs) and receptor-activated Ca2+ channels or a combination of the two. Although the contributions of internal ER-Ca2+ stores to neuronal Ca2+ dynamics are well recognized, the study of how STIM and subsequently, SOCE-mediated by it, influences neuronal functioning, is as yet a nascent field (Megha, 2019).

Mammals have two isoforms of STIM, STIM1 and STIM2, both which are widely expressed in the brain. As mammalian neurons also express multiple isoforms of Orai and IP3R, it follows that STIM-mediated SOCE might occur in them. Support for this comes from studies in mice, where STIM1-mediated SOCE has been reported for cerebellar granule neurons and isolated Purkinje neurons, while STIM2-mediated SOCE has been shown in cortical and hippocampal neurons. STIM can also have SOCE-independent roles in excitable cells, that are in contrast to its role via SOCE. In rat cortical neurons and vascular smooth muscle cells, Ca2+ release from ER-stores prompts the translocation of STIM1 to ER-plasma membrane junctions, and binding to the L-type VGCC, CaV1.2. Here STIM1 inhibits CaV1.2 directly and causes it to be internalized, reducing the long-term excitability of these cells. In cardiomyocyte-derived HL1 cells, STIM1 binds to a T-type VGCC, CaV1.3, to manage Ca2+ oscillations during contractions. These studies indicate that STIM regulates cytosolic Ca2+ dynamics in excitable cells, including neurons and that an array of other proteins determines if STIM regulation results in activation or inhibition of neurons. Despite knowledge of the expression of STIM1 and STIM2 in the hypothalamus, the major neuroendocrine centre in vertebrates, studies on STIM in neuroendocrine cells are scarce. This study therefore used Drosophila melanogaster to address this gap (Megha, 2019).

Neuroendocrine cells possess elaborate machinery for the production, processing and secretion of neuropeptides (NPs), which perhaps form the largest group of evolutionarily conserved signalling agents. Inside the brain, NPs typically modulate neuronal activity and consequently, circuits; when released systemically, they act as hormones. Drosophila is typical in having a vast repertoire of NPs that together play a role in almost every aspect of its behaviour and physiology. Consequently, NP synthesis and release are highly regulated processes. As elevation in cytosolic Ca2+ is required for NP release, a contribution for STIM-mediated SOCE to NE function was hypothesized (Megha, 2019).

Drosophila possess a single gene for STIM, IP3R and Orai, and all three interact to regulate SOCE in Drosophila neurons. In dopaminergic neurons, dSTIM is important for flight circuit maturation, with dSTIM-mediated SOCE regulating expression of a number of genes, including Ral, which controls neuronal vesicle exocytosis. In glutamatergic neurons, dSTIM is required for development under nutritional stress and its' loss results in down-regulation of several ion channel genes which ultimately control neuronal excitability. Further, dSTIM over-expression in insulin-producing NE neurons could restore Ca2+ homeostasis in a non-autonomous manner in other neurons of an IP3R mutant, indicating an important role for dSTIM in NE cell output, as well as compensatory interplay between IP3R and dSTIM. At a cellular level, partial loss of dSTIM impairs SOCE in Drosophila neurons as well as mammalian neural precursor cells. Additionally, reducing dSTIM in Drosophila dopaminergic neurons attenuates KCl-evoked depolarisation and as well as vesicle release. Because loss of dSTIM specifically in dimm+ NE cells results in a pupariation defect on nutrient restricted (NR) media, this study used the NR paradigm as a physiologically relevant context in which to investigate STIM's role in NE cells from the cellular as well as systemic perspective (Megha, 2019).

This study employed an in vivo approach coupled to a functional outcome, in order to broaden understanding of how STIM regulates neuropeptides. A role for dSTIM-mediated SOCE in Drosophila neuroendocrine cells for survival on NR was previously established. The previous study offered the opportunity to identify SOCE-regulated peptides, produced in these neuroendocrine cells, that could be investigated in a physiologically relevant context (Megha, 2019).

In Drosophila, both Crz and sNPF have previously been attributed roles in many different behaviours. Crz has roles in adult metabolism and stress responses, sperm transfer and copulation, and regulation of ethanol sedation. While, sNPF has been implicated in various processes including insulin regulation circadian behaviour, sleeping and feeding. Thus, the identification of Crz and sNPF in coping with nutritional stress is perhaps not surprising, but a role for them in coordinating the larval to pupal transition under NR is novel (Megha, 2019).

A role for Crz in conveying nutritional status information is supported by this study. In larvae, Crz+ DLPs are known to play a role in sugar sensing and in adults, they express the fructose receptor Gr43a. Additionally, they express receptors for neuropeptides DH31, DH44 and AstA, which are made in the gut as well as larval CNS. Together, these observations and are strongly indicative of a role for Crz+ DLPs in directly or indirectly sensing nutrients, with a functional role in larval survival and development in nutrient restricted conditions (Megha, 2019).

Several neuropeptides and their associated signalling systems are evolutionarily conserved. The similarities between Crz and GnRH (gonadotrophin-releasing hormone), and sNPF and PrRP (Prolactin-releasing peptide), at the structural, developmental and receptor level therefore, is intriguing. Structural similarity of course does not imply functional conservation, but notably, like sNPF, PrRP has roles in stress response and appetite regulation. This leads to the conjecture that GnRH and PrRP might play a role in mammalian development during nutrient restriction (Megha, 2019).

dSTIM regulates Crz and sNPF at the levels of peptide release and likely, peptide synthesis upon NR. It is speculated that neuroendocrine cells can use these functions of STIM, to fine tune the amount and timing of peptide release, especially under chronic stimulation (such as 24hrs NR), which requires peptide release over a longer timeframe. Temporal regulation of peptide release by dSTIM may also be important in neuroendocrine cells that co-express peptides with multifunctional roles, as is the case for Crz and sNPF. It is conceivable that such different functional outcomes may require distinct bouts of NP release, varying from fast quantile release to slow secretion. As elevation in cytosolic Ca2+ drives NP vesicle release, neurons utilise various combinations of Ca2+ influx mechanisms to tune NP release. For example, in Drosophila neuromuscular junction, octopamine elicits NP release by a combination of cAMP signalling and ER-store Ca2+, and the release is independent of activity-dependent Ca2+ influx. In the mammalian dorsal root ganglion, VGCC activation causes a fast and complete release of NP vesicles, while activation of TRPV1 causes a pulsed and prolonged release. dSTIM-mediated SOCE adds to the repertoire of mechanisms that can regulate cytosolic Ca2+ levels and therefore, vesicle release. This has already been shown for Drosophila dopaminergic neurons and this study extends the scope of release to peptides. Notably, dSTIM regulates exocytosis via Ral in neuroendocrine cells, like in dopaminergic neurons (Megha, 2019).

In Drosophila larval Crz+ DLPs, dSTIM appears to have a role in both fed, as well as NR conditions. On normal food, not only do Crz+ DLPs exhibit small but significant levels of neuronal activity but also, loss of dSTIM in these neurons reduced Crz signalling. Thus, dSTIM regulates Ca2+ dynamics and therefore, neuroendocrine activity, under basal as well as stimulated conditions. This is consistent with observations that basal SOCE contributes to spinogenesis, ER-Ca2+ dynamics as well as transcription. This regulation appears to have functional significance only in NR conditions as pupariation of larvae, with reduced levels of dSTIM in Crz+ neurons, is not affected on normal food. In a broader context, STIM is a critical regulator of cellular Ca2+ homeostasis as well as SOCE, and a role for it in the hypothalamus has been poorly explored. Because STIM is highly conserved across the metazoan phyla, this study predicts a role for STIM and STIM-mediated SOCE in peptidergic neurons of the hypothalamus. There is growing evidence that SOCE is dysregulated in neurodegenerative diseases. In neurons derived from mouse models of familial Alzheimer's disease and early onset Parkinson's, reduced SOCE has been reported. How genetic mutations responsible for these diseases manifest in neuroendocrine cells is unclear. If they were to also reduce SOCE in peptidergic neurons, it's possible that physiological and behavioural symptoms associated with these diseases, may in part stem from compromised SOCE-mediated NP synthesis and release (Megha, 2019).

Sensory integration and neuromodulatory feedback facilitate Drosophila mechanonociceptive behavior

Nociception is an evolutionarily conserved mechanism to encode and process harmful environmental stimuli. Like most animals, Drosophila melanogaster larvae respond to a variety of nociceptive stimuli, including noxious touch and temperature, with stereotyped escape responses through activation of multimodal nociceptors. How behavioral responses to these different modalities are processed and integrated by the downstream network remains poorly understood. By combining trans-synaptic labeling, ultrastructural analysis, calcium imaging, optogenetics and behavioral analyses, this study uncovered a circuit specific for mechanonociception but not thermonociception. Notably, integration of mechanosensory input from innocuous and nociceptive sensory neurons is required for robust mechanonociceptive responses. It was further shown that neurons integrating mechanosensory input facilitate primary nociceptive output by releasing short neuropeptide F, the Drosophila neuropeptide Y homolog. These findings unveil how integration of somatosensory input and neuropeptide-mediated modulation can produce robust modality-specific escape behavior (Hu, 2017).

Sensing noxious stimuli and responding with appropriate nociceptive responses is essential for avoiding potentially harmful environments. The somatosensory system of vertebrates and invertebrates features distinct neuronal subtypes sensing different modalities (e.g., innocuous or nociceptive touch, temperature) and conveys converging and diverging information to higher brain centers. This requires a multi-layered hierarchical neuronal network that reliably detects and integrates innocuous or nociceptive stimuli and translates them into appropriate behavioral responses. Moreover, neuromodulation by neuropeptides may add additional complexity to somatosensory information processing in both vertebrates and invertebrates, either by acting locally as co-neurotransmitters or globally via widespread tonic release. In Drosophila, over 40 neuropeptides and corresponding G protein-coupled neuropeptide receptors have been identified so far and shown to regulate various behaviors including thermo-nociceptive sensitisation. How different somatosensory modalities are integrated at the circuit level and modulated by neuropeptide signaling to affect nociceptive behavior is thus of great interest (Hu, 2017).

The Drosophila larval peripheral nervous system (PNS) provides a genetically tractable model to address these questions. It features type l ciliated neurons (chordotonal and external sensory neurons), bipolar (bd) and four classes of dendritic arborization (da) neurons (C1da-C4da). While C2da and C3da neurons respond to innocuous touch, C4da neurons are multimodal nociceptors responding to harsh mechanical touch, noxious temperatures and strong UV and blue light. Nociceptive stimuli or optogenetic activation of C4da neurons in Drosophila larvae elicit a nocifensive response characterized by stereotyped rolling behavior followed by locomotion speedup. The role of C4da neurons as primary nociceptors is well established, yet the complexity of their downstream circuitry has just started to emerge. An extensive hierarchical network was found to integrate nociceptive and high-frequency vibration cues which enhance nociceptive behavioral responses. Indeed, most natural stimuli elicit multisensory responses that enhance the selection of specific behaviors. However, it is still elusive how the network processes modality-specific sensory input to produce stimulus-dependent actions (Hu, 2017).

This study identified novel nociceptive network components and a neuromodulatory feedback mechanism specifically required for C4da neuron-mediated mechano-nociceptive responses (Hu, 2017).

This study identified and characterized a novel and modality-specific node of the nociceptive network required for mechanically induced escape behavior. C4da neurons form functional synaptic contacts with A08n neurons, which have recently been suggested to be involved in nociceptive behavior. It was further shown that they respond to mechano-nociceptive stimuli (relayed via C4da neurons) and are necessary and sufficient for nociceptive behavior. Anatomically, the results suggest that A08n neurons are core relay neurons as they receive input from all C4da neurons and thus potentially convey nociceptive information from anywhere on the body wall (Hu, 2017).

Moreover, robust escape responses to mechano-nociceptive stimuli require the activity of C2da and C3da sensory neurons, which normally respond to innocuous touch. Intriguingly, this study found that neuropeptide-producing DP-ilp7 neurons integrate mechano-sensory input from C2/C3/C4da neurons to facilitate nociceptive escape behavior. DP-ilp7 neurons receive C4da neuron input along the entire VNC (even though such input alone was not sufficient to elicit physiological responses) and can respond to input from touch-responsive C2da neuron as well as coincident activation of C3da and C4da neuron. Thus, innocuous and nociceptive mechanical cues can be integrated -- at least in part -- by converging onto DP-ilp7 neurons (Hu, 2017).

Previous results have suggested that C2da and C3da but not C1da neurons respond to mechanical stimulation; further, C3da neuron activity and the mechanosensitive channel NompC are required for innocuous touch. This study found that, curiously, C2da neurons are required in mechano-nociceptive behavior but not innocuous touch responses and that their activation induces C-shape bending and slow rolling which depends partly on ilp7 neuron function. Thus C2da neurons mediate aspects of nociceptive behaviors (Hu, 2017).

In vertebrates, nociceptive and innocuous touch inputs converge both mono- and polysynaptically within the dorsal horn. Interestingly, VGLUT3 is specifically expressed in C-low threshold mechanoreceptors and required for injury-induced hypersensitivity to mechanical stimuli, suggesting that vertebrate innocuous touch receptors participate in nociception as well. Consistently, alleviation of mechanically-induced pain require low-threshold mechanoreceptor function in mice. It should be interesting to investigate the mechanism of innocuous and nociceptive touch integration at the network level in higher organisms in more detail (Hu, 2017).

Similarly to most vertebrate nociceptors, C4da are multimodal neurons detecting nociceptive mechanical, thermal and aversive light stimuli. Both thermo- and mechano-nociceptive stimulation elicit C4da neuron-dependent rolling and escape responses, while short-wavelength light triggers an avoidance response. How activation of C4da neurons (by different stimuli) produces distinct or seemingly identical nociceptive behaviors is not fully understood. A recent study showed that light and thermal stimulation evoke low-frequency firing trains vs. rapid bursting of C4da neurons, respectively, suggesting that modality-specific physiological responses might be one mechanism. These data adds another differentiating mechanism: mechano-nociceptive behavior relies on recruitment of discrete network components of the nociceptive network (innocuous touch and DP-ilp7 neurons), which are not required for thermal responses. Interestingly, TrpA1-expressing neurons in the larval CNS respond to steep temperature gradients and can induce nociceptive rolling. These cells might be connected to C4da neurons suggesting another modality specific subset of the circuit encodes thermo-nociception (Hu, 2017).

The findings that inactivation of C2da, C3da or DP-ilp7 neurons virtually completely blocked mechano- but not thermo-nociceptive behavior suggests that the integration of mechanosensory neurons is critical for modality specific escape behavior. While non-nociceptive stimuli alone are not sufficient to trigger nociceptive responses, vibrational stimulation of chordotonal neurons has been shown to enhance C4da-mediated escape behavior. Both sensory pathways are integrated at the 2nd-order level by local basin neurons and further converging on command-like neurons (termed goro) at the 4th level. Similarly, DP-ilp7 neurons provide a neural substrate for innocuous and nociceptive touch integration. Integration of multiple mechanical modalities at multiple levels might protect against predators like the parasitoid wasp L. boulardi, which provide multisensory cues (wing beat, innocuous and nociceptive touch) that are very potent in eliciting larval nocifensive responses (Hu, 2017).

While many components of the nociceptive network in Drosophila larvae have been described, a role of neuromodulation has not been extensively investigated. This study showed that DP-ilp7 neuron activation was sufficient to facilitate mechano-nociceptive behavior. Curiously, sNPF, but not ilp7 peptide expressed in DP-ilp7 neurons plays a critical role in mechano-nociception. sNPF is dendritically localized in DP-ilp7 neurons in direct apposition to sensory nerve terminals. The corresponding sNPF-R is expressed in C2da, C3da and C4da neurons suggesting that sNPF provides an activity dependent regulatory feedback signal within this circuit. Both, reducing functions of sNPF in DP-ilp7 or sNPF-R in sensory C2-4da neurons impaired mechano-nociceptive behavior. Interestingly, sNPF is also localized to C4da neuron axon terminals and its knockdown there leads to thermo-nociceptive hypersensitivity. This raises the possibility that sNPF has distinct functions in nociception depending on when and where it is released (Hu, 2017).

Drosophila sNPF is involved in feeding and sleep behavior. Interestingly, food search behavior in starved animals relies on sNPF dependent presynaptic facilitation in olfactory receptor neurons. It is proposed that DP-ilp7 neuron activation by sensory neurons results in local sNPF release and feedback facilitation of sensory neuron output via sNPF-R signaling. The data show that DP-ilp7 activity and sNPF are necessary for full C4da and A08n neuron activation after mechano-nociceptive stimulation suggesting activity-dependent sNPF release is critical for facilitating nociceptive output. Consistently, this study found that sNPF-R function in C4da neuron was necessary for A08n neuron activation suggesting that sNPF signaling regulates synaptic output. Surprisingly, sNPF-R was functionally and behaviorally required not only in C4da, but also in C2/3da neurons, although they are not directly connected to A08n neurons. It is possible that C2/3da neuron output on DP-ilp7 neurons might be reduced upon sNPF-R inactivation, which in turn reduces sNPF release and C4da neuron output. Alternatively, C2/3da neuron output to additional network components might indirectly regulate A08n activation (Hu, 2017).

Taken together, this study has uncovered a mechanism that encodes modality-specific nociceptive behavior through recruitment of specific network components, multisensory integration and neuromodulatory feedback signaling. Interestingly, the closest mammalian homolog of sNPF-R, NPY receptor 2, is expressed in mechano-nociceptive A-fibers, which mediate paw withdrawal in mice. Moreover, NPY is highly expressed in the dorsal spinal cord and can act to alleviate chronic pain. Thus modality-specific network regulation by NPY and other neuropeptides may have an evolutionarily conserved role in regulating network function under both physiological and pathological conditions (Hu, 2017).

Identified peptidergic neurons in the Drosophila brain regulate insulin-producing cells, stress responses and metabolism by coexpressed short neuropeptide F and corazonin

Insulin/IGF-like signaling regulates the development, growth, fecundity, metabolic homeostasis, stress resistance and lifespan in worms, flies and mammals. Eight insulin-like peptides (DILP1-8) are found in Drosophila. Three of these (DILP2, 3 and 5) are produced by a set of median neurosecretory cells (insulin-producing cells, IPCs) in the brain. Activity in the IPCs of adult flies is regulated by glucose and several neurotransmitters and neuropeptides. One of these, short neuropeptide F (sNPF), regulates food intake, growth and Dilp transcript levels in IPCs via the sNPF receptor (sNPFR1) expressed on IPCs. This study identified a set of brain neurons that utilizes sNPF to activate the IPCs. These sNPF-expressing neurons (dorsal lateral peptidergic neurons, DLPs) also produce the neuropeptide corazonin (CRZ) and have axon terminations impinging on IPCs. Knockdown of either sNPF or CRZ in DLPs extends survival in flies exposed to starvation and alters carbohydrate and lipid metabolism. Expression of sNPF in DLPs in the sNPF mutant background is sufficient to rescue wild-type metabolism and response to starvation. Since CRZ receptor RNAi in IPCs affects starvation resistance and metabolism, similar to peptide knockdown in DLPs, it is likely that also CRZ targets the IPCs. Knockdown of sNPF, but not CRZ in DLPs decreases transcription of Dilp2 and 5 in the brain, suggesting different mechanisms of action on IPCs of the two co-released peptides. These findings indicate that sNPF and CRZ co-released from a small set of neurons regulate IPCs, stress resistance and metabolism in adult Drosophila (Kapan, 2012).

A large population of diverse neurons in the Drosophila central nervous system expresses short neuropeptide F, suggesting multiple distributed peptide functions

Insect neuropeptides are distributed in stereotypic sets of neurons that commonly constitute a small fraction of the total number of neurons. However, some neuropeptide genes are expressed in larger numbers of neurons of diverse types suggesting that they are involved in a greater diversity of functions. One of these widely expressed genes, snpf, encodes the precursor of short neuropeptide F (sNPF). To unravel possible functional diversity the distribution of transcript of the snpf gene and its peptide products wer mapped in the central nervous system (CNS) of Drosophila in relation to other neuronal markers. There are several hundreds of neurons in the larval CNS and several thousands in the adult Drosophila brain expressing snpf transcript and sNPF peptide. Most of these neurons are intrinsic interneurons of the mushroom bodies. Additionally, sNPF is expressed in numerous small interneurons of the CNS, olfactory receptor neurons (ORNs) of the antennae, and in a small set of possibly neurosecretory cells innervating the corpora cardiaca and aorta. A sNPF-Gal4 line confirms most of the expression pattern. None of the sNPF immunoreactive neurons co-express a marker for the transcription factor DIMMED, suggesting that the majority are not neurosecretory cells or large interneurons involved in episodic bulk transmission. Instead a portion of the sNPF producing neurons co-express markers for classical neurotransmitters such as acetylcholine, GABA and glutamate, suggesting that sNPF is a co-transmitter or local neuromodulator in ORNs and many interneurons. Interestingly, sNPF is coexpressed both with presumed excitatory and inhibitory neurotransmitters. A few sNPF expressing neurons in the brain colocalize the peptide corazonin and a pair of dorsal neurons in the first abdominal neuromere coexpresses sNPF and insulin-like peptide 7 (ILP7). It is likely that sNPF has multiple functions as neurohormone as well as local neuromodulator/co-transmitter in various CNS circuits, including olfactory circuits both at the level of the first synapse and at the mushroom body output level. Some of the sNPF immunoreactive axons terminate in close proximity to neurosecretory cells producing ILPs and adipokinetic hormone, indicating that sNPF also might regulate hormone production or release (Nässel, 2008).

Intrinsic neurons of Drosophila mushroom bodies express Short Neuropeptide F: Relations to extrinsic neurons expressing different neurotransmitters

Mushroom bodies constitute prominent paired neuropils in the brain of insects, known to be involved in higher olfactory processing and learning and memory. In Drosophila there are about 2,500 intrinsic mushroom body neurons, Kenyon cells, and a large number of different extrinsic neurons connecting the calyx, peduncle, and lobes to other portions of the brain. The neurotransmitter of the Kenyon cells has not been identified in any insect. This study shows expression of the gene snpf and its neuropeptide products (short neuropeptide F; sNPFs) in larval and adult Drosophila Kenyon cells by means of in situ hybridization and antisera against sequences of the precursor and two of the encoded peptides. Immunocytochemistry displays peptide in intrinsic neuronal processes in most parts of the mushroom body structures, except for a small core in the center of the peduncle and lobes and in the alpha'- and beta'-lobes. Weaker immunolabeling is seen in Kenyon cell bodies and processes in the calyx and initial peduncle and is strongest in the more distal portions of the lobes. Different antisera and Gal4-driven green fluorescent proteins were used to identify Kenyon cells and different populations of extrinsic neurons defined by their signal substances. Thus, neurotransmitter systems converging on Kenyon cells were displayed: neurons likely to utilize dopamine, tyramine/octopamine, glutamate, and acetylcholine. Attempts to identify other neurotransmitter components (including vesicular glutamate transporter) in Kenyon cells failed. However, it is likely that the Kenyon cells utilize an additional neurotransmitter, yet to be identified, and that the neuropeptides described in this study may represent cotransmitters (Johard, 2008).

What is the function of the snpf gene in Kenyon cells and is there more than one sNPF produced of the four peptides predicted by the precursor? Several peptide products from the sNPF neuropeptide precursor have been identified by mass spectrometry from different portions of the Drosophila nervous system (Predel, 2004; Baggerman, 2005; Garczynski, 2006; Wegener, 2006). Of these only sNPF-1, sNPF-14-11 and sNPF-212-19 were unequivocally identified (Predel, 2004; Wegener, 2006) and sNPF-14-11 was purified and sequenced in the present paper. Thus, the evidence for sNPF-3 and -4 (RLRWamides) is weaker because no fragmentation analysis was performed (Garczynski, 2006). However, the specific antiserum to sNPF-3 used in this study does label the same neurons as seen with that to the sNPF precursor. Therefore, unless the sNPF-3 antiserum recognizes the uncleaved peptide within the precursor, it is possible that one or both of the RWamides are also produced. In spite of their RLRWamide C-termini, both sNPF-3 and -4 can activate the sNPF receptor (Mertens, 2002; Feng, 2003; Garczynski, 2006; Johard, 2008 and references therein).

The identification of SPSLRLRFamide as the major immunoreactive product identifiable from whole fruit flies suggests that this is likely to be a predominant final product from this neuropeptide gene. In summary, this study considers the snpf-expressing neurons as producers of sNPF1 AQRSPSLRLRFamide and the sNPF1- and -2-derived peptides, SPSLRLRFamide, confirmed by peptide isolation and mass spectrometry. It is interesting in this respect to note that both vertebrate and insect types of convertases can be expected to cleave this peptide twice from the sNPF precursor (Johard, 2008).

So what are the functions of sNPFs? The first identification of an sNPF (designated Aedes head peptides) was in the mosquito Aedes aegypti and subsequently related peptides have been identified in several other insect specie. Given the widespread expression of this gene and its products in both interneurons and neurosecretory cells within the Drosophila central nervous system, it can be expected that these peptides play a variety of functional roles as central neuromodulators (or cotransmitters) and as hormones released in the circulation. Earlier studies of bioactivities of sNPF in different insects revealed that they are myostimulatory on various visceral muscles, and in locusts they stimulate ovarian growth and induce increases in vitellogeninin levels in the circulation. In female mosquitoes the sNPFs induce host seeking behavior (Johard, 2008 and references therein).

More recently, interference with snpf expression in Drosophila has shown that sNPFs are involved in regulation of feeding (Lee, 2004). In some insects sNPFs are also expressed in endocrine cells of the midgut, suggesting further regulatory roles (e.g., Veenstra, 1995). As shown in this study, one exciting role of sNPFs may be as a neuroactive compound (neuromodulator or cotransmitter) in Drosophila mushroom bodies. Thus they may play a role in higher olfactory processing or even in olfactory learning and memory or in other functions proposed for this brain center (Johard, 2008).

In summary, this study has provided a detailed mapping of the distribution of the products of the gene encoding the sNPF precursor based on in situ hybridization and immunocytochemistry. A novel finding is that snpf-derived peptides are present in a major subpopulation of intrinsic neurons of the mushroom bodies in Drosophila. Thus this study has identified the first putative neuroactive compound in intrinsic neurons of this brain center, which is known to play a major role in olfactory learning and memory. It is suspected that the sNPF-expressing Kenyon cells produce an additional fast neurotransmitter, yet to be identified (Johard, 2008).

Minibrain/Dyrk1a regulates food intake through the Sir2-FOXO-sNPF/NPY pathway in Drosophila and mammals

Feeding behavior, one of the most essential activities in animals, is tightly regulated by neuroendocrine factors. Drosophila short neuropeptide F (sNPF) and the mammalian functional homolog neuropeptide Y (NPY) regulate food intake. Understanding the molecular mechanism of sNPF and NPY signaling is critical to elucidate feeding regulation. This study found that minibrain (mnb) and the mammalian ortholog Dyrk1a, target genes of sNPF and NPY signaling, regulate food intake in Drosophila and mice. In Drosophila neuronal cells and mouse hypothalamic cells, sNPF and NPY modulated the mnb and Dyrk1a expression through the PKA-CREB pathway. Increased Dyrk1a activated Sirt1 to regulate the deacetylation of FOXO, which potentiated FOXO-induced sNPF/NPY expression and in turn promoted food intake. Conversely, AKT-mediated insulin signaling suppressed FOXO-mediated sNPF/NPY expression, which resulted in decreasing food intake. Furthermore, human Dyrk1a transgenic mice exhibited decreased FOXO acetylation and increased NPY expression in the hypothalamus, and increased food intake. These findings demonstrate that Mnb/Dyrk1a regulates food intake through the evolutionary conserved Sir2-FOXO-sNPF/NPY pathway in Drosophila and mammals (Hong, 2012).

The production of sNPF and NPY in sNPFnergic and hypothalamic neurons of flies and mammals respectively, is increased during fasting. These neuropeptides are secreted to produce paracrine and endocrine effects but also feedback upon their synthesizing neurons where they respectively induce mnb and Dyrk1a gene expression through the PKA-CREB pathway. This Mnb/Dyrk1a kinase phosphorylates and activates the Sir2/Sirt1 deacetylase, which in turn deacetylates and activates the FOXO transcription factor. Among its many potential targets, FOXO then increases sNPF/NPY mRNA expression. Negative controls modulate the positive feedback of sNPF/NPY. Feeding activates the insulin receptor-PI3K-AKT pathway. FOXO becomes phosphorylated and transcriptionally inactivated by translocation to the cytoplasm. In this state the induction of sNPF/NPY by FOXO is decreased. Because sNPF and NPY are orexogenic, their positive feedback during fasting should reinforce the propensity for food intake whereas the negative regulation of sNPF and NPY mRNA during feeding condition would then contribute to satiety (Hong, 2012).

FOXO family transcriptional factors are involved in metabolism, longevity, and cell proliferation. FOXO is in part regulated in these processes by post-transcriptional modifications including phosphorylation and acetylation. In many model systems, the ligand activated Insulin-PI3K-AKT pathway phosphorylates FOXO to inactivate this transcription factor by moving it to the cytoplasm. The cytoplasmic localization of FOXO is mediated by 14-3-3 chaperone proteins in Drosophila and mammals. FOXO may also be acetylated, as is FoxO1 of mice, by the CREB-binding protein (CBP)/p300 acetylase and this inhibits FOXO transcriptional function by suppressing its DNA-binding affinity. Such FoxO1 acetylation can be reversed by SirT1 to help activate the FoxO1 transcription factor. This study describes for Drosophila how dFOXO in sNPFR1 neurons regulates the expression of sNPF and food intake. This mechanism parallels how hypothalamic FoxO1 regulates food intake through its control of orexigenic NPY and Agrp in rodents. Post-transcriptional modification of FOXO is central to these controls in both animals. sNPF and NPY expression is increased when FOXO is deacetylated by Sir2/Sirt1, while sNPF and NPY are decreased when FOXO is phosphorylated via the Insulin-PI3K-AKT pathway. Post-transcriptional modifications of FOXO proteins play a critical role for controlling food intake through the sNPF and NPY expression in flies and rodents (Hong, 2012).

Mnb/Dyrk1a participate in olfactory learning, circadian rhythm, and the development of the nervous system and brain. Mnb and Dyrk1a proteins contain a nuclear targeting signal sequence, a protein kinase domain, a PEST domain, and a serine/threonine rich domain. The kinase domains are evolutionary well-conserved from flies to humans. In Down syndrome (DS), chromosome 21 trisomy gives patients three copies of a critical region that includes the Mnb/Dyrk1a; trisomy of this region is associated with anomalies of both the nervous and endocrine systems. DS patients often show high Body Mass Index due to the increased fat mass. Children with DS have elevated serum leptin coupled with leptin resistance, both of which contribute to the obesity risk common to DS patients. This study found a novel function of Mnb/Dyrk1a that may underlay this metabolic condition of DS patients. Mnb/Dyrk1a regulates food intake in flies and mice. This is controlled by sNPF/NPY-PKA-CREB upstream signaling and thus produces downstream affects upon Sir2/Sirt1-FOXO-sNPF/NPY. Fasting not only increases the expression of mnb, but also of sNPF, suggesting that Mnb kinase activates a positive feedback loop where Sir2-dFOXO induces sNPF gene expression. Notably, fasting increases Sirt1 deacetylase activity and localizes FoxO1 to the nucleus in the orexogenic AgRP neurons of the mouse hypothalamus. Increased dosage of Dyrk1a in DS patients may reinforce the positive feedback by NPY and disrupt the balance between hunger and satiety required to maintain a healthy body mass (Hong, 2012).

Insulin produced in the pancreas affects the hypothalamus to regulate feeding in mammals. Insulin injected into the intracerebroventrical of the hypothalamus reduces food intake while inhibiting insulin receptors of the hypothalamic ARC nucleus causes hyperphasia and obesity in rodent models. This study showed a similar pattern for Drosophila where overexpression of insulin-like peptide (Dilp2) at insulin producing neurons decreased food intake while food intake was increased by inhibiting the insulin receptor in sNPFR1 expressing neurons. Likewise, during fasting, serum insulin and leptin levels are decreased in mammals, as is mRNA for insulin-like peptides of Drosophila. Thus, the mechanism by which insulin and insulin receptor signaling suppresses food intake is conserved from fly to mammals in at least some important ways (Hong, 2012).

Previous work has shown how sNPF signaling regulates Dilp expression through ERK in IPCs and controls growth in Drosophila (Lee, 2008). This study shows that sNPF signaling regulates mnb expression through the PKA-CREB pathway in non-IPC neurons and controls food intake. Since sNPF works through the sNPFR1 receptor, sNPFR1 in IPCs and non-IPCs neurons might transduce different signals and thereby modulate different phenotypes. Four Dilps (Dilp1, 2, 3, and 5) are expressed in the IPCs of the brain. Interestingly, levels of Dilp1 and 2 mRNA are reduced in the sNPF mutant, which has small body size, but this study finds only Dilp3 and 5 mRNA levels are reduced upon 24 h fasting. Likewise, only Dilp5 is reduced when adult flies are maintained on yeast-limited diets. In addition, Dilp1 and 2 null mutants show slight reduced body weights but Dilp3 and Dilp5 null mutants do not. These results suggest that Dilp1 and 2 behave like a mammalian insulin growth factor for size regulation while Dilp3 and 5 act like a mammalian insulin for the regulation of metabolism. However, in the long term starvation, Dilp2 and Dilp5 mRNA levels are reduced and Dilp3 mRNA expression is increased (Hong, 2012).

During fasting, sNPF but not sNPFR1 mRNA expression was increased in samples prepared from fly heads increasing food intake. In contrast, in feeding, the high level of insulin signaling reduced sNPF but not sNPFR1 mRNA expression and suppresses food intake. Interestingly, in the antenna of starved flies, sNPFR1 but not sNPF mRNA expression is increased and induces presynaptic facilitation, which results in effective odor-driven food search. However, high insulin signaling suppresses sNPFR1 mRNA expression and prevents presynaptic facilitation in DM1 glomerulus. These results indicate that starvation-mediated or insulin signaling-mediated sNPF-sNPFR1 signaling plays a critical role in Drosophila feeding behavior including food intake and food search even though the fine tuning is different (Hong, 2012).

This study presents a molecular mechanism for how sNPF and NPY regulate food intake in Drosophila and mice. A system of positive feedback regulation for sNPF and NPY signaling is described that increases food intake and a mode of negative regulation for sNPF and NPY by the insulin signaling that suppresses food intake. Modifications of the FOXO protein play a critical role for regulating sNPF and NPY expression, resulting in the control of food intake (Hong, 2012).

The Drosophila neuropeptides PDF and sNPF have opposing electrophysiological and molecular effects on central neurons

Neuropeptides have widespread effects on behavior, but how these molecules alter the activity of their target cells is poorly understood. A new model system was employed in Drosophila to assess the electrophysiological and molecular effects of neuropeptides, recording in situ from larval motor neurons which transgenically express a receptor of choice. Focus was placed on two neuropeptides, Pigment-dispersing factor (PDF) and short neuropeptide F (sNPF), which play important roles in sleep/rhythms and feeding/metabolism. PDF treatment depolarized motor neurons expressing the PDF receptor (PDFR), increasing excitability. sNPF treatment had the opposite effect, hyperpolarizing neurons expressing the sNPF receptor (sNPFR). Live optical imaging using a genetically encoded FRET-based sensor for cyclic AMP (cAMP) showed that PDF induced a large increase in cAMP, whereas sNPF caused a small but significant decrease in cAMP. Co-expression of pertussis toxin or RNAi interference to disrupt the G-protein Galphao blocked the electrophysiological responses to sNPF, showing that sNPFR acts via Galphao signaling. Using a fluorescent sensor for intracellular calcium, it was observed that sNPF-induced hyperpolarization blocked spontaneous waves of activity propagating along the ventral nerve cord, demonstrating that the electrical effects of sNPF can cause profound changes in natural network activity in the brain. This new model system provides a platform for mechanistic analysis of how neuropeptides can affect target cells at the electrical and molecular level, allowing for predictions of how they regulate brain circuits that control behaviors such as sleep and feeding (Vecsey, 2014).

Drosophila short neuropeptide F regulates food intake and body size

Neuropeptides regulate a wide range of animal behavior including food consumption, circadian rhythms, and anxiety. Recently, Drosophila neuropeptide F, which is the homolog of the vertebrate neuropeptide Y, was cloned, and the function of Drosophila neuropeptide F in feeding behaviors was well characterized. However, the function of the structurally related short neuropeptide F (sNPF) was unknown. This paper report the cloning, RNA, and peptide localizations, and functional characterizations of the Drosophila sNPF gene. The sNPF gene encodes the preprotein containing putative RLRF amide peptides and was expressed in the nervous system of late stage embryos and larvae. The embryonic and larval localization of the sNPF peptide in the nervous systems revealed the larval central nervous system neural circuit from the neurons in the brain to thoracic axons and to connective axons in the ventral ganglion. In the adult brain, the sNPF peptide was localized in the medulla and the mushroom body. However, the sNPF peptide was not detected in the gut. The sNPF mRNA and the peptide were expressed during all developmental stages from embryo to adult. From the feeding assay, the gain-of-function sNPF mutants expressed in nervous systems promoted food intake, whereas the loss-of-function mutants suppressed food intake. Also, sNPF overexpression in nervous systems produced bigger and heavier flies. These findings indicate that the sNPF is expressed in the nervous systems to control food intake and regulate body size in Drosophila melanogaster (Lee, 2004. Full text of article).

A receptor for Short Neuropeptide F - Neuropeptide F-like Receptor 76F

A seven transmembrane G-protein coupled receptor has been cloned from Drosophila melanogaster. This receptor shows structural similarities to vertebrate Neuropeptide Y2 receptors and is activated by endogenous Drosophila peptides, recently designated as short neuropeptide Fs (sNPFs). sNPFs have so far been found in neuroendocrine tissues of four other insect species and of the horseshoe crab. In locusts, they accelerate ovarian maturation, and in mosquitoes, they inhibit host-seeking behavior. Expression analysis by RT-PCR shows that the sNPF receptor (Drm-sNPF-R) is present in several tissues (brain, gut, Malpighian tubules and fat body) from Drosophila larvae as well as in ovaries of adult females. All 4 Drosophila sNPFs clearly elicited a calcium response in receptor expressing mammalian Chinese hamster ovary cells. The response is dose-dependent and appeared to be very specific. The short NPF receptor was not activated by any of the other tested arthropod peptides, not even by FMRFamide-related peptides (also ending in RFamide), indicating that the Arg residue at position 4 from the amidated C-terminus appears to be crucial for the response elicited by the sNPFs (Mertens, 2002).

Since the Drosophila genome encodes at least 4 receptors belonging to the NPY subgroup of receptors, the Drosophila EST database was searched for the presence of EST clones, encoding one of the NPY-type receptors. PCR amplification of the EST clone (GH23382) with oligonucleotide primers specific for the predicted ORF of CG7395 (Neuropeptide F-like Receptor 76F) produced a single product of approximately 1800 bp. Sequence determination of the TA-cloned PCR product revealed a DNA insert of 1803 bp, corresponding to the sequence and size of the predicted receptor in the cDNA database of BDGP. The deduced protein encoded by the ORF of Drm-sNPF-R is 600 amino acids long. Analysis by the TMHMM program revealed that this protein is predicted to have seven transmembrane domains along with the intracellular and extracellular loops, consistent with the known G-protein coupled receptors. The N-terminal extracellular region exhibits no O-glycosylation, 2 N-glycosylation sites, along with 7 Ser/Thr phosphorylation sites (Mertens, 2002).

A phylogenetic tree based on Clustal W alignment of Drm-sNPF-R and various known NPY receptors indicates that Drm-sNPF-R is most closely related to the vertebrate neuropeptide Y2 receptors, i.e., of the domestic guinea pig (33% identity and 49% homology), humans (33% identity and 49% homology), the domestic pig (33% identity and 48% homology), and the rat (33% identity and 48% homology). An NPY-like orphan GPCR of C. elegans (C53C7.1) displays 33% identity and 47% homology. Sequence conservation among Drm-sNPF-R, the human Y2 receptor, and the C. elegans orphan receptor is depicted by similarities shown in their alignment by the AlignX program (Mertens, 2002).

Short NPFs and 'head' peptides display substantial sequence similarities and appear to belong to the same family. All NPFs have a typical R(K)–X₁–R–X₂amide motif, where the first amino acid of this motif is always a basic amino acid residue such as Arg or Lys. X₁ can be L, T or P and X₂ is always an aromatic amino acid residue such as Phe or Trp. It is proposed to (re)name all peptides with the R(K)–X₁–R–X₂amide C-terminal motif, as short NPFs; these peptides do not only occur in the head or central nervous system, but instead reach 10 times higher amounts in the abdomen and the midgut. In addition, their precursor in the Drosophila genome is annotated as the short NPF precursor. The present identification of a specific short NPF receptor in Drosophila is in favor of the presence of functional short NPFs. Several reports indicate that short NPFs have a hormonal function in insects, associated with reproduction and digestion. The expression of the short NPF receptor not only in the nervous system, but also in peripheral targets (ovaries, gut) is in agreement with a hormonal function of short NPFs. Hemolymph from sugar-fed mosquito females contains 414 fmol/μl immunoreactive short NPF. Short NPFs are also abundantly present in endocrine cells of the midgut, suggesting that they might have a function in digestion. The demonstration of the presence of the short NPF receptor transcript in the midgut favors this hypothesis (Mertens, 2002).

Functional characterization of a neuropeptide F-like receptor from Drosophila melanogaster

A cDNA clone encoding a seven-transmembrane domain, G-protein-coupled receptor (Neuropeptide F-like Receptor 76F, NPFR76F, or GPCR60), has been isolated from Drosophila melanogaster. Deletion mapping showed that the gene encoding this receptor is located on the left arm of the third chromosome at position 76F. Northern blotting and whole mount in situ hybridization have shown that this receptor is expressed in a limited number of neurons in the central and peripheral nervous systems of embryos and adults. Analysis of the deduced amino acid sequence suggests that this receptor is related to vertebrate neuropeptide Y receptors. This Drosophila receptor shows 62%-66% similarity and 32%-34% identity to type 2 neuropeptide Y receptors cloned from a variety of vertebrate sources. Coexpression in Xenopus oocytes of NPFR76F with the promiscuous G-protein Galpha16 showed that this receptor is activated by the vertebrate neuropeptide Y family to produce inward currents due to the activation of an endogenous oocyte calcium-dependent chloride current. Maximum receptor activation was achieved with short, putative Drosophila neuropeptide F peptides (Drm-sNPF-1, 2 and 2s). Neuropeptide F-like peptides in Drosophila have been implicated in a signalling system that modulates food response and social behaviour. The identification of this neuropeptide F-like receptor and its endogenous ligand by reverse pharmacology will facilitate genetic and behavioural studies of neuropeptide functions in Drosophila (Feng, 2003).

Since the NPFR76F receptor was maximally activated by a short insect NPF-like sequence from Leptinotarsa, genome mining was used to search for Drosophila peptides which might be functionally equivalent to (or better than) the Leptinotarsa I peptide at activating NPFR76F. Initially, one precursor sequence, a gene (npf) encoding Drosophila NPF was identified at chromosome location 89D3. This gene was found by blasting an incomplete version of the Drosophila genome (October 1999) with the amino acid sequence of Aplysia NPY. This revealed a precursor molecule encoding a 36 amino acid peptide with sequence similarity to NPF. The completed Drosophila genome sequence has now been searched and no other potential NPF precursors were identified. Since this peptide (NPF-A1) contained a potential dibasic amino acid cleavage site within its sequence, both the shorter 28 amino acid form (NPF-A2) and the full-length peptide (NPF-A1) were sequenced for testing (Feng, 2003).

A second open reading frame in the Drosophila genome encodes a precursor peptide for two short NPF-like peptides (Drm-sNPF-1, AQRSPSLRLRFamide and Drm-sNPF-2, WFGDVNQKPIRSPSLRLRFamide). The precursor for these peptides is encoded by the short NPF precursor (sNPF) gene (CG13968) and maps to position 38A7 on the left arm of Drosophila chromosome 2. The WFGDVNQKPIRSPSLRLRFamide peptide (Drm-sNPF-2) contains a potential single basic amino acid-processing site. This peptide (Drm-sNPF-2), its shorter form (peptide Drm-sNPF-2 s, PIRSPSLRLRFamide) and the Drm-sNPF-1 peptide (all encoded by the sNPF gene) were synthesized. The same precursor was predicted to include the sequences for two other short peptides, PQRLRWamide and PMRLRWamide, which have been designated Drm-sNPF-3 and Drm-sNPF-4, respectively. These peptides were synthesized and tested (Feng, 2003).

When tested at 1 µm, the shorter NPF-like peptides derived from the sNPF gene (peptides Drm-sNPF-1 and Drm-sNPF-2) were more effective than the original Leptinotarsa I NPF-like sequence at inducing inward currents in Xenopus oocytes expressing NPFR76F and G_alpha16. Shortening of peptide Drm-sNPF-2 to the Drm-sNPF-2s form may slightly increase its effectiveness. The longer Drosophila peptides derived from the precursor gene npf at 89D3 were much less effective than the original Leptinotarsa I NPF-like sequence at inducing inward currents. In addition, the PQRLRWamide (Drm-sNPF-3) and PMRLRWamide (Drm-sNPF4) peptides were much less effective than the original Leptinotarsa I sequence at inducing inward currents (Feng, 2003).

Dose-response curves for the shorter endogenous Drosophila NPF-like peptides encoded by the sNPF gene at 38A7 reveal that AQRSPSLRLRFamide (peptide Drm-sNPF-1) is the most potent peptide tested (pEC50 = -8.84). It showed a threshold for the generation of inward currents between 100 pm and 1 nm and a maximal effect at 100 nm. The second putative endogenous Drosophila NPF-like peptide encoded by the sNPF gene, PIRSPSLRFamide (peptide Drm-sNPF-2 s) (pEC50 = -7.62), and the original Leptinotarsa I NPF-like sequence, ARGPQLRLRFamide (pEC50 = -7.83), were an order of magnitude less potent than AQRSPSLRLRFamide. These results justify the classification of NPFR76F as a NPF-like receptor and suggest that the short peptide AQRSPSLRLRFamide (sNPF-1) may be the endogenous agonist for this receptor. The other two short peptides encoded by the sNPF gene, PQRLRWamide (pEC50 = -6.1) and PMRLRWamide (pEC50 = -7.30), were 2.7 and 1.5 orders of magnitude, respectively, less potent than the AQRSPSLRLRFamide sequence, leading to questioning of their designation as true short NPF-like peptides (Feng, 2003).

Expression of NPFR76F transcripts was assessed by Northern blot analysis of poly(A)⁺ RNA prepared from adult body parts. A single transcript of 6.5 kb was detected in both heads and appendages (legs and antennae), suggesting that NPFR76F is expressed in both the central and peripheral nervous systems. In addition to finding transcript in heads and appendages, trace amounts were also seen in bodies. When compared with the amount of RNA loaded from each body part (as indicated by the ubiquitous rp49 loading control), the relative abundance of transcript in bodies is very low. This distribution of the NPFR76F transcript is consistent with a role for this NPF-like receptor in the Drosophila nervous system (Feng, 2003).

To further refine the NPFR76F receptor transcript expression, in situ hybridization with a digoxigenin-labelled antisense RNA probe to whole mounts of the mature embryos was used. The central nervous system in embryos is composed of two dorsal brain hemispheres and a fused ventral ganglion. The NPFR76F receptor is expressed both in the dorsal brain and in the ventral ganglion. In the brain the receptor is strongly expressed in the specific cells in the dorsal posterior region. It is estimated that there are 22-24 cells in each brain lobe expressing NPFR76F, including the strongly expressing cells. In the ventral ganglion, pairs of cells along the ventral midline, as well as cells found in a bilaterally symmetric pattern in a more lateral position from the midline, also express receptor mRNA. In each full segment of the ventral ganglion, the receptor is strongly expressed in eight to 12 cells, including a pair of cells at the midline in each segment (Feng, 2003).

In the peripheral nervous system the receptor is expressed in a subset of sensilla and in the anterior sensory complex. It is estimated that there are 10-14 cells in the anterior sensory complex, including the antennomaxillary complex, the labral sensory complex and the labial sensory complex, that express NPFR76F. Finally, in the posterior sensilla, there are eight cells that express NPFR76F. The expression pattern of the NPFR76F receptor in many specific cells in the dorsal brain, the ventral ganglion, lateral sensilla, the anterior sensory complex and the posterior sensilla suggests that this receptor is involved in a widespread modulation of neuronal activity (Feng, 2003).

The activation of G-protein gated inwardly rectifying K+ channels by a cloned Drosophila melanogaster neuropeptide F-like receptor

A Drosophila melanogaster G-protein-coupled receptor (NPFR76F) that is activated by neuropeptide F-like peptides has been expressed in Xenopus oocytes to determine its ability to regulate heterologously expressed G-protein-coupled inwardly rectifying potassium channels. The activated receptor produced inwardly rectifying potassium currents by a pertussis toxin-sensitive G-protein-mediated pathway and the effects were reduced in the presence of proteins, such as the betaARK 1 carboxy-tail fragment and alpha-transducin, which bind G-protein betagamma-subunits. Short Drosophila NPF-like peptides are more potent than long NPF-like peptides at coupling the receptor to the activation of inwardly rectifying potassium channels. The putative endogenous short Drosophila NPF-like peptides showed agonist-specific coupling depending on whether their actions were assessed as the activation of the inwardly rectifying potassium channels or as the activation of endogenous inward chloride channels through a co-expressed promiscuous G-protein, Galpha16. As inwardly rectifying potassium channels are known to be encoded in the Drosophila genome and the NPFR76F receptor is widely expressed in the Drosophila nervous system, the receptor could function to control neuronal excitability or slow wave potential generation in the Drosophila nervous system (Reale, 2004).

Drosophila short neuropeptide F signalling regulates growth by ERK-mediated insulin signalling

Neuropeptides regulate a wide range of animal behavior including food consumption, circadian rhythms, and anxiety. Recently, Drosophila neuropeptide F, which is the homolog of the vertebrate neuropeptide Y, was cloned, and the function of Drosophila neuropeptide F in feeding behaviors was well characterized. However, the function of the structurally related short neuropeptide F (sNPF) was unknown. This study reports the cloning, RNA, and peptide localizations, and functional characterizations of the Drosophila sNPF gene. The sNPF gene encodes the preprotein containing putative RLRF amide peptides and was expressed in the nervous system of late stage embryos and larvae. The embryonic and larval localization of the sNPF peptide in the nervous systems revealed the larval central nervous system neural circuit from the neurons in the brain to thoracic axons and to connective axons in the ventral ganglion. In the adult brain, the sNPF peptide was localized in the medulla and the mushroom body. However, the sNPF peptide was not detected in the gut. The sNPF mRNA and the peptide were expressed during all developmental stages from embryo to adult. From the feeding assay, the gain-of-function sNPF mutants expressed in nervous systems promoted food intake, whereas the loss-of-function mutants suppressed food intake. Also, sNPF overexpression in nervous systems produced bigger and heavier flies. These findings indicate that the sNPF is expressed in the nervous systems to control food intake and regulate body size in Drosophila melanogaster (Lee, 2004).

Various evidence suggests that sNPF and dNPF peptides have different functions. The sNPF peptide is found only in nervous systems, whereas the neuropeptide F (dNPF) is found as the Drosophila brain-gut peptide. The expression patterns of sNPF and dNPF differ in the larval brain. For example, the sNPF expression is found in the anterior dorsal neurons of the brain, whereas the dNPF expression is detected in the four neurons of larval brain. In the feeding behavior analysis, overexpression of sNPF in wandering larvae did not extend the feeding period, contrary to the extension of the feeding period in the wandering larval stage by overexpression of dNPF. At the receptor level, each peptide works in different receptors; for example, the NPFR76F receptor is for sNPF peptides, and the DmNPFR1 receptor is for the dNPF. These differences indicate that the dNPF and sNPF peptides function in different neurons and may regulate different aspects of feeding behaviors in Drosophila (Lee, 2004).

Like other neuropeptides, the sNPF peptide may be involved in regulating various physiological processes other than regulating food intake because the sNPF peptide and transcript were expressed during all developmental stages, and the sNPF is localized in the mushroom body calyx and medulla of the adult brain. The mushroom body is involved in learning and memory. These unknown multi-functions of the sNPF peptide in various biological processes are the subjects of future studies (Lee, 2004).

Structural studies of Drosophila short neuropeptide F: Occurrence and receptor binding activity

Among insects, short neuropeptide Fs (sNPF) have been implicated in regulation of reproduction and feeding behavior. For Drosophila melanogaster, the nucleotide sequence for the sNPF precursor protein encodes four distinctive candidate sNPFs. In the present study, all four peptides were identified by mass spectrometry in body extracts of D. melanogaster; some also were identified in hemolymph, suggesting potential neuroendocrine roles. Actions of sNPFs in D. melanogaster are mediated by the G protein-coupled receptor Drm-NPFR76F. Mammalian CHO-K1 cells were stably transfected with the Drm-NPFR76F receptor for membrane-based radioreceptor studies. Binding assays revealed that longer sNPF peptides comprised of nine or more amino acids are clearly more potent than shorter ones of eight or fewer amino acids. These findings extend understanding of the relationship between structure and function of sNPFs (Garczynski, 2006).

The sNPFs of D. melanogaster differ in their interactions with the sNPF receptor Drm-NPFR76F, as analyzed directly by radioreceptor assay. A wide variety of D. melanogaster sNPFs were assayed for their ability to inhibit the binding of ¹²⁵I-[D-Y¹]-Drm-sNPF1 to membranes prepared from cells stably transfected with Drm-NPFR76F. Two distinctive classes of activity of sNPFs were readily apparent. The sNPF peptides containing nine or more amino acids typically exhibited high affinity, as judged by an IC₅₀ < 1 nM, whereas peptides containing eight for fewer amino acids exhibited IC₅₀ values of >5 nM. Those exhibiting lower affinity included sNPF3 and sNPF4, peptides with the C-terminal RLRWa sequence. The minimum length of the highly active group was represented by sNPF2_11–19. The sequence of this sNPF2_11–19 (RSPSLRLRFa) differs from sNPF1_4–11 (SPSLRLRFa) only by a single arginine residue, which appears to confer a substantial increase in binding affinity. To test this hypothesis, an alanine substituted analog, Drm-sNPF2_11–19R11A, was assayed and found to exhibit a substantial drop in activity, with an IC₅₀ of only 12.5 nM, indicating the crucial role of this arginine residue (Garczynski, 2006).

For further tests of these apparent structure-function relations, putative sNPFs identified in the genomes of A. gambiae and A. aegypti also were examined in the Drm-NPFR76F radioreceptor assay. Each of these mosquito sNPF peptides conformed to the distinctive pattern of length-associated activity established previously for those of D. melanogaster. In contrast, the A. aegypti head peptides which partly resemble sNPFs were either weakly active, Aea-HP-I, or inactive, Aea-HP-III, despite being of sufficient length and having the requisite arginine. Accordingly, additional structural features in the C-terminus common to sNPF peptides appear important for high affinity binding (Garczynski, 2006).

Search PubMed for articles about Drosophila sNPF

Asahina, K., Watanabe, K., Duistermars, B. J., Hoopfer, E., Gonzalez, C. R., Eyjolfsdottir, E. A., Perona, P. and Anderson, D. J. (2014). Tachykinin-expressing neurons control male-specific aggressive arousal in Drosophila. Cell 156: 221-235. PubMed ID: 24439378

Baggerman, G., Liu, F., Wets, G. and Schoofs, L. (2005). Bioinformatic analysis of peptide precursor proteins. Ann. N. Y. Acad. Sci. 1040: 59-65. PubMed ID: 15891006

Bhukel, A., Beuschel, C. B., Maglione, M., Lehmann, M., Juhasz, G., Madeo, F. and Sigrist, S. J. (2019). Autophagy within the mushroom body protects from synapse aging in a non-cell autonomous manner. Nat Commun 10(1): 1318. PubMed ID: 30899013

Emeson, R. B. and Morabito, M. V. (2005). Food fight: the NPY-serotonin link between aggression and feeding behavior. Sci. STKE pe12. PubMed ID: 15798100

Feng, G., et al. (2003). Functional characterization of a neuropeptide F-like receptor from Drosophila melanogaster. Eur. J. Neurosci. 18: 227-238. PubMed ID: 12887405

Garczynski, S. F., Brown, M. R. and Crim, J. W. (2006). Structural studies of Drosophila short neuropeptide F: Occurrence and receptor binding activity. Peptides 27(3): 575-82. PubMed ID: 16330127

Hong, S. H., Lee, K. S., Kwak, S. J., Kim, A. K., Bai, H., Jung, M. S., Kwon, O. Y., Song, W. J., Tatar, M. and Yu, K. (2012). Minibrain/Dyrk1a regulates food intake through the Sir2-FOXO-sNPF/NPY pathway in Drosophila and mammals. PLoS Genet 8: e1002857. PubMed ID: 22876196

Hu, C., Petersen, M., Hoyer, N., Spitzweck, B., Tenedini, F., Wang, D., Gruschka, A., Burchardt, L. S., Szpotowicz, E., Schweizer, M., Guntur, A. R., Yang, C. H. and Soba, P. (2017). Sensory integration and neuromodulatory feedback facilitate Drosophila mechanonociceptive behavior. Nat Neurosci 20(8):1085-1095. PubMed ID: 28604684

Inagaki, H. K., Panse, K. M. and Anderson, D. J. (2014). Independent, reciprocal neuromodulatory control of sweet and bitter taste sensitivity during starvation in Drosophila. Neuron 84: 806-820. PubMed ID: 25451195

Johard, H. A., et al. (2008). Intrinsic neurons of Drosophila mushroom bodies express short neuropeptide F: relations to extrinsic neurons expressing different neurotransmitters. J. Comp. Neurol. 507(4): 1479-96. PubMed ID: 18205208

Kapan, N., Lushchak, O. V., Luo, J. and Nassel, D. R. (2012). Identified peptidergic neurons in the Drosophila brain regulate insulin-producing cells, stress responses and metabolism by coexpressed short neuropeptide F and corazonin. Cell Mol Life Sci 69(23): 4051-4066. PubMed ID: 22828865

Ko, K. I., Root, C. M., Lindsay, S. A., Zaninovich, O. A., Shepherd, A. K., Wasserman, S. A., Kim, S. M. and Wang, J. W. (2015). Starvation promotes concerted modulation of appetitive olfactory behavior via parallel neuromodulatory circuits. Elife 4. PubMed ID: 26208339

Lee, K. S., You, K. H., Choo, J. K., Han, Y. M. and Yu, K. (2004). Drosophila short neuropeptide F regulates food intake and body size. J. Biol. Chem. 279(49): 50781-9. PubMed ID: 15385546

Lee, K. S., et al. (2008). Drosophila short neuropeptide F signalling regulates growth by ERK-mediated insulin signalling. Nat. Cell Biol. 10(4): 468-75. PubMed ID: 18344986

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date revised: 15 December 2019

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