InteractiveFly: GeneBrief

Adenosine receptor: Biological Overview | References

BIOLOGICAL OVERVIEW

Metabolites are increasingly appreciated for their roles as signaling molecules. To dissect the roles of metabolites, it is essential to understand their signaling pathways and their enzymatic regulations. From an RNA interference (RNAi) screen for regulators of intestinal stem cell (ISC) activity in the Drosophila midgut, this study identified adenosine receptor (AdoR) as a top candidate gene required for ISC proliferation. Ras/MAPK and Protein Kinase A (PKA) signaling act downstream of AdoR and Ras/MAPK mediates the major effect of AdoR on ISC proliferation. Extracellular adenosine, the ligand for AdoR, is a small metabolite that can be released by various cell types and degraded in the extracellular space by secreted adenosine deaminase. Interestingly, down-regulation of adenosine deaminase-related growth factor A (Adgf-A) from enterocytes is necessary for extracellular adenosine to activate AdoR and induce ISC overproliferation. As Adgf-A expression and its enzymatic activity decrease following tissue damage, this study provides important insights into how the enzymatic regulation of extracellular adenosine levels under tissue-damage conditions facilitates ISC proliferation (Xu, 2020).

The Drosophila midgut epithelium consists of multipotent intestinal stem cell (ISCs), their immediate progenies known as enteroblasts (EBs, which are progenitor cells primed for differentiation), and differentiated cells including enterocytes (ECs, which is the major cell type in number), and enteroendocrine cells (EEs). ISCs/EBs can adjust their proliferation and differentiation activities by deploying conserved core pathways such as JAK/Stat, Notch, Ras/MAPK, JNK, and Hippo. The dynamic responses of adult ISCs/EBs to different regenerative demands under physiological or pathological conditions depend on the machineries to detect microenvironment cues and modulate the activity of aforementioned core pathways, which have not been investigated in vivo systematically (Xu, 2020).

To understand how ISCs/EBs sense their microenvironment, an RNAi screen was performed to identify receptor-coding genes that regulate ISC activity, among which Adenosine Receptor (AdoR) emerged as a top candidate required for ISC self-renewal and proliferation. Characterization of the AdoR-signaling pathway revealed the role of AdoR downstream pathways in regulating different aspects of ISC activity. Importantly, this study demonstrated that the mitogenic activity of the AdoR ligand, adenosine, is inhibited by adenosine deaminase-related growth factor A (Adgf-A) from ECs and that Adgf-A activity decreases following tissue damage. Altogether, this study demonstrates how an EC-derived metabolic enzyme modulates ISC activity by restricting extracellular adenosine (Xu, 2020).

An RNAi screen was performed for regulators of ISC activity and AdoR was identified as a gene required for Ras/MAPK and PKA signaling in the ISCs/EBs. Characterization of AdoR and its ligand revealed that, in the healthy midgut, EC-derived Adgf-A limits the bioavailability of extracellular adenosine and restricts AdoR signaling in ISCs/EBs to a baseline level that supports ISC maintenance. However, the damaged midgut lacks sufficient levels of Adgf-A to restrict extracellular adenosine, thus allowing the activation of AdoR and its downstream pathways to stimulate the regenerative activity of ISCs (Xu, 2020).

Purines not only are required for nucleic acid synthesis and the cellular energy supply, but also represent the most primitive and common extracellular chemical messengers. Extracellular adenosine acts on P1-type purinergic receptors, i.e., AdoRs. The effects of AdoR signaling on cell growth are context-dependent. For example, adenosine inhibits the growth of imaginal disk cells, and Adgf-A was initially identified as a growth factor that stimulates the proliferation of Drosophila imaginal disk and embryonic cells in vitro (Zurovec, 2002). In contrast, in both larval lymph gland (Mondal, 2011) and adult midgut, AdoR supports proliferation and differentiation in the stem/progenitor cells whereas AdgfA from a nonautonomous source suppresses AdoR activity. Despite the remarkably similar roles of AdoR in controlling behaviors of 2 different types of stem/progenitor cells, AdoR activation leads to hemapoietic progenitor exhaustion but ISC expansion. Furthermore, Ras/MAPK activity, rather than PKA (as in the hemapoietic progenitors), functions as a necessary and sufficient downstream component mediating AdoR-induced ISC overproliferation (Xu, 2020).

Identification of AdoR as an ISC regulator led to a dissection of the function of its downstream pathways, i.e., PKA and Ras/MAPK. Although earlier studies reported that EC-like differentiation in Caco2 colorectal cancer cells correlates with PKA activation and that pharmacological induction of cAMP/PKA suppresses the migration of mammalian intestinal or colorectal cancer cells, this study implicates PKA signaling in controlling ISC behaviors in vivo. This study found that PKA activation in ISCs/EBs induces ISC-EC differentiation and EB membrane elongation, whereas PKA activation in ECs nonautonomously stimulates ISC proliferation. PKA regulates cytoskeletal organizing proteins such as Rac, Cdc42, Rho, and PAK. Interestingly, PKA antagonizes Rac to induce morphological changes in neurons. A similar mechanism might explain how PKA affects EB morphology (Xu, 2020).

Ras/MAPK activity in the ISCs/EBs is responsive to a wide spectrum of inputs, including the EGFR pathway, the PDGF- and VEGF-receptor-related pathway, and cytosolic Ca2+ levels. This study confirmed AdoR as another upstream signal that can affect Ca2+ and Ras/MAPK activity. Since earlier studies suggested that GPCRs might affect intracellular Ca2+ levels, whereas high levels of cytosolic Ca2+ levels can induce Ras/MAPK activity in ISCs/EBs, it is likely that the detailed mechanism for AdoR to activate Ras/MAPK implicates the regulation of Ca2+ levels (Xu, 2020).

Following AdoR activation, both Ras/MAPK and PKA signaling are induced to facilitate ISC overproliferation and accelerated production of ECs, whereas the perdurance of PKA activity in a massive number of newly produced ECs has a synergistic effect with Ras/MAPK activity in ISCs/EBs in accelerating proliferation. Since human AdoRs are often highly expressed in carcinomas, a similar paradigm of PKA and Ras/MAPK synergy might fuel oncogenic growth in epithelial tissues (Xu, 2020).

Mammalian AdoRs and human ADA2 have been extensively studied in the hematopoietic and immune systems where ADA2 is produced by differentiating monocytes to stimulate T cell and macrophage proliferation (Hasco, 2008; Zavialov, 2010). Although mammalian AdoRs are expressed in human digestive epithelial cells, their functions remain elusive. Different groups have reported contradictory results suggesting either a protective or a pathological role of AdoR signaling during tissue damage in the mouse intestine, which could be due to the differences in mouse culture conditions, genetic backgrounds, damage models, or inflammation responses. Therefore, this study in Drosophila might help clarify the function of AdoR signaling in the digestive epithelium and in epithelial stem cells (Xu, 2020).

In carcinomas, ADA2 is focally and frequently deleted, based on copy number analysis. Deleterious ADA2 mutations have been identified in colorectal cancers in The Cancer Genome Atlas (TCGA) and Catalogue of Somatic Mutations in Cancer projects. Moreover, ADA2 expression is significantly down-regulated in colorectal cancers, according to microarray studies and RNA-seq datasets from TCGA. Further, anti-ADA2 stainings were detected in the normal digestive epithelium but not in colorectal cancers. Therefore, the down-regulation of ADA2 in colorectal carcinomas has been observed at DNA, RNA, and protein levels. Unfortunately, ADA2 cannot be studied in a mouse model because of a rodent-specific gene loss event during evolution. Moreover, murine developmental and physiological programs have adapted to the loss of ADA2, as transgenic expression of human ADA2 in mice results in abnormal development and embryonic/neonatal lethality. Therefore, these findings describe a striking case in which flies are uniquely suited for understanding the function and regulation of an important disease-related gene (Xu, 2020).

Gliotransmission and adenosine signaling promote axon regeneration

How glia control axon regeneration remains incompletely understood. This study investigated glial regulation of regenerative ability differences of closely related Drosophila larval sensory neuron subtypes. Axotomy elicits Ca(2+) signals in ensheathing glia, which activates regenerative neurons through the gliotransmitter adenosine and mounts axon regenerative programs. However, non-regenerative neurons do not respond to glial stimulation or adenosine. Such neuronal subtype-specific responses result from specific expressions of adenosine receptors in regenerative neurons. Disrupting gliotransmission impedes axon regeneration of regenerative neurons, and ectopic adenosine receptor expression in non-regenerative neurons suffices to activate regenerative programs and induce axon regeneration. Furthermore, stimulating gliotransmission or activating the mammalian ortholog of Drosophila adenosine receptors in retinal ganglion cells (RGCs) promotes axon regrowth after optic nerve crush in adult mice. Altogether, the findings demonstrate that gliotransmission orchestrates neuronal subtype-specific axon regeneration in Drosophila and suggest that targeting gliotransmission or adenosine signaling is a strategy for mammalian central nervous system repair (Wang, 2023).

Axon regeneration failure is a major hurdle for functional recovery after CNS lesion. In addition, Wallerian axon degeneration is associated with neurodegenerative diseases, hence promoting that axon regeneration in these diseases is of great interest. By parallel investigation of closely related Drosophila neuronal subtypes, gliotransmission is proposed as a previously unknown mechanism by which glia could control axon regeneration. These findings expand the existing glial roles in axon regeneration. In a working model that summarizes these Drosophila studies, ensheathing glia actively respond to axotomy and the resulting glial Ca2+ spikes lead to the release of ATP/adenosine. Although adenosine could interact with all sensory neurons, the AdoR expression pattern ensures that C4da, but not C3da, neurons respond to adenosine. This glia-to-neuron signaling then mounts pro-regenerative programs in C4da neurons, including burst firing, Ca2+ spikes, and Ras activity to promote axon regrowth of C4da neurons. Moreover, ectopic AdoR expression or activation of those regenerative programs in C3da neurons is sufficient to induce their axon regeneration (Wang, 2023).

How could axotomy elicit glial Ca2+ signals in Drosophila larvae? Glia are known to express neurotransmitter receptors, allowing them to "listen to" neuronal activity and provide feedback modulation of neuronal activity through gliotransmitters. To assess whether the neuronal activity could have an effect on glial Ca2+ signals, AITC was applied to activate C4da neurons. However, Allyl isothiocyanate (AITC) did not evoke Ca2+ signals in ensheathing glia, arguing against the neuronal activity as a trigger of glial Ca2+ signals. These results support unidirectional signaling from glia to sensory neurons. They also suggest that glial Ca2+ spikes are produced either cell-autonomously or alternatively through a cell-non-autonomous mechanism that involves glial interactions with non-neuronal cells. Burst firing observed in Drosophila larval C4da neurons is reminiscent of the ectopic discharges of injured dorsal root ganglion (DRG) neurons, the mammalian counterpart of Drosophila larval PNS sensory neurons. As a hallmark of neuropathic pain, the ectopic discharges of DRG neurons are also regulated by gliotransmission. This suggests that burst firing of C4da neurons could reflect nociceptive hypersensitivity after axotomy. Hence, one possible role of gliotransmission could be to sensitize injured C4da neurons. Taken together, gliotransmission appears to be related to both axon regeneration and nociceptive hypersensitivity. Future work is needed to determine whether gliotransmission could regulate DRG axon regeneration (Wang, 2023).

By stimulating Drosophila larval C3da neurons with different firing patterns, this study provides direct evidence that firing patterns, instead of excitability, dictate the axon regenerative strength. A possible mechanism is that neuronal activity patterns are associated with different Ca2+ signals. For example, burst, but not tonic, firing evokes strong Ca2+ signals in C3da neurons, likely due to prolonged depolarization associated with burst firing. Previous studies indicate that the stimulation of neuronal activity promotes mammalian axon regeneration, but the reported effects are variable. One possibility could be that the activity patterns are not controlled in these studies. In the future, it will be of interest to deliver precisely controlled neuronal activity patterns to determine their effects on axon regeneration in other experimental models (Wang, 2023).

It is proposed that the Ca2+ spikes in Drosophila larval C4da neurons are pro-regenerative signal with the following characteristics: they are neuronal-type-specific, correlate with burst firing, exhibit delayed onset, persist throughout axon regeneration, and are evoked by axonal but not dendritic injury. Optogenetic stimulation of C3da neurons starting at ~6 hpa (i.e., after immediate Ca2+ transients) further indicates that Ca2+ spikes are sufficient to induce axon regeneration. Because Ca2+ signals with different spatiotemporal patterns activate diverse neuronal pathways, it is likely that Ca2+ spikes and immediately Ca2+ transients regulate different aspects of axon regeneration. Although immediate Ca2+ transients could regulate growth cone formation, persistent Ca2+ spikes could maintain Ras activity at a high level for sustained axon regrowth. It will be interesting to determine whether axotomy could trigger Ca2+ spikes in other systems, and if so, their roles in axon regeneration (Wang, 2023).

Unlike CNS, mammalian PNS neurons such as DRG can regenerate their axons. Therefore, mammalian CNS and PNS neurons could be broadly defined as two neuronal groups bearing axon regenerative differences, although each group is highly heterogeneous and contains multiple neuronal subtypes. In this regard, the differences between mammalian CNS and PNS are much larger than those between Drosophila C4da and C3da neurons. To explore whether Adora2b signaling could underlie the regenerative differences of mammalian CNS and PNS neurons, the published single-cell RNA-seq dataset was search, but it was found that Adora2b was expressed at low levels in both CNS and DRG neurons. Hence, other mechanisms must exist to explain their regenerative differences. One such mechanism could be related to intrinsic growth abilities. For example, the injury of mouse RGCs causes a reduction of mTOR activity; by contrast, no reduction of mTOR activity was found after the injury of DRG neurons (Wang, 2023).

Glial cells in the mammalian retina could release ATP, glutamate, and D-serine. The mouse study indicates that the stimulation of the gliotransmitter release alone by designer receptor exclusively activated by designer drugs (DREADDs) is ineffective, but combining glial DREADD stimulation and neuronal Adora2b expression greatly enhanced axon regeneration. Because Adora2b is barely expressed in the mouse retina, these results suggest that engineering RGCs to respond to the gliotransmitter adenosine is a prerequisite for the observed effect. Similarly, Drosophila C3da neurons do not express AdoR, and engineering them to respond to adenosine induces axon regeneration. These analyses suggest that stimulation of gliotransmission could promote axon regeneration across species if Adora2b orthologs are expressed in neurons. Notably, Adora2b overexpression in RGCs alone moderately increased axon regeneration, suggesting that extracellular adenosine exists in the injured retina. These results further suggest that in the absence of glial DREADD stimulation, axotomy could trigger basal adenosine release (Wang, 2023).

Adenosine exerts protective functions in different mammalian tissues including the nervous system, by triggering diverse signaling events. Extracellular adenosine is normally low but is rapidly increased after injury. As a classic neuromodulator, adenosine controls essential homeostatic functions such as sleep. However, whether adenosine could play a role in axon regeneration is largely unknown. Pharmacological Adora3 activation is shown to promote neurite outgrowth of cultured RGCs. In the current study, Adora2b was pharmacologically activated, and this was found to promote axon regeneration and survival of RGCs in adult mice. The magnitude of RGC axon regeneration induced by Adora2b activation was comparable with that after PTEN knockdown, and combining Adora2b activation with PTEN knockdown further enhanced axon regeneration. These findings suggest that targeting Adora2b signaling, either alone or in combination with other pro-regenerative pathways, is likely a strategy for mammalian CNS repair (Wang, 2023).

In the mouse study, DREADDs were used to stimulate the gliotransmitter release. However, the spatiotemporal dynamics of Ca2+ elevations and gliotransmitter release are yet unknown and will be the focus of future studies. Moreover, the current study has focused on mouse retinal glial cells that are positive for GFAP, which is normally found in astrocytes. Future studies are needed to determine whether the stimulation of gliotransmission from other retinal glial types such as Müller glia or microglia could have a similar role in RGC axon regeneration (Wang, 2023).

Adenosine receptor and its downstream targets, Mod(mdg4) and Hsp70, work as a signaling pathway modulating cytotoxic damage in Drosophila

Adenosine (Ado) is an important signaling molecule involved in stress responses. Studies in mammalian models have shown that Ado regulates signaling mechanisms involved in "danger-sensing" and tissue-protection. Yet, little is known about the role of Ado signaling in Drosophila. This study observed lower extracellular Ado concentration and suppressed expression of Ado transporters in flies expressing mutant huntingtin protein (mHTT). Ado signaling was altered using genetic tools; the overexpression of Ado metabolic enzymes, as well as the suppression of Ado receptor (AdoR) and transporters (ENTs) were found to minimize mHTT-induced mortality. The downstream targets of the AdoR pathway were identified, the modifier of mdg4 (Mod(mdg4)) and heat-shock protein 70 (Hsp70), which modulated the formation of mHTT aggregates. Finally, a decrease in Ado signaling affects other Drosophila stress reactions, including paraquat and heat-shock treatments. This study provides important insights into how Ado regulates stress responses in Drosophila (Lin, 2021).

Tissue injury, ischemia, and inflammation activate organismal responses involved in the maintenance of tissue homeostasis. Such responses require precise coordination among the involved signaling pathways. Adenosine (Ado) represents one of the key signals contributing to the orchestration of cytoprotection, immune reactions, and regeneration, as well as balancing energy metabolism (Borea, 2016). Under normal conditions, the Ado concentration in blood is in the nanomolar range; however, under pathological circumstances the extracellular Ado (e-Ado) level may dramatically change. Ado has previously been considered a retaliatory metabolite, having general tissue protective effects. Prolonged adenosine signaling, however, can exacerbate tissue dysfunction in chronic diseases. As suggested for the nervous system in mammals, Ado seems to act as a high pass filter for injuries by sustaining viability with low insults and bolsters the loss of viability with more intense insults (Cunha, 2016; Lin, 2021 and references therein).

Adenosine signaling is well-conserved among phyla. The concentration of Ado in the Drosophila melanogaster hemolymph is maintained in the nanomolar range, as in mammals, and increases dramatically in adenosine deaminase mutants or during infections (Dolezelova, 2005; Novakova, 2011). Unlike mammals, D. melanogaster contains only a single Ado receptor (AdoR) isoform (stimulating cAMP) and several proteins that have Ado metabolic and transport activities involved in the fine regulation of adenosine levels. D. melanogaster adenosine deaminase-related growth factors (ADGFs), which are related to human ADA2, together with adenosine kinase (AdenoK) are the major metabolic enzymes converting extra- and intra-cellular adenosine to inosine and AMP, respectively. The transport of Ado across the plasma membrane is mediated by three equilibrative and two concentrative nucleoside transporters (ENTs and CNTs, respectively) similar to their mammalian counterparts. Ado signaling in Drosophila has been reported to affect various physiological processes, including the regulation of synaptic plasticity in the brain, proliferation of gut stem cells, hemocyte differentiation, and metabolic adjustments during the immune response (Knight, 2010; Mondal, 2011; Bajgar, 2015; Xu, 2020; Lin, 2021 and references therein).

The present study examined the role of Drosophila Ado signaling on cytotoxic stress and aimed to clarify the underlying mechanism. Earlier reports have shown that expression of the expanded polyglutamine domain from human mutant huntingtin protein (mHTT) induces cell death in both Drosophila neurons and hemocytes. In this study, the low-viability phenotype of mHTT-expressing larvae and it was observed that such larvae display a lower level of e-Ado in the hemolymph. Furthermore, this study used genetic tools and altered the expression of genes involved in Ado metabolism and transport to find out whether changes in Ado signaling can modify the phenotype of mHTT-expressing flies. Finally, a downstream mechanism was uncovered of the Drosophila Ado pathway, namely mod(mdg4) and heat-shock protein 70 (Hsp70), which modify both the formation of mHTT aggregates and the stress response to heat-shock and paraquat treatments (Lin, 2021).

Adenosine signaling represents an evolutionarily conserved pathway affecting a diverse array of stress responses. As a ubiquitous metabolite, Ado has evolved to become a conservative signal among eukaryotes. In previous studies, Drosophila adoR mutants (Dolezelova, 2007; Wu, 2009) and mice with a knockout of all four adoRs (Xiao, 2019) both displayed minor physiological alteration under normal conditions. This is consistent with the idea that Ado signaling more likely regulates the response to environmental changes (stresses) rather than being involved in maintaining fundamental homeostasis in both insect and mammalian models (Cunha, 2019). This study examined the impact of altering the expression of genes involved in Ado signaling and metabolism on the cytotoxicity and neurodegeneration phenotype or Q93 mHTT-expressing flies. A novel downstream target of this pathway, mod(mdg4), was discovered and showed its effects on the downregulation of Hsp70 proteins, a well-known chaperone responsible for protecting cells against various stress conditions, including mHTT cytotoxicity, as well as thermal or oxidative stress (Lin, 2021).

The low level of Ado observed in da-Gal4>mHTT flies suggests that it might have a pathophysiological role; lowering of the Ado level might represent a natural response to cytotoxic stress. Consistently, experimentally decreased Ado signal rescued the mHTT phenotype, while an increased Ado signal had deleterious effects. Interestingly, a high level of Ado in the hemolymph has previously been observed in Drosophila infected by a parasitoid wasp. A raised e-Ado titer has not only been shown to stimulate hemocyte proliferation in the lymph glands (Mondal 2011), but also to trigger metabolic reprogramming and to switch the energy supply toward hemocytes (Bajgar, 2015). In contrast, the experiments show that a lowered e-Ado titer results in increased Hsp70 production. Increased Hsp70 has previously been shown to protect the cells from protein aggregates and cytotoxicity caused by mHTT expression, as well as some other challenges including oxidative stress (paraquat treatment) or heat-shock. The fine regulation of extracellular Ado in Drosophila might mediate the differential Ado responses via a single receptor isoform. Earlier experiments on Drosophila cells also suggested that different cell types have different responses to Ado signaling (Fleischmannov, 2012; Lin, 2021 and references therein).

The data also showed that altered adenosine signaling through the receptor is closely connected to Ado transport, especially to ent2 transporter function. It was observed that adoR and ent2 knockdowns provide the most prominent rescue of mHTT phenotypes. In addition, the overexpression of adoR and ent2 genes results in effects that are opposite to their knockdowns, thus supporting the importance of these genes as key regulators of mHTT phenotypes. A previous report showed that responses to adoR and ent2 mutations cause identical defects in associative learning and synaptic transmission (Knight, 2010). The present study shows that the phenotypic response of mHTT flies to adoR and ent2 knockdowns are also identical. The results suggest that the source of e-Ado for inducing AdoR signaling is mainly released by ent2. Consistently, the knockdown of ent2 has previously been shown to block Ado release from Drosophila hemocytes upon an immune challenge (Bajgar, 2015), as well as from wounded cells stimulated by scrib-RNAi (Poernbacher, 2018) or bleomycin feeding (Xu, 2020). These data support the idea that both adoR and ent2 work in the same signaling pathway (Lin, 2021).

The results revealed that lower AdoR signaling has a beneficial effect on mHTT-expressing flies, including increasing their tolerance to oxidative and heat-shock stresses. The effect of lower Ado signaling in mammals has been studied by pharmacologically blocking AdoRs, especially by the non-selective adenosine receptor antagonist caffeine. Interestingly, caffeine has beneficial effects on both neurodegenerative diseases and oxidative stress in humans. In contrast, higher long-term Ado concentrations have cytotoxic effects by itself in both insect and mammalian cells (Schrier, 2001; Merighi, 2002). Chronic exposure to elevated Ado levels has a deleterious effect, causing tissue dysfunction, as has been observed in a mammalian system. Extensive disruption of nucleotide homeostasis has also been observed in mHTT-expressing R6/2 and Hdh150 mice (Lin, 2021).

This study identified a downstream target of the AdoR pathway, mod(mdg4), which modulates mHTT cytotoxicity and aggregations. This gene has previously been implicated in the regulation of position effect variegation, chromatin structure, and neurodevelopment. The altered expression of mod(mdg4) has been observed in flies expressing untranslated RNA containing CAG and CUG repeats. In addition, mod(mdg4) has complex splicing, including trans-splicing, producing at least 31 isoforms. All isoforms contain a common N-terminal BTB/POZ domain which mediates the formation of homomeric, heteromeric, and oligomeric protein complexes. Among these isoforms, only two [including mod(mdg4)-56.3 (isoform H) and mod(mdg4)-67.2 (isoform T)] have been functionally characterized. mod(mdg4)-56.3 is required during meiosis for maintaining chromosome pairing and segregation in males. mod(mdg4)-67.2 interacts with suppressor of hairy wing [Su(Hw)] and Centrosomal protein 190 kD (CP190) forming a chromatin insulator complex which inhibits the action of adjacent enhancers on the promoter, and is important for early embryo development and oogenesis. This study showed that mod(mdg4) is controlled by AdoR which consecutively works as a suppressor of Hsp70 chaperone. The downregulation of adoR or mod(mdg4) leads to the induction of Hsp70, which in turn suppresses mHTT aggregate formation and other stress phenotypes. Although the results showed that silencing all mod(mdg4) isoforms decreases cytotoxicity and mHTT inclusion formation, it was not possible clarify which of the specific isoforms is involved in such effects, since AdoR seems to regulate the transcriptions of multiple isoforms. Further study will be needed to identify the specific mod(mdg4) isoform(s) connected to Hsp70 production (Lin, 2021).

In summary, the data suggest that the cascade (ent2)-AdoR-mod(mdg4)-Hsp70 might represent an important general Ado signaling pathway involved in the response to various stress conditions, including reaction to mHTT cytotoxicity, oxidative damage, or thermal stress in Drosophila cells. The present study provides important insights into the molecular mechanisms of how Ado regulates mHTT aggregate formation and stress responses in Drosophila; this might be broadly applicable for understanding how the action of Ado affects disease pathogenesis (Lin, 2021).

Caffeine Delays Ethanol-Induced Sedation in Drosophila

Caffeine and ethanol are among the most widely available and commonly consumed psychoactive substances. Both interact with adenosine receptor-mediated signaling which regulates numerous neurological processes including sleep and waking behaviors. In mammals, caffeine is an adenosine receptor antagonist and thus acts as a stimulant. Conversely, ethanol is a sedative because it promotes GABAergic neurotransmission, inhibits glutamatergic neurotransmission, and increases the amount of adenosine in the brain. Despite seemingly overlapping interactions, not much is known about the effect of caffeine on ethanol-induced sedation in Drosophila. In this study, using Drosophila melanogaster as a model, it was shown that caffeine supplementation in food delays the onset of ethanol-induced sedation in males and females of different strains. The resistance to sedation reverses upon caffeine withdrawal. Heterozygous adenosine receptor mutant flies are resistant to sedation. These findings suggest that caffeine and adenosine receptors modulate the sedative effects of ethanol in Drosophila (Tremblay, 2022).

Drosophila is an excellent model to study the effects of caffeine, adenosine receptor signaling, and alcohol on a variety of behaviors such as circadian rhythm, locomotion, and cognition. This study is the first to examine interplay among caffeine, Drosophila adenosine receptor, and ethanol-induced sedation in Drosophila. The results demonstrate that exposure to caffeine prolongs the onset of sedation in both male and female w¹¹¹⁸ and OR-R flies, commonly used control and wild type Drosophila strains, respectively. Sex differences in ethanol-induced sedation time are observed in both vertebrates and invertebrates including Drosophila. Female flies are reported to have a shorter sedation time than males. However, in w¹¹¹⁸ sexual dimorphism of sedation does not resolve when exposed to 100% ethanol. It was observed that w¹¹¹⁸ females showed a trend (not statistically significant) for a higher ST50 than males in control at all concentrations of caffeine tested (Tremblay, 2022).

The effect of caffeine on sedation time when sedated with 100% ethanol was most robust and statistically significant at 0.5 mg/mL dose and did not hold at 1 mg/mL dose. The effect of caffeine on sedation time was more prominent with 50% and 75% ethanol possibly because the effect of caffeine manifests more effectively when the rate of onset of sedation is less drastic at lower doses of ethanol. Other studies in flies have examined the effects of caffeine, at a similar dose range, on adenosine receptor expression, fecundity, egg laying, and life span. It is possible that at higher doses, flies are generally weaker or less viable, as caffeine appears to begin to confer mortality in flies at 1 mg/mL concentration. Significant widespread mortality was observed at 3 mg/mL and 5 mg/mL dose after one-week exposure. At higher caffeine doses, a weakened state is possible either because flies are avoiding consuming highly caffeinated food due to its bitter taste and/or due to the direct physiological effects of caffeine. There is support for a direct effect of caffeine on physiological functions leading to mortality in flies as shown by the correlation between caffeine-induced mortality and reduced transcript levels of neuromodulators and adenosine receptors. The effect of caffeine on sedation is reversible and wears off within three days of caffeine withdrawal, likely due to its metabolic clearance. Caffeine is metabolized by the Cytochrome P-450 enzymes in both Drosophila and humans. Caffeine metabolites (theophylline, theobromine, paraxanthine) are also detected within 3 h of caffeine ingestion in flies. The half-life of caffeine in humans is 3-7 h (Tremblay, 2022).

Caffeine primarily acts as a stimulant in mammals because of its antagonism of adenosine receptor signaling, which promotes sleep. This study addressed the relationship between adenosine receptor gene dosage and sedation in flies. Heterozygous adenosine receptor mutant flies were found to be resistant to sedation. It was not possible to conduct sedation assays on homozygous mutant flies for the AdoR^MB04401 allele because they generally do not survive to adulthood. This finding is in accordance with a previous study in which adenosine A2A receptor knockout mice were shown to be resistant to ethanol-induced hypnotic effects. Mammalian adenosine A2A receptor has the highest homology to the only adenosine receptor isoform in Drosophila. However, in Drosophila, caffeine may also act through additional pathways, such as the dopamine receptor-mediated signaling. The fly dopamine/ecdysteroid receptor (DopEcR) mediates ethanol-induced sedation. The Drosophila dopamine receptor (dDA1) and dopamine signaling have been independently shown to modulate the wake-promoting effect of caffeine, suggesting a potential adenosine receptor-independent mechanism of action for caffeine in flies (Tremblay, 2022).

Additionally, the effects of caffeine on sleep does not depend on adenosine activity. Further, caffeine was unable to inhibit adenosine receptor-mediated signaling in Drosophila neuroblast cell line in vitro. Therefore, it appears that caffeine and adenosine receptor either through coordination and/or independently play a role in ethanol-induced sedation (Tremblay, 2022).

The data suggests that pharmacological (caffeine exposure) and genetic (adenosine receptor mutation) disruption of adenosine receptor function delays ethanol sedation. Therefore, it can be hypothesized that agonists of the adenosine receptor, especially the endogenous ligand, adenosine, will promote sedation. Ethanol elevates extracellular adenosine levels which in turn activate the adenosine receptors. In mammalian systems, the ethanol-mediated elevation of adenosine modulates ethanol-induced behaviors primarily through adenosine A1 and A2 receptors. In future studies, it will be interesting to determine effects of adenosine receptor agonists on sedation and the cellular and molecular characteristics of signaling pathways downstream of adenosine receptors that mediate ethanol-induced sedation in flies (Tremblay, 2022).

Broadly, this study further supports the use of Drosophila as a model to study complex human behavior and to examine the colloquial notion of the negative implications of mixing caffeine with alcohol. Human correlational studies have found that individuals concurrently consuming caffeinated energy drinks and alcohol are more likely to consume more alcohol and have more severe negative consequences due to alcohol consumption [41]. When both substances are consumed in conjunction, caffeine reduces perceived inebriation, which leads to further consumption, thereby promoting binge drinking and risky behavior such as driving under the influence of alcohol. Further studies on the interaction between caffeine and ethanol will improve understanding of the biochemical and behavioral consequences of their consumption and aid in creating awareness of this public health crisis, especially for adolescents and young adults (Tremblay, 2022).

Mutation in the Drosophila melanogaster adenosine receptor gene selectively decreases the mosaic hyperplastic epithelial outgrowth rates in wts or dco heterozygous flies

Adenosine (Ado) is a ubiquitous metabolite that plays a prominent role as a paracrine homeostatic signal of metabolic imbalance within tissues. It quickly responds to various stress stimuli by adjusting energy metabolism and influencing cell growth and survival. Ado is also released by dead or dying cells and is present at significant concentrations in solid tumors. Ado signaling is mediated by Ado receptors (AdoR) and proteins modulating its concentration, including nucleoside transporters and Ado deaminases. This study examined the impact of genetic manipulations of three Drosophila genes involved in Ado signaling on the incidence of somatic mosaic clones formed by the loss of heterozygosity (LOH) of tumor suppressor and marker genes. Genetic manipulations with the AdoR, equilibrative nucleoside transporter 2 (Ent2), and Ado deaminase growth factor-A (Adgf-A) were shown to cause dramatic changes in the frequency of hyperplastic outgrowth clones formed by LOH of the warts (wts) tumor suppressor, while they have almost no effect on control yellow (y) clones. In addition, the effect of AdoR is dose-sensitive and its overexpression leads to the increase in wts hyperplastic epithelial outgrowth rates. Consistently, the frequency of mosaic hyperplastic outgrowth clones generated by the LOH of another tumor suppressor, discs overgrown (dco), belonging to the wts signaling pathway is also dependent on AdoR. These results provide interesting insight into the maintenance of tissue homeostasis at a cellular level (Sidorov, 2015).

Characterization of the Drosophila adenosine receptor: the effect of adenosine analogs on cAMP signaling in Drosophila cells and their utility for in vivo experiments

Adenosine receptors (AR) belonging to the G protein-coupled receptor family influence a wide range of physiological processes. Recent elucidation of the structure of human A2AR revealed the conserved amino acids necessary for contact with the Ado moiety. However, the selectivity of Ado analogs for AR subtypes is still not well understood. Previous work has shown that the Drosophila adenosine receptor (AdoR) evokes an increase in cAMP and calcium concentration in heterologous cells. This study has characterized the second-messenger stimulation by endogenous AdoR in a Drosophila neuroblast cell line and examined a number of Ado analogs for their ability to interact with AdoR. Ado can stimulate cAMP but not calcium levels in Drosophila cells. One full and four partial AdoR agonists, as well as four antagonists, were found. The employment of the full agonist, 2-chloroadenosine, in flies mimicked in vivo the phenotype of AdoR over-expression, whereas the antagonist, SCH58261, rescued the flies from the lethality caused by AdoR over-expression. Differences in pharmacological effect of the tested analogs between AdoR and human A2AR can be partially explained by the dissimilarity of specific key amino acid residues disclosed by the alignment of these receptors (Kucerova, 2012).

Interaction between differentiating cell- and niche-derived signals in hematopoietic progenitor maintenance

Maintenance of a hematopoietic progenitor population requires extensive interaction with cells within a microenvironment or niche (see Hematopoetic progenitor maintenance in the Drosophila blood system). In the Drosophila hematopoietic organ, niche-derived Hedgehog signaling maintains the progenitor population. This study shows that the hematopoietic progenitors also require a signal mediated by Adenosine deaminase growth factor A (Adgf-A) arising from differentiating cells that regulates extracellular levels of adenosine. The adenosine signal opposes the effects of Hedgehog signaling within the hematopoietic progenitor cells and the magnitude of the adenosine signal is kept in check by the level of Adgf-A secreted from differentiating cells. These findings reveal signals arising from differentiating cells that are required for maintaining progenitor cell quiescence and that function with the niche-derived signal in maintaining the progenitor state. Similar homeostatic mechanisms are likely to be utilized in other systems that maintain relatively large numbers of progenitors that are not all in direct contact with the cells of the niche (Mondal, 2011).

The mammalian hematopoietic niche displays complex interactions between populations of HSCs and progenitors to maintain their numbers. The relative in vivo contributions of cues emanating from the microenvironment in regulating stem cell versus progenitor maintenance remains unclear. Several stem cell and progenitor populations demonstrate slow cell cycling and this property of 'quiescence' is critical for maintaining their integrity over a period of time (Mondal, 2011).

In vivo genetic analysis in Drosophila allows for the study of stem cell properties in their endogenous microenvironment. Drosophila blood cells, or hemocytes, develop within an organ called the lymph gland, where differentiating hemocytes, their progenitors, and the cells of the signaling microenvironment or niche, are found. Differentiated blood cells in Drosophila are all myeloid in nature and are located along the outer edge of the lymph gland, in a region termed the cortical zone (CZ. These arise from a group of progenitors located within an inner core of cells termed the medullary zone (MZ). The MZ cells are akin to the common myeloid progenitors (CMP) of the vertebrate hematopoietic system. They quiesce, lack differentiation markers, are multipotent, and give rise to all Drosophila blood lineage. MZ progenitors are maintained by a small group of cells, collectively termed the posterior signaling center (PSC), that function as a hematopoietic niche. Clonal analysis has suggested the existence of a niche-bound population of hematopoietic stem cells, although such cells have not yet been directly identified (Mondal, 2011).

The PSC cells express Hedgehog (Hh), which is required for the maintenance of the MZ progenitors. Cubitus interruptus (Ci) is a downstream effector of Hh signaling similar to vertebrate Gli proteins; it is maintained in its active Ci¹⁵⁵ form in the presence of Hh and degraded to the repressor Ci⁷⁵ form in the absence of Hh. PSC-derived Hh signaling causes MZ cells to exhibit high Ci¹⁵⁵ (Mondal, 2011).

Proliferation of circulating larval hemocytes is also regulated by Adenosine Deaminase Growth Factor-A (Adgf-A), which is similar to vertebrate adenosine deaminases (ADAs). Adgf-A is a secreted enzyme that converts extracellular adenosine into inosine by deamination. Two distinct adenosine deaminases, ADA1 and ADA2/CECR1, are found in humans. CECR1 is secreted by monocytes as they differentiate into macrophages. In Drosophila, mutation of Adgf-A causes increased adenosine levels and increase in circulating blood cells (Mondal, 2011 and references therein).

Extracellular adenosine is sensed by the single Drosophila adenosine receptor (AdoR) that generates a mitogenic signal through the G protein/adenylate cyclase/cAMP-dependent Protein Kinase A (PKA) pathway (Dolezelova, 2007). A target of PKA is the transcription factor Ci, which also transduces the Hedgehog signal. This study explored the potential link between adenosine and Hedgehog signaling, both through PKA mediated regulation of Ci, and a model was proposed that the niche signal and the CZ signal interact to maintain the progenitor population in a quiescent and undifferentiated state within the MZ of the lymph gland (Mondal, 2011).

The first cells that express differentiation markers appear stereotypically at the peripheral edge of the lymph gland. These differentiating cells will eventually populate an entire peripheral compartment that will comprise the CZ. The timing of the first signs of differentiation matches closely with the onset of quiescence among the precursor population, eventually giving rise to the medullary zone (MZ) (Mondal, 2011).

The close temporal synchronization of CZ formation and the quiescence of MZ progenitors raised the intriguing possibility that the onset of differentiation might regulate the proliferation profile of the progenitors. To test this hypothesis, cell death was induced by expressing the pro-apoptotic proteins Hid and Reaper in the differentiating hemocytes, and the effect of their loss was assayed in the progenitor population. Loss of CZ cells was found to induce proliferation of the adjacent progenitor cells, which are normally quiescent at this stage (Mondal, 2011).

Candidate ligands in the lymph gland were knocked down by RNA interference (RNAi) and monitored for a loss of progenitor quiescence. This survey identified Pvf1 as a signaling molecule that is required for the maintenance of quiescence within the lymph gland. Expressing Pvf1^RNAi using Gal4 drivers specific to either niche (PSC) cells using Antp-gal4, progenitor cells using dome-gal4, or differentiating cells using Hml-gal4 showed that PSC-specific knockdown is sufficient to induce progenitor proliferation, whereas Pvf1 knockdown in progenitors or differentiating cells has no effect on the lymph gland. These results indicate that Pvf1 synthesized in the PSC is required for progenitor quiescence (Mondal, 2011).

To determine the site of Pvf1 function, its receptor Pvr was knocked down in the lymph gland using a similar approach. Interestingly, it was found that Pvr^RNAi expressed under the control of drivers specific to differentiating cells (Hml-gal4 and pxn-gal4) causes a loss of progenitor quiescence. The BrdU incorporating cells do not express differentiation markers. Thus, differentiation follows the proliferative event. Lymph glands are not similarly affected when Pvr function is downregulated in the progenitors themselves. These results indicate that Pvf1 originates in the niche and activates Pvr in maturing hemocytes, and that this signaling system is important for the quiescence of MZ progenitors. These results did not explain, though, how maturing cells might signal back to the progenitors causing them to maintain quiescence (Mondal, 2011).

Given the previously known role of Adgf-A in the control of hemocyte number in circulation (Dolezal, 2005), whether this protein plays a similar role in the lymph gland was investigated. Remarkably, downregulation of the secreted Adgf-A protein in the differentiating hemocytes of the CZ, achieved by expressing Adgf-A^RNAi under Hml-gal4 control, induces loss of quiescence of MZ progenitors, similar to that seen with loss of Pvr in the CZ. This suggests that Adgf-A may act as a signal originating from differentiating hemocytes that is required for maintaining progenitor quiescence. In support of this idea, while overexpression of Adgf-A in differentiating hemocytes alone does not affect normal zonation, it suppresses the induced progenitor proliferation caused by downregulation of Pvr. For loss of signaling molecules, it is the break in the signaling network necessary for reducing adenosine that causes continued proliferation and eventual differentiation. For rpr/hid the signaling cell itself has been removed, thereby causing a lack in a backward signal. Quantitative analysis of the data is consistent with a role for Adgf-A downstream of Pvr (Mondal, 2011).

The role of a niche signal is well established in many developmental systems that involve stem cell/progenitor populations. In the Drosophila lymph gland the niche expresses Hh and maintains a group of progenitor cells (Mandal, 2007). This current study establishes an additional mechanism, parallel to the niche signal that originates from differentiating cells, which also regulates quiescence of hematopoietic progenitors (Mondal, 2011).

The cells of the lymph gland proliferate at early stages, from embryo to mid second instar. At this stage, cells farthest from the PSC initiate differentiation and the rest enter a quiescent phase defining a MZ. In wild-type, the cells of the MZ remain quiescent and in progenitor form throughout the third instar, and this process requires a combination of the PSC and CZ signals. If either signal is removed, the progenitor population will eventually be lost due to differentiation. In many different genetic backgrounds, if quiescence is lost, the progenitor population initially continues to incorporate BrdU during the second instar without expressing any maturation markers. The differentiation phenotype, characterized by the expression of such markers, follows this abnormal proliferation. The net result is that whenever the progenitors accumulate BrdU (but not express any markers of differentiation) in the second instar, all cells of the lymph gland are differentiated and no MZ remains in the third instar. While the nature of the signal that triggers hemocyte differentiation is not known, withdrawal of Wingless may play a role in this process (Mondal, 2011).

Experimental analysis has demonstrated a novel role for Pvr in maturing hemocytes and its ligand, Pvf1, in the cells of the PSC. Pvf1 expression increases at a stage when the lymph gland is highly proliferative. At this critical window in development, Pvf1 originating from the PSC is transported to the differentiating hemocytes, binds to its receptor Pvr, and activates a STAT-dependent signaling cascade. At this stage, Pvf1 is sensed by all cells but it is only in the differentiating hemocytes that it activates Adgf-A in an AdoR/Pvr-dependent manner. This secreted factor Adgf-A is required for regulating extracellular adenosine levels. High adenosine would signal through AdoR and PKA to inactivate Ci and reduce the effects of the niche-derived Hedgehog signal leading to differentiation of the progenitor cells. The function of the Adgf-A signal is to reduce this adenosine signal and therefore reinforce the maintenance of progenitors by the Hedgehog signal. Thus, the Adgf-A and Hh signals work in the same direction but Adgf-A does so by negating a proliferative signal due to adenosine. In wild-type, equilibrium is reached through a signal that does not originate from the niche that opposes this proliferative process. The attractive step in this model is that the CZ and niche (in this case Hh-dependent) signals both impinge on common downstream elements allowing for control of the progenitor population relative to the niche and the differentiated cells. Most importantly, this is a mechanism for maintaining quiescence within a moderately large population of cells that is not in direct contact with a niche. By the time the three zone PSC/MZ/CZ system is set up in the late second instar all the cells of the MZ express high levels of E-cadherin, become quiescent and are maintained as progenitors and are capable of giving rise to all blood cell lineages. Under such circumstances, the interaction between a niche-derived signal and an equilibrium signal originating from differentiating cells can maintain homeostatic control of the progenitor population. Several vertebrate stem cell/progenitor scenarios such as during bone morphogenesis and hematopoiesis or in the Drosophila intestine have progenitors and differentiating cells in close proximity that could pose an opportunity for a similar niche and differentiating cell-derived signal interaction. In fact, evidence for such interactions have recently been provided for vertebrate skin cells (Mondal, 2011).

The role of small molecules such as adenosine has not yet been adequately addressed in vertebrate progenitor maintenance. A small molecule such as extracellular adenosine is unlikely to form a gradient over the population of cells and maintain such a gradient over a developmental time scale. It is much more likely that this system operates similar to the 'quorum sensing' mechanisms described for prokaryotes. A critical level of adenosine is required for proliferation and by expressing the Adgf-A signal this threshold amount is lowered, causing quiescence in the entire population (Mondal, 2011).

This study describes a developmental mechanism that is relevant to the generation of an optimal number of blood cells in the absence of any overt injury or infection. However, a system that utilizes such a mechanism to maintain a progenitor population could potentially sense a disruption upon induction of various metabolic stresses to cause differentiation of myeloid cells. Various mitochondrial and cellular stresses can cause an increase in extracellular adenosine (Fredholm, 2007), but whether they are relevant to this system remains to be studied. In the past, dual use has been observed of reactive oxygen species (ROS) as well as Hypoxia Inducible Factor-a (HIF-a) in both development and stress response of the Drosophila hematopoietic system. Responses to injury have been described in the Drosophila intestine, and in satellite cells that respond during injury, a stress related signal could be the initiating factor that overrides a maintenance signal. Thus, the equilibrium generated through developmental interactions is disrupted to promote a cellular response to stress signals (Mondal, 2011).

Equilibrative nucleoside transporter 2 regulates associative learning and synaptic function in Drosophila

Nucleoside transporters are evolutionarily conserved proteins that are essential for normal cellular function. This study examined the role of equilibrative nucleoside transporter 2 (ent2) in Drosophila. Null mutants of ent2 are lethal during late larval/early pupal stages, indicating that ent2 is essential for normal development. Hypomorphic mutant alleles of ent2, however, are viable and exhibit reduced associative learning. RNA interference was used to knock down ent2 expression in specific regions of the CNS and show that ent2 is required in the alpha/beta lobes of the mushroom bodies and the antennal lobes. To determine whether the observed behavioral defects are attributable to defects in synaptic transmission, transmitter release at the larval neuromuscular junction (NMJ) was studied. Excitatory junction potentials were significantly elevated in ent2 mutants, whereas paired-pulse plasticity was reduced. An increase in stimulus dependent calcium influx was also observed in the presynaptic terminal. The defects observed in calcium influx and transmitter release probability at the NMJ were rescued by introducing an adenosine receptor mutant allele (AdoR¹) into the ent2 mutant background. The results of the present study provide the first evidence of a role for ent2 function in Drosophila and suggest that the observed defects in associative learning and synaptic function may be attributable to changes in adenosine receptor activation (Knight, 2010).

A Drosophila adenosine receptor activates cAMP and calcium signaling

Adenosine receptors (AdoR) are members of the G protein-coupled receptor superfamily and mediate extracellular adenosine signaling, but the mechanism of adenosine signaling is still unclear. This study reports the first characterization of an insect AdoR, encoded by the Drosophila gene CG9753. Adenosine stimulation of Chinese hamster ovary cells carrying transiently expressed CG9753 led to a dose-dependent increase of intracellular cAMP and calcium, but untransfected controls showed no such response, showing that CG9753 encodes a functional AdoR. Endogenous CG9753 transcripts were detected in the brain, imaginal discs, ring gland and salivary glands of third-instar Drosophila larvae, and CG9753 overexpression in vivo caused lethality or severe developmental anomalies. These developmental defects were reduced by adenosine depletion, consistent with the proposed function of the CG9753 product as an AdoR. Overexpression of the G protein subunit Galpha(s) or of the catalytic subunit of protein kinase A (PKA) partially mimicked and enhanced the defects caused by ectopic expression of AdoR. These results suggest that AdoR is an essential part of the adenosine signaling pathway and Drosophila offers a unique opportunity to use genetic analysis to study conserved aspects of the adenosine signaling pathway (Dolezelova, 2007).

A role for adenosine deaminase in Drosophila larval development

Adenosine deaminase (ADA) is an enzyme present in all organisms that catalyzes the irreversible deamination of adenosine and deoxyadenosine to inosine and deoxyinosine. Both adenosine and deoxyadenosine are biologically active purines that can have a deep impact on cellular physiology; notably, ADA deficiency in humans causes severe combined immunodeficiency. This study has established a Drosophila model to study the effects of altered adenosine levels in vivo by genetic elimination of adenosine deaminase-related growth factor-A (ADGF-A), which has ADA activity and is expressed in the gut and hematopoietic organ. This study show that the hemocytes (blood cells) are the main regulator of adenosine in the Drosophila larva, as was speculated previously for mammals. The elevated level of adenosine in the hemolymph due to lack of ADGF-A leads to apparently inconsistent phenotypic effects: precocious metamorphic changes including differentiation of macrophage-like cells and fat body disintegration on one hand, and delay of development with block of pupariation on the other. The block of pupariation appears to involve signaling through the Adenosine receptor (AdoR), but fat body disintegration, which is promoted by action of the hemocytes, seems to be independent of the AdoR. The existence of such an independent mechanism has also been suggested in mammals (Dolezal, 2005).

Adenosine A2A receptors contribute to the radial migration of cortical projection neurons through the regulation of neuronal polarization and axon formation

Cortical interneurons born in the subpallium reach the cortex through tangential migration, whereas pyramidal cells reach their final position by radial migration. Purinergic signaling via P2Y1 receptors controls the migration of intermediate precursor cells from the ventricular zone to the subventricular zone. It was also reported that the blockade of A2A receptors (A2AR) controls the tangential migration of somatostatin+ interneurons. This study found that A2AR control radial migration of cortical projection neurons. In A2AR-knockout (KO) mouse embryos or naive mouse embryos exposed to an A2AR antagonist, an accumulation of early-born migrating neurons was observed in the lower intermediate zone at late embryogenesis. In utero knockdown of A2AR also caused an accumulation of neurons at the lower intermediate zone before birth. This entails the presently identified ability of A2AR to promote multipolar-bipolar transition and axon formation, critical for the transition of migrating neurons from the intermediate zone to the cortical plate. This effect seems to require extracellular ATP-derived adenosine since a similar accumulation of neurons at the lower intermediate zone was observed in mice lacking ecto-5'-nucleotidase (CD73-KO). These findings frame adenosine as a fine-tune regulator of the wiring of cortical inhibitory and excitatory networks (Alcada-Morais, 2021).

Homo-oligomerization of the human adenosine A2a receptor is driven by the intrinsically disordered C-terminus

G protein-coupled receptors (GPCRs) have long been shown to exist as oligomers with functional properties distinct from those of the monomeric counterparts, but the driving factors of oligomerization remain relatively unexplored. This study focused on the human adenosine A2A receptor (A2AR), a model GPCR that forms oligomers both in vitro and in vivo. Combining experimental and computational approaches, the intrinsically disordered C-terminus of A2AR was discovered to drive receptor homo-oligomerization. The formation of A2AR oligomers declines progressively with the shortening of the C-terminus. Multiple interaction types are responsible for A2AR oligomerization, including disulfide linkages, hydrogen bonds, electrostatic interactions, and hydrophobic interactions. These interactions are enhanced by depletion interactions, giving rise to a tunable network of bonds that allow A2AR oligomers to adopt multiple interfaces. This study uncovers the disordered C-terminus as a prominent driving factor for the oligomerization of a GPCR, offering important insight into the effect of C-terminus modification on receptor oligomerization of A2AR and other GPCRs reconstituted in vitro for biophysical studies (Nguyen, 2021).

CRISPR/Cas9 mediated deletion of the adenosine A2A receptor enhances CAR T cell efficacy

Adenosine is an immunosuppressive factor that limits anti-tumor immunity through the suppression of multiple immune subsets including T cells via activation of the adenosine A2A receptor (A2AR). Using both murine and human chimeric antigen receptor (CAR) T cells, this study showed that targeting A2AR with a clinically relevant CRISPR/Cas9 strategy significantly enhances their in vivo efficacy, leading to improved survival of mice. Effects evoked by CRISPR/Cas9 mediated gene deletion of A2AR are superior to shRNA mediated knockdown or pharmacological blockade of A2AR. Mechanistically, human A2AR-edited CAR T cells are significantly resistant to adenosine-mediated transcriptional changes, resulting in enhanced production of cytokines including IFNgamma and TNF, and increased expression of JAK-STAT signaling pathway associated genes. A2AR deficient CAR T cells are well tolerated and do not induce overt pathologies in mice, supporting the use of CRISPR/Cas9 to target A2AR for the improvement of CAR T cell function in the clinic (Giuffrida, 2021).

Physiology and effects of nucleosides in mice lacking all four adenosine receptors

Adenosine is a constituent of many molecules of life; increased free extracellular adenosine indicates cell damage or metabolic stress. The importance of adenosine signaling in basal physiology, as opposed to adaptive responses to danger/damage situations, is unclear. This study generated mice lacking all four adenosine receptors (ARs), Adora1-/-;Adora2a-/-;Adora2b-/-;Adora3-/- (quad knockout [QKO]), to enable investigation of the AR dependence of physiologic processes, focusing on body temperature. The QKO mice demonstrate that ARs are not required for growth, metabolism, breeding, and body temperature regulation (diurnal variation, response to stress, and torpor). However, the mice showed decreased survival starting at about 15 weeks of age. While adenosine agonists cause profound hypothermia via each AR, adenosine did not cause hypothermia (or bradycardia or hypotension) in QKO mice, indicating that AR-independent signals do not contribute to adenosine-induced hypothermia. The hypothermia elicited by adenosine kinase inhibition (with A134974), inosine, or uridine also required ARs, as each was abolished in the QKO mice. The proposed mechanism for uridine-induced hypothermia is inhibition of adenosine transport by uridine, increasing local extracellular adenosine levels. In contrast, adenosine 5'-monophosphate (AMP)-induced hypothermia was attenuated in QKO mice, demonstrating roles for both AR-dependent and AR-independent mechanisms in this process. The physiology of the QKO mice appears to be the sum of the individual knockout mice, without clear evidence for synergy, indicating that the actions of the four ARs are generally complementary. The phenotype of the QKO mice suggests that, while extracellular adenosine is a signal of stress, damage, and/or danger, it is less important for baseline regulation of body temperature (Xiao, 2019).

Epithelial cells release adenosine to promote local TNF production in response to polarity disruption

Disruption of epithelial integrity contributes to chronic inflammatory disorders through persistent activation of stress signalling. This study uncovered a mechanism whereby disruption of apico-basal polarity promotes stress signalling. Depletion of Scribbled (Scrib), a baso-lateral determinant, causes epithelial cells to release adenosine through equilibrative channels into the extracellular space. Autocrine activation of the adenosine receptor leads to transcriptional upregulation of TNF, which in turn boosts the activity of JNK signalling. Thus, disruption of cell polarity feeds into a well-established stress pathway through the intermediary of an adenosine signalling branch. Although this regulatory input could help ensuring an effective response to acute polarity stress, it is suggested that it becomes deleterious in situations of low-grade chronic disruption by provoking a private inflammatory-like TNF-driven response within the polarity-deficient epithelium (Poernbacher, 2018).

Adenosine receptors as mediators of both cell proliferation and cell death of cultured human melanoma cells

Adenosine displays contradictory effects on cell growth: it improves cell proliferation, but it may also induce apoptosis and impair cell survival. Following the pharmacologic characterization of adenosine receptor expression on the human melanoma cell line A375, A375 was chosen as a cellular model to define how the extracellular adenosine signals are conveyed from each receptor. By using selective adenosine receptor agonists or antagonists, this study found that A2A stimulation reduced cell viability and cell clone formation, whereas, at the same time, it improved cell proliferation. In support of this finding it was demonstrated that the stimulation of A2A adenosine receptors stably expressed in Chinese hamster ovary cell clone reproduced deleterious effects observed in human melanoma cells. A3 stimulation counteracted A2A-induced cell death but also reduced cell proliferation. Furthermore, it was found that A3 stimulation ensures cell survival. This study has demonstrated that adenosine triggers a survival signal via A3 receptor activation and it kills the cell through A2A receptor inducing a signaling pathway that involves protein kinase C and mitogen-activated protein kinases (Merighi, 2002).

Search PubMed for articles about Drosophila AdoR

Alcada-Morais, S., Goncalves, N., Moreno-Juan, V., Andres, B., Ferreira, S., Marques, J. M., Magalhaes, J., Rocha, J. M. M., Xu, X., Partidario, M., Cunha, R. A., Lopez-Bendito, G. and Rodrigues, R. J. (2021). Adenosine A2A Receptors Contribute to the Radial Migration of Cortical Projection Neurons through the Regulation of Neuronal Polarization and Axon Formation. Cereb Cortex. PubMed ID: 34184030 b

Bajgar, A., Kucerova, K., Jonatova, L., Tomcala, A., Schneedorferova, I., Okrouhlik, J. and Dolezal, T. (2015). Extracellular adenosine mediates a systemic metabolic switch during immune response. PLoS Biol 13(4): e1002135. PubMed ID: 25915062

Borea, P. A., Gessi, S., Merighi, S. and Varani, K. (2016). Adenosine as a multi-signalling guardian angel in human diseases: when, where and how does it exert its protective effects? Trends Pharmacol Sci 37(6): 419-434. PubMed ID: 26944097

Cunha, R. A. (2016). How does adenosine control neuronal dysfunction and neurodegeneration? J Neurochem 139(6): 1019-1055. PubMed ID: 27365148

Dolezal, T., Dolezelova, E., Zurovec, M. and Bryant, P. J. (2005). A role for adenosine deaminase in Drosophila larval development. PLoS Biol 3(7): e201. PubMed ID: 15907156

Dolezelova, E., Nothacker, H. P., Civelli, O., Bryant, P. J. and Zurovec, M. (2007). A Drosophila adenosine receptor activates cAMP and calcium signaling. Insect Biochem Mol Biol 37(4): 318-329. PubMed ID: 17368195

Fredholm, B. B. (2007). Adenosine, an endogenous distress signal, modulates tissue damage and repair. Cell Death Differ 14(7): 1315-1323. PubMed ID: 17396131

Giuffrida, L., Sek, K., Henderson, M. A., Lai, J., Chen, A. X. Y., Meyran, D., Todd, K. L., Petley, E. V., Mardiana, S., Molck, C., Stewart, G. D., Solomon, B. J., Parish, I. A., Neeson, P. J., Harrison, S. J., Kats, L. M., House, I. G., Darcy, P. K. and Beavis, P. A. (2021). CRISPR/Cas9 mediated deletion of the adenosine A2A receptor enhances CAR T cell efficacy. Nat Commun 12(1): 3236. PubMed ID: 34050151

Hasko, G., Linden, J., Cronstein, B. and Pacher, P. (2008). Adenosine receptors: therapeutic aspects for inflammatory and immune diseases. Nat Rev Drug Discov 7(9): 759-770. PubMed ID: 18758473

Knight, D., Harvey, P. J., Iliadi, K. G., Klose, M. K., Iliadi, N., Dolezelova, E., Charlton, M. P., Zurovec, M. and Boulianne, G. L. (2010). Equilibrative nucleoside transporter 2 regulates associative learning and synaptic function in Drosophila. J Neurosci 30(14): 5047-5057. PubMed ID: 20371825

Kucerova, L., Broz, V., Fleischmannova, J., Santruckova, E., Sidorov, R., Dolezal, V. and Zurovec, M. (2012). Characterization of the Drosophila adenosine receptor: the effect of adenosine analogs on cAMP signaling in Drosophila cells and their utility for in vivo experiments. J Neurochem 121(3): 383-395. PubMed ID: 22353178

Lin, Y. H., Maaroufi, H. O., Kucerova, L., Rouhova, L., Filip, T. and Zurovec, M. (2021). Adenosine receptor and its downstream targets, Mod(mdg4) and Hsp70, work as a signaling pathway modulating cytotoxic damage in Drosophila. Front Cell Dev Biol 9: 651367. PubMed ID: 33777958

Merighi, S., Mirandola, P., Milani, D., Varani, K., Gessi, S., Klotz, K. N., Leung, E., Baraldi, P. G. and Borea, P. A. (2002). Adenosine receptors as mediators of both cell proliferation and cell death of cultured human melanoma cells. J Invest Dermatol 119(4): 923-933. PubMed ID: 12406340

Mandal, L., Martinez-Agosto, J. A., Evans, C. J., Hartenstein, V. and Banerjee, U. (2007). A Hedgehog- and Antennapedia-dependent niche maintains Drosophila haematopoietic precursors. Nature 446(7133): 320-324. PubMed ID: 17361183

Mondal, B. C., Mukherjee, T., Mandal, L., Evans, C. J., Sinenko, S. A., Martinez-Agosto, J. A. and Banerjee, U. (2011). Interaction between differentiating cell- and niche-derived signals in hematopoietic progenitor maintenance. Cell 147(7): 1589-1600. PubMed ID: 22196733

Nguyen, K. D. Q., Vigers, M., Sefah, E., Seppala, S., Hoover, J. P., Schonenbach, N. S., Mertz, B., O'Malley, M. A. and Han, S. (2021). Homo-oligomerization of the human adenosine A2a receptor is driven by the intrinsically disordered C-terminus. Elife 10. PubMed ID: 34269678

Novakova, M. and Dolezal, T. (2011). Expression of Drosophila adenosine deaminase in immune cells during inflammatory response. PLoS One 6(3): e17741. PubMed ID: 21412432

Poernbacher, I. and Vincent, J. P. (2018). Epithelial cells release adenosine to promote local TNF production in response to polarity disruption. Nat Commun 9(1): 4675. PubMed ID: 30405122

Schrier, S. M., van Tilburg, E. W., van der Meulen, H., Ijzerman, A. P., Mulder, G. J. and Nagelkerke, J. F. (2001). Extracellular adenosine-induced apoptosis in mouse neuroblastoma cells: studies on involvement of adenosine receptors and adenosine uptake. Biochem Pharmacol 61(4): 417-425. PubMed ID: 11226375

Sidorov, R., Kucerova, L., Kiss, I. and Zurovec, M. (2015). Mutation in the Drosophila melanogaster adenosine receptor gene selectively decreases the mosaic hyperplastic epithelial outgrowth rates in wts or dco heterozygous flies. Purinergic Signal 11(1): 95-105. PubMed ID: 25528157

Tremblay, S., Zeng, Y., Yue, A., Chabot, K., Mynahan, A., Desrochers, S., Bridges, S. and Ahmad, S. T. (2022). Caffeine Delays Ethanol-Induced Sedation in Drosophila. Biology (Basel) 12(1). PubMed ID: 36671755

Xiao, C., Liu, N., Jacobson, K. A., Gavrilova, O. and Reitman, M. L. (2019). Physiology and effects of nucleosides in mice lacking all four adenosine receptors. PLoS Biol 17(3): e3000161. PubMed ID: 30822301

Xu, C., Franklin, B., Tang, H. W., Regimbald-Dumas, Y., Hu, Y., Ramos, J., Bosch, J. A., Villalta, C., He, X. and Perrimon, N. (2020). An in vivo RNAi screen uncovers the role of AdoR signaling and adenosine deaminase in controlling intestinal stem cell activity. Proc Natl Acad Sci U S A 117(1): 464-471. PubMed ID: 31852821

Wang, F., Ruppell, K. T., Zhou, S., Qu, Y., Gong, J., Shang, Y., Wu, J., Liu, X., Diao, W., Li, Y. and Xiang, Y. (2023). Gliotransmission and adenosine signaling promote axon regeneration. Dev Cell 58(8): 660-676. PubMed ID: 37028426

Zavialov, A. V., Gracia, E., Glaichenhaus, N., Franco, R., Zavialov, A. V. and Lauvau, G. (2010). Human adenosine deaminase 2 induces differentiation of monocytes into macrophages and stimulates proliferation of T helper cells and macrophages. J Leukoc Biol 88(2): 279-290. PubMed ID: 20453107

Zurovec, M., Dolezal, T., Gazi, M., Pavlova, E. and Bryant, P. J. (2002). Adenosine deaminase-related growth factors stimulate cell proliferation in Drosophila by depleting extracellular adenosine. Proc Natl Acad Sci U S A 99(7): 4403-4408. PubMed ID: 11904370

date revised: 10 December 2024

Home page: The Interactive Fly © 2011 Thomas Brody, Ph.D.