InteractiveFly: GeneBrief

hangover: Biological Overview | References

BIOLOGICAL OVERVIEW

The hangover gene defines a cellular stress pathway that is required for rapid ethanol tolerance in Drosophila melanogaster. To understand how cellular stress changes neuronal function, Hangover function was analyzed on a cellular and neuronal level. Evidence is provided that Hangover acts as a nuclear RNA binding protein and the phosphodiesterase 4d ortholog dunce as a target RNA. A transcript-specific dunce mutant was generated that is impaired not only in ethanol tolerance but also in the cellular stress response. At the neuronal level, Dunce and Hangover are required in the same neuron pair to regulate experience-dependent motor output. Within these neurons, two cyclic AMP (cAMP)-dependent mechanisms balance the degree of tolerance. The balance is achieved by feedback regulation of Hangover and dunce transcript levels. This study provides insight into how nuclear Hangover/RNA signaling is linked to the cytoplasmic regulation of cAMP levels and results in neuronal adaptation and behavioral changes (Ruppert, 2017).

In mammals and insects alike, repetitive ethanol exposure leads to the development of tolerance. Tolerance is defined as an increased resistance to the behavioral effects of ethanol upon previous exposure. Tolerance is also used as a criterion to diagnose alcohol use disorders. Previous work has established Drosophila melanogaster as a suitable model system to analyze the molecular and neural bases of rapid ethanol tolerance. At least two different mechanisms contribute to the rapid development of tolerance. One requires the monoamine octopamine, which shares functional similarities to noradrenaline in vertebrates, whereas the other relies on a cellular stress response uncovered by the phenotypic characterization of hangover (hang) mutants (Scholz, 2000, Scholz, 2005). The hang gene encodes a large, approximately 210-kDa nuclear zinc finger protein that is expressed in most, if not all, neurons in the adult brain (Scholz, 2005). A Hang-related protein, ZNF699, is associated with alcoholism in humans (Riley, 2006), supporting the idea that a cellular stress mechanism underlying ethanol tolerance is evolutionarily conserved between humans and D. melanogaster. hang mutants develop reduced ethanol tolerance and show defects in their response to oxidative stress and heat shock-induced ethanol resistance. However, the cellular signaling process upon which hang acts remains unclear, and how the broadly expressed hang protein mediates specific behavioral changes in response to global increases of cellular stressors such as ethanol remains an open question (Ruppert, 2017).

In neuronal cell lines, ethanol exposure reduces cyclic AMP (cAMP) levels, which results in cellular tolerance. cAMP-dependent phosphodiesterases (PDEs) are essential components of the cAMP signaling cascade and fine-tune cyclic nucleotide signaling. In Drosophila, three different cAMP-dependent PDEs have been identified; two of them play a dual role in cyclic guanosine monophosphate (cGMP) and cAMP signaling, whereas only Dunce (Dnc) is cAMP specific. The dnc gene encodes for at least eight isoforms with high homology to the human phosphodiesterase 4 class of PDEs. In general, PDE4s can be divided into two classes based on the presence or absence of the two highly conserved N-terminal domains. The long forms contain the upstream conserved region 1 and 2 (UCR1 and UCR2, respectively) domains, whereas the short forms lack the UCR1 domain and have either a partial or complete UCR2 domain. The dnc isoforms share a common PDE domain but differ in their N termini, suggesting that they may exhibit different regulatory and cellular functions. This hypothesis has been further supported by the phenotypic characterization of various dnc mutants. For example, dnc¹ and dnc^M11 share similar initial learning and memory defects but differ at the level of the neuroanatomical structure of the larval neuromuscular junction. Nerve terminal growth is increased in dnc¹ mutants, whereas the nerve terminals grow normally in dnc^M11 mutants. These phenotypic differences correlate with differences in PDE activity. dnc¹ mutants show reduced PDE activity, and dnc^M11 mutants display no detectable PDE activity; as a result, the cAMP level is elevated in both mutants. While the function of PDE4s has been analyzed in terms of the cellular stress response to oxidative stress and in non-neuronal tissues such as pulmonary endothelial cells, the function of PDE4s in ethanol-induced cellular stress in neurons has not been addressed (Ruppert, 2017 and references therein).

This study provides evidence that Hang functions as a nuclear RNA binding protein and dnc is identified as a target gene. To confirm the role of the PDE4 ortholog Dnc in ethanol tolerance, the consequence of altered cAMP signaling was investigated by analyzing the dnc¹ mutant for defects in ethanol-induced behaviors. Because multiple Dnc isoforms are altered in this dnc mutant, a new dnc^RA transcript-specific mutant, dnc^Δ143, was generated, enabling isoform-specific analysis of ethanol tolerance. To identify the neurons required for Dnc-dependent ethanol tolerance, a dnc^RA-specific promoter-GAL4 driver (dnc^RA-GAL4). Phenotypic characterization revealed that both the dnc^Δ143 and hang mutants exhibited similar impairment in the cellular stress response to ethanol. Both genes were required in the same neurons, enabling them to participate in the same cellular signaling process. In addition, Dnc^PA regulates Hang function during the development of ethanol tolerance. Furthermore, cytoplasmic Dnc, rather than nuclear Dnc, is required for tolerance development. The identification of the PDE4 ortholog Dnc as an interaction partner of Hang links nuclear Hang function to the regulation of cytoplasmic cAMP levels. These results substantiate the conserved nature of the signaling process underlying the neuronal stress response to ethanol (Ruppert, 2017).

This study shows how the nuclear hang protein, which is broadly expressed in nearly all neurons of the CNS, mediates specific behavioral changes in response to a global increase in the stressor ethanol by identifying the neurons required for Hang-dependent ethanol tolerance. In these neurons, hang can bind RNA and modify the transcript levels of the phosphodiesterase 4d ortholog dnc, linking nuclear signaling to the regulation of cAMP levels. Consistently, hang mutants show increased cAMP levels. In addition, this study shows that tolerance development requires the presence of the specific dnc isoform Dnc^RA in the cytoplasm of the same neurons. The functional relationship between dnc and hang depends on feedback regulation that balances the degree of tolerance formed (Ruppert, 2017).

Although emerging evidence in mice suggests that pharmacological inhibition of PDE4s reduces alcoholism-associated behaviors such as ethanol intake and preference (Blednov, 2014), what type of cellular process this inhibition of PDE4 activity is embedded in and whether this intervention acts on a neuronal level remains completely unclear. Every cell in organisms uses cAMP signaling as a second messenger system, and during ethanol exposure, the concentration of ethanol increases more or less everywhere in the brain. Nevertheless, the specificity of cAMP signaling for Hang- and Dnc^PA-dependent tolerance development can be ascribed to a pair of F1 neurons. In the neurons, other participants in the cAMP signaling cascade, such as the adenylyl cyclase Rutabaga, are present and functional, as shown by the fact that Rutabaga expression in the F1 neurons ameliorates visual learning defects in those mutants. The specific requirement of cAMP signaling in these neurons for tolerance might be linked to the more general function of the F1 neurons in motor control. At first glance, visual memory learning, particularly contour learning, and ethanol tolerance may not seem to have much in common because their sensory input differs. However, learning is measured as a change in the direction of flight of a tethered fly, linking visual information processing to motor output. In addition, there is evidence that the activity of the F1 neurons is required for the maintenance of high walking motivation, again linking the function of these neurons to motor output regulation. Behavioral ethanol tolerance is also measured by analyzing changes in motor output, such as the ability to maintain or regain posture after a previous ethanol experience (Scholz, 2000). The dnc^Δ143 mutants showed no difference in ethanol sensitivity, and therefore, the basic requirements of the behavior are normal. Considering the other observed functions of F1 neurons in locomotion, a more general function for F1 neurons is proposed that involves linking previous motor experience to new information and changes in behavioral output. All cells within the brain are exposed to an increasing ethanol concentration and might respond with a cellular stress response, but only defects in neurons in the specific neuronal networks uncovered here impact performance on the assayed task (Ruppert, 2017).

At the behavioral level, the cAMP second messenger system has been implicated in the regulation of ethanol sensitivity in both mammals and invertebrates. The Drosophila cAMP mutant rutabaga ethanol sensitivity increase (Moore, 1998) correlates with reduced cAMP levels (Cheung, 1999). Therefore, the increase in cAMP levels caused by the loss of PDE function should result in increased resistance to ethanol; however, neither the dnc¹ nor the dnc^Δ143 mutants, which have increased cAMP levels, showed a defect in ethanol sensitivity. In addition, other dnc mutants, such as dnc^M11, show normal ethanol sensitivity (Moore, 1998). To date, a role for dnc in sensitivity seems likely, given the increased sensitivity of rutabaga mutants; however, this role might not depend on the analyzed isoforms, as the tested dnc mutants showed normal ethanol sensitivity. In addition to its potential role in the regulation of ethanol sensitivity, Dnc-dependent cAMP function is required for the regulation of rapid ethanol tolerance. The dnc^Δ143-like dnc¹ mutants showed reduced ethanol tolerance but normal ethanol sensitivity, supporting the dissociation between cAMP function in ethanol sensitivity and tolerance. On a cellular level, differences in cAMP regulation might result from subcellular differences in PDE activity due to differences in PDE localization (Conti, 2007). The mouse PDE4D gene, which exhibits the highest homology with dnc, encodes at least four isoforms that are expressed in different subcellular domains. In addition, in the mollusk Aplysia, three different PDE4 isoforms localize to different subcellular membranes. Dnc^PA function in a specific subcellular compartment during tolerance development is consistent with the finding that nuclear Dnc^PG did not improve the reduced tolerance of the dnc^Δ143 mutants. Dnc^PA appears functionally similar to Dnc^all because both isoforms restored or improved the ethanol tolerance of the dnc^Δ143 mutants (Ruppert, 2017).

At first glance, the prolongation of cAMP signaling due to the elevation of cAMP levels seems to reduce ethanol tolerance. Consistently, the dnc^Δ143 and hang^AE10 mutants, with reduced tolerance, had elevated cAMP levels. These observations indicate that under normal circumstances, the termination of cAMP signaling plays a critical role in determining the level of tolerance formed. However, the true situation is more complex because the level of tolerance in the hang^AE10, dnc^Δ143 double mutants with increased cAMP levels was normal. Therefore, the dynamics of the cAMP changes are more crucial than the absolute cAMP level. The importance of dynamic changes of cAMP levels was already implicated by the observed similar impairments of learning and memory in rutabaga mutants with reduced cAMP levels and dnc mutants with increased cAMP levels. The dynamical change in ethanol tolerance might be achieved through the regulation of each gene's transcript level. In the hang^AE10 mutants, dnc was reduced, whereas temporally restricted expression of hang in the adult brain in a dnc^RA-GAL4-dependent manner increased tolerance and dnc levels, consistent with the idea that hang is a positive regulator of dnc transcript levels. In contrast, Dnc^RA is a negative regulator of hang expression because in the dnc^Δ143 mutants, hang was upregulated. The existence of a feedback regulation between dunce and hang is further supported by the observation that overexpression of hang in a dnc^RA-GAL4 dependent manner produces similar changes in dnc^RA- transcript levels and tolerance as found in the dnc^Δ143; hang^AE10 double mutants. However, the interaction is even more complicated. Overexpressing hang throughout development was lethal, and temporally restricted hang expression in the adult increased tolerance. That hang function is extremely dose sensitive was also demonstrated by previous results showing that the loss or gain of hang function at the NMJ results in similar morphological defects (Schwenkert, 2008). Taken together, these results indicate that there is complex feedback regulation between hang and dnc (Ruppert, 2017).

In summary, at the cellular/systems level, a reduction in cAMP level is required for behavioral tolerance. Crucial regulators at the cellular level include two conserved proteins, the PDE4d ortholog dnc and the cellular stress regulator Hang. The subcellular function of dnc and spatially and temporally controlled cAMP levels is important. cAMP regulation is linked via a feedback mechanism to the nuclear RNA binding protein hang under tight temporal control. Furthermore, the identification of the requirement of hang and the PDE4d ortholog dnc in the same neurons for regulation of experience-dependent motor output in Drosophila provides insight into how specific behavioral changes are achieved in response to global increases in cellular stressors such as ethanol (Ruppert, 2017).

The hangover gene negatively regulates bouton addition at the Drosophila neuromuscular junction

The synaptic growth of neurons during the development and adult life of an animal is a very dynamic and highly regulated process. During larval development in Drosophila new boutons and branches are added at the glutamatergic neuromuscular junction (NMJ) until a balance between neuronal activity and morphological structures is reached. Analysis of several Drosophila mutants suggest that bouton number and size might be regulated by separate signaling processes. This study show a new role for Hangover as a negative regulator of bouton number at the NMJ. The hangover gene (hang) encodes a nuclear zinc finger protein. It has a function in neuronal plasticity mediating ethanol tolerance, a behavior that develops upon previous experience with ethanol. hang^AE10 mutants have more boutons and an extended synaptic span. Moreover, hang expression in the motoneuron is required for the regulation of bouton number and the overall length of muscle innervation. However, the increase in bouton number does not correlate with a change in synaptic transmission, suggesting a mechanism independent from neuronal activity leads to the surplus of synaptic boutons. In contrast, expression levels of the cell adhesion molecule Fasciclin II (FASII) are reduced in the hang mutant. This finding suggests that the increase in bouton number in hang mutants is caused by a reduction in FASII expression, thus, linking the regulation of nuclear gene expression with the addition of boutons at the NMJ regulated by cell adhesion molecules (Schwenkert, 2008).

The hangover gene defines a stress pathway required for ethanol tolerance development

Repeated alcohol consumption leads to the development of tolerance, simply defined as an acquired resistance to the physiological and behavioural effects of the drug. This tolerance allows increased alcohol consumption, which over time leads to physical dependence and possibly addiction. Previous studies have shown that Drosophila develop ethanol tolerance, with kinetics of acquisition and dissipation that mimic those seen in mammals. This tolerance requires the catecholamine octopamine, the functional analogue of mammalian noradrenaline. This study describes a new gene, hangover, which is required for normal development of ethanol tolerance. hangover flies are also defective in responses to environmental stressors, such as heat and the free-radical-generating agent paraquat. Using genetic epistasis tests, this study shows that ethanol tolerance in Drosophila relies on two distinct molecular pathways: a cellular stress pathway defined by hangover, and a parallel pathway requiring octopamine. hangover encodes a large nuclear zinc-finger protein, suggesting a role in nucleic acid binding. There is growing recognition that stress, at both the cellular and systemic levels, contributes to drug- and addiction-related behaviours in mammals. These studies suggest that this role may be conserved across evolution (Scholz, 2005).

Search PubMed for articles about Drosophila Hangover

Blednov, Y. A., Benavidez, J. M., Black, M. and Harris, R. A. (2014). Inhibition of phosphodiesterase 4 reduces ethanol intake and preference in C57BL/6J mice. Front Neurosci 8: 129. PubMed ID: 24904269

Cheung, U. S., Shayan, A. J., Boulianne, G. L. and Atwood, H. L. (1999). Drosophila larval neuromuscular junction's responses to reduction of cAMP in the nervous system. J Neurobiol 40(1): 1-13. PubMed ID: 10398067

Conti, M. and Beavo, J. (2007). Biochemistry and physiology of cyclic nucleotide phosphodiesterases: essential components in cyclic nucleotide signaling. Annu Rev Biochem 76: 481-511. PubMed ID: 17376027

Moore, M. S., DeZazzo, J., Luk, A. Y., Tully, T., Singh, C. M. and Heberlein, U. (1998). Ethanol intoxication in Drosophila: Genetic and pharmacological evidence for regulation by the cAMP signaling pathway. Cell 93(6): 997-1007. PubMed ID: 9635429

Riley, B. P., Kalsi, G., Kuo, P. H., Vladimirov, V., Thiselton, D. L., Vittum, J., Wormley, B., Grotewiel, M. S., Patterson, D. G., Sullivan, P. F., van den Oord, E., Walsh, D., Kendler, K. S. and Prescott, C. A. (2006). Alcohol dependence is associated with the ZNF699 gene, a human locus related to Drosophila hangover, in the Irish Affected Sib Pair Study of Alcohol Dependence (IASPSAD) sample. Mol Psychiatry 11(11): 1025-1031. PubMed ID: 16940975

Ruppert, M., Franz, M., Saratsis, A., Velo Escarcena, L., Hendrich, O., Gooi, L. M., Schwenkert, I., Klebes, A. and Scholz, H. (2017). Hangover links nuclear RNA signaling to cAMP regulation via the phosphodiesterase 4d ortholog dunce. Cell Rep 18(2): 533-544. PubMed ID: 28076795

Scholz, H., Franz, M. and Heberlein, U. (2005). The hangover gene defines a stress pathway required for ethanol tolerance development. Nature 436(7052): 845-847. PubMed ID: 16094367

Schwenkert, I., Eltrop, R., Funk, N., Steinert, J. R., Schuster, C. M. and Scholz, H. (2008). The hangover gene negatively regulates bouton addition at the Drosophila neuromuscular junction. Mech Dev 125(8): 700-711. PubMed ID: 18524547

date revised: 12 April 2022

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