Alcohol and Alcoholism Advance Access first published online on May 25, 2008
This version published online on August 1, 2008
Alcohol and Alcoholism, doi:10.1093/alcalc/agn045
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The Drosophila homolog of jwa is required for ethanol tolerance
1 Department of Molecular CellBiology and Toxicology, School of Public Health, Nanjing Medical University, 140Hanzhong Road, Nanjing 210029, China
2 Model Animal ResearchCenter, Nanjing University,Nanjing 210023, China
Corresponding to: Jianwei Zhou, MD, PhD,Department of Molecular Cell Biology and Toxicology, School of PublicHealth, Nanjing Medical University, 140 Hanzhong Rd, Nanjing 210029,China. Tel: +86-25-8666-2961; fax:+86-25-8686-2050; E-mail:jwzhou{at}njmu.edu.cn
Received 20 December 2007; first review notified 7 April 2008; in revised form 20 April 2008; accepted 6 May 2008
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Aims: Alcohol abuse poses a serious public health problem, and repeated ingestion can produce tolerance, leading to dependence and addiction. However, the mechanisms underlying alcohol tolerance and addiction are not fully understood. Drosophilae have been employed as a suitable model to study the molecular mechanisms underlying ethanol tolerance. JWA, a newly identified microtubule-binding protein, was shown to regulate cell stress responses, transportation of intracellular excitatory amino acids, and the MAPK signal transduction pathway. The JWA mouse homologue addicsin, was postulated to play a role in the development of morphine tolerance and dependence. This study was designed to determine whether JWA participates in ethanol tolerance in Drosophila. Methods: The jwa homologous gene in Drosophila, CG10373 (djwa) was cloned and the anti-djwa and cDNA-djwa transgenic fly strains, which exhibit a reduced and elevated djwa expression respectively were constructed. Real-time PCR was used to measure the djwa levels in the resulting fly strains. Rapid tolerance experiments including inebriation exposure and recovering assay were employed. Results: The djwa and the human jwa genes share a significant sequence similarity. Their genomic nucleotide and deduced amino acid sequence identities are 41.4% and 53.6%, respectively. In inebriation tests, the wild type w1118 flies and the cDNA-djwa flies acquired ethanol tolerance after several exposures whereas the anti-djwa flies did not. Conclusions: The JWA genes are evolutionarily conserved. The djwa function is required for acquiring ethanol tolerance in Drosophila. JWA is likely a novel molecule playing an important role in ethanol tolerance and drug addiction. Our results present a new direction for research related to alcohol tolerance and addiction.
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Introduction |
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There is an urgent need for a better understanding of mechanisms responsible for alcohol tolerance and addiction in order to prevent and control this public health problem. Tolerance to the acute intoxicating effects of ethanol is a risk factor for the development of alcoholism and is at least partially, genetically determined (Schuckit and Smith, 2000
). In general, the acute effects of alcohol on the nervous system include potentiation of inhibitory ion channels, such as 
-aminobutyric acid (GABA)A and glycine receptors, and inhibition of excitatory ion channels, such as NMDA receptors and L-type voltage-gated calcium channels (Harris, 1999). In recent years, growth factors including insulin, glial cell line-derived neurotrophic factor (GDNF), and brain-derived neurotrophic factor (BDNF) were found to play a critical role in manifestation of behavioral effects of alcohol in adults (Janak et al., 2006). These findings expand the area of investigating the neural mechanisms that underlie the development and maintenance of alcohol addiction.
Studies showed that Drosophila may serve as a suitable model to understand and resolve this problem. For instance, as measured by an increase in the mean elution time (MET) between the first and second ethanol exposure adult Drosophila develop ethanol tolerance as soon as 4 hr after the first exposure (Scholz et al., 2000). Behaviors induced by acute exposure in Drosophila are similar to those observed in mammals; with low ethanol doses inducing a state of increased activity, while higher quantities produce sedation (Heberlein, 2000; Janak et al., 2006). Recently, it was reported that the hangover (hang) gene plays essential roles in development of the tolerance and the gene product was shown to mediate cellular stress pathway (Scholz et al., 2000, 2005). Heat stress was found to induce tolerance to a subsequent exposure to ethanol and implicated a nucleic acid binding zinc finger protein encoded by the hangover gene in both the response to heat stress and induction of ethanol tolerance (Scholz et al., 2005). It was also demonstrated that flies lacking the neuromodulator octopamine, owing to a mutation in the gene encoding tyramine β hydroxylase (Tbh), showed a reduction in ethanol tolerance (Scholz et al., 2000). Furthermore, induction of ethanol tolerance was completely abolished in flies carrying both null mutations in hang and in the gene encoding Tbh, which suggested that both hang and the neurotransmitter octopamine were markedly involved in the induction of ethanol tolerance (Scholz et al., 2005). However, the molecular mechanisms remain unclear.
The human jwa gene was first identified as a retinoic acid-responsive and cytoskeleton-associated gene (Zhou et al., 1999). ADP-ribosylation-like factor 6 interacting protein 5 (ARL6IP5) and prenylated Rab acceptor 1 (PRA1) domain family 3 (PRAF3), and its several homologues [e.g. glutamate-transporter associated protein 3–18 (GTRAP3–18) and addicsin] have also been studied (Fo et al., 2006; Ingley et al., 1999; Lin et al., 2001). The rat protein GTRAP3–18, which has 95% of amino acids identical to JWA, is a negative regulator of excitatory amino acid carrier 1 (EAAC1) that functions in transmembrane glutamate transport (Butchbach et al., 2003; Lin et al., 2001). In PC12 cells, JWA modulates the intracellular amino acid homeostasis, not only for excitatory amino acids such as glutamate, but also for inhibitory amino acids such as taurine (Li et al., 2003). The mouse addicsin gene (98% identical to GTRAP3–18) is involved in the development of morphine tolerance and dependence, possibly through modulation of glutamate uptake by EAAC1 (Huerta et al., 2006; Ikemoto et al., 2002). We have previously found that the jwa gene responds to environmental stimuli, showing increased expression in response to oxidative and heat-shock stresses (Cao et al., 2002; Xia et al., 2001). In cultured cell models, the expression of JWA correlates with the hsp70 response to oxidative stress (Chen et al., 2005; Zhu et al., 2006). Ethanol induced cellular responses similar to those elicited by heat shock stresses (Piper, 1995; Wilke et al., 1994), and exposure to heat utilizes an unidentified pathway involved in ethanol tolerance (Scholz et al., 2005). In response to environmental stresses, the hang gene functions by reducing the damage produced by reactive oxygen species (ROS) (Scholz et al., 2005). Similarly, the jwa gene serves as an environmental stress sensor to protect cells against ROS induced DNA damage (Chen et al., 2007). These findings led us to postulate that jwa is essential for development of ethanol tolerance in Drosophila.
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Materials and Methods |
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Fly stocks
Flies were raised at 25°C on standard cornmeal/molasses/agar medium with a 12 /12 hr light/dark cycle. Newly derived flies were collected over a 2-day period and studied when they were 5 to 7 days old. All experiments were started at same time of day to minimize the effect of circadian rhythms. Genotypes of stocks were as follows: w1118; T (2; 3) apXa /CyO, P [Act GFP, w-] CC2; TM6C, Sb1, Tb1 and tub P-Gal4/ TM3, Sb. The w1118 stock was used for injection and was the background stock in which the anti-djwa and cDNA-djwa lines of transgenic flies were made. For all assays, only male flies were used.
Plasmid construction and generation of transgenic flies
djwa/CG10373 cDNA was used to generate various P-element transformants, and the cDNA was derived from w1118 flies. The plasmid pUAST-cDNA-djwa was constructed by subcloning the full-length djwa cDNA (649 bp) into the pUAST vector. The plasmid pUAST-anti-djwa was constructed by subcloning the -73 to 178bp fragment (252 bp) of djwa cDNA into the pUAST vector in the opposite orientation. The following primers were used for constructing the two vectors: 5'-GGAAGATCTCCCAAAAACCATGACAACG-3' (forward) and 5'-CCGCTCGAGTCAAACTGGTCAGTCTAATT-3' (reverse) for cDNA-djwa, and 5'-GCTCTAGAACGTCAACC-GAGTTATCAGAA-3' (forward), and 5'-CGGAATT-CTGGTAATAGAGCAGGTTCTTC-3' (reverse) for anti-djwa. To obtain transgenic flies, embryos from w1118 were obtained, and a pUAST plasmid and a helper plasmid, 
2–3, were microinjected into embryos. After microinjection, UAS-anti-djwa and UAS-cDNA-djwa flies were obtained. The P element was inserted by crossing transgenic line with T (2; 3) apXa /CyO, P [Act GFP, w-] CC2; TM6C, Sb1, Tb1 to identify the chromosome. To drive the expression of UAS constructs, tub P-Gal4/ TM3, Sb were used, which led to expression throughout the body. The heterozygote tub-GAL4; uas-jwa and tub-GAL4; uas-anti-jwa were used for the behavioral assays.
RNA analysis
Total RNA was extracted from about 30 w1118 and anti-djwa flies, 5–7-days old, with Trizol reagent (Gibco BRL, Gaithersburg MD, USA). About 1 µg of RNA was used for the reverse transcription reaction with OligodT (18T) (Invitrogen, Shanghai, China). The cDNA was amplified with the following primers: 5'-TCCGTGACATCAAGGAGAAGC-3' (forward) and 5'-CAAGAACGAGGGCTGGAACA-3' (reverse) for actin, 5'-TCATAGGCGTTATCTGGC-3' (forward) and 5'-GCTTGTTCTTGATGTTTCG-3' (reverse) for djwa. The reaction cycles for semi-quantitative RT-PCR was 20, and the annealing temperature was 60°C for both djwa and action. The fragment size was 176 bp for djwa and 258 bp for actin. Quantitative real-time PCR was performed with EvaGreen (Biotium Hayward, CA), a DNA intercalating dye that is more stable and sensitive than SYBR Green I (Ihrig et al., 2006; Wang et al., 2006). The following thermal cycling conditions were used: denaturation, 94°C for 5 min followed by 44 cycles of denaturation at 94°C for 35 sec, annealing 60°C for 30 sec, extension 72°C for 35 sec, and plate read. The cycles were followed by a final extension step at 72°C for 8 min, and melting curve from 70°C to 90°C. The data was analyzed using the 2CT method (Livak and Schmittgen, 2001), and the mean, and SD were determined from quadruple samples.
Inebriator
The inebriator was constructed based on the model described by Bellen (1998). Air was passed through a water flask (100 ml of distilled, deionized water), through a bubbler containing 10 ml of 100% ethanol in a 60 °C water bath, through a trap to collect any condensed ethanol, and then into a column consisting of 12 50-ml centrifuge tubes without bottoms. In each tube, there were three meshes serving as baffles. The flies held on to the baffles until they were inebriated, at which time they fell and eluted from the column. The length of the column was 120 cm, which prevented flies from moving out of the column freely.
Tolerance Assay
All behavioral experiments described here were performed with age and sex-matched flies (5 to 7-day old males). For each inebriation test, approximately 100 flies were collected and placed in a vial with fresh food for 24 hr at 25°C prior to testing. In the inebriator, flies were exposed to ethanol vapor; those that eluted out of the inebriator were collected each min. Between exposures, flies were allowed to recover at 25°C in food vials closed with humidified cotton plugs. The second inebriator exposure was initiated 4hr after the start of the first exposure; the third exposure occurred 24hr after the first exposure. In all tests, the air flow, quantity of 100% ethanol, and the temperature of the water bath were the same to ensure that the ethanol vapor concentration was invariable. The MET corresponds to the sum of the number of flies eluting each min multiplied by the time of elution divided by the total number of flies (Moore et al., 1998). All experiments were accomplished at least in duplicate.
For the recovering assay, about 60 male flies were placed in perforated 50-ml tubes and exposed to ethanol vapor until all were sedated. The sedated flies were moved to a flat plate and observed until all had recovered. Sedated flies were scored as those that were lying on their backs or sides or those ‘facedown’ with their legs extended in a non-standard posture (Cowmeadow et al., 2005). Recovered flies were scored as those regaining postural control. After the flies had recovered from sedation, they were transferred to food vials closed with humidified cotton plugs. Four hr later, flies were exposed to the ethanol vapor for second time. The number of flies recovering from sedation in each experiment was noted each min during the recovery phase. The % flies recovering was plotted against time.
Statistical Analysis
Differences in METs were analyzed using the Student's t test (two-tailed test assuming equal variance of the mean). For the recovering tests described above, the log rank test was used to determine whether there was a significant difference between the recovery curves for once- or twice-sedated flies.
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Results |
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Homologous gene in fly and identification of transgenic flies
To test our hypothesis, the Drosophila homologue of human jwa was initially identified by sequence comparison. The Drosophila gene with the highest homology in protein levels is CG10373, and its genomic region is 2L:18704570–18705699. The gene encodes a putative protein of 27.8 kD, and the protein shares strong homology with JWA. As determined with the clustal-x program (Thompson et al., 1997
), 41.4% of the amino acids and 53.6% of the DNA bases were identical between the two genes (http://www.ncbi.nlm.nih.gov/sites/entrez?db=homologene, Fig. 1). The alignment revealed two regions "NLLYYQTNY" and "FIHASLRLRN", both of which are highly conserved. These regions were previously identified by analyzing GTRAP3–18 sequences from different species (Butchbach et al., 2003).
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The level of the djwa expression was altered by using the UAS/GAL4 system (Brand and Perrimon, 1993
). The tub-GAL4 driver line was used to increase or decrease expression djwa in all cells of the adult fly. The transcript levels of djwa were determined by RT-PCR and real-time-PCR analysis, and the results showed that the djwa transcript present in w1118 Drosophilae was approximately 3.2-fold higher than that in the anti-djwa counterparts (Fig. 2). For cDNA-djwa Drosophilae, the transcript was present in amounts 2-fold greater than that for w1118 counterparts (Table 1).
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Sedation of flies using ethanol vapor
In the inebriator study, ethanol-saturated air stream has been an effective method to deliver a reproducible ethanol dose and to rapidly sedate flies (Cowmeadow et al., 2005
; Moore et al., 1998). During the first few min following ethanol exposure, the flies enter a hyperactivity phase, in which they walk around at a faster speed. This hyperkinetic phase persists for a few min, and then their movements gradually slow down and eventually stop. After 10–20 min following ethanol exposure, the flies become uncoordinated, and lose their ability to stand on the baffles and gradually tumble downward. This phenomenon was described by Rothenfluh and Heberlein (2002), and also observed in all of our experiments.
Knockdown of djwa gene eliminate rapid tolerancer
In inebriation tests, the METs as previously described were measured (Moore et al., 1998). As the control, the wild-type w1118 flies eluted from the inebriator with a normal distribution, with the distribution for the second elution being longer (Fig. 3a). The METs were 14.2 ± 0.3, 18.1± 0.5 and 22.5 ± 0.7 min for the first, second, and third exposures respectively, representing asignificant increase of 27.5% between the first and second exposures and a marked rise of 58.5% between the first and third exposures (Fig. 3b). Data suggest that, with prior exposure, the w1118 flies acquired ethanol tolerance.
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100 flies were placed in the inebriator. Graphs (on the left) show the normal distribution for elution of the three kinds of flies after first and second exposures to ethanol vapor. Bars (on the right) show their mean elution times (METs) after one, two, or three exposures. a, b: w1118 flies showed an increase of 58.5% in ethanol tolerance after the third exposure, and significant differences of the second MET to the first MET was found (P < 0.0001), n = 17 experiments; c, d: cDNA-djwa flies showed an increase of 85.4% in ethanol tolerance after the third exposure, and significant differences of the second MET to the first MET was found (P < 0.0001), n = 15 experiments; e, f: anti-djwa flies showed no increase in ethanol tolerance after a second or third exposure, and no significant differences of second MET to the first MET was found (P = 0.2230), n = 20 experiments. Error bars in all figures correspond to the SEM.Similar results were noted for cDNA-djwa flies. The normal distribution shifted on the second exposure (Fig. 3c). The METs were 15.1± 0.4, 20.1± 0.7, and 28.0 ± 1.0 min for the first, second and third exposures, respectively. These times are quantitatively longer than those for w1118 flies. For cDNA-djwa flies, there was a significant increase of 33.1% between the first and second exposures and a marked rise of 85.4% between the first and third exposures (Fig. 3d). For anti-djwa flies, the tolerance to alcohol exposure disappeared (Fig. 3e). There were no significant changes in the METs: 11.1 ± 0.3, 11.6 ± 0.3, and 10.8 ± 0.3 min for the first, second, and third exposures, respectively (Fig. 3f), indicating that decrease in djwa function prevents the development of alcohol tolerance.
We also measured the recovery time for sedated flies, using the methods developed by (Cowmeadow et al. 2005). For the w1118 andcDNA-djwa flies, the recovery curves for a second exposure were significantly higher than those for a first exposure (Fig. 4a,b). However, foranti-djwa flies, there was no significant difference between the two curves (Fig. 4c). The time needed for 50% of flies recovering from sedation (t1/2 recovering) is 30 min and 22.5 min for w1118 first and second exposure, respectively; 33 min and 20 min for cDNA-djwa flies; 64 min and 67 min foranti-djwa flies, respectively. It is apparent that t1/2anti-djwa >t1/2 w1118 >t1/2cDNA-djwa. In addition, about 20% of theanti-djwa flies died in the second recovery experiment, indicating that theanti-djwa flies were more sensitive to ethanol exposure, further demonstrating the importance of djwa in the development of alcohol tolerance.
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ADH and hangover gene transcription level
In addiction, similar to humans, alcohol dehydrogenase (ADH) is predominantly responsible for ethanol metabolism in flies. It was reported that ethanol consumption increased ADH expression in adults (Geer et al., 1988
). However, to evaluate whether different transgenes might influence the expression of ADH, ADH mRNA levels were determined prior to and after alcohol exposure. The results showed that there is no significant difference in ADH mRNA levels before and after ethanol exposure (Table 2). To determine the relationship between djwa and hangover, the transcription levels of the two genes in both anti-djwa flies and w1118 flies were measured, and it was found that hangover gene transcription levels were down-regulated when djwa was knockdown in flies. As shown in Table 3, the hangover gene transcription level in w1118 flies was 4.2 fold higher compared with anti-djwa flies.
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Discussion |
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Drosophila provides a reliable model system for the study of drugs abuse (Janak et al., 2006
; Wen et al., 2005). It was shown that many drugs of abuse (such as morphine, cocaine, and ethanol) act through the same neurotransmitter system in both Drosophila and mammals (Bainton et al., 2000; Porzgen et al., 2001). A number of novel genes which may be effective for understanding the effect of ethanol were screened (Berger et al., 2004; Morozova et al., 2006). Here evidence indicates that a novel gene, djwa/CG10373, plays an important role in the response to ethanol in Drosophila.
Thejwa gene encodes a large PRA1 domain, which was thought to form a new family of PRAF proteins that play a role in intracellular protein transport (Fo et al., 2006). It was shown that JWA (GTRAP3–18) might regulate glutamate transport (Butchbach et al., 2003; Lin et al., 2001), and glutamate transport by EAAC1 is important for drug tolerance (Mao et al., 2002; Wang et al., 2006). Furthermore, there is direct evidence that addicsin gene is involved in the development of morphine tolerance and dependence (Ikemoto et al., 2002). Recently, studies showed that inhibition of JWA almost completely abolished addiction induced by morphine in rats involving enhanced expression of neurotropin 3 (NT-3) associated mechanisms (unpublished data). Other investigators also found that the ethanol-tolerance was responsive to some critical supportive neurotropin factors, including NT-3 and brain-derived neurotropic factor (BDNF) (Dohrman et al., 1997; Heaton et al., 2004). BDNF is a member of the nerve growth factor family of neurotropins and plays a critical role in synaptic plasticity (Carter et al., 2002). Increases in BDNF were reported to reduce alcohol intake, whereas decreases in BDNF were associated with increased alcohol intake (Janak et al., 2006).
The action of BDNF is mediated by binding to TrkB receptors and subsequently activating of the MAPK signal transduction pathway (Impey et al., 1999; Janak et al., 2006). It is well known that MAPK pathway plays an important role in the molecular mechanisms underlying drug addiction (Girault et al., 2007). Extracellular signal-regulated kinase (ERK), one of the highly conserved mitogen-activated protein kinase (MAPK) modules, is activated by drugs of abuse in a subset of neurons in reward-related brain regions (Valjent et al., 2000).This activation, necessary for the expression of immediate early genes, depends upon dopamine D1 and glutamate receptors (Valjent et al., 2005). Chen et al (2007) found that JWA was essential for activation of the MAPK pathway, and knock-down of JWA blocked ERK phosphorylation. Blockade of ERK activation prevents long-lasting behavioural changes, including psychomotor sensitization and conditioned place preference (Gerdjikov et al., 2004; Valjent et al., 2006). Similarly, in rat morphine addiction, ERK phosphorylation was inhibited by knock-down of JWA, and the withdrawal reactions were also attenuated (unpublished data).
The above results suggest that djwa is necessary for the acquisition of ethanol tolerance in Drosophila. The tolerance may involve the octopaminergic system and the Hang -mediated cellular stress pathway (Scholz et al., 2005). In invertebrates, octopamine acts as a neurohormone, neuromodulator, and neurotransmitter (Roeder, 1999). Since djwa might function as an amino acid transporter (http://flybase.bio.indiana.edu/) and, in mammals, JWA is a microtubule-associated protein (Chen et al., 2004; Li et al., 2003), it is postulated that djwa is a transporter in octopaminergic systems. In flies, the hang gene is required for development of ethanol tolerance, and also functions in responses to environmental stress such as heat shock (Scholz et al., 2005). The jwa gene also responds to environmental stress (heat shock and oxidative stress) (Chen et al., 2007; Zhu et al., 2005, 2006). Jwa participates in the signal transduction pathways associated with hydrogen peroxide(H2O2)-associated oxidative stress through enhancing intracellular defence and preventing H2O2-induced DNA damage (Chen et al., 2007; Zhu et al., 2006). Furthermore, it was found that the t1/2 of anti-djwa flies was increased more than 2 fold compared to wild flies, which indicated that anti-djwa flies were more sensitive to ethanol, and djwa might play an important role in development of alcohol tolerance. Taken together, djwa apparently functions in the development of ethanol tolerance in Drosophila by possibly participating in the cellular stress pathway defined by the hang gene or in the transport activity associated with octopaminergic systems. Since these pathways are conserved in evolution (Scholz et al., 2005), further analyses need to be performed in other cell or animal models.
In conclusion, Drosophila gene djwa plays an essential role in ethanol tolerance. As demonstrated with an inebriation test and a recovery assay, flies with a djwa knockdown did not acquire ethanol tolerance after repeated exposures to ethanol. Our results suggest that the human jwa gene may also effectively participate in the development of ethanol tolerance, providing a new research approach model to examine prevention and treatment of alcohol abuse and addiction.
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ACKNOWLEDGEMENTS |
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We thank Dr. Ruiwen Zhang and Dr. Donald L. Hill in Department of Pharmacology and Toxicology and Comprehensive Cancer Centre, University of Alabama at Birmingham for their discussion and insightful suggestions. This work was funded in part by the Foundation of Jiangsu Province Key Lab of Modern Pathogen Biology (8833, 8834); the National Natural Science Foundation of China (30500412 to JZ, 30570910 to JC) and the key basic research project of Education Department, Jiangsu Province (06KJA33023).
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