Introduction

Sphingolipids, important components of eukaryotic cell membranes, play an essential role in maintaining the structural and functional integrity of the biological membrane systems [1]. In addition, sphingolipids serve as a reservoir of many signaling molecules, such as ceramides, sphingosine (SPH), and sphingosine-1-phosphate (S1P), all of which have been implicated in a variety of cellular responses, such as cell proliferation, survival, differentiation, senescence, and apoptosis [2].

Ceramidases are a group of enzymes that catalyze the hydrolysis of ceramides to generate SPH, which is phosphorylated to form S1P. Ceramidases have been cloned from various organisms, including bacteria [3, 4], yeast [5, 6], Drosophila melanogaster [7], zebra fish [8], mice [9], rats [10], and humans [11]. Ceramidases have been shown to play an important role in regulating ceramides, sphingosine, and S1P, and thereby cellular responses mediated by these sphingolipid metabolites.

Five human ceramidases encoded by five distinct genes were cloned: the acid ceramidase (ASAH1), neutral ceramidase (ASAH2), alkaline ceramidase 1 (ACER1), alkaline ceramidase 2 (ACER2), and alkaline ceramidase 3 (ACER3). Each human ceramidase has a mouse counterpart. ACERs have similar protein sequences, but they have no homology to ASAH1 and ASAH2, which also have distinct protein sequences [2]. These human ceramidases have been shown to play an important role in the metabolism of sphingolipids and biological responses. Genetic deficiency in ASAH1 causes Farber disease, characterized by accumulation of ceramides and sphingolipids in lysosomes [12]. The human ASAH2 has been implicated in cell survival against TNF-α [13]. ACER1 is highly expressed in the epidermis and has a role in the differentiation of epidermal keratinocytes likely by regulating the metabolism of ceramides, SPH, and S1P [14]. In vitro studies suggest that ACER2 expression is important for cell proliferation and survival, likely by enhancing S1P signaling and/or reducing that of ceramides, but its overexpression may lead to cell growth arrest and/or cell death because of accumulation of cytotoxic levels of SPH in cells [11]. Our recent studies suggest that ACER3 may act as a house-keeping enzyme that maintains unsaturated long-chain ceramides, such as d-erythro(e)-C18:1- and C20:1-ceramides, at low levels in cells. Because of the existence of multiple isozymes in mammals, studies on the physiological and pathological roles of mammalian ceramidases are challenging.

The metabolic pathway of sphingolipids appears to be conserved between Drosophila and mammals. As in mammals, a tight regulation of the metabolism of sphingolipids in Drosophila is required for a variety of biological processes, such as reproduction and development [1, 15, 16]. However, the number of enzymes for each metabolic step is much smaller than that in mammals. Because of this simplicity, we define the physiological role of alkaline ceramidases using this model organism. By analyzing the sequence of the genome of Drosophila melanogaster, we found that only one putative protein encoded by the Drosophila brainwashing gene (BWA) shares similarity in protein sequence to known alkaline ceramidases. The BWA gene was identified as a gene whose mutation causes an abnormality in the structure of the corpora pedunculata (mushroom bodies) in the brain, although its biochemical properties remain unclear. In this study, we demonstrate that the protein product (Dacer) of the BWA gene has alkaline ceramidase activity and that Dacer plays an important role in the metabolism of ceramides in Drosophila. We further show that compared to the wild-type Drosophila, the Drosophila mutant deficient in Dacer had delayed pre-adult development, increased anti-oxidative stress capacity, and an extended lifespan, suggesting that Dacer plays an important role in various biological processes during the development and aging process of the fruit fly.

Materials and methods

Drosophila melanogaster stocks and maintenance

Wild-type w1118 and bwa e02081 (BL-18012) lines were obtained from the Bloomington Drosophila melanogaster Stock Center (Indiana University, Bloomington, IN). The annotated brainwashing gene (CG13969) localizes to the 38B2-3 region of the left arm of the second chromosome. A P-element insertion in this gene was identified (Fig. 1) and was subsequently mobilized to generate a mutant. Stocks were maintained at 25°C on a 12:12 h light:dark cycle at constant humidity by using standard cornmeal medium consisting of 8.6% sucrose, 1% yeast, 11.3% cornmeal, 1% agar, and 1% propionic acid.

Fig. 1
figure 1

Transposon location of the dacer mutant. The Dacer/BWA gene localizes to the 38B2-3 region of the left arm of the second chromosome. A P-element insertion (PBac(43)-bwa e02081) was isolated and was subsequently mobilized to generate a dacer/bwa mutant that carries a transposon within the 4th extron of the Dacer/BWA gene

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Lipid preparation

d-e-Sphingosine (C18SPH) with 18 carbons and C17SPH were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). C14SPH, C16SPH, and d-e-dihydrosphingosine with 14 (C14DHS) or 16 (C16DHS) carbons were from Matreya, LLC (Pleasant Gap, PA). All ceramides used in this study were synthesized as described [17] in the lipidomics core at the Medical University of South Carolina (MUSC), Charleston, SC.

cDNA cloning

A BLAST search of the Drosophila melanogaster genomic database using human alkaline ceramidases as queries revealed a putative protein encoded by the BWA gene (GenBank accession number: AF323976). To amplify the BWA coding sequence or open reading frame (ORF), total RNA was isolated from whole Drosophila adults using Trizol (Invitrogen) according to the manufacturer’s instructions. RNA was reverse-transcribed to cDNA using the SMART™ PCR cDNA synthesis kit (Clontech, TaKaRa). A small portion of transcribed cDNAs was subjected to PCR amplifications using the primer pair BWAF (5′-AGGATCCATGGGCGGGATGGGGGGCGGCGGGCT-3′) and BWAR (5′-AAAGCTTTTAGATGGTGTTG CGCAGAGCCTTAC-3′) under the condition of one cycle of 94°C for 60 s, 34 cycles of 94°C for 30 s, 63°C for 60 s, and 72°C for 60 s. The PCR products were cloned into the vector pGEM-T easy (Promega, USA). The resulting BWA ORF construct pGEMT-Dacer was sequenced to confirm the correctness of the BWA ORF.

Plasmid construction

pGEMT-Dacer was digested with the restriction enzymes BamHI and HindIII to release the Dacer ORF, which was subcloned in-frame with the 6 × histidine (HIS) tag into the baculovirus transfer vector pFastBacHT. DH10 Escherichia coli Bac cells (Gibco-BRL) were then transformed with the plasmid pFastBacHT-Dacer. Site-specific transposition of the expression cassette from the pFastBacHT-Dacer plasmid to the bacmid DNA of DH10 Bac cells disrupts the lacZ gene, and the recombinant clones are lacZ− and gentamycin resistant. The positive colonies (white) containing the recombinant bacmid (Bac-Dacer) and wild-type (WT) bacmid (Bac-WT) were cultured to isolate the bacmid DNA.

Dacer overexpression in High Five cells

High Five cells (Tn5B-1-4) were cultured in TNM-FH insect medium (Sigma–Aldrich) supplemented with 10% fetal bovine serum (Gibco, BRL). The bacmid DNA of Bac-Dacer and Bac-WT were transfected by Lipofectamine 2000 (Invitrogen) into High Five cells to yield the Autographa californica nuclear polyhedrosis virus (AcNPV) carrying the Dacer ORF and control bacmid. A baculoviral stock with a suitable titer was generated and used to infect High Five cells. Forty-eight hours after infection, the cells were harvested. After being washed three times with PBS, the cells were resuspended in buffer A (20 mM Tris–HCl, pH 7.4), containing 0.25 M sucrose and 20 g/ml protease inhibitor mixture (CLAP; Roche Applied Science), and then were sonicated for 5 s at a power level of 35% on a microtip-equipped Sonic Dismembrator. The total cell lysates were centrifuged at 1,000 ×g for 10 min. The post-nuclear supernatants were centrifuged for 10 min at 10,000×g to obtain the Mr = 10,000 membrane fraction. The resulting supernatant was centrifuged for 1 h at 100,000 ×g to obtain microsomes (the Mr = 100,000 membrane fraction), which were washed three times with buffer A. Microsomes (40 μg protein per lane) prepared from the High Five cells containing the bacmid of Bac-Dacer as well as Bac-WT were resolved by SDS-PAGE and then transferred to a nitrocellulose membrane. The HIS-tagged Dacer was detected by an anti-HIS antibody using an ECL Plus detection kit (Amersham Pharmacia Biotech) according to the manufacturer’s instructions.

Protein concentration determination

Protein concentrations were determined with BSA as a standard using a bicinchoninic acid (BCA) protein determination kit (Beyotime) according to the manufacturer’s instructions.

Ceramidase activity assay

Ceramidase activity was determined by the release of sphingosine from ceramides according to Xu et al. [11]. Briefly, microsomes (20 μg protein) were incubated with ceramide substrate (75 μM) in 40 μl assay buffer (25 mM Tris-HCl, pH 6–9, containing 5 mM CaCl2, 0.15% Triton X-100) at 37°C for 30 min. The reactions were stopped by extraction with chloroform and methanol. Sphingoid bases in the lipid extracts were determined by HPLC with C17SPH as an internal standard as described [17].

ESI/MS/MS lipid analysis

Electrospray ionization (ESI)/MS/MS analysis of sphingolipids was performed on a Thermo Finnigan TSQ 7000 triple quadrupole mass spectrometer, operating in a multiple reaction monitoring (MRM) positive ionization mode, according to published methods [18]. Each of the samples of 48-h-old Drosophila pupae and 3-day-old adult flies was suspended in 4 ml of the ethyl acetate/isopropanol/water (60/30/10%; v/v) solvent system in a 15-ml tube, to which was added 50 µl of a mixture (1 µM) of internal sphingolipid standards (ISs) including C17SPH, d-e-C16-ceramide (d17:1/16:0), and d-e-C18-ceramide (d17:1/18:0). Drosophila pupae or adults were then homogenized for 5 min using a homogenizer set at the maximal power mode, followed by 1 h sonication in a water bath sonicator. After centrifugation, 1 ml of the lipid extract was used for phospholipid (Pi) determination as described [19], and the remaining was used for ESI/MS/MS. The lipid extracts were dried under a stream of nitrogen gas. For ESI/MS/MS, dried lipid extracts for each sample were dissolved in 100 μl of acidified (0.2% formic acid) methanol, and were injected on the HP1100/TSQ 7000 LC/MS system and gradient-eluted from the BDS Hypersil C8, 150 × 3.2 mm, 3-μm particle size column, with a 1.0 mM methanolic ammonium formate/2 mM aqueous ammonium formate mobile phase system. Peaks corresponding to the target analytes and IS were collected and processed using Xcalibur software. Quantitative analyses of endogenous sphingolipids (SPLs) were based on calibration curves generated by spiking an artificial matrix with known amounts of the target analyte synthetic standards and an equal amount of the IS. The target analyte/IS peak area ratios were compared to the calibration curves using a linear regression model. Levels of particular SPLs were normalized to Pi and expressed as SPLs/Pi (pmol/μmol).

Developmental time analysis

Fly eggs were laid on 90-mm petri dishes containing 20 ml of standard cornmeal medium. The dishes were monitored at 12-h intervals, and the numbers of embryos, larvae, and pupae were recorded; emerging adults were collected, sexed, and counted. The data recoding continued until no flies emerged for 3 consecutive days. The developmental time of egg, larval, and pupal stages of immature flies were calculated.

Juvenile hormone acid methyltransferase (JHAMT) activity assay

Juvenile hormone III (JH III) was purchased from Sigma, and [3H]-SAM was from PerkinElmer. JH III acid (JHA) was produced from their corresponding methylesters by saponification [20]. Purified JHA and [3H]-SAM were dissolved in sodium phosphate solution (0.1 M Na2HPO4-NaH2PO4, pH 7.2). Corpus allatums (CA) were isolated from ten wandering larvae, which were homogenized in the sodium phosphate buffer. After centrifugation, the supernatant (50 μl) was transferred to an equal volume of reaction buffer containing JHA and [3H]-SAM. For negative controls, only the phosphate solution buffer was added to the same reaction mixtures. After incubation at 28°C for 90 min, the enzymatic reaction was stopped by the addition of an equal volume of methanol. The radiolabeled product was extracted with 1 ml of hexane and counted with a liquid scintillation counter [21].

Lifespan analysis

Eclosing (insects emerging from pupal case) adults (0–12 h) were collected. Approximately 200 male and female flies were maintained separately in individual vials (with 20 flies per vial) and transferred to fresh vials every 3 days, and survivors were counted every 24 h. For statistical analysis, the mean and maximum lifespan of each strain was calculated from the time (in days) at which survival reached 50 and 10% of the starting population.

Oxidative stress assay

Three-day-old flies (100 male and 100 female of each strain) were transferred to vials containing standard cornmeal medium supplemented with 20 mM paraquat (with 20 flies per vial) [22]. To measure the lifespan on oxidative stress, the numbers of dead flies were counted every 6 h.

Mitochondrial hydrogen peroxide measurement

Hydrogen peroxide production was measured in isolated fly mitochondria under normal respiration conditions as described [23]. Adult flies at different ages living on normal food or paraquat-containing food were collected on ice before mitochondria were isolated using Tissue Mitochondria Isolation Kit (Beyotime) according to the manufacturer’s instructions. Protein concentration was determined using a BCA Protein Determination Kit (Beyotime) with bovine serum albumin as standard. Mitochondria (about 0.15 μg protein/μl) were incubated at 25°C in 50 μl assay medium containing 20 mM sn-glycerol 3-phosphate (Sigma) as substrate. Mitochondrial hydrogen peroxide production was determined using the Hydrogen Peroxide Assay Kit (Beyotime) according to the manufacturer’s instructions. The absorbance at 560 nm was recorded on a μQuantMicroplate spectrophotometer (Biotek Instruments). All tests were done in triplicate, and more than 100 flies were used in each assay.

ATP measurement

ATP levels in the flies were quantified by a luciferin-luciferase system using the ATP Assay Kit (Beyotime) according to the manufacturer’s instructions. Newly eclosed flies were sexed and reared at a density of 20 flies per vial. The flies were aged to different days at 25°C on the normal diet. The flies were washed thoroughly in PBS and homogenized in the same buffer. The levels of ATP were determined by luminometry using the SpectraMax M5 luminometer.

Statistic analyses

Statistical significance of the data from the qRT-PCR were analyzed using Tukey’s student range test. Differences in development time, JHAMT activity, and ATP levels between w1118 and dacer mutant flies were tested using Student’s t tests. Data were handled using SAS, and the significance level was P < 0.05 in all tests. Asterisk (*) indicates the significant difference between two means.

Results

BWA has alkaline ceramidase activity

A BLAST search of the Drosophila melanogaster genomic database using human alkaline ceramidases as queries revealed a putative protein encoded by the BWA gene (GenBank accession number: AF323976). This putative protein exhibited a 35, 46, and 26% identity in protein sequence to the human alkaline ceramidases ACER1, ACER2, and ACER3, respectively. The BWA gene product was also found to contain multiple domains conserved among these alkaline ceramidases (Fig. 2a). This putative ORF was amplified by RT-PCR from adult Drosophila RNA as described in “Materials and methods.” DNA sequencing indicated that the cloned ORF has the predicted sequence. The pSORTII program predicted that the BWA gene consists of five putative transmembrane domains (TM1-5) (Fig. 2b). According to its homology to the human alkaline ceramidases, we postulated that the BWA gene product may be a Drosophila alkaline ceramidase (Dacer).

Fig. 2
figure 2

Coding and protein sequences of Dacer. a The Dacer coding sequence (GenBank accession number: AF323976) was cloned from adult Drosophila flies. The amino acid sequence was derived from the coding sequence using the DNAman software. Putative transmembrane domains (TMs1–5) of Dacer were predicted by the pSORT II program. b Protein sequence alignment of Dacer and ACER2 was performed using the DNAman software. Identical amino acid residues among the aligned proteins are shaded in dark grey and similar amino acid residues light grey

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To determine whether Dacer indeed encodes alkaline ceramidase activity, the Dacer coding sequence was cloned into a Baculoviral vector (pFastBacHT B), and the recombinant bacmid DNA (Bac-Dacer) or the WT bacmid DNA (Bac-WT) was transfected into High Five cells. The microsomes were isolated from High Five cells transfected with Bac-Dacer or Bac-WT. Western blot analysis with the anti-6xHIS tag antibody detected a protein band (apparent molecular mass ~34 kDa) in the microsomes isolated from the cells transfected with Bac-Dacer but not in the microsomes isolated from cells transfected with Bac-WT. In vitro activity assays demonstrated that, compared to the Bac-WT microsomes, the Bac-Dacer microsomes had a ~twofold increase in ceramidase activity on d-e-C6, d-e-C12, d-e-C16, d-e-C18, and d-e-C24:1-ceramide at pH 9.0 (Fig. 3b), suggesting that Dacer indeed possesses ceramidase activity.

Fig. 3
figure 3

Dacer encodes alkaline ceramidase activity. a Microsomes were prepared from Bac-Dacer and Bac-WT cells. A portion of the microsomes was subjected to Western blot analysis with the anti-HIS antibody (1:1,000). The relative molecular mass (Mr) (~34 kDa) was estimated according to standard proteins. b Another portion of the above microsomes was assayed for ceramidase activity at pH 9.0 using indicated ceramides as substrates. c Microsomal ceramidase activity was determined using d-e-C16-ceramide as substrate at different pH values. Ceramidase activity of the recombinant Dacer at each pH was computed by subtracting ceramidase activity in Bac-WT microsomes from that in Bac-Dacer microsomes. The Dacer ceramidase activity at pH 8 is highest and set as 100%, and ceramidase activity at other pH values is expressed as % of the maximal activity. All data represent the mean value of three independent experiments performed in duplicate

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To determine whether, similar to its mammalian homologues, Dacer has an alkaline pH optimum for its in vitro activity, we measured ceramidase activity of the recombinant Dacer at different pH values. The activity of the recombinant Dacer was determined by subtracting endogenous ceramidase activity of High Five cells in Bac-WT microsomes from ceramidase activity in Bac-Dacer microsomes. We found that recombinant Dacer has the highest activity at pH 8.0 (Fig. 3c), suggesting that Dacer has an alkaline pH optimum for its in vitro activity.

Dacer/BWA is highly expressed in the midgut and at the pupal stage of the life cycle

To better understand the physiological roles of Dacer, qRT-PCR was performed to quantify the temporal and spatial expression of Dacer based on the normalized relative quantification 2−ΔΔCt method using Drosophila tubulin cDNA as a control. Dacer mRNA levels were much higher in the midgut than in other organs of Drosophila (Fig. 4a). We also found that Dacer mRNA levels are upregulated at the pupal stage compared to the larval or adult stage of the life cycle. These results suggest that Dacer mRNA is expressed highly in the midgut, moderately in the brain, and only slightly in other organs, and that Dacer expression is upregulated at the pupal stage during the development of Drosophila (Fig. 4b).

Fig. 4
figure 4

Dacer is highly expressed in the ovary and at the pupal stage. qRT-PCR analysis was performed with cDNAs reversely transcribed from RNA isolated from different Drosophila organs (a) or from flies at different developmental stages (b). Data represent the mean value ± SE of three independent experiments performed in duplicate

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Dacer inactivation increases Drosophila ceramide levels

Dacer/bwa mutant flies have been reported to have a fusion of the beta lobes in the central brain [24]. The mutant flies from the Bloomington Drosophila Stock Center were identified to harbor a transposon within the 4th extron of the Dacer/BWA gene (designated bwae02081) (Fig. 5a). Because Dacer/BWA is a Drosophila alkaline ceramidase that catalyzes the hydrolysis of ceramides, we examined ceramide levels in 48-h-old pupae and 3-day-old adults of wild-type w1118 flies and dacer mutant flies. ESI/MS/MS demonstrated that compared to w1118 controls, both dacer mutant pupae (Fig. 5b, c) and adults (Fig. 5d, e) had much higher levels of most ceramide species containing either C14SPH or C16SPH, suggesting that Dacer plays a role in controlling the levels of ceramides in Drosophila at both pupal and adult stages.

Fig. 5
figure 5

Dacer inactivation increases Drosophila ceramides. a Total RNA isolated from dacer mutant and control flies was reversely transcribed into cDNAs. The cDNAs were subjected to RT-PCR analysis for Dacer mRNA using the primer pair BWA-5′ and BWA-3′ corresponding to the 5′ and 3′ ends of the coding sequence, respectively. Note that the full-length coding sequence (840 bp) was amplified from controls' RNA, but not from mutants' RNA. b, c. Pupae of mutant and control flies were harvested at 48 h and subjected to ESI/MS/MS analysis for C14SPH- and C16SPH-ceramides. d, e. Adults of mutant and control flies were harvested at day 3 and subjected to ESI/MS/MS analysis for C14SPH- and C16SPH-ceramides. The contents of sphingolipids were normalized to total phospholipids (Pi) in flies. Data represent mean value ± SE of three independent experiments performed in duplicate

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Dacer inactivation delays Drosophila pre-adult development

We showed that Dacer mRNA levels are markedly increased at the pupal stage, indicating that Dacer may have a role in the pre-adult development of the Drosophila. To investigate this possibility, the pre-adult development time of w1118 or dacer mutant flies was determined. We found that like w1118 control embryos, the embryos of the mutants developed normally and eventually emerged as adults without obvious abnormalities. However, the development time from egg to adult eclosion increased significantly (P < 0.05) in dacer mutants (10.49 ± 0.06 days; n = 138), compared to w1118 flies (8.85 ± 0.06 days; n = 136) (Table 1). Data analysis further showed that the larval development time was 6.92 ± 0.08 (n = 242) days for dacer mutants and 4.69 ± 0.04 (n = 179) days for w1118 controls, suggesting that differences in the total immature development rate result in the longer larval development time of dacer mutants compared to w1118 controls. In addition, the immature development time was 8.85 ± 0.08 days for both w1118 male (n = 90) and female flies (n = 48), but small differences were observed between immature mutant male (10.69 ± 0.08 days; n = 68) and female flies (10.29 ± 0.09 days; n = 68). These results demonstrate that the Dacer inactivation significantly delays the pre-adult development, especially the larval development in Drosophila.

Table 1 Dacer inactivation delays pre-adult development in Drosophila
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Dacer inactivation increases JHAMT activity

Juvenile hormones (JHs) are a family of sesquiterpenoid hormones that play a central role in the control of insect’s development and growth [25]. Juvenile hormone acid methyltransferase (JHAMT) of the silkworm Bombyx mori methylates the carboxyl group of JH I, II, and III acids (JHAs) to generate hormonally active JHs, and it has been shown to be critical in JH biosynthesis [20, 26]. JHAMT activity positively correlates with levels of JHs in insects at the larval stage [27]. Based on these observations, we determined whether Dacer inactivation affected JHAMT activity in Drosophila larvae. As shown in Fig. 6, JHAMT activity was significantly increased in dacer mutant flies (0.66 ± 0.04 fmol/CA/h) compared with w1118 control flies (0.49 ± 0.05 fmol/CA/h), suggesting that Dacer inactivation may increase the levels of JHs in the Drosophila at the larval stage.

Fig. 6
figure 6

The dacer mutant flies have enhanced JHAMT activity. Purified JH III acid and [3H]-SAM were dissolved in sodium phosphate solution. CAs were isolated from wandering larvae and homogenized in the sodium phosphate buffer. After centrifugation, the supernatant was transferred to an equal volume of reaction buffer containing JHA and [3H]-SAM. The enzymatic reaction was incubated at 28°C for 90 min and then was stopped by the addition of an equal volume of methanol. The radiolabeled product was extracted with 1 ml of hexane and counted with a liquid scintillation counter. Data represent the mean value ± SE of JHAMT activity (fmol/CA/h). Three independent experiments were performed in duplicate

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Dacer inactivation increases Drosophila lifespan

To get more insight in the physiological role of Dacer in mature flies, we analyzed the effect of Dacer inactivation on the lifespan in Drosophila. The rationale for this study is that ceramides have been shown to be important for Drosophila longevity [28]. We found that the average lifespan (50% survival) and maximum lifespan (10% survival) were 38.07 ± 0.85 (n = 213) and 53.19 ± 0.27 (n = 21) days, respectively, for the dacer mutant female flies, and 24.65 ± 0.73 (n = 232) and 44.00 ± 0.58 (n = 23) days, respectively, for the w1118 female flies (Fig. 7a). The average lifespan and maximum lifespan were 41.27 ± 1.04 (n = 182) and 55.44 ± 0.23 (n = 18) days, respectively, for the dacer mutant male flies, and 27.82 ± 0.73 (n = 173) and 41.82 ± 0.85 (n = 17) days, respectively, for the w1118 male flies (Fig. 7b). These results clearly indicate that Dacer inactivation significantly increases the Drosophila lifespan (Student's t test, P < 0.0001 for all comparisons).

Fig. 7
figure 7

The dacer mutant flies have extended lifespans; 213 mutant and 232 control female as well as 182 mutant and 173 control male eclosion flies were collected. Male and female flies were maintained separately in individual vials on normal food and transferred to fresh vials every 3 days. Survivors were scored every 24 h. Longevity curves of w1118 control flies (open circles) and of dacer mutant flies (filled circles) are shown. For statistical analysis, the mean and maximum lifespan of each strain was calculated from the time (in days) at which survival reached 50 and 10% of the starting population

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The dacer mutant flies are resistant to oxidative stress damage

Enhanced resistance to various stresses is often associated with interventions that extend lifespan. We then examined the ability of w1118 and mutant flies to resist oxidative stress. The rationale behind this study is that ceramides have been shown to be important in anti-oxidative stress in Drosophila [28]. Because paraquat, upon uptake by Drosophila, was shown to kill insects by generating reactive oxygen species (ROS) [29], the period that an insect survives paraquat should correlate to the insect’s capacity for anti-oxidative stress. Based on this assumption, we determined whether there is a difference in the survival time between w1118 and dacer mutants. We demonstrated that in the presence of 20 mM paraquat in the food, the average lifespan and maximum lifespan were 154.4 ± 6.7 (n = 100) and 267.0 ± 7.8 (n = 10) h, respectively, for the mutant female flies, 78.2 ± 3.9 (n = 100) and 156.0 ± 11.0 (n = 10) h, respectively, for the w1118 female flies (Fig. 8a). The average and maximum lifespan were 128.0 ± 6.0 (n = 100) and 235.8 ± 4.7 (n = 10) h, respectively, for the mutant male flies and 60.5 ± 2.1 (n = 10) and 97.2 ± 1.2 (n = 10) h, respectively, for w1118 male flies (Fig. 8b). These results indicated that Dacer inactivation imparts resistance to oxidative stress for both male and female flies.

Fig. 8
figure 8

The dacer mutant flies are resistant to oxidative stress. Three-day-old flies were transferred to vials containing standard cornmeal medium added with 20 mM paraquat (20 flies per vial). To measure the survival rate under oxidative stress, the lifespan of control and mutant flies on a paraquat diet was analyzed. Longevity curves of control w1118 flies (open circles) and of dacer mutant flies (filled circles) are shown. For statistical analysis, the mean and maximum lifespan of each strain was calculated from the time (in hours) at which survival reached 50 and 10% of the starting population

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Dacer inactivation reduces stress-induced ROS production in the mitochondria

Increasing studies demonstrated that ROS production is one of the key determinants of Drosophila’s lifespan [30]. Therefore, we measured the rate of production of hydrogen peroxide (H2O2) in live mitochondria from dacer and control adult flies living on normal diets at different ages. As shown in Fig. 9a, at day 1, the amount of H2O2 produced by dacer and control mitochondria was similar. However, by day 5, the amount of H2O2 was slightly lower in the dacer mitochondria as compared with control mitochondria. This trend continued until at least day 20. This suggests that during the aging process, the ROS production is slightly reduced in long-lived dacer mutant flies as compared to that in w1118 controls.

Fig. 9
figure 9

The dacer mutant flies have decreased ROS production in the mitochondria. a Mutant and control eclosion flies were collected and maintained on normal food. Flies at different ages (1-, 5-, 10-, 15-, and 20-day-old) were kept on ice before isolation of mitochondria. Mitochondria isolated by using Tissue Mitochondria Isolation Kit were incubated at 25°C with 20 mM sn-glycerol 3-phosphate as substrate. Mitochondrial hydrogen peroxide production was measured at 560 nm using the Hydrogen Peroxide Assay Kit. The 100 value refers to control H2O2 production of mitochondria that was obtained from 1-day-old w1118 flies, and other values represent % of the control H2O2 production. b Three-day-old flies were collected and maintained on 20 mM paraquat-containing food. Mitochondria were isolated from dacer and control flies at 36 h after paraquat treatment. Mitochondrial hydrogen peroxide production was measured at 560 nm. The 100 value refers to control H2O2 production of mitochondria that was obtained from w1118 flies, and other values represent % of the control H2O2 production. All data represent the mean value of three independent experiments performed in duplicate

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We showed that dacer mutants survived paraquat better than wild-type flies. To determine whether dacer mutants produce less ROS in response to paraquat than wild-type controls do, we compared the rates of H2O2 production in live mitochondria from dacer and control flies at 36 h after paraquat treatment. As shown in Fig. 9b, the production of mitochondrial H2O2 was significantly reduced in dacer mutants compared to w1118 controls, suggesting that Dacer inactivation reduces the stress-induced production of ROS in mitochondria.

Dacer inactivation alters mitochondria metabolic function involved in motor activity

The mutant had greater vial wall-climbing ability than the wild type (data not shown). A recent study indicated that accumulating oxidative stress leads to compromised metabolic functions that induce the progressive decline on motor activity and early death [28]. Because dacer mutant flies have lower ROS production and an increased resistance to oxidative stress, we predicted that Dacer inactivation may alter mitochondrial metabolic functions that are involved in motor activity. Mitochondria are important organelles for cellular respiration and energy supply through ATP generation [31]. ATP levels have been routinely used as an indicator of metabolic function in Drosophila [32, 33]. Thus, ATP levels were measured in w1118 or dacer mutant adult flies. As shown in Fig 10, ATP levels were higher in both young and old dacer mutant flies compared to age-matched w1118 flies, suggesting that Dacer inactivation enhances Drosophila metabolic function.

Fig. 10
figure 10

The dacer mutant flies have increased ATP levels. ATP levels in the flies were determined by luciferin-luciferase system. Young (day 1) and old flies (day 20) on the normal diet were washed thoroughly in PBS and homogenized. The levels of ATP in homogenates were estimated by luminometer. The ATP levels in control (open bars) and mutant flies (filled bars) are shown. The error bars of the ATP levels in young dacer mutant and old w1118 flies are too small to see

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Discussion

The BWA gene was identified as a gene whose mutations cause an abnormality in the structure of the mushroom body in Drosophila. However, the biochemical properties and physiological function of the product of the BWA gene have been unclear. Protein sequence alignment revealed that the protein product of the BWA gene shares sequence similarity with the alkaline ceramidases that we previously cloned from yeast (YPC1p and YDC1p), mice (Acer1, Acer2, and Acer3), and humans (ACER1, ACER2, and ACER3). BWA is closest in protein sequence to ACER2 and Acer2. Similar to ACER2, we demonstrated that BWA has the highest in vitro ceramidase activity at pH values around 8. Like ACER2 and other alkaline ceramidases, BWA has multiple putative transmembrane domains, suggesting that it is a membrane-bound protein. Based on its similarity in protein sequence and enzymatic activity to known alkaline ceramidases, we concluded that BWA is a Drosophila alkaline ceramidase, which we renamed Dacer. Unlike mammals that express three alkaline ceramidase genes, Drosophila melanogaster has no other homologues of Dacer. This simplicity makes Drosophila an ideal model for studying the physiological and pathological roles of alkaline ceramidases.

Our previous studies demonstrated that mammalian alkaline ceramidases play an important role in regulating the metabolism of sphingolipids [11]. In this study, we showed that Dacer inactivation leads to an increase in ceramides in both pupae and adult flies, suggesting that, similar to mammalian alkaline ceramidase, the Drosophila alkaline ceramidase also plays an important role in controlling the metabolism of Drosophila ceramides. Because ceramides can be converted to glycosphingolipids, ceramide-phosphoethanolamines (sphingomyelin analogues), or ceramide-1-phosphate, Dacer inactivation may also result in increases in the levels of these ceramide-containing sphingolipids. This possibility is currently under investigation. Interestingly, Dacer inactivation did not cause a decrease in the levels of its products, free sphingoid bases, in either pupae or adult flies (data not shown). This indicates that Dacer inactivation may increase the generation of free sphingoid bases by a different ceramidase, such as the neutral ceramidase, or decreases the conversion of free sphingoid bases to their phosphates.

By qPCR analysis, we demonstrated that Dacer mRNA is highly expressed at much higher levels in pupae than in adult flies. One would expect that the levels of ceramides should be lower in pupae than in adult flies, but the opposite is true. This suggests that pupae may have a much higher capacity in the de novo synthesis of ceramides than adult flies do. The high expression of Dacer may prevent ceramides from being accumulated in the cellular compartment(s) where Dacer resides. This role of Dacer may be physiologically relevant because too much ceramide may cause an organelle stress.

It was shown that the structure of the ellipsoid body of Drosophila melanogaster was altered in dacer mutants [24]. Our preliminary studies with electron microscopy did not show obvious abnormalities in the structure of the brain in the dacer mutant (data not shown), although with qPCR we demonstrated that Dacer mRNA is expressed in the Drosophila brain. The effect of the Dacer mutation on the ellipsoid body and other functional zones warrants more thorough investigation.

In this study, we showed that the development time of the immature stages from egg to adult eclosion increased significantly in dacer mutants compared to w1118 controls. In Drosophila, the timing of adult emergence depends upon ecological factors, including temperature, photoperiod, humidity, food, intrinsic factors, and hormones [34]. JHs are synthesized de novo in specialized endocrine glands, corpus allatums (CA). A strict regulation of the titer of JHs throughout the insect’s life is critical to its successful development. Previous studies showed that the titer of ecdysone and JHs in the developing insect control its developmental stage. At the development stage, as long as there are enough JHs, the ecdysone promotes larva-to-larva molts [35]. With lower amounts of JHs, the ecdysone promotes pupation, and complete absence of JH results in the formation of the adult [36]. JHAMT activity has been shown to positively correlate with levels of JHs in larval insects [27]. We found that JHAMT activity was significantly increased in dacer mutant larvae compared to the wild-type larvae, suggesting that Dacer inactivation may significantly increase JHs in larvae. These observations suggest that Dacer inactivation delays pre-adult development probably because of a delay in lowering JHs in developing insects. How Dacer inactivation increases JHAMT activity remains unknown.

In this study, we found that the lifespan of the dacer mutant flies was greatly extended compared to wild-type flies. Interestingly, the yeast mutants deficient in alkaline ceramidase YDC1, the yeast homologue of Dacer, also have an increased chronological lifespan compared to wild-type yeast strains [37], whereas YDC1 overexpression leads to reduced chronological lifespan [38]. These results suggest that the role of alkaline ceramidase in controlling lifespan is conserved from low eukaryotes to high eukaryotes. We demonstrated that the inactivation of Dacer caused a significant increase in the levels of ceramides. Because the inactivation of Dacer leads to increased ceramides in Drosophila without affecting the levels of free sphingoid bases, the increased lifespan of the dacer mutant may be associated with altered levels of ceramides and/or ceramide-containing sphingolipids (gluosylceramides, ceramide-phosphoethanolamines, and ceramide-1-phosphate). In line with this concept, Rao et al. [28] reported that an inactivation of the ceramide transfer protein gene (Dcert) reduces levels of ceramides and ceramide-phosphoethanolamines and significantly decreases the Drosophila lifespan.

The mechanism by which ceramides and/or ceramide-containing sphingolipids affect Drosophila lifespan is complex. Our data suggest that increasing the levels of ceramides and/or ceramide-containing sphingolipids, via inactivation of the alkaline ceramidase, extends Drosophila’s lifespan, probably via reducing the oxidative damage to mitochondria. We found that, during the natural aging process, the mitochondrial ROS production was lower in long-lived dacer mutant flies than in age-matched control flies. The difference in ROS production was more significant when the flies were challenged with paraquat-induced oxidative stress. These results suggest that an increase of ceramides and ceramide-containing sphingolipids reduces the production of mitochondrial ROS, especially in response to stress. Mitochondria have been suggested to be the main targets of ROS-induced oxidative damage [31]; thus, there is an inverse relationship between the production of mitochondrial ROS and mitochondrial function [39]. In line with this notion, we demonstrated that the ATP levels decline much more slowly during the aging process in dacer mutant flies than in wild-type flies. This suggests that increasing ceramides and ceramide-related sphingolipids attenuates the age-related decline in mitochondrial function by reducing ROS production. The production of ATP in mitochondria declines with age during the human aging process [40]. This also holds true for Drosophila [41]. Therefore, a decline in mitochondrial function due to an increase in ROS production may limit an organism’s lifespan.

In addition to a reduction in mitochondrial damage by ROS, Rao et al. [28] showed that reduced ceramides in dcert1 mutants increase plasma membrane fluidity, resulting in enhanced oxidative damage to cellular proteins. Because Dacer inactivation may increase the content of ceramides and ceramide-containing sphingolipids in the plasma membrane, it is plausible that Dacer inactivation increases the stiffness of the plasma membrane, thus resulting in increased resistance to ROS. Therefore, increasing the levels of ceramide and ceramide-related sphingolipids due to Dacer inactivation increases Drosophila’s lifespan also by reducing oxidative damage to other organelles.

It is generally believed that ceramides are stressful molecules that induce cell apoptosis or cell death. However, our data support the notion that ceramides, especially those targeted to degradation by Dacer, have a protective role in stress response in Drosophila. Indeed, emerging studies suggest that ceramides could be important anti-stress molecules, especially in invertebrates. Menuz et al. [42] demonstrated that loss of one of the ceramide synthase genes, hyl2, makes Caenorhabditis elegans more susceptible to anoxia and heat shock, suggesting that ceramide species synthesized by hyl2 have a protective role for animals under the stresses of oxygen deprivation and heat. Even in mammals, some ceramide species may have an anti-stress role. A very recent study by Senkal et al. [43] showed that tumor cells with decreased levels of ceramide carrying 16-carbon acyl chain (d-erythro-C16-ceramide) die because of ER stress, suggesting that this ceramide species has a protective role in ER stress.

In conclusion, we demonstrate that the product of the BWA is a Drosophila alkaline ceramidase that plays an important role in the metabolism of ceramide, Drosophila pre-adult development, the aging process, and oxidative stress. This study may suggest new directions for experiments designed to unravel the physiological role of mammalian alkaline ceramidases.