Fungal-derived semiochemical 1-octen-3-ol disrupts dopamine packaging and causes neurodegeneration

Exposure to 1-Octen-3-ol Shortens Survival Span and Affects Locomotion of Wild-Type D. melanogaster.

To evaluate the toxic effects of a low dose (0.5 ppm) of 1-octen-3-ol, we exposed wild-type w¹¹¹⁸ flies to a pure form of volatile-phase 1-octen-3-ol. The time to 50% mortality of adult flies exposed to 0.5 ppm of 1-octen-3-ol was ∼16.9 d (Fig. 1A). Furthermore, exposure of wild-type flies to 0.5 ppm of 1-octen-3-ol caused locomotor defects as assessed by a negative geotaxis assay as early as 24 h, with a further decline in locomotor activity upon continuous exposure to 1-octen-3-ol until day 7 (Fig. 1B).

Exposure to 1-Octen-3-ol Induces Loss of Dopaminergic Neurons and Decreases Dopamine Levels in Head Extracts.

To test whether 0.5 ppm of 1-octen-3-ol causes loss of dopaminergic neurons, we used transgenic TH-GAL4; UAS-GFP adults (18). The morphology and number of posteriorly located dopaminergic neurons in these transgenic fly brains were assessed after a 24-h exposure to 0.5 ppm of 1-octen-3-ol. Exposure to 1-octen-3-ol significantly decreased the number of all of the subgroups of dopaminergic neurons, except for PPL1 (Fig. 2 A and B). We then performed HPLC to quantify the effect of 0.5 ppm of 1-octen-3-ol on levels of dopamine and its metabolites. A significant decrease in dopamine levels (28%) was observed concomitantly with an increase in DOPAC levels (40%) compared with control flies. These data suggested that a potential impairment of dopamine handling is associated with exposure to 1-octen-3-ol (Fig. 2C).

1-Octen-3-ol Exposure Inhibits Dopamine Uptake in HEK-Dopamine Transporter/VMAT Ortholog Cells via Its Action on the Dopamine Transporter.

To determine whether the increased DOPAC observed following exposure to 1-octen-3-ol was the result of impaired dopamine transport, we measured the effect of 1-octen-3-ol on [³H] dopamine (DA) uptake in human embryonic kidney (HEK) cells stably expressing human dopamine transporter (DAT) and human VMAT ortholog VMAT2. We exposed 1, 3, and 10 ppm of 1-octen-3-ol for 2 h to these cell lines and found no significant effect on DAT/VMAT activity at 1 ppm, but 3 and 10 ppm reduced the uptake to a significant extent. Exposure to 10 ppm of 1-octen-3-ol for 2 h reduced uptake by 95% in HEK-DAT cells. The experiment was repeated in HEK-DAT/VMAT2 cells that express human DAT and VMAT2 with 10 ppm, and similar results were observed (Fig. 3 A and B). Protein assays confirmed that the loss of uptake was not due to a loss of cells after exposure to 1-octen-3-ol. The near-complete inhibition of DAT prevents the assay from detecting any effect of 1-octen-3-ol on VMAT2. Additionally, methods to measure VMAT2 uptake were not compatible with the airborne exposure setup. Therefore, we took a more direct approach to assessing the putative role of VMAT in the effects of 1-octen-3-ol.

VMAT Mutants Are Sensitive Whereas Transgenic Strains for VMAT Are Resistant to 0.5 ppm of 1-Octen-3-ol.

The Drosophila VMAT gene contains two splice variants, dVMAT-A and -B, where dVMAT-A is expressed in all dopaminergic, serotonergic, and octopaminergic neurons and functions similarly to mammalian VMAT2 (19, 20). To assess the potential for 1-octen-3-ol to exert dopaminergic toxicity through disruption of dopamine handling in vivo, we first exposed two loss-of-function mutant lines for dVMAT-A, l(2)SH0459/+ and Delta14/+, to 0.5 ppm of 1-octen-3-ol (21). The survival span was significantly reduced to 8 and 10 d in l(2)SH0459/+ and Delta14/+ dVMAT mutants, respectively, compared with 17 d for w¹¹¹⁸ wild-type flies (Fig. 4A). The unexposed wild-type flies (^+/+) and dVMAT mutant strains l(2)SH0459/+ and Delta14/+ used as controls for these experiments failed to show any death during this period. We then overexpressed UAS-dVMAT in dopaminergic neurons using the dopamine driver TH-GAL4. Upon exposure to 0.5 ppm of 1-octen-3-ol, average survival of overexpressed transgenic flies TH-GAL4-4; UAS-dVMAT was about 3.5 d longer than that of control flies TH-GAL4 and UAS-dVMAT. Similarly, the overexpression of UAS-dVMAT using the pan-neuronal driver elav-GAL4 led to an extension of survival duration by 4 d compared with control flies (Fig. 4B).

HPLC analysis of dopamine and DOPAC levels in the head extracts of flies overexpressing dVMAT in dopaminergic neurons (TH-GAL4; UAS-dVMAT) exposed to 0.5 ppm of 1-octen-3-ol failed to show the alteration of DA and DOPAC levels (Fig. 4C) that was observed in wild-type flies (Fig. 2C). We further determined the protective effect of dVMAT against 1-octen-3-ol by evaluating the status of dopaminergic neurons overexpressing dVMAT. The overexpression of dVMAT prevented the 1-octen-3-ol–mediated loss of dopaminergic neurons (Fig. 4 D and E) that was seen in wild-type flies (Fig. 2 A and B).

Heterozygous tyrosine hydroxylase Mutant Flies Are Sensitive to 1-Octen-3-ol and Demonstrate Defective Locomotion Compared with Wild-Type Flies.

Considering that 1-octen-3-ol exposure led to alteration of dopamine homeostasis and dopamine neuron loss, we then investigated whether 1-octen-3-ol mediates the variation in the activity of the gene responsible for dopamine biosynthesis, tyrosine hydroxylase (TH), encoded by pale (ple) locus in Drosophila. We exposed two heterozygous loss-of-function mutant strains of the ple gene, ple² and ple⁴, known to have mutations in the tyrosine hydroxylase gene and to possess low dopamine pools (22) to 0.5 ppm of 1-octen-3-ol. Heterozygous mutant strains ple² and ple⁴ exhibited reduced tolerance to 0.5 ppm of 1-octen-3-ol with a 50% mortality obtained at 13.8 and 12.4 d, respectively, compared with wild-type flies exposed to the same concentration of 1-octen-3-ol. Unexposed control flies did not show any death during the observed time period (Fig. 5A).

We also measured movement deficits in wild-type w¹¹¹⁸, ple², and ple⁴ mutant flies after 1 and 7 d of exposure to 0.5 ppm of 1-octen-3-ol. After 1 d of exposure to 0.5 ppm of 1-octen-3-ol, there was no significant difference in the average time for exposed wild-type and ple mutants to cross a 5-cm distance. However, after 7 d of continuous exposure to 1-octen-3-ol, the average time required for ple mutants was about 1.5-fold higher than for wild-type flies (Fig. 5B). In the absence of 1-octen-3-ol, both ple mutants demonstrated climbing ability similar to wild type at both time points.

Drosophila Strains.

All stocks were cultured on Ward’s instant Drosophila medium (blue), and all experiments were performed at 25 °C. Wild-type y¹, w¹¹¹⁸, a yellow-body and white-eyed strain that carries otherwise wild-type genes, was used for all of the experiments unless otherwise stated. The following mutant fly strains were used: for tyrosine hydroxylase, pale²/+ and pale⁴/+ (22); for dVMAT mutants, l(2)SH0459/+ and Delta14/+ (21). The transgenic reporter strains used were TH-GAL4; UAS-GFP, elav-GAL4; UAS-GFP, and TH-GAL4; UAS-dVMAT. Except for TH-GAL4, elav-GAL4, and dVMAT mutant and transgenic lines, which were gifts from Janis O’Donnell (University of Alabama, Tuscaloosa, AL), Venugopal Reddy (Rutgers, The State University of New Jersey, Piscataway, NJ), and David Krantz (University of California, Los Angles, CA), respectively, all mutant and transgenic strains were obtained from the Bloomington Stock Center.

Exposure of Flies to 1-Octen-3-ol.

Adult 48-h posteclosed flies were fed on 1% (wt/vol) agar media supplemented with 5% (wt/vol) sucrose in 250-mL glass flasks. The racemic form of 1-octen-3-ol (98%) was purchased from Sigma-Aldrich (O5284-25G) in liquid form. The exposure of 1-octen-3-ol was carried out using the method published in Inamdar et al. (16) with minor modifications. The flies were exposed to vapors from undiluted aqueous 1-octen-3-ol at 0.5 ppm (vol/vol) concentration.

Mobility Assay.

The mobility assays were based on negative geotaxis behavior. The assays were performed using a published protocol (16). The time taken by a single fly to cross a 5-cm distance in 8 s was recorded. Control and pale mutant flies exposed to 0.5 ppm of 1-octen-3-ol were tested at 1 and 7 d of exposure. The average value was calculated from three trials in both assays with 1 min of rest between the trials.

Confocal Microscopy.

Confocal microscopy experiments were done to monitor the effect of 1-octen-3-ol on the dopaminergic neurons in D. melanogaster using the transgenic lines TH-GAL4; UAS-eGFP and TH-GAL4; UAS-dVMAT. The brains from age-matched controls and 1-octen-3-ol–exposed flies were dissected and mounted on slides to examine the number and status of GFP-expressing dopaminergic neurons. The final image was obtained as an average of Z sections of at least 10–12 sections using a Zeiss LSM 710 confocal microscope. The number of dopaminergic neurons was scored directly under the microscope by visualizing GFP expression to acquire the quantitative data on the status of dopaminergic neurons exposed to 1-octen-3-ol.

HPLC Analysis.

One hundred heads from frozen w¹¹¹⁸ flies were homogenized in 100 µL of 0.1 N perchloric acid and then centrifuged at 18,000 × g for 10 min at 4 °C. The pellets were kept for protein assay and the supernatants were filtered through 0.22-µm filters. Ten microliters of supernatant was injected onto an HPLC with electrochemical detection (Waters) for neurochemical analysis of DA and its metabolite DOPAC. The components were separated on a cation-exchange column (MD-150, 150 × 3.2 mm column; ESA Biosciences) using an isocratic mobile phase (MD-TM mobile phase; ESA Biosciences) containing 2.2 mM NaCl pumped at a constant flow rate of 0.5 mL/min. The compounds were quantified by electrochemical detection using a glassy carbon working electrode, 2.0-mm diameter in situ silver reference electrode (Flow Cell, 2mm GC WE, ISAAC; Waters). The pellets in the bottom of the tubes were dried overnight in an oven at 30 °C. The dried pellets were dissolved in 100 μL of 0.5 N NaOH in a sonicating water bath for 1 h at 37 °C, and then 400 µL of H₂O was added to bring the final concentration of NaOH to 0.1 N. The protein concentration for each sample was determined with a bicinchoninic acid assay reagent kit (Pierce) at 562 nm with a SpectraMax microplate reader (Molecular Devices) using BSA as a standard. The data were expressed in ng/mg of protein.

Whole-Cell [³H]DA Uptake.

The human kidney transgenic cell lines HEK-DAT and HEK-DAT/VMAT2 were used. Our protocol was modified from Bernstein et al. (57). Cells were plated in Transwell inserts for 24-well plates 1 d before the experiments were performed. Cells were exposed to 1-octen-3-ol by the airborne exposure method previously described (15) for 2 h. After exposure, cells were washed with 100 µL of uptake buffer (4 mM Tris, 6.25 mM Hepes, 120 mM NaCl, 5 mM KCl, 1.2 mM CaCl₂, 1.2 mM MgSO₄, 5.6 mM d-glucose, 1.7 mM ascorbic acid, 1 µM pargyline, pH 7.4). Cells were incubated with 90 µL of uptake buffer with or without 10 µM nomifensine for 15 min. After incubation, 10 µL of uptake buffer containing [³H]DA and DA for a final concentration of 20 nM [³H]DA and 1 µM DA was added. Cells were incubated at 37 °C for 10 min. Nonspecific uptake was determined in the presence of 10 µM nomifensine. Uptake was terminated by aspirating uptake buffer and washing each well twice with 100 µL of ice-cold uptake buffer. Cells were lysed in 0.1 N NaOH and transferred to vials containing 3 mL of scintillation mixture. Radioactivity was counted using a Beckman LS6500 liquid scintillation counter.

Statistical Analysis.

All of the graphs were plotted using Prism 5 (GraphPad), and data were analyzed using a one-tail Student t test or one-way/two-way ANOVA (Dunnett’s posttest or Bonferroni posttest) wherever appropriate. We have described the details of the statistical analysis in the figure legends.