Behavioral and metabolic effects of the atypical antipsychotic ziprasidone on the nematode Caenorhabditis elegans

Abstract

Atypical antipsychotics are associated with metabolic syndrome, primarily associated with weight gain. The effects of Ziprasidone, an atypical antipsychotic, on metabolic syndrome has yet to be evaluated. Here in, we evaluated lipid accumulation and behavioral changes in a new experimental model, the nematode Caenorhabditis elegans (C. elegans). Behavioral parameters in the worms were evaluated 24 h after Ziprasidone treatment. Subsequently, lipid accumulation was examined using Nile red, LipidTox green and BODIPY labeling. Ziprasidone at 40 µM for 24 h effectively decreased the fluorescence labeling of all markers in intestinal cells of C. elegans compared to control (0.16% dimethyl sulfoxide). Ziprasidone did not alter behaviors related to energetic balance, such as pharynx pumping, defecation cycles and movement. There was, however, a reduction in egg-production, egg-laying and body-length in nematodes exposed to Ziprasidone without any changes in the progression of larval stages. The serotoninergic pathway did not appear to modulate Ziprasidone's effects on Nile red fluorescence. Additionally, Ziprasidone did not alter lipid accumulation in daf-16 or crh-1 deletion mutants (orthologous of the transcription factors DAF-16 and CREB, respectively). These results suggest that Ziprasidone alters reproductive behavior, morphology and lipid reserves in the intestinal cells of C. elegans. Our results highlight that the DAF-16 and CREB transcription factors are essential for Ziprasidone-induced fat store reduction.

Figure 1. Nile Red Fluorescence reduction in Caenorhabditis elegans wild-type (N2) after Ziprasidone treatment for 24h.

(A) Representative images of Ziprasidone treatment; (B) dose-response curve to Ziprasidone. A set of optical sections of 2.52 mm through the specimen (called a "Z series") of the first intestinal pair cells. The control group correspond to DMSO 0.16% in Acetic acid (1:10 000). The results were expressed in percentage of control (mean of fluorescence, standard deviation (SD), n>15). * p<0.05; ***p<0.0001 statistically different when compared to control 24h by one way ANOVA with Bonferroni correction for post hoc multiple comparisons.

Figure 2. Fluorescence decrease induced by Ziprasidone exposition in Caenorhabditis elegans wild-type (N2) for 24 h.

(A) Representative images of fat stores evidenced by BODIPY labeling and (B) fluorescence quantification; (C) Illustrative images and (D) densitometric quantification follow the fluorophore LipidTox Green labeling. The treatment with Ziprasidone (40 µM) or control (DMSO 0.16% in Acetic acid (1:10 000)) began in L4 larval stage until adult for 24 h. **p<0.01; ***p<0.0001, statistically different compared to the control 24h by one way ANOVA with Bonferroni correction for post hoc multiple comparisons, mean, SD, n= 10-25). The drug was poured and precipitated on agar plates and experiment performed two times at different days.

Figure 3. Ziprasidone activity-time curve on Nile red fluorescence in Caenorhabditis elegans.

(A) Nile Red fluorescence in wild-type adult worms (N2) after Ziprasidone (40 µM) treatment; (B) Activity-time curve for 2, 4, 6, 8 and 24 hours. At the image acquisition time, all worms were in the same larval stage (adult). Data were normalized in percentage of each time respective control. Just in 24h hours, Ziprasidone significantly reduced the Nile Red fluorescence (***p<0.001, by ANOVA with Bonferroni correction for post hoc multiple comparisons, mean, SD, n>20-30).

Figure 4. Caenorhabditis elegans wild-type behavior after Ziprasidone treatment for 24h.

The worms were observed as the pharynx pumping/ min (A) and intervals of defecation cycles (s) (B) on treatment plates. The treatment with Ziprasidone (40 µM) or control (DMSO 0.16% in Acetic acid (1:10 000)) started in L4 larval stage for 24 h (adult). No statistic difference was found compared to control (0.16% DMSO) (mean, SD, n=20). The drug was poured and precipitated on agar plates.

Figure 5. C. elegans wild-type movement behavior after Ziprasidone treatment for 24h.

(A) Distance (µm), (B) speed (µm/s) and (C) acceleration (µm/s²) were quantified during 30 s after 24 h of Ziprasidone (40 µM) or vehicle exposition (DMSO 0.16% in Acetic acid (1:10 000)) started in L4 larval stage for 24 h (adult). No statistic difference was found compared to control (0.16% DMSO), mean, SD, n>6. The experiment was repeated three times in distinct days.

Figure 6. Reproductive behavior of the nematode C. elegans under Ziprasidone exposition.

(A) The egg production and (B) egg laying were observed after 24, 48 and 72 hours of Ziprasidone treatment (40 µM) or vehicle as control (DMSO 0.16% in Acetic acid (1:10 000)) (mean, SD, n> 20)). The results represent the mean of eggs inside the worm (A) and the mean of the eggs released during 2 h in different time of treatment (24, 48 and 72 hours). ***p<0.0001, statistically different when compared to respective control of each exposition time by one way ANOVA with Bonferroni correction for post hoc multiple comparisons.

Figure 7. Caenorhabditis elegans wild-type (N2) perimeter after Ziprasidone treatment for 24, 48 and 72 hours.

(A) Representative images of worms and (B) the perimeter quantification in µm after 24, 48 and 72 hours of Ziprasidone (40 µM) or vehicle control (DMSO 0.16% in Acetic acid (1:10 000)) exposition. After the determined times, the worms were removed of the plates and the images acquired with a stereoscope (40x, Nikon SMZ 1500 Stereoscope microscope). Statistically different compared to the respective control of each time (**p<0.001; ***p<0.0001, by Student’s T-test, mean, SD, n>20). The assays were confirmed twice.

Figure 8. C. elegans larval development after Ziprasidone exposition.

The worms were treated with Ziprasidone (40 µM) or control (DMSO 0.16% in Acetic acid (1:10 000)) after L1 larval stage until gravid adult. The results were evaluated by two way ANOVA no significant differences between groups were found (% worms in the larval stage, n=20).

Figure 9. Ziprasidone effect on Nile Red fluorescence of C. elegans mutant to the serotoninergic.

(A) Representative images of Nile Red fluorescence on C. elegans wild-type and mutants; (B) Fluorescence profile of Nile Red label on mutants exposed to the vehicle (0.16% DMSO in Acetic acid (1:10 000)) and (C) percentage of loss of Nile Red fluorescence on mutants exposed to Ziprasidone (40µM) compared to the wild-type worm (N2). The treatment began in the L4 larval stage until adult. ##p<0.0001 statistically different compared with wild-type worm exposed to the vehicle; ***p<0.0001 statistically different compared with wild-type worm under Ziprasidone (40µM) treatment (% of control, SD, n> 15, one way ANOVA with Bonferroni correction for post hoc multiple comparisons).

Figure 10. Effects on lipid stores after exogenous exposition to the monoamines and Ziprasidone in wild-type worms (N2).

The nematodes were exposed to the monoamines (A) tyramine (TA, 5 mM), (B) dopamine (DA, 5 mM), (C) octopamine (AO, 5 mM) and (D) serotonin (5-HT, 5mM) isolated or concomitant to the Ziprasidone (40 µM). The fat amount was evaluated using Nile Red labeling follow density quantification in the first intestinal pair cells. The results were expressed in percentage of respective control: HCl 0.1N (compared to the monoamines), DMSO (0.16% in Acetic acid (1:10 000), compared to the Ziprasidone group) and HCl + DMSO (compared to the monoamine + Ziprasidone association). *p<0.05; **p<0.01; ***p<0.001, statistically different compared to the respective control by one way ANOVA with Bonferroni correction for post hoc multiple comparisons (% of control, SD, n=20).

Figure 11. Recover of lipid profile through Nile Red labeling in mutants to daf-16 and crh-1 transcription factors after Ziprasidone treatment (40 µM).

(A) Representative images of Nile Red fluorescence and (B) fluorescence amount in C. elegans mutants (daf-16, crh-1) and wild-type (N2). The results were expressed in percentage of control (DMSO 0.16% in Acetic acid (1:10 000)). ***p<0.0001, statistically different compared to the control by one way ANOVA with Bonferroni correction for post hoc multiple comparisons (mean of fluorescence, SD, n>25).