Effect of serotonin modulation on dystrophin-deficient zebrafish

Abstract

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease caused by mutation of the dystrophin gene. Pharmacological therapies that function independently of dystrophin and complement strategies aimed at dystrophin restoration could significantly improve patient outcomes. Previous observations have suggested that serotonin pathway modulation ameliorates dystrophic pathology, and re-application of serotonin modulators already used clinically would potentially hasten availability to DMD patients. In our study, we used dystrophin-deficient sapje and sapje-like zebrafish models of DMD for rapid and easy screening of several classes of serotonin pathway modulators as potential therapeutics. None of the candidate drugs tested significantly decreased the percentage of zebrafish exhibiting the dystrophic muscle phenotype in the short-term birefringence assay or lengthened the lifespan in the long-term survival assay. Although we did not identify an effective drug, we believe our data is of value to the DMD research community for future studies, and there is evidence that suggests serotonin modulation may still be a viable treatment strategy with further investigation. Given the widespread clinical use of selective serotonin reuptake inhibitors, tricyclic antidepressants and reversible inhibitors of monoamine oxidase, their reapplication to DMD is an attractive strategy in the field's pursuit to identify pharmacological therapies to complement dystrophin restoration strategies.

Keywords: Drug screening; Duchenne muscular dystrophy; Serotonin; Zebrafish.

Fig. 1.

Experimental design of the short-term zebrafish birefringence assay. (A) Heterozygous sapje or sapje-like pairs were mated and their respective embryos were collected and pooled. Drug treatment was initiated on 1 dpf and continued through 4 dpf when birefringence was analyzed. (B) Representative images of the patchy muscle birefringence pattern characteristic of sapje and sapje-like homozygous mutants compared to the highly organized sarcomere structure of (+/+) and (+/−) siblings. Given that the sapje and sapje-like dystrophin mutations are recessive, 25% of untreated offspring are expected to exhibit the affected muscle phenotype.

Fig. 2.

Short-term assay of serotonin, serotonin precursors, products and receptor agonists. (A–E) Treatment with serotonin, 5-hydroxy-L-tryptophan (5-HTP), tryptophan, melatonin and cisapride did not significantly decrease the percentage of zebrafish exhibiting the affected muscle phenotype detected by birefringence. Treatment with 2.5 μg/ml aminophylline significantly decreased the percentage of affected fish. Data represent means±s.e.m.; *P<0.05 versus paired control by one-way ANOVA and Bonferroni post-hoc test. Values above each column indicate the number of sapje (N) and sapje-like (n) fish treated with the respective drug. (F) Both affected and unaffected zebrafish treated with ≤16.5 μM cisapride exhibited abnormal body morphology.

Fig. 3.

Short-term assay of SSRIs. (A–F) Treatment with citalopram, escitalopram, fluoxetine, fluvoxamine, paroxetine and sertraline did not significantly decrease the percentage of zebrafish exhibiting the affected muscle phenotype detected by birefringence. Treatment with 2.5 μg/ml aminophylline significantly decreased the percentage of affected fish. (G) Zebrafish treated with fluoxetine exhibited dose-dependent toxicity. Data represent means±s.e.m.; *P<0.05 versus paired control by one-way ANOVA and Bonferroni post-hoc test. Values above each column indicate the number of sapje (N) and sapje-like (n) fish treated with the respective drug.

Fig. 4.

Short-term assay of tricyclic antidepressants and RIMAs. (A–F) Treatment with amitriptyline, clomipramine, imipramine, moclobemide, pirlindole and toloxatone did not significantly decrease the percentage of zebrafish exhibiting the affected muscle phenotype detected by birefringence. Treatment with 2.5 μg/ml aminophylline significantly decreased the percentage of affected fish. Data represent means±s.e.m.; *P<0.05 versus paired control by one-way ANOVA and Bonferroni post-hoc test. Values above each column indicate the number of sapje (N) and sapje-like (n) fish treated with the respective drug.

Fig. 5.

Long-term zebrafish survival assay. (A) Experimental design of the long-term survival assay. Cohorts of sapje or sapje-like offspring were screened as affected or unaffected on 4 dpf, at which time drug treatment was initiated and continued through 30 dpf. The water was changed and surviving fish were counted every other day. (B–G) Treatment with 33 μM serotonin, 66 μM 5-HTP, 16.5 μM tryptophan, 33 μM melatonin, 8.25 μM cisapride or 33 μM moclobemine did not significantly improve the survival of affected fish. 8.25 μM cisapride was toxic to both affected and unaffected fish beginning on 14 dpf. For each condition, 30–40 fish were tested in three replicate experiments. Data represent means±s.e.m. †P<0.05 affected versus respective unaffected, *P<0.05 drug-treated versus respective control by two-way ANOVA and Bonferroni post-hoc test. AF, affected; UA, unaffected. (H) Affected fish treated with 2.5 μg/ml aminophylline significantly increased survival versus affected controls. †P<0.05 affected versus respective unaffected, *P-values are for the closed blue circles and indicate significant difference between drug-treated AF versus control AF by two-way ANOVA and Bonferroni post-hoc test.