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Gene-specific differential response to anti-apoptotic therapies in zebrafish models of ocular coloboma
Cheryl Y Gregory-Evans1, Mariya Moosajee, Xianghong Shan
1Department of Ophthalmology and Visual Sciences, University of British Columbia, Vancouver BC, Canada. cge30@interchange.ubc.ca
Purpose:
We recently demonstrated that molecular therapy using aminoglycosides can overcome the underlying genetic defect in two zebrafish models of ocular coloboma and showed abnormal cell death to be a key feature associated with the optic fissure closure defects. In further studies to identify molecular therapies for this common congenital malformation, we now examine the effects of anti-apoptotic compounds in zebrafish models of ocular coloboma in vivo.
Methods:
Two ocular coloboma zebrafish lines (pax2.1/noi(tu29a) and lamb1/gup(m189)) were exposed to diferuloylmethane (curcumin) or benzyloxycarbonyl-Val-Ala-Asp(Ome)-fluoromethylketone (zVAD-fmk; a pan-caspase inhibitor) for up to 8 days post-fertilization. The effects of these compounds were assessed by morphology, histology, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and western blot analysis.
Results:
The size of the coloboma in gup zebrafish mutants treated with diferuloylmethane was greatly reduced. In treated mutants a reduction in TUNEL staining and a 67% decrease in activated caspase-3 protein were observed. The release of cytochrome c from the mitochondria into the cytosol was reduced fourfold by in vivo diferuloylmethane treatment, suggesting that the drug was acting to inhibit the intrinsic apoptotic pathway. Inhibition of caspases directly with zVAD-fmk also resulted in a similar reduction in coloboma phenotype. Treatment with either diferuloylmethane or zVAD-fmk resulted in a statistically significant 1.4 fold increase in length of survival of these mutant zebrafish (p<0.001), which normally succumb to the lethal genetic mutation. In contrast, the coloboma phenotype in noi zebrafish mutants did not respond to either diferuloylmethane or zVAD-fmk exposure, even though inhibition of apoptotic cell death was observed by a reduction in TUNEL staining.
Conclusions:
The differential sensitivity to anti-apoptotic agents in lamb1-deficient and pax2.1-deficient zebrafish models, suggests that apoptotic cell death is not a final common pathway in all ocular coloboma genotypes. When considering anti-cell death therapies for ocular colobomatous defects attention should be paid to the genotype under investigation.
Insights
Anti-apoptotic compounds like curcumin and zVAD-fmk reduced ocular coloboma in some zebrafish models by inhibiting cell death. However, effectiveness varied by genetic cause, indicating genotype-specific therapeutic approaches are crucial for ocular coloboma treatment.
Area of Science:
- Developmental Biology
- Genetics
- Pharmacology
Background:
- Ocular coloboma is a common congenital malformation.
- Abnormal cell death is a key feature in optic fissure closure defects.
- Previous studies showed aminoglycosides can correct genetic defects in zebrafish models.
Purpose of the Study:
- To investigate the efficacy of anti-apoptotic compounds in zebrafish models of ocular coloboma.
- To determine if inhibiting apoptosis can correct ocular coloboma defects in vivo.
- To explore molecular therapies for congenital ocular malformations.
Main Methods:
- Two zebrafish lines with ocular coloboma (pax2.1/noi and lamb1/gup) were treated with diferuloylmethane (curcumin) or zVAD-fmk (pan-caspase inhibitor).
- Effects were assessed using morphology, histology, TUNEL staining, and western blot analysis.
- Changes in apoptosis markers like activated caspase-3 and cytochrome c release were measured.
Main Results:
- Diferuloylmethane significantly reduced coloboma size and TUNEL staining in gup mutants, decreasing activated caspase-3 and cytochrome c release.
- zVAD-fmk also reduced coloboma phenotype in gup mutants.
- Both treatments increased survival in gup mutants but did not affect the coloboma in noi mutants, despite reducing apoptosis.
Conclusions:
- Apoptotic cell death is not a universal pathway in all ocular coloboma genotypes.
- The effectiveness of anti-apoptotic therapies for ocular coloboma depends on the underlying genetic defect.
- Genotype-specific considerations are essential when developing anti-cell death therapies for ocular coloboma.
