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Susan Brockerhoff, Eva Ma, Alaron Lewis, George Stearns; Calcium fluxes in degenerating pde6cw59 mutant zebrafish cones indicate a non-apoptotic mechanism of cell death. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4193.
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© ARVO (1962-2015); The Authors (2016-present)
More than 10% of the mutations that cause photoreceptor degeneration induce cell death by interfering with normal cGMP metabolism. The molecular signaling cascade underlying cGMP-mediated photoreceptor death is unknown. In this study, we examined in vivo intracellular Ca2+ fluxes occurring in pde6cw59 mutant zebrafish cones during degeneration caused by elevated cGMP.
Transposon transgenesis constructs were injected into 1-cell zebrafish embryos to generate mosaics and transgenic lines. Live pde6cw59 transgenic mutant larvae at 5 and 6 days postfertilization were anesthetized and embedded in low melt agarose prior to imaging. Cones were imaged for up to 9 hours using an Olympus FV1000-MPE multiphoton microscope equipped with a 25X water immersion objective. Image J was used to process and analyze data.
To simultaneously analyze cone cell morphology and Ca2+ fluxes, we generated double transgenic zebrafish that express the Ca2+ indicator GCAMP3 in all cones [Tg(TαCP::GCAMP3)] and tdTomato selectively in long-wavelength cones [Tg(Trβ2::tdTomato)]. Electroretinograms indicate that expression of GCAMP3 in cones does not alter their response to light. To show that GCAMP3 works in photoreceptors, we induced death by excessive infrared laser and observed dramatic increases in fluorescence during degeneration. In contrast, large sustained Ca2+ increases were not observed in pde6cw59 mutant cones undergoing the morphological changes associated with cell death. Interestingly, we detect transient increases (“bursts”) in intracellular Ca2+ in mutant cones. These Ca2+ bursts are rapid and occur with varying frequency among the mutant cones. However, the presence of these transient increases did not correlate with the morphology changes that occur in degenerating mutant photoreceptors.
In contrast to what is observed during canonical apoptosis, large intracellular Ca2+ increases are not detected in pde6cw59 mutant zebrafish cones degenerating due to elevated cGMP. The rapid transient Ca2+ bursts may represent distressed cellular homeostasis prior to degeneration. These studies provide the first in vivo analysis of Ca2+ changes during the death of photoreceptors within a host animal. Understanding the molecular events triggered during cGMP-mediated photoreceptor degeneration will help treat and cure this disease.
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