Chapter 2 22 Cystine accumulation in ctns ‒/‒ larvae and organs of adult ctns ‒/‒ zebrafish Being a major pathologic feature of cystinosis, we measured cystine levels in both homogenized larvae and adult organs of mutant fish compared to the wt. ctns‒/‒ larvae at 6 dpf accumulated cystine about ten times higher compared to wt larvae (Fig. 2b). We also had a similar increase in homogenates of morphant larvae (data not shown). Furthermore, cystine levels in ctns‒/‒ larvae gradually decreased in response to increasing concentrations of cysteamine in the swimming water (Fig. 2b). Other thiol compounds related to cystine metabolism such as oxidized glutathione (GSSG), total glutathione (GSH) and free cysteine were also evaluated in homogenates of wt and mutant larvae (Fig. 2c-e). ctns‒/‒ larvae showed significantly higher levels of both GSSG and free cysteine compared to the wt; however, GSH was not significantly different. Cysteamine treatment significantly reduced abnormally high GSSG levels. In adult fish, the kidneys of 8 months ctns‒/‒ zebrafish demonstrated cystine concentrationsover 50 times of that detected in wt kidneys. Similarly, the ctns‒/‒ brain accumulated 10 times higher cystine, while the liver and heart accumulated double the amount of cystine in the wt (Fig. 2f-i). Thus, the results show that the inactivation of ctnsgene in zebrafish leads to the failure of cystine metabolism, recapitulating the human phenotype. ctns‒/‒ zebrafish show growth retardation and higher rates of embryonic mortality Next, we investigated if the defects in cystine metabolism had other effects on zebrafish. Therefore, we monitored the developmental stages of both ctns‒/‒ and wt zebrafish embryos in four independent crossings over the first three days of maturation at predetermined time points (3h, 6h, 24h, 48h and 72 hpf). ctns‒/‒ embryos showed significant delay in development at all time points investigated, although the difference was more striking at early time points (≤ 24h)(Fig. 3). Additionally, the percentage of dead embryos during the first 3 days post fertilization was significantly higher in ctns‒/‒ zebrafish (101/363 (27.8%)), when compared to wt (33/322 (10.2%)), P<0.001. Hatching was also relatively delayed in ctns‒/‒ embryos at both 48hpf and 72hpf time points. In a different set of experiments, mortality rates were improved with therapeutic doses of cysteamine (Fig. 3f). Hatching rates were also partially normalized by cysteamine therapy (Supplementary Fig. S2 online). Thus, the deregulation of ctns gene led to overall developmental delay, and increased embryonic mortality that are partially restored by cysteamine.
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