Chapter 2 24 (bars=1 mm). (b) Cystine content in homogenates of 6 dpf wt or ctns‒/‒ zebrafish larvae. ctns‒/‒ larvae were either free of treatment (N=133) or subjected to 0.1 or 1.0 mM of cysteamine in the swimming water (N=111 and 121 larvae, respectively). Comparison was performed with wt larvae (N=191). (c) Oxidized glutathione (GSSG) content in homogenates of 6 dpf wt or ctns‒/‒ zebrafish larvae (same conditions and larval numbers as cystine). (d) Total glutathione (GSH) content in homogenates of 6 dpf wt or ctns‒/‒ zebrafish larvae.ctns‒/‒ larvae were either free of treatment (N=80) or subjected to 0.1 or 1.0 mM of cysteamine in the swimming water (N=108 and 104 larvae, respectively). Comparison was performed with wt larvae (N=158). (e) Free cysteine content in homogenates of 6 dpf wt or ctns‒/‒zebrafish larvae (same conditions and larval numbers as GSH). (f-i) Cystine content in homogenates of 8-month-old adults (Kidney, brain, heart and liver, respectively) (N=3 of each genotype). Concentrations of cystine and other thiol compounds were expressed as nmol/mg protein. * P<0.05, ** P<0.01, *** P<0.001. ctns‒/‒ zebrafish larvae have increased apoptosis rate that can be ameliorated by cysteamine We further addressed whether, as reported previously in human and mouse tissues,7,11,12 cystinosis triggered apoptosis in zebrafish larvae. Apoptosis was investigated in surviving wt and ctns‒/‒ larvae at 5 dpf using the Acridine Orange (AO) fluorescent dye which binds to DNA of apoptotic cells and spares necrotic cells. Cystinotic larvae were naïve to treatment or treated with 0.1 mM of cysteamine (N=10 for each condition). The apoptotic spots in the untreated ctns‒/‒ larvae were clearly visible and significantly increased compared to wt larvae. Interestingly, the low dose of cysteamine significantly reduced both number and intensity of apoptotic spots, P<0.001 (Fig. 4a-d). We further confirmed the increased rate of apoptosis through the detection of positive staining for caspase-3 by immunohistochemistry in 5 dpf ctns‒/‒ larvae. Apoptotic signals in immunohistochemistry were not restricted to skeletal structures but were also present in internal organs especially in proximal tubules and in the liver (Fig. 4e,f). A higher caspase-3 enzyme activity performed by a luciferase based assay was detected in the homogenates of ctns‒/‒ larvae compared to the wt at 5 dpf, P<0.001 (Fig. 4g). ctns‒/‒ zebrafish larvae have normal locomotor activity In order to evaluate if there are any early behavioural or kinetic abnormalities in the cystinotic zebrafish larvae, we monitored the locomotor activity of 5 dpf ctns‒/‒ larvae (N=56) in comparison to wt larvae (N=52) under light and dark conditions. The quantification of locomotor activity (in actinteg units) did not reveal any significant difference between the two genotypes regardless of the lighting conditions (P=0.416 in light and P=0.279 in dark) (Supplementary Fig. S3 online). Figure 2. Continued
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