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. 2019 Apr 2:10:122.
doi: 10.3389/fphar.2019.00122. eCollection 2019.

S-Sulfocysteine Induces Seizure-Like Behaviors in Zebrafish

Jennifer Plate  1 , Wiebke A Sassen  2 , Ahmed H Hassan  1 , Franziska Lehne  1 , Reinhard W Köster  2 , Tobias Kruse  1
Affiliations

S-Sulfocysteine Induces Seizure-Like Behaviors in Zebrafish

Jennifer Plate et al. Front Pharmacol. .

Abstract

Sulfite is a neurotoxin, which is detoxified by the molybdenum cofactor (Moco)-dependent enzyme sulfite oxidase (SOX). In humans, SOX deficiency causes the formation of the glutamate analog S-Sulfocysteine (SSC) resulting in a constant overstimulation of ionotropic glutamatergic receptors. Overstimulation leads to seizures, severe brain damage, and early childhood death. SOX deficiency may be caused either by a mutated sox gene or by mutations in one of the genes of the multi-step Moco biosynthesis pathway. While patients affected in the first step of Moco biosynthesis can be treated by a substitution therapy, no therapy is available for patients affected either in the second or third step of Moco biosynthesis or with isolated SOX deficiency. In the present study, we used a combination of behavior analysis and vital dye staining to show that SSC induces increased swimming, seizure-like movements, and increased cell death in the central nervous system of zebrafish larvae. Seizure-like movements were fully revertible upon removal of SSC or could be alleviated by a glutamatergic receptor antagonist. We conclude that in zebrafish SSC can chemically induce phenotypic characteristics comparable to the disease condition of human patients lacking SOX activity.

Keywords: NMDA receptors; S-Sulfocysteine; molybdenum cofactor; sulfite oxidase; zebrafish.

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Figures

Figure 1
Figure 1
Analysis of S-Sulfocysteine induced movement in 3 dpf zebrafish larvae. (A) For each experiment, routinely five larvae per well were transferred in fresh 30% Danieau and allowed to habituate 10 min prior to S-Sulfocysteine (SSC) application. SSC was added to a final concentration of 2 mM (right, II). As a control (left, I), an equivolume of 30% Danieau was added. Larvae were allowed to accommodate for 30 s and then imaged for 9 min. The recorded tracks for each of the larvae analyzed are shown in different colors. The starting points of non-treated larvae are illustrated by asterisks with the corresponding numbers of the larvae. (B–D) Seizure-like behavior of SSC-treated larvae was quantified as described (Baraban et al., 2005). (B) For stage I, the total distances traveled for all larvae were determined and plotted. (C) Percentage time larvae spent with movement over the time span of the experiment. In an additional experiment, larvae were transferred to fresh medium after 10 min incubation with SSC. All data obtained for the transfer experiment were summarized in one bar chart. Error bars indicate the standard error of the mean. (D) Percentage of larvae showing increased activity (stage I), circling swim behavior (stage II), and clonus-like convulsions (stage III). For all experiments, three full independent biological replicates (rep.) were analyzed; *, significant with p < 0.01.
Figure 2
Figure 2
An N-methyl-d-aspartate (NMDA) receptor antagonist partially rescues the S-Sulfocysteine induced seizure-like behavior in zebrafish. (A) Glutamate and S-Sulfocysteine (SSC) are structurally highly similar (Schwarz, 2016). (B–E) The effect of the NMDA receptor antagonist MK801 on SSC-induced zebrafish movement was characterized by the addition of 2 mM MK801 to 2 and 3 dpf larvae treated with 2 mM SSC. (B) and (C) The total distance traveled by SSC-treated 2 dpf and 3 dpf larvae was determined and compared to the distance traveled in the presence of SSC + MK801. As controls, larvae were treated with MK801 and water, respectively. (D) and (E) Percentage of time SSC-treated and SSC + MK801-treated larvae spent with movement. The controls were the same as for (B) and (C). Three full independent biological replicates were analyzed. Error bars represent the standard error of the mean. NS, not significant; *, significant with p < 0.01.
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