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bioRxiv · 10.1101/2025.06.01.657248

γ-glutamyl-cysteine is a critical intermediate in glutathione-led amelioration of PFOS-neurotoxicity

Sammi, S. R.·Boonpraman, N.·Kuhn, N. C.·Cannon, J. R.
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Abstract

Perfluorooctane sulfonate (PFOS) is one of the most prevalent PFAS. Earlier studies using Caenorhabditis elegans indicated that glutathione (GSH) ameliorates PFOS-induced neurodegeneration. This study investigates the GSH synthesis pathway to elucidate critical neuroprotective mechanisms. We assessed the effects of GSH precursors, cysteine, glutamic acid, and cysteine + glutamic acid, and of the intermediate {gamma}-glutamyl-cysteine ({gamma}-Glu-Cys), on PFOS neurotoxicity. While cysteine showed slight neuroprotection, other GSH precursors, glutamate, and cysteine + glutamate did not demonstrate beneficial effects. However, the crucial GSH synthesis intermediate, {gamma}-Glu-Cys, conferred neuroprotection comparable to GSH. Notably, no changes were observed in the gene or protein expression of GSH synthesis enzymes in C. elegans and SH-SY5Y cells, respectively. Further, testing of the alternative pathway for 5-oxoproline-mediated GSH synthesis revealed that it is not involved in {gamma}-glutamyl-cysteine-mediated neuroprotection. Paradoxically, 5-oxoproline supplementation was neurotoxic, plausibly due to glutamate toxicity. Further, we tested the effect of acute PFOS exposure on mitochondrial complexes I, II, III, and IV. We observed significant inhibition of enzyme activity in mitochondrial complexes II, III, and IV. Next, we tested whether GSH and {gamma}-Glu-Cys could rescue PFOS-induced enzyme inhibition. We found that while both GSH and {gamma}-Glu-Cys could rescue enzyme inhibition in complex III, the rescuing effect was altogether absent for complex IV. Overall, our findings confirmed that both GSH and {gamma}-Glu-Cys can rescue PFOS-induced neurodegeneration and mitochondrial enzyme affliction. This study effectively uncovers a novel mechanism that addresses existing knowledge gaps pertaining to the role of the GSH pathway in curtailing PFOS neurotoxicity. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/657248v3_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1050eeborg.highwire.dtl.DTLVardef@11668e0org.highwire.dtl.DTLVardef@3f51ddorg.highwire.dtl.DTLVardef@1178865_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract {gamma}-glutamyl-cysteine is the critical intermediate in glutathione-modulated amelioration of PFOS-neurotoxicity Perfluorooctanesulfonate (PFOS) leads to dopaminergic cell loss in C. elegans. Glutathione (GSH) confers neuroprotection against PFOS neurotoxicity. Further investigation of the critical components involved in GSH biosynthesis identified that while precursors of GSH, Cysteine and glutamate fail to confer neuroprotection, only {gamma}-glutamyl-cysteine alleviates neuronal loss similar to GSH. C_FIG

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Sammi, S. R., Boonpraman, N., Kuhn, N. C., Cannon, J. R.. 2025-06-03. γ-glutamyl-cysteine is a critical intermediate in glutathione-led amelioration of PFOS-neurotoxicity. https://doi.org/10.1101/2025.06.01.657248

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