Limits...
γ-Glutamylcysteine detoxifies reactive oxygen species by acting as glutathione peroxidase-1 cofactor.

Quintana-Cabrera R, Fernandez-Fernandez S, Bobo-Jimenez V, Escobar J, Sastre J, Almeida A, Bolaños JP - Nat Commun (2012)

Bottom Line: Reactive oxygen species regulate redox-signaling processes, but in excess they can cause cell damage, hence underlying the aetiology of several neurological diseases.In primary neurons, endogenously synthesized γ-glutamylcysteine fully prevents apoptotic death in several neurotoxic paradigms and, in an in vivo mouse model of neurodegeneration, γ-glutamylcysteine protects against neuronal loss and motor impairment.Thus, γ-glutamylcysteine takes over the antioxidant and neuroprotective functions of glutathione by acting as glutathione peroxidase-1 cofactor.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Institute of Neurosciences of Castile and Leon, University of Salamanca, Campus Miguel de Unamuno, 37007 Salamanca, Spain.

ABSTRACT
Reactive oxygen species regulate redox-signaling processes, but in excess they can cause cell damage, hence underlying the aetiology of several neurological diseases. Through its ability to down modulate reactive oxygen species, glutathione is considered an essential thiol-antioxidant derivative, yet under certain circumstances it is dispensable for cell growth and redox control. Here we show, by directing the biosynthesis of γ-glutamylcysteine-the immediate glutathione precursor-to mitochondria, that it efficiently detoxifies hydrogen peroxide and superoxide anion, regardless of cellular glutathione concentrations. Knocking down glutathione peroxidase-1 drastically increases superoxide anion in cells synthesizing mitochondrial γ-glutamylcysteine. In vitro, γ-glutamylcysteine is as efficient as glutathione in disposing of hydrogen peroxide by glutathione peroxidase-1. In primary neurons, endogenously synthesized γ-glutamylcysteine fully prevents apoptotic death in several neurotoxic paradigms and, in an in vivo mouse model of neurodegeneration, γ-glutamylcysteine protects against neuronal loss and motor impairment. Thus, γ-glutamylcysteine takes over the antioxidant and neuroprotective functions of glutathione by acting as glutathione peroxidase-1 cofactor.

Show MeSH

Related in: MedlinePlus

mitoGCL down-modulates mitochondrial superoxide in primary neurons.(a) Transfection of rat primary neurons with mitoGCL, but not with its inactive form (mitoGCL (E103A)), significantly decreased basal levels of mitochondrial O2·–, as quantified by MitoSox fluorescence in the transfected neurons (identified by GFP+ fluorescence); mitoGCL—but not mitoGCL (mut)—fully prevented glutamate (100 μM per 5 min)-induced mitochondrial O2·–, as quantified 24 h after glutamate treatment, by MitoSox fluorescence. (b) Silencing glutathione peroxidase-1 (siGPx1), but not glutathione synthetase (siGSS) or glutathione reductase (siGSR), significantly enhanced glutamate (100 μM per 5 min)-mediated mitochondrial O2·–, as judged by MitoSox fluorescence 24 h after treatment, in mitoGCL-expressing neurons; such enhancement was not affected by GSS co-silencing. (c) Expression of mitoGCL rescued the increase, observed after 24 h, in active caspase-3 in GFP+-neurons triggered by glutamate (100 μM per 5 min), as assessed by flow cytometry. (d) mitoGCL expression prevented neuronal apoptotic death, as revealed by annexin V+/7-AAD− neurons 24 h after glutamate treatment (100 μM per 5 min). (e) mitoGCL expression prevented the increase in mitochondrial O2·– (as assessed by MitoSox fluorescence) caused by incubation of neurons with rotenone (Rot, 10 μM), antimycin A (AA, 10 μM) or 3-nitropropionic acid (3NP, 2 mM) for 15 min. NS, not significant; *P<0.05; #P<0.05 versus untreated (analysis of variance; n=3 independent culture preparations). All data are expressed as mean±S.E.M.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3316877&req=5

f3: mitoGCL down-modulates mitochondrial superoxide in primary neurons.(a) Transfection of rat primary neurons with mitoGCL, but not with its inactive form (mitoGCL (E103A)), significantly decreased basal levels of mitochondrial O2·–, as quantified by MitoSox fluorescence in the transfected neurons (identified by GFP+ fluorescence); mitoGCL—but not mitoGCL (mut)—fully prevented glutamate (100 μM per 5 min)-induced mitochondrial O2·–, as quantified 24 h after glutamate treatment, by MitoSox fluorescence. (b) Silencing glutathione peroxidase-1 (siGPx1), but not glutathione synthetase (siGSS) or glutathione reductase (siGSR), significantly enhanced glutamate (100 μM per 5 min)-mediated mitochondrial O2·–, as judged by MitoSox fluorescence 24 h after treatment, in mitoGCL-expressing neurons; such enhancement was not affected by GSS co-silencing. (c) Expression of mitoGCL rescued the increase, observed after 24 h, in active caspase-3 in GFP+-neurons triggered by glutamate (100 μM per 5 min), as assessed by flow cytometry. (d) mitoGCL expression prevented neuronal apoptotic death, as revealed by annexin V+/7-AAD− neurons 24 h after glutamate treatment (100 μM per 5 min). (e) mitoGCL expression prevented the increase in mitochondrial O2·– (as assessed by MitoSox fluorescence) caused by incubation of neurons with rotenone (Rot, 10 μM), antimycin A (AA, 10 μM) or 3-nitropropionic acid (3NP, 2 mM) for 15 min. NS, not significant; *P<0.05; #P<0.05 versus untreated (analysis of variance; n=3 independent culture preparations). All data are expressed as mean±S.E.M.

Mentions: Neurons are particularly vulnerable against excess ROS, hence requiring continuous supply and regeneration of GSH for survival15. We therefore investigated the possible efficacy of γ-glutamylcysteine at detoxifying ROS in (patho) physiologically relevant neuronal death models. Glutamate treatment increased mitochondrial O2·– in rat primary neurons (Fig. 3a; Supplementary Fig. S2a), an effect that was abolished by the N-methyl-D-aspartate receptor antagonist16, MK801 (Supplementary Fig. S2b). Expression of mitoGCL in neurons was sufficient to decrease basal mitochondrial O2·– (Fig. 3a), which contrasts with the lack of effect in HEK293T cells (Fig. 1f). The different response of these cells to mitoGCL expression is likely due to the above-mentioned vulnerability of neurons to oxidative stress15 versus the resistance of HEK293T cells (Fig. 2b). Furthermore, mitoGCL, but not its inactive E103A mutant form, prevented the increase in mitochondrial O2·– induced by glutamate-receptor stimulation (Fig. 3a). GPx1—but not GSS or GSR—knockdown (Supplementary Fig. S2c) significantly enhanced O2·– (Fig. 3b), despite neurons expressed mitoGCL, indicating that GPx1 use of γ-glutamylcysteine is essential for γ-glutamylcysteine-mediated O2·– detoxification in this model of excitotoxicity. Glutamate triggered an increase in the proportion of neurons with active caspase-3 (Fig. 3c) and with annexin V+/7-AAD− staining (Fig. 3d), indicating an intrinsic (mitochondrial) mode of apoptotic death; this was prevented by antagonizing the N-methyl-D-aspartate receptors (Supplementary Fig. S2d,e). Notably, mitoGCL largely—but not fully—prevented the rise in the percentage of neurons with the apoptotic phenotype (Fig. 3c,d). In addition, mitoGCL abolished O2·– enhancement triggered by other mitochondrial ROS-inducing agents1718, such as rotenone, antimycin and 3-nitropropionic acid (3NP; Fig. 3e).


γ-Glutamylcysteine detoxifies reactive oxygen species by acting as glutathione peroxidase-1 cofactor.

Quintana-Cabrera R, Fernandez-Fernandez S, Bobo-Jimenez V, Escobar J, Sastre J, Almeida A, Bolaños JP - Nat Commun (2012)

mitoGCL down-modulates mitochondrial superoxide in primary neurons.(a) Transfection of rat primary neurons with mitoGCL, but not with its inactive form (mitoGCL (E103A)), significantly decreased basal levels of mitochondrial O2·–, as quantified by MitoSox fluorescence in the transfected neurons (identified by GFP+ fluorescence); mitoGCL—but not mitoGCL (mut)—fully prevented glutamate (100 μM per 5 min)-induced mitochondrial O2·–, as quantified 24 h after glutamate treatment, by MitoSox fluorescence. (b) Silencing glutathione peroxidase-1 (siGPx1), but not glutathione synthetase (siGSS) or glutathione reductase (siGSR), significantly enhanced glutamate (100 μM per 5 min)-mediated mitochondrial O2·–, as judged by MitoSox fluorescence 24 h after treatment, in mitoGCL-expressing neurons; such enhancement was not affected by GSS co-silencing. (c) Expression of mitoGCL rescued the increase, observed after 24 h, in active caspase-3 in GFP+-neurons triggered by glutamate (100 μM per 5 min), as assessed by flow cytometry. (d) mitoGCL expression prevented neuronal apoptotic death, as revealed by annexin V+/7-AAD− neurons 24 h after glutamate treatment (100 μM per 5 min). (e) mitoGCL expression prevented the increase in mitochondrial O2·– (as assessed by MitoSox fluorescence) caused by incubation of neurons with rotenone (Rot, 10 μM), antimycin A (AA, 10 μM) or 3-nitropropionic acid (3NP, 2 mM) for 15 min. NS, not significant; *P<0.05; #P<0.05 versus untreated (analysis of variance; n=3 independent culture preparations). All data are expressed as mean±S.E.M.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3316877&req=5

f3: mitoGCL down-modulates mitochondrial superoxide in primary neurons.(a) Transfection of rat primary neurons with mitoGCL, but not with its inactive form (mitoGCL (E103A)), significantly decreased basal levels of mitochondrial O2·–, as quantified by MitoSox fluorescence in the transfected neurons (identified by GFP+ fluorescence); mitoGCL—but not mitoGCL (mut)—fully prevented glutamate (100 μM per 5 min)-induced mitochondrial O2·–, as quantified 24 h after glutamate treatment, by MitoSox fluorescence. (b) Silencing glutathione peroxidase-1 (siGPx1), but not glutathione synthetase (siGSS) or glutathione reductase (siGSR), significantly enhanced glutamate (100 μM per 5 min)-mediated mitochondrial O2·–, as judged by MitoSox fluorescence 24 h after treatment, in mitoGCL-expressing neurons; such enhancement was not affected by GSS co-silencing. (c) Expression of mitoGCL rescued the increase, observed after 24 h, in active caspase-3 in GFP+-neurons triggered by glutamate (100 μM per 5 min), as assessed by flow cytometry. (d) mitoGCL expression prevented neuronal apoptotic death, as revealed by annexin V+/7-AAD− neurons 24 h after glutamate treatment (100 μM per 5 min). (e) mitoGCL expression prevented the increase in mitochondrial O2·– (as assessed by MitoSox fluorescence) caused by incubation of neurons with rotenone (Rot, 10 μM), antimycin A (AA, 10 μM) or 3-nitropropionic acid (3NP, 2 mM) for 15 min. NS, not significant; *P<0.05; #P<0.05 versus untreated (analysis of variance; n=3 independent culture preparations). All data are expressed as mean±S.E.M.
Mentions: Neurons are particularly vulnerable against excess ROS, hence requiring continuous supply and regeneration of GSH for survival15. We therefore investigated the possible efficacy of γ-glutamylcysteine at detoxifying ROS in (patho) physiologically relevant neuronal death models. Glutamate treatment increased mitochondrial O2·– in rat primary neurons (Fig. 3a; Supplementary Fig. S2a), an effect that was abolished by the N-methyl-D-aspartate receptor antagonist16, MK801 (Supplementary Fig. S2b). Expression of mitoGCL in neurons was sufficient to decrease basal mitochondrial O2·– (Fig. 3a), which contrasts with the lack of effect in HEK293T cells (Fig. 1f). The different response of these cells to mitoGCL expression is likely due to the above-mentioned vulnerability of neurons to oxidative stress15 versus the resistance of HEK293T cells (Fig. 2b). Furthermore, mitoGCL, but not its inactive E103A mutant form, prevented the increase in mitochondrial O2·– induced by glutamate-receptor stimulation (Fig. 3a). GPx1—but not GSS or GSR—knockdown (Supplementary Fig. S2c) significantly enhanced O2·– (Fig. 3b), despite neurons expressed mitoGCL, indicating that GPx1 use of γ-glutamylcysteine is essential for γ-glutamylcysteine-mediated O2·– detoxification in this model of excitotoxicity. Glutamate triggered an increase in the proportion of neurons with active caspase-3 (Fig. 3c) and with annexin V+/7-AAD− staining (Fig. 3d), indicating an intrinsic (mitochondrial) mode of apoptotic death; this was prevented by antagonizing the N-methyl-D-aspartate receptors (Supplementary Fig. S2d,e). Notably, mitoGCL largely—but not fully—prevented the rise in the percentage of neurons with the apoptotic phenotype (Fig. 3c,d). In addition, mitoGCL abolished O2·– enhancement triggered by other mitochondrial ROS-inducing agents1718, such as rotenone, antimycin and 3-nitropropionic acid (3NP; Fig. 3e).

Bottom Line: Reactive oxygen species regulate redox-signaling processes, but in excess they can cause cell damage, hence underlying the aetiology of several neurological diseases.In primary neurons, endogenously synthesized γ-glutamylcysteine fully prevents apoptotic death in several neurotoxic paradigms and, in an in vivo mouse model of neurodegeneration, γ-glutamylcysteine protects against neuronal loss and motor impairment.Thus, γ-glutamylcysteine takes over the antioxidant and neuroprotective functions of glutathione by acting as glutathione peroxidase-1 cofactor.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Institute of Neurosciences of Castile and Leon, University of Salamanca, Campus Miguel de Unamuno, 37007 Salamanca, Spain.

ABSTRACT
Reactive oxygen species regulate redox-signaling processes, but in excess they can cause cell damage, hence underlying the aetiology of several neurological diseases. Through its ability to down modulate reactive oxygen species, glutathione is considered an essential thiol-antioxidant derivative, yet under certain circumstances it is dispensable for cell growth and redox control. Here we show, by directing the biosynthesis of γ-glutamylcysteine-the immediate glutathione precursor-to mitochondria, that it efficiently detoxifies hydrogen peroxide and superoxide anion, regardless of cellular glutathione concentrations. Knocking down glutathione peroxidase-1 drastically increases superoxide anion in cells synthesizing mitochondrial γ-glutamylcysteine. In vitro, γ-glutamylcysteine is as efficient as glutathione in disposing of hydrogen peroxide by glutathione peroxidase-1. In primary neurons, endogenously synthesized γ-glutamylcysteine fully prevents apoptotic death in several neurotoxic paradigms and, in an in vivo mouse model of neurodegeneration, γ-glutamylcysteine protects against neuronal loss and motor impairment. Thus, γ-glutamylcysteine takes over the antioxidant and neuroprotective functions of glutathione by acting as glutathione peroxidase-1 cofactor.

Show MeSH
Related in: MedlinePlus