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Synaptic NMDA receptor activity is coupled to the transcriptional control of the glutathione system.

Baxter PS, Bell KF, Hasel P, Kaindl AM, Fricker M, Thomson D, Cregan SP, Gillingwater TH, Hardingham GE - Nat Commun (2015)

Bottom Line: How the brain's antioxidant defenses adapt to changing demand is incompletely understood.This tunes antioxidant capacity to reflect the elevated needs of an active neuron, guards against future increased demand and maintains redox balance in the brain.Notably, these activity-dependent cell-autonomous mechanisms were found to cooperate with non-cell-autonomous Nrf2-driven support from astrocytes to maintain neuronal GSH levels in the face of oxidative insults.

View Article: PubMed Central - PubMed

Affiliation: Centre for Integrative Physiology, University of Edinburgh School of Biomedical Sciences, Hugh Robson Building, George Square, Edinburgh EH8 9XD, UK.

ABSTRACT
How the brain's antioxidant defenses adapt to changing demand is incompletely understood. Here we show that synaptic activity is coupled, via the NMDA receptor (NMDAR), to control of the glutathione antioxidant system. This tunes antioxidant capacity to reflect the elevated needs of an active neuron, guards against future increased demand and maintains redox balance in the brain. This control is mediated via a programme of gene expression changes that boosts the synthesis, recycling and utilization of glutathione, facilitating ROS detoxification and preventing Puma-dependent neuronal apoptosis. Of particular importance to the developing brain is the direct NMDAR-dependent transcriptional control of glutathione biosynthesis, disruption of which can lead to degeneration. Notably, these activity-dependent cell-autonomous mechanisms were found to cooperate with non-cell-autonomous Nrf2-driven support from astrocytes to maintain neuronal GSH levels in the face of oxidative insults. Thus, developmental NMDAR hypofunction and glutathione system deficits, separately implicated in several neurodevelopmental disorders, are mechanistically linked.

No MeSH data available.


Related in: MedlinePlus

Deleterious effects of NMDAR blockade in vivo are due to Gclc transcriptional repression.(a) Blockade of NMDARs causes a reduction of cortical GSH content in vivo. Cortical GSH levels measured in P6 rat pups 24 h after the first injection. *P=0.0495, one-tailed t-test (n=4). (b,c) Blockade of NMDARs reduces GCL enzyme activity and Gclc expression in vivo. Rat pups treated as in Fig. 4a and cortical GCL activity (b) and Gclc expression (c) were measured at 12 h and expressed relative to the mean of the control group. P=0.0006 (b), 0.0042 (c) (n=4). (d) GCEE sustains GSH levels in the presence of GCL inhibitor. Neurons were treated with GCEE (1 mM) for 1 h, followed by 200 μM BSO for 24 h, followed by MCB assay. *P=0.0002 (n=9 (con), n=5 (BSO)). (e) GCEE attenuates GSH depletion by oxidative insult. Neurons were treated for 1 h with GCEE then rate of decline in GS-bimane fluorescence induced by 250 μM H2O2 measured. *P=0.0070 (n=4). (f,g) GCEE attenuates oxidative stress-induced Puma mRNA expression and cell death. Neurons were preincubated ±GCEE for 1 h, then treated with 100 μM H2O2 and either Puma levels. *P=0.0086, 0.047 (f) n=9 (con), n=5 (GCEE)) or cell death analysed.*P=0.0110, 0.0217 (n=4 (H2O2, n=3 (Con)). (h) GCEE rescues NMDAR blockade dependent forebrain GSH depletion in vivo. P6 Rat pups were injected twice at 0 and 8 h and cortical GSH levels measured at 24 h, normalized to protein content and expressed relative to MK-801 treated samples. *P=<0.0001, 0.0105, 0.0087, 1WA-Fph (n=8,9,9 for Veh, MK, MK+GCEE respectively). (i,j) GCEE rescues neurons from MK-801 induced cell death in vivo. P6 Rat pups were injected twice at 0 and 8 h and killed 24 h post first injection, followed by Fluoro-Jade staining-based analysis of neurodegeneration within the hippocampus. *P=<0.0001, 0.0105, 0.0087, 1WA-Fph (n=9, 11, 11 for Veh, MK and MK+GCEE, respectively). Scale bar, 30 μm.
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f5: Deleterious effects of NMDAR blockade in vivo are due to Gclc transcriptional repression.(a) Blockade of NMDARs causes a reduction of cortical GSH content in vivo. Cortical GSH levels measured in P6 rat pups 24 h after the first injection. *P=0.0495, one-tailed t-test (n=4). (b,c) Blockade of NMDARs reduces GCL enzyme activity and Gclc expression in vivo. Rat pups treated as in Fig. 4a and cortical GCL activity (b) and Gclc expression (c) were measured at 12 h and expressed relative to the mean of the control group. P=0.0006 (b), 0.0042 (c) (n=4). (d) GCEE sustains GSH levels in the presence of GCL inhibitor. Neurons were treated with GCEE (1 mM) for 1 h, followed by 200 μM BSO for 24 h, followed by MCB assay. *P=0.0002 (n=9 (con), n=5 (BSO)). (e) GCEE attenuates GSH depletion by oxidative insult. Neurons were treated for 1 h with GCEE then rate of decline in GS-bimane fluorescence induced by 250 μM H2O2 measured. *P=0.0070 (n=4). (f,g) GCEE attenuates oxidative stress-induced Puma mRNA expression and cell death. Neurons were preincubated ±GCEE for 1 h, then treated with 100 μM H2O2 and either Puma levels. *P=0.0086, 0.047 (f) n=9 (con), n=5 (GCEE)) or cell death analysed.*P=0.0110, 0.0217 (n=4 (H2O2, n=3 (Con)). (h) GCEE rescues NMDAR blockade dependent forebrain GSH depletion in vivo. P6 Rat pups were injected twice at 0 and 8 h and cortical GSH levels measured at 24 h, normalized to protein content and expressed relative to MK-801 treated samples. *P=<0.0001, 0.0105, 0.0087, 1WA-Fph (n=8,9,9 for Veh, MK, MK+GCEE respectively). (i,j) GCEE rescues neurons from MK-801 induced cell death in vivo. P6 Rat pups were injected twice at 0 and 8 h and killed 24 h post first injection, followed by Fluoro-Jade staining-based analysis of neurodegeneration within the hippocampus. *P=<0.0001, 0.0105, 0.0087, 1WA-Fph (n=9, 11, 11 for Veh, MK and MK+GCEE, respectively). Scale bar, 30 μm.

Mentions: We next investigated the extent to which synaptic NMDAR activity regulates the GSH system in vivo in the developing brain. We studied the effects of inducing NMDAR hypoactivity by administrating the NMDAR antagonist MK-801 to P7 rats. MK-801 administration led to a reduction in total glutathione levels in the forebrain (Fig. 5a). Moreover, analysis of the activity of GSH pathway enzymes revealed that NMDAR hypoactivity led to a strong reduction in GCL activity in the brain (Fig. 5b), which was also associated with a strong reduction in Gclc mRNA expression (Fig. 5c). Expression of Gsr mRNA was also repressed by MK-801 administration (Supplementary Fig. 4a) but GR enzyme activity was not repressed (Supplementary Fig. 4b), potentially due to changes in mRNA not yet reflected at the protein level. Collectively these data suggest that NMDAR hypoactivity leads to a deficit in the GSH biosynthetic pathway in developing neurons due to a requirement for NMDAR activity to support expression of Gclc. Administration of NMDAR antagonists to rodents within the first two postnatal weeks has been consistently shown to induces an increase in apoptosis in certain brain regions, including the hippocampus38. Our observations regarding the role of the NMDAR in coupling synaptic activity to activation of the GSH biosynthetic pathway, via transcriptional induction of Gclc raised the possibility that dysregulation of GSH biosynthesis underlies some of the deleterious effects of NMDAR hypoactivity in the developing brain. We next tested this hypothesis.


Synaptic NMDA receptor activity is coupled to the transcriptional control of the glutathione system.

Baxter PS, Bell KF, Hasel P, Kaindl AM, Fricker M, Thomson D, Cregan SP, Gillingwater TH, Hardingham GE - Nat Commun (2015)

Deleterious effects of NMDAR blockade in vivo are due to Gclc transcriptional repression.(a) Blockade of NMDARs causes a reduction of cortical GSH content in vivo. Cortical GSH levels measured in P6 rat pups 24 h after the first injection. *P=0.0495, one-tailed t-test (n=4). (b,c) Blockade of NMDARs reduces GCL enzyme activity and Gclc expression in vivo. Rat pups treated as in Fig. 4a and cortical GCL activity (b) and Gclc expression (c) were measured at 12 h and expressed relative to the mean of the control group. P=0.0006 (b), 0.0042 (c) (n=4). (d) GCEE sustains GSH levels in the presence of GCL inhibitor. Neurons were treated with GCEE (1 mM) for 1 h, followed by 200 μM BSO for 24 h, followed by MCB assay. *P=0.0002 (n=9 (con), n=5 (BSO)). (e) GCEE attenuates GSH depletion by oxidative insult. Neurons were treated for 1 h with GCEE then rate of decline in GS-bimane fluorescence induced by 250 μM H2O2 measured. *P=0.0070 (n=4). (f,g) GCEE attenuates oxidative stress-induced Puma mRNA expression and cell death. Neurons were preincubated ±GCEE for 1 h, then treated with 100 μM H2O2 and either Puma levels. *P=0.0086, 0.047 (f) n=9 (con), n=5 (GCEE)) or cell death analysed.*P=0.0110, 0.0217 (n=4 (H2O2, n=3 (Con)). (h) GCEE rescues NMDAR blockade dependent forebrain GSH depletion in vivo. P6 Rat pups were injected twice at 0 and 8 h and cortical GSH levels measured at 24 h, normalized to protein content and expressed relative to MK-801 treated samples. *P=<0.0001, 0.0105, 0.0087, 1WA-Fph (n=8,9,9 for Veh, MK, MK+GCEE respectively). (i,j) GCEE rescues neurons from MK-801 induced cell death in vivo. P6 Rat pups were injected twice at 0 and 8 h and killed 24 h post first injection, followed by Fluoro-Jade staining-based analysis of neurodegeneration within the hippocampus. *P=<0.0001, 0.0105, 0.0087, 1WA-Fph (n=9, 11, 11 for Veh, MK and MK+GCEE, respectively). Scale bar, 30 μm.
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f5: Deleterious effects of NMDAR blockade in vivo are due to Gclc transcriptional repression.(a) Blockade of NMDARs causes a reduction of cortical GSH content in vivo. Cortical GSH levels measured in P6 rat pups 24 h after the first injection. *P=0.0495, one-tailed t-test (n=4). (b,c) Blockade of NMDARs reduces GCL enzyme activity and Gclc expression in vivo. Rat pups treated as in Fig. 4a and cortical GCL activity (b) and Gclc expression (c) were measured at 12 h and expressed relative to the mean of the control group. P=0.0006 (b), 0.0042 (c) (n=4). (d) GCEE sustains GSH levels in the presence of GCL inhibitor. Neurons were treated with GCEE (1 mM) for 1 h, followed by 200 μM BSO for 24 h, followed by MCB assay. *P=0.0002 (n=9 (con), n=5 (BSO)). (e) GCEE attenuates GSH depletion by oxidative insult. Neurons were treated for 1 h with GCEE then rate of decline in GS-bimane fluorescence induced by 250 μM H2O2 measured. *P=0.0070 (n=4). (f,g) GCEE attenuates oxidative stress-induced Puma mRNA expression and cell death. Neurons were preincubated ±GCEE for 1 h, then treated with 100 μM H2O2 and either Puma levels. *P=0.0086, 0.047 (f) n=9 (con), n=5 (GCEE)) or cell death analysed.*P=0.0110, 0.0217 (n=4 (H2O2, n=3 (Con)). (h) GCEE rescues NMDAR blockade dependent forebrain GSH depletion in vivo. P6 Rat pups were injected twice at 0 and 8 h and cortical GSH levels measured at 24 h, normalized to protein content and expressed relative to MK-801 treated samples. *P=<0.0001, 0.0105, 0.0087, 1WA-Fph (n=8,9,9 for Veh, MK, MK+GCEE respectively). (i,j) GCEE rescues neurons from MK-801 induced cell death in vivo. P6 Rat pups were injected twice at 0 and 8 h and killed 24 h post first injection, followed by Fluoro-Jade staining-based analysis of neurodegeneration within the hippocampus. *P=<0.0001, 0.0105, 0.0087, 1WA-Fph (n=9, 11, 11 for Veh, MK and MK+GCEE, respectively). Scale bar, 30 μm.
Mentions: We next investigated the extent to which synaptic NMDAR activity regulates the GSH system in vivo in the developing brain. We studied the effects of inducing NMDAR hypoactivity by administrating the NMDAR antagonist MK-801 to P7 rats. MK-801 administration led to a reduction in total glutathione levels in the forebrain (Fig. 5a). Moreover, analysis of the activity of GSH pathway enzymes revealed that NMDAR hypoactivity led to a strong reduction in GCL activity in the brain (Fig. 5b), which was also associated with a strong reduction in Gclc mRNA expression (Fig. 5c). Expression of Gsr mRNA was also repressed by MK-801 administration (Supplementary Fig. 4a) but GR enzyme activity was not repressed (Supplementary Fig. 4b), potentially due to changes in mRNA not yet reflected at the protein level. Collectively these data suggest that NMDAR hypoactivity leads to a deficit in the GSH biosynthetic pathway in developing neurons due to a requirement for NMDAR activity to support expression of Gclc. Administration of NMDAR antagonists to rodents within the first two postnatal weeks has been consistently shown to induces an increase in apoptosis in certain brain regions, including the hippocampus38. Our observations regarding the role of the NMDAR in coupling synaptic activity to activation of the GSH biosynthetic pathway, via transcriptional induction of Gclc raised the possibility that dysregulation of GSH biosynthesis underlies some of the deleterious effects of NMDAR hypoactivity in the developing brain. We next tested this hypothesis.

Bottom Line: How the brain's antioxidant defenses adapt to changing demand is incompletely understood.This tunes antioxidant capacity to reflect the elevated needs of an active neuron, guards against future increased demand and maintains redox balance in the brain.Notably, these activity-dependent cell-autonomous mechanisms were found to cooperate with non-cell-autonomous Nrf2-driven support from astrocytes to maintain neuronal GSH levels in the face of oxidative insults.

View Article: PubMed Central - PubMed

Affiliation: Centre for Integrative Physiology, University of Edinburgh School of Biomedical Sciences, Hugh Robson Building, George Square, Edinburgh EH8 9XD, UK.

ABSTRACT
How the brain's antioxidant defenses adapt to changing demand is incompletely understood. Here we show that synaptic activity is coupled, via the NMDA receptor (NMDAR), to control of the glutathione antioxidant system. This tunes antioxidant capacity to reflect the elevated needs of an active neuron, guards against future increased demand and maintains redox balance in the brain. This control is mediated via a programme of gene expression changes that boosts the synthesis, recycling and utilization of glutathione, facilitating ROS detoxification and preventing Puma-dependent neuronal apoptosis. Of particular importance to the developing brain is the direct NMDAR-dependent transcriptional control of glutathione biosynthesis, disruption of which can lead to degeneration. Notably, these activity-dependent cell-autonomous mechanisms were found to cooperate with non-cell-autonomous Nrf2-driven support from astrocytes to maintain neuronal GSH levels in the face of oxidative insults. Thus, developmental NMDAR hypofunction and glutathione system deficits, separately implicated in several neurodevelopmental disorders, are mechanistically linked.

No MeSH data available.


Related in: MedlinePlus