Figure 4: Cooperation between synaptic activity and astrocytic Nrf2 activators in supporting neuronal GSH levels.
From: Synaptic NMDA receptor activity is coupled to the transcriptional control of the glutathione system
(a,b) The Nrf2 activator CDDOTFEA induces Gclc expression and GCL activity in astrocytes but not in neurons. Regular astrocyte-containing (AC) cultures (5–10% astrocytes), astrocyte-free (AF) neuronal cultures and astrocyte cultures were treated with CDDOTFEA (250 nM) for 24 h after which Gclc mRNA (P=0.0033, 2WA-Fph, n=4) and GCL activity (P=0.0002, 2WA, Fph, n=5) were assayed. (c) CDDOTFEA protects neuronal cultures against H2O2-induced GSH loss via an astrocyte-dependent mechanism. AC- and AF-neuronal cultures were treated ±CDDOTFEA for 24 h after which the rate of GSH loss induced by 100 μM H2O2 was measured by MCB assay. *P=0.0274, 2WA-Fph (n=5). (d) Neurons were treated ±CDDOTFEA±MRP1 inhibitor (MK571, 10 μM) and H2O2 (100 μM)-induced neuronal death induced 24 h later. *P=0.0027, 0.003, 0.0017, 2WA-Fph (n=4). (e) Astrocyte-free neurons still exhibit activity-dependent protection against oxidative stress but display elevated overall vulnerability. AC- and AF-neuronal cultures were treated ±BiC/4-AP for 24 after which the indicated concentrations of H2O2 were applied and cell death analysed 24 h later. *P<0.0001 for all; #P=0.0007, 0.0002 comparing AF-neuronal death with equivalent AC-neuronal death level, 1WA-Fph (n=6). (f) Synaptic activity and Nrf2 activation by CDDOTFEA cooperate to prevent GSH depletion in astrocyte-containing neuronal cultures. Regular astrocyte-containing neuronal cultures were treated ±BiC/4-AP±CDDOTFEA as indicated for 24 h after which the rate of GSH loss induced by 200 μM H2O2 was measured by MCB assay. *P=0.0005, 0.0207, <0.0001, 0.0003, 0.0006, 1WA-Fph (n=4–12). (g) Synaptic activity and Nrf2 activation by CDDOTFEA cooperate to prevent oxidative stress-induced death in astrocyte-containing neuronal cultures. Regular astrocyte-containing neuronal cultures were treated ±BiC/4-AP±CDDOTFEA as indicated for 24 h after which 200 μM H2O2 was applied and cell death analysed 24 h later. *P=<0.0001, <0.0001, <0.0001, 0.0096, 0.0003, 2WA-Fph (n=8).
