Synaptic NMDA receptor activity boosts intrinsic antioxidant defenses

Abstract

Intrinsic antioxidant defenses are important for neuronal longevity. We found that in rat neurons, synaptic activity, acting via NMDA receptor (NMDAR) signaling, boosted antioxidant defenses by making changes to the thioredoxin-peroxiredoxin (Prx) system. Synaptic activity enhanced thioredoxin activity, facilitated the reduction of overoxidized Prxs and promoted resistance to oxidative stress. Resistance was mediated by coordinated transcriptional changes; synaptic NMDAR activity inactivated a previously unknown Forkhead box O target gene, the thioredoxin inhibitor Txnip. Conversely, NMDAR blockade upregulated Txnip in vivo and in vitro, where it bound thioredoxin and promoted vulnerability to oxidative damage. Synaptic activity also upregulated the Prx reactivating genes Sesn2 (sestrin 2) and Srxn1 (sulfiredoxin), via C/EBPbeta and AP-1, respectively. Mimicking these expression changes was sufficient to strengthen antioxidant defenses. Trans-synaptic stimulation of synaptic NMDARs was crucial for boosting antioxidant defenses; chronic bath activation of all (synaptic and extrasynaptic) NMDARs induced no antioxidative effects. Thus, synaptic NMDAR activity may influence the progression of pathological processes associated with oxidative damage.

Fig. 1. Synaptic NMDAR activity promotes resistance to oxidative insults and prevents ROS accumulation

A) TUNEL-positive apoptosis in the cortex of P7 mice subjected to IP-injection of MK-801 at P6 (exposure for 24 h). Quantitation (upper) and representative sections (lower-scale bar 200 μm). B,C) Analysis of carbonyl content of 2-D separated proteins from the cortices of P7 mice subjected to IP-injection of MK-801. Blotted 2-DE gels were stained with silver staining to reveal protein spots (right). C) Comparison of intensity of representative protein spots *p<0.05 (n=6) 2-tailed T-test (in this and subsequent cases unless stated). D) Cell death due to 24 h H₂O₂ insult in the face of the indicated treatments, applied 12 h before insult. BiC/4-AP stimulation is labelled as “BiC” in this and subsequent figures. (Lower) Examples pictures, scale bar=40 μm. *p<0.05 compared to control H₂O₂ treated (n=4), mean ± s.e.m shown in this and all cases. E) Cell death measured by analyzing ATP levels. Treatments as for (D). *p<0.05 compared to control, H₂O₂ treated, n=3. F) Cell death due to 24 h H₂O₂ insult in the face of the indicated treatments, applied 12 h before insult. All activity was terminated prior to H₂O₂ exposure (by TTX + MK-801). *p<0.05 compared to control (H₂O₂ treated), n=4. G,H). ROS accumulation following H₂O₂ treatment and in control conditions measured within neurons treated as indicated. Two ROS probes used as indicated. Fluorescence levels were normalized to cell number (as measured using the Celltiter Glo assay, Promega, *p<0.05, n=5).

Fig. 2. Synaptic activity prevents the overoxidation of peroxiredoxins in response to an oxidative insult, and negatively regulates the Thioredoxin inhibitor Txnip

A) Western analysis of Prx overoxidation using an anti-PrxSO_2/3H specific antibody. Analysis normalized to appropriate Prx band intensity. *p<0.05 compared to control, H₂O₂-treated neurons (n=5). Note for upper (PrxIV) band, a higher exposure is often taken to more accurately assess loading (as is the case in the example shown). B,C) Thioredoxin, Thioredoxin reductase and 2-Cys Prx levels analyzed by Western blot after 24 h of the indicated treatments (n=3). D) q-RT-PCR of Txnip RNA from rat cortical neurons treated as indicated (this and all subsequent q-RT-PCR data are normalized to Gapdh levels. *p<0.05 compared to control (n=4)). E) Western analysis of Txnip protein expression in response to the indicated treatments (24 h, *p<0.05 compared to control, n=3). This and all quantitation of Western blots involves normalisation to β-tubulin or other suitable control (such as Akt in the case of Pi-Akt, Prx in the case of PrxSO_2/3H). F) Co-immunoprecipitation of Thioredoxin with an anti-Txnip antibody in samples from neurons experiencing different levels of synaptic NMDAR activity for 24 h (*p<0.05, n=5). G) Thioredoxin activity (insulin-reducing assay) in neurons treated with MK-801, expressed as a % of the activity observed in BiC/4-AP-treated neurons (*p<0.05 compared to BiC-stimulated neurons, n=6).

Fig. 3. Txnip and Thioredoxin can regulate neuronal vulnerability to oxidative stress

A) Loss of GFP-positive neurons expressing Txnip, Thioredoxin (Txn) or control plasmid (beta-globin) in the face of 24 h 100 μM H₂O₂. *p<0.05 (n=4). B) Neurons transfected with a constitutively active vector (SV40-Luc) and Txnip or control plasmid, treated with BiC/4-AP prior to oxidative insult for 24 h, followed by luciferase activity measurement *p<0.05 (n=5). C(left) q-RT-PCR of Txnip mRNA expression in the murine cortex in vivo in response to MK-801 treatment at P0 and P7. *p<0.05 (n=4). C(right) Western blot showing up-regulation of Txnip protein in the cerebral cortex of individual mice in vivo by MK-801 treatment of P7 mice. Each lane represents a different mouse. D) RNA from post-mortem human frontal cortices was extracted and TXNIP mRNA abundance assessed by quantitative RT-PCR. TXNIP is shown normalized to GAPDH and 28S, as their expression in the frontal cortex does not change significantly with age(n=16). E) Neurons transfected with rTxnip plus control or one of two Txnip-directed siRNAs. Protein harvested at 24 h and subjected to Western analysis for Txnip protein. Untransfected sample (U/T) shown for comparison. F) Loss of GFP-positive neurons transfected with control or one of two Txnip siRNAs in the face of 24 h 100 μM H₂O_2. Scale bar 40 μm. Data from 4 independent experiments.

Fig. 4. Synaptic activity promotes reduction of Prx-SO_2/3H and induces neuroprotective expression of the Prx-SO_2/3H-reducing genes Sesn2 and Srxn1

A) Recovery of PrxII-SO_2/3H following a 1 h exposure to high H₂O₂ (200 μM). Neurons stimulated as indicated for 12 h prior to H₂O₂ exposure. Washout period was 16 h (*p<0.05 compared to pre-washout level, n=5). B,C) q-RT-PCR of Sestrin2 (Sesn2, B) and Sulfiredoxin (Srxn1, C) in response to the indicated treatments (BiC stimulation: 4 h). *p<0.01 (n=7). Lower panels show regulation of Sesn2 and Srxn1 protein. D) H₂O₂-induced loss of GFP-positive neurons transfected as indicated. Scale bar 60 μm.*p<0.05 (one-way ANOVA followed by Fisher’s LSD post-hoc test for this and subsequent siRNA experiments, n=4). Right panels show example pictures (arrows indicate transfected neurons whose fate is studied, Scale bar 60 μm). E) Effect of control- or Sesn2-directed siRNA on BiC/4-AP (24 h) induced Sesn2 expression. F) Effect of control- or Srxn1-directed siRNA on production of rat Srxn1 driven from an expression vector. G) Effect of knock-down of Sesn2 and Srxn1 on activity-dependent protection against an oxidative insult. Neurons were transfected with control siRNA or one of two pairs of siRNAs which target both Sesn2 and Srxn1. Neurons were stimulated where indicated with BiC/4-AP and then treated with 100 μM H₂O_2.. *p<0.05 unpaired T-test (n=4). H) Effect of knock-down of Sesn2 and Srxn1 on long-lasting activity-dependent protection. Experimental details as for (G) except that BiC/4-AP-induced synaptic activity was terminated after 12 h by the addition of TTX + MK-801. After this, the oxidative insult (100 μM H₂O₂) was applied. *p<0.05 (n=6).

Fig. 5. Txnip is a FOXO target gene

A) Schematic showing conservation of a FOXO binding site in the proximal 5′ promoter region of Txnip, positions given relative to start of protein coding region (NB. 5′UTR is 222 nt long). B) PI3K inhibition by LY294002 (30 μM) induces Txnip expression, and suppression of Txnip protein and mRNA expression by BiC treatment is blocked by LY294002 *p<0.05 compared to control (n=3). C) Txnip-Luc and Txnip-(mut)-Luc (FOXO site mutated) activity assayed 16 h after the indicated treatments (n=5). In all reporter assays, firefly luciferase based reporter signal is normalized to expression of a cotransfected renilla luciferase control plasmid, pTK-RL. D) Txnip-Luc and Txnip-(mut)-Luc activity assayed in neurons co-transfected with Control or FOXO expression plasmids. *p<0.05 compared to control plasmid (n=3-5). E) Western analysis of FOXO1 phosphorylation in response to the indicated treatments (30 min stimulation, example Western shown below analysis). *p<0.05 compared to control (n=3). F) Subcellular distribution of transfected myc-tagged FOXO1 analyzed by immunofluorescence (examples on the right; scale bar 30 μm.). G,H) Chromatin immunoprecipitation with an anti-FOXO1 antibody, followed by PCR of the Txnip promoter region containing the consensus FOXO binding site (compared to PCR of the input). (G) shows analysis of ChIP band intensity (Normalized to input) relative to MK-801-treated neurons *p<0.05 (n=5). (H) shows example individual ChIP experiments, with (H, right) showing an anti-phospho-FOXO1 negative control ChIP.

Fig. 6. Sesn2 is a C/EBP target gene, Srxn1 is an AP-1 target gene

All experiments performed in cortical neurons. A) Deletion analysis of a luciferase-based reporter of the Sesn2 promoter. B) Effect of an interfering mutant of C/EBP on activity-dependent induction of the Sesn2 reporter construct. *p<0.05 compared to control plasmid (n=5). C) Effect of putative C/EBP binding site mutation on activity-dependent induction of the Sesn2 promoter (distal site ATTTCACACC mutated to ATTTCGGCCC; proximal site TTTGCAGCATC mutated to TTTGCGGCCTC). NB. C/EBP sites have consensus (A/T)TTGCG(C/T)AA(C/T), although quite substantial variations are tolerated. D) q-RT-PCR of C/EBPβ induction by synaptic activity at 4 h (n=6). E) Deletion analysis of a luciferase-based reporter of the Srxn1 promoter. F) Effect of AP-1 (cfos+cjun) expression on activity of the Srxn1 reporter construct, both wild-type and that containing mutations in the two putative AP-1 binding sites (distal site TGAGTCA mutated to TAAGCTT, proximal site TGAGTCA mutated to TGGGCCC). G) Effect of AP-1 site mutation on activity-dependent induction of the Srxn1 promoter.

Fig. 7. Extrasynaptic NMDARs do not promote antioxidative effects

A) Phospho-(Serine 473)-Akt kinetics in cortical neurons in response to a range of glutamate concentration (normalized to Akt levels, n=3). The dashed line indicates the level of phospho-Akt induced by BiC/4-AP treatment at 2 h. Example Western also shown. B) Subcellular distribution of transfected myc-tagged FOXO1 analyzed by immunofluorescence, and stimulated as indicated for either 30 min or 2 h (n=3). C-F) q-RT-PCR of the indicated genes in response to a range of glutamate concentrations (4 h stimulation). Dashed line indicates the level of expression following 4 h BiC/4-AP stimulation (n=3). G,H) Lack of recovery of PrxII-SO_2/3H following a 1 h exposure to high [H₂O₂] (200 μM). Neurons treated with glutamate for 12 h prior to H₂O₂ exposure. Washout (w/o) period is 16 h. Prx-SO_2/3H levels normalized to Prx loading (n=3). I) Cell death due to 24 h H₂O₂ insult in the face of the indicated glutamate treatments, applied 12 h before insult (n=3).

Fig. 8. Memantine, but not NR2B antagonists, discriminate between pro-survival and pro-death NMDAR signaling

A) Ifenprodil (3 μM) sensitivity of whole-cell and extrasynaptic currents (n=8) isolated using the “quantal block” protocol. B) Cell-death induced by 1 h exposure to 50 μM NMDA ± NMDAR antagonists MK-801 (10 μM), ifenprodil (3 μM), Ro 25-6981 (500 nM) *p<0.05 (n=3). C) Cell death due to 24 h H₂O₂ in the face of the indicated treatments, applied 12 h before insult. *p<0.05 (n=4). D) Txnip mRNA expression in neurons treated as indicated (24 h). *p<0.05 (n=3). E) Cell death due to 24 h H₂O₂ in the face of the indicated treatments. *p<0.05 (n=3). F) Effect of ifenprodil on BiC/4-AP induction of Sesn2-Luc and Srxn1-Luc. *p<0.05 (n=4). G) Effect of memantine (5 or 10 μM) on NMDA (50 μM)-induced cell death. *p<0.05 compared to control NMDA treatment (n=3). H) Cell death due to 24 h H₂O₂ insult in the face of the indicated treatments. *p<0.05 (n=3). I) Subcellular distribution of transfected myc-tagged FOXO1 analyzed by immunofluorescence. J) Txnip mRNA expression in neurons treated for 24 h as indicated. K-M) Effect of memantine on BiC/4-AP-induced changes in Sesn2 (K), Srxn1 (L) and Txnip (M) expression (n=3). N) Effect of memantine on BiC/4-AP-induced protection against H₂O₂ treatment. O) TUNEL analysis of the cortex of P7 mice subjected to IP-injection of memantine (20 mgkg^-1) at P6 (for 24 h, n=6). Dotted line indicated the level of death observed with MK-801 injection (Fig. 1a).

Fig. 9. An ischemic episode, followed by reperfusion, induces overoxidation of peroxiredoxins

Western analysis of Prx-SO_2/3H formation in homogenate from the MCA territory (cortex and striatum) of mice subjected to 60 min MCAO followed by 3 h reperfusion, compared to the equivalent region from sham-operated mice. Analysis (left) of the Western (right) of 6 ischemic and 5 control samples.