Mild oxidative stress activates Nrf2 in astrocytes, which contributes to neuroprotective ischemic preconditioning.

Haskew-Layton et al. (1) reported that subtoxic doses of H2O2 fails to activate nuclear factor erythroid 2-related factor (Nrf2) in astrocytes and triggers Nrf2-independent responses that protect cocultured neurons. Contrary to this, we show that mild oxidative insults, including subtoxic H2O2, strongly activate astrocytic Nrf2/antioxidant response element (ARE)-dependent gene expression, which, moreover, contributes to neuroprotective ischemic preconditioning. In mixed neuron/astrocyte cultures (2, 3), treatment with physiologically relevant H2O2 doses (25–100 μM, similar/less than those recorded postischemia) (4) induced Nrf2-target genes sulfiredoxin (Srxn1) and heme-oxygenase 1 (Hmox1) in wild-type but not Nrf2 cultures (Fig. 1). Similarly, exposing cultures to oxygen–glucose deprivation (OGD; an in vitro ischemia model), followed by reoxygenation, also induced Nrf2target genes. Induction of Hmox1 in mixed cultures was restricted to astrocytes (Fig. 1), and Nrf2-target gene induction was not observed in enriched neuronal cultures (<0.2% astrocytes) (Fig. 1), strongly suggesting that astrocytes are the sole locus for Nrf2 activation by oxidative stress. Furthermore, study of enriched Nrf2 and Nrf2 astrocyte cultures showed clear H2O2 (and OGD)-induced Nrf2-dependent gene activation, contrary to that reported in ref. 1. One possible explanation for this discrepancy lies in their Nrf2 assay: a luciferase reporter incorporating the ARE of the NQO1 promoter (1). Different AREs can have different Nrf2 dependencies for basal and/or inducible activity, and we observe relatively weak Nqo1 induction by 100 μM H2O2 (2.1± 0.06fold; n = 5). Basal Nrf2 activity seems sufficient for strong Nqo1 expression in astrocytes: basal Nqo1 expression in Nrf2 cultures is only 14 ± 2% of that in WT. Another potential explanation is that H2O2 doses >30 μM were not studied, because 100 μM were reportedly toxic based on 3-(4,5-Dimethylthiazol-2-yl)2,5-diphenyltetrazolium (MTT) assay (1). However, the MTT assay may overstate toxicity, because it measures cellular NAD (P)H-dependent reducing activity, which could drop if subtoxic stress causes NAD(P)H levels to fall. We see no evidence of H2O2 toxicity in astrocytes up to 200 μM, as assessed by either ATP assay or nuclear integrity (Fig. 1). In contrast to the reported Nrf2-independence of adaptive astrocytic neuroprotective responses acting on immature neurons (48 h in culture) (1), we find that astrocytic Nrf2 contributes to adaptive neuroprotective responses in more mature neurons (Fig. 2). A brief, nonneurotoxic episode of OGD (90 min) applied to mixed cultures preconditions neurons against a subsequent neurotoxic OGD episode 24 h later (Fig. 2). This preconditioning episode activates Nrf2 in mixed cultures (Srxn1: 1.97± 0.06-fold; Hmox1: 1.48± 0.08-fold; n = 6) but not in pure neuronal cultures, and Hmox1 induction is restricted to astrocytes. In Nrf2 cultures, neuronal vulnerability to OGD was similar to Nrf2 cultures (Fig. 2). However, the brief OGDinduced preconditioning effect was substantially lower in Nrf2 cultures (Fig. 2), strongly implicating Nrf2 activation in ischemic preconditioning. This response may also be relevant in vivo: a standard preconditioning inducing stimulus in adult mice (15min occlusion of the middle cerebral artery) triggered Nrf2-target gene induction in the ipsilateral cortical hemisphere (Fig. 2). Finally, we observe that subtoxic H2O2 also induces Hmox1/Srxn1 expression in human ES cell-derived astrocytes (Fig. 2), suggesting that human Nrf2 is activated by mild oxidative stress. Thus, in addition to Nrf2-independent pathways (1), astrocytic Nrf2dependent pathways are likely to be important mediators of neuroprotective adaptive responses to oxidative stress.