Proceedings of The Physiological Society
King's College London (2011) Proc Physiol Soc 22, C21
Astrocytic Nrf2 is a mediator of ischemic preconditioning
K. Bell1, B. Mubarak1, J. Fowler2, P. Baxter1, K. Gupta4, T. Tsujita3, S. Chowdhry3, R. Patani4, S. Chandran4, K. Horsburgh2, J. Hayes3, G. Hardingham1
1. Centre for Integrative Physiology, University of Edinburgh, Edinburgh, United Kingdom. 2. Centre for Cognitive and Neural Systems, University of Edinburgh, Edinburgh, United Kingdom. 3. Biomedical Research Institute, University of Dundee, Dundee, United Kingdom. 4. Euan MacDonald Centre for MND Research, University of Edinburgh, Edinburgh, United Kingdom.
The transcription factor Nrf2 regulates the Phase II antioxidant response, enhancing resistance to oxidative insult. Whether Nrf2 forms part of an endogenous protective response in the brain however, is not clear. Nrf2 has been reported to be activated following mild oxidative stress in non-neural tissues, we therefore sought to determine whether the same is the case in neural tissue. To achieve this, Nrf2 target gene expression was quantified by qrtPCR in DIV 9-11 primary mouse cortical neuron/astrocyte cultures (90%NeuN+/10%GFAP+), which were exposed to either peroxide (0, 25, 50uM) or oxygen glucose deprivation (OGD, 3 hour, treatments same throughout). Nrf2 target genes sulfiredoxin (Srxn1) and heme-oxygenase (Hmox1) were both significantly induced by peroxide (n=4, p<0.05 student t-test throughout unless otherwise specified) and OGD (n=5, p<0.05). To confirm Nrf2-specific induction, experiments were repeated in Nrf2 -/- neurons, revealing no induction post-OGD or post-peroxide (n=4-8, p>0.05), demonstrating Nrf2-dependent gene induction. Astrocytes appear to be the sole locus for Nrf2 activation as peroxide or OGD exposure fail to induce gene induction in pure neuronal cultures (>98%NeuN+, n=4-5, p>0.05), but significantly increase both Srxn1 and Hmox1 expression in pure astrocytic cultures (>96%GFAP+, n=3, p<0.05). Moreover, Hmox1 protein induction in mixed cultures is localized to astrocytes post-insult. To determine whether astrocytic Nrf2 activation could protect against a subsequent insult a preconditioning protocol was established. Exposure to 1.5 hour OGD significantly protected cells from a subsequent 3 hour insult 24 hours later (cell death assessed by DAPI-assisted quantification, n=5, p<0.05, ANOVA + post-hoc). Both Srxn1 and Hmox1 are upregulated following 1.5 hour OGD (n=4, p<0.05), indicating that the preconditioning stimulus is sufficient to recruit Nrf2. Applying the preconditioning protocol to both Nrf2 wild type and -/- neurons (n=5,11 respectively), revealed a significant decrease in protection in the absence of Nrf2 (p<0.05, ANOVA + post-hoc), highlighting the central role of astrocytic Nrf2 in mediating an acquired neuroprotective response. Nrf2 may also have a role in acquired neuroprotection in vivo as transient occlusion of the middle cerebral artery in adult mice, following anesthesia induction and maintenance with isoflurane (2%) in a mixture of 30% O2 and 70% N2O by face mask, for a duration known to trigger preconditioning, led to a significant increase in Srxn1 and HO-1 gene expression in the preconditioned hemisphere (n=6, p<0.01, paired t-test). The observed Nrf2-dependent recruitment of distinct and complimentary antioxidant pathways following transient episodes of oxidative stress both in vitro and in vivo1 suggests the presence of an endogenous protective response and highlights the importance of Nrf2 as a therapeutic target.
Where applicable, experiments conform with Society ethical requirements