Induction of ischemic preconditioning (IPC) represents a potential therapy against cerebral ischemia by activation of adaptive pathways and modulation of mitochondria to induce ischemic tolerance to various cells and tissues. Mitochondrial dysfunction has been ascribed to contribute to numerous neurodegenerative conditions and cerebral ischemia. Nuclear erythroid 2-related factor 2 (Nrf2) is a transcription factor that has traditionally been involved in upregulating cellular antioxidant systems to combat oxidative stress in the brain; however, the association of Nrf2 with mitochondria in the brain remains unclear. In the present study, we investigated the effects of Nrf2 on (i) IPC-induced protection of astrocytes; (ii) OXPHOS protein expression; and (iii) mitochondrial supercomplex formation.
Oxygen-glucose deprivation (OGD) was used as an in vitro model of cerebral ischemia and IPC in cultured rodent astrocytes derived from WT C57Bl/6J and Nrf2−/− mice. OXPHOS proteins were probed via western blotting, and supercomplexes were determined by blue native gel electrophoresis.
IPC-induced cytoprotection in wild-type, but not Nrf2−/− mouse astrocyte cultures following a lethal duration of OGD. In addition, our results suggest that Nrf2 localizes to the outer membrane in non-synaptic brain mitochondria, and that a lack of Nrf2 in vivo produces altered supercomplex formation in mitochondria.
Our findings support a role of Nrf2 in mediating IPC-induced protection in astrocytes, which can profoundly impact the ischemic tolerance of neurons. In addition, we provide novel evidence for the association of Nrf2 to brain mitochondria and supercomplex formation. These studies offer new targets and pathways of Nrf2, which may be heavily implicated following cerebral ischemia.
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This study was funded by grants from the National Institutes of Health, National Institute of Neurological Disease and Stroke (NINDS) NS45676, NS054147, NS34773, F31 NS080344.
Conflict of Interest
There authors declare that they have no conflicts of interest.
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. This article does not contain any studies with human participants performed by any of the authors.
Electronic Supplementary Materials
Supplementary Fig. 1 Genotyping of Nrf2−/− and WT C57Bl/6J mice. LAC Z denotes the portion of the Nrf2 locus that has been replaced with a LAC Z and Neomycin resistance gene cassette. Presence of LAC Z indicates animals that have this gene in place of exon 4 and exon 5 of the Nrf2 gene and denotes a knockout animal. Presence of WT allele denotes presence of Nrf2 gene. Presence of both LAC Z and WT gene indicate a heterozygous knockout animal. Genotyping was performed by Transnetyx, Inc. (Cordova, TN, USA). (TIFF 306 kb)
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Narayanan, S.V., Dave, K.R. & Perez-Pinzon, M.A. Ischemic Preconditioning Protects Astrocytes against Oxygen Glucose Deprivation Via the Nuclear Erythroid 2-Related Factor 2 Pathway. Transl. Stroke Res. 9, 99–109 (2018). https://doi.org/10.1007/s12975-017-0574-y
- Reactive oxygen species