Astroglial NF-κB mediates oxidative stress by regulation of NADPH oxidase in a model of retinal ischemia reperfusion injury - PubMed (original) (raw)

Comparative Study

Astroglial NF-κB mediates oxidative stress by regulation of NADPH oxidase in a model of retinal ischemia reperfusion injury

David J Barakat et al. J Neurochem. 2012 Feb.

Abstract

Astrocytes undergo rapid activation after injury, which is mediated in part by the transcription factor nuclear factor-kappaB (NF-κB). Consequently, activated astrocytes have been shown to induce the NF-κB regulated phagocyte NADPH oxidase (PHOX), resulting in elevated production of reactive oxygen species. We investigated the regulatory mechanisms of PHOX-induced oxidative stress in astrocytes and its non-cell-autonomous effects on retinal ganglion cell loss following retinal ischemia-reperfusion (IR) injury. To study PHOX activity and neurotoxicity mediated by glial NF-κB, we employed GFAP-IκBα-dn transgenic mice, where the NF-κB canonical pathway is suppressed specifically in astrocytes. Our analysis showed that NF-κB activation in astrocytes correlated with an increased expression of PHOX and reactive oxygen species production in primary cells and whole retinas subjected to oxygen-glucose deprivation or IR injury. Selective blockade of NF-κB in astrocytes or application of NADPH oxidase inhibitors suppressed retinal ganglion cell loss in co-cultures with astroglia challenged by oxygen-glucose deprivation. Furthermore, genetic suppression of astroglial NF-κB reduced oxidative stress in ganglion layer neurons in vivo in retinal IR. Collectively, our results suggest that astroglial NF-κB-regulated PHOX activity is a crucial toxicity pathway in the pathogenesis of retinal IR injury.

Published 2011. This article is a US Government work and is in the public domain in the USA.

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Figures

Figure 1

Figure 1. Inhibition of astroglial NF-κB reduces oxidative stress following retinal IR injury

(A) Representative images showing DNA/RNA damage localizing to NeuN-positive cells detected by 8-OH-guanosine (red) immunofluoresent labeling. Double-labeled cells are abundant in the GCL of the central retina 3 days after IR injury. Scale bar, 100 mm (B) Quantification of DNA/RNA damage; total counts of 8-OH-guanosine/NeuN/Hoechst-positive cells were averaged from the sum of 4 standard fields in each retinal region. Values are means ± SEM, n=6; *p<0.05.

Figure 2

Figure 2. Inhibition of aNF-κB promotes survival of retinal ganglion cells in vitro after OGD

(A–C) Quantification of active caspase-3 positive RGCs in co-culture with astrocytes 24 hours after OGD challenge (OGD) vs. normoxic controls (Control) performed by confocal microscopy. The total quantities of NeuN or beta-III tubulin/Hoechst positive RGCs observed in 5 randomly selected fields of each experimental plate are compared. Values are means ± SEM, n=5; *p<0.05. (A) Activated caspase-3 positive RGCs in co-culture with WT and TG astrocytes; (B) Activated caspase-3 positive WT RGC in co-cultures with astrocytes treated with the NADPH oxidase inhibitors DPI (1µM) or apocynin (100µM); (C) Activated caspase-3 positive RGCs in co-cultures of WT astrocytes with WT or p47null RGCs (D) Cell death rates in WT and p47null RGCs challenged by astrocyte-conditioned media (ACM) from primary astrocytes subjected to OGD challenge (OGD ACM) or from normoxic control astrocytes (Control ACM). The percentages of dead (PI/Hoechst positive) and apoptotic (Annexin V/Hoechst positive) RGCs in ACM-treated cultures were calculated relative to total quantities of Hoechst positive cell in 5 randomly selected fields. Values are means ± SEM, n=5; *p<0.05.

Figure 3

Figure 3. Transcriptional regulation of NADPH oxidase following OGD in primary astrocytes

(A–B) Gene expression analysis of the p47PHOX and gp91PHOX genes 6, 9 and 12 hours after OGD challenge. Changes were calculated as percentages of untreated control (Control) value. (C) Analysis of p47PHOXmRNA decay in actinomysin D (5µg/ml) treated cells with or without cycloheximide (5µg/ml) 12 hours after OGD treatment. (D) Relative abundance of p47PHOX transcript levels in astrocytes treated with vehicle or cycloheximide (5µg/ml). Gene expression levels were normalized to β-actin. Values are means ± SEM, n=4 and *p<0.05.

Figure 4

Figure 4. Regulation of homeostatic levels of PHOX is NF-κB dependent

(A) Gene expression analysis of gp91PHOX and p47PHOX transcripts 12 hours after OGD. Changes were calculated as percentages of untreated WT control (Control) values (n=5, *p<0.05). (B) Time course analysis of gp91PHOXand p47PHOXprotein levels after OGD by quantitative western blot. (C) Quantification of the data shown in (B): the gp91PHOX and p47PHOX band intensities were normalized to β-actin (n=3, *p<0.05). (D) Relative expression levels of genes encoding PHOX subunit in untreated WT and TG astrocytes (n=5; p<0.01).

Figure 5

Figure 5. ROS production by PHOX in astrocytes is regulated by NF-κB

(A) WT and TG astrocytes were assayed for superoxide production by Diogenes luminescence in control cultures and 24 hours after OGD challenge (OGD) (RLU = relative light units; n=5, p<0.05). (B) Transfection of p47PHOX siRNA reduces p47PHOX protein levels. Cell lysates were analyzed by western blot 24 hours post-transfection. (C) ROS production in live astrocytes was detected by Diogenes chemiluminescence 24 hours following OGD in non-targeting control siRNA (control) and p47PHOXsiRNA transfected cells. Astrocytes were transfected with siRNA 24 hours prior to OGD treatment. (n=5, *p<0.01). (D) Analysis of ROS production in WT and TG astrocytes 20 minutes after addition of PMA (1ng/ml) in control cultures and 24 hours after OGD (n=5, *p<0.01). (E) Representative recordings of ROS production from (D) in WT vs TG cells. Chemiluminesence was quenched by the addition of SOD (20U/ml). (F) Analysis of p47PHOX phosphorylation status by western blot in primary astrocyte extracts. OGD challenged and control astrocytes were treated with DMSO vehicle or PMA (1ng/ml) for 20 minutes prior to cell lysis.

Figure 6

Figure 6. The effects of NF-κB and PHOX suppression on OGD-induced cell death in primary astrocytes

(A–B) Cell death rates in cultures determined by quantification of cells with PI (red) and AnnexinV (green) labeling 24 hours after OGD (n=3, *p<0.05). (B) Representative images from experiment A showing abundant dead (PI-positive or PI/AnnexinV-positive) cells in WT but not in TG cultures; Hoechst dye (blue) labels nuclei for total cell counts. (C) Cell death rates (% of PI- positive among Hoechst- positive) cells in vehicle-treated control and 1µM DPI-treated astrocytes cultures 24 hours after OGD (n=4, *p<0.05). Scale bar 50µm.

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