Article Figures & Data
Figures
- Fig. 1.
Schematic explanation of neuron–glial coculture setups used in this study. A, For a simple neuron–glial coculture setup the virus-infected glia were transplanted directly into naive (no contact with virus) mixed neuronal–glial cultures after 24 hr of transgene expression. B, Some experiments required a setup by which glia were separated physically from neurons (membrane-delimited coculture). This system consisted of naive cultures prepared in 24-well plates; infected glia were separated by a culture plate insert. Both cocultures were maintained for 24 hr and then exposed to 3 mm glutamate (Glu) for a further 24 hr, followed by quantitation of neuronal viability.
- Fig. 2.
Cortical glia have higher basal Nrf2 expression and ARE promotor activity than neurons. A, Western blot of heterologously expressed Nrf2 (105 kDa) and Nrf2DN (28 kDa) in HEK293 cells. B, A comigrating 105 kDa band corresponding to Nrf2 is observed in enriched cortical glial, but not neuronal, cultures. Densitometric analysis reveals an ∼12-fold difference in Nrf2 protein (n = 3); *p < 0.05. C, Coexpression of Nrf2 cDNA with a hPAP-encoding reporter of ARE-mediated gene expression (rQR51) greatly increases neuronal hPAP expression. Reporter constructs carrying a mutation within the core ARE consensus sequence (rQR51Mut) were not inducible. Coexpression with Nrf2DN cDNA suppresses both neuronal and glial hPAP expression. *p < 0.05, neuron comparison to pEF (empty vector) control; #p< 0.05, glial comparison to pEF control. D, E,Representative hPAP-stained astrocyte-like cells with coexpression of pEF vector only. F, G, With Nrf2 overexpression cells of both neuronal and glial morphology show ARE-driven hPAP expression. Data from hPAP experiments represent the mean ± SEM number of cells counted from triplicate coverslips over four independent experiments.
- Fig. 3.
Ad-Nrf2-infected cultures exhibit enhanced antioxidant potential. A, Time course evaluation of Nrf2 protein overexpression with parallel induction of HO-1 expression.B, Histochemical staining revealed increased NQO1 activity in ad-Nrf2-infected neurons, but not neighboring uninfected neurons, visible under DIC optics (bottom panels). Neuronal NQO1 staining was not observed in the ad-GFP group (top panels). Scale bar, 20 μm. C, xCT mRNA levels increase with Nrf2 overexpression as shown by RT-PCR. Nrf2 mRNA derived from infection was detected by using selective mouse primers. D, Nrf2 overexpression increases total intracellular GSH/GSSG levels. Sublethal glutamate exposure (6 hr) leads to partial depletion of intracellular GSH in all groups (open bars). Control groups represent a separate group of cultures with no glutamate exposure but that were vehicle-treated (filled bars). Culture were given a total of 48 hr for expression before being harvested for GSH analysis. E,Increase in mCBi staining is primarily enriched in glia of ad-Nrf2-infected mixed cultures. Cultures are depicted in phase-contrast (Phase), fluorescence immunostaining for anti-GFAP (GFAP+) and anti-GFP (Infected), and 60 μm mCBi staining (mCBi). mCBi staining images show selected areas with high numbers of glial clusters and are not representative of actual mixed culture composition. HO-1 and NQO1 images are representative of at least three separate experiments. GSH data represent mean ± SEM of four separate experiments performed in duplicate; *p < 0.05, compared with GFP control.
- Fig. 4.
Nrf2 overexpression in a subpopulation of cells confers widespread neuronal protection from oxidative glutamate toxicity. A, Immunocytochemistry for eGFP (green, identifying infected cells) and NSE (red marker, a neuron-selective marker). Within a typical ad-GFP-infected culture the infected neurons (yellow, red + green), uninfected neurons (red), and infected glia (green) can be observed. B, Group data evaluating the vulnerability of infected neurons to oxidative glutamate toxicity. Data are expressed as a percentage of GFP+NSE+ cells (presumed infected neurons) in the indicated glutamate treatment group as compared with the ad-GFP control group. VE, Vitamin E (α-tocopherol), 100 μm.C, Viability of GFP+NSE− cells (presumed infected glia) present per image was not affected significantly with glutamate treatment.D, Uninfected neurons within cultures containing Nrf2-infected cells are more resistant to oxidative glutamate toxicity. Data represent the mean ± SEM number of cells counted over triplicate wells from at least three independent experiments; *p < 0.05, compared with the GFP control no-glutamate group.
- Fig. 5.
Nrf2 overexpression in mixed immature cortical cultures protects neurons from H2O2-mediated toxicity, but not staurosporine-induced apoptosis. Ad-GFP- and ad-Nrf2-infected cultures were allowed to express transgenes for 48 hr before exposure to 0.3–30 μm H2O2 (A;n = 3) or 0.1–10 μm staurosporine (B; n = 4) for a further 24 hr. Cells were stained for NSE to evaluate neuronal viability. Data represent the mean ± SEM from the indicated number of experiments performed in quadruplicate; *p < 0.05, compared with ad-GFP-infected control.
- Fig. 6.
A small fraction of infected glial cells is sufficient to protect neurons from oxidative glutamate toxicity.A, Representative images from glial–neuron coculture setup (see Fig. 1 A). Within a typical coculture infected glia (green) and uninfected neurons (red) can be observed. Uninf, Uninfected glia transplanted. B, Group data obtained from plate scanning for NSE (red) fluorescence.C, Decreased neuronal viability is demonstrated by a loss of red fluorescence. The addition of Nrf2-overexpressing glia restores NSE expression to levels found in an ad-GFP-infected group that was not exposed to glutamate. Data represent the mean ± SEM from three separate experiments performed in quadruplicate; *p < 0.05. Scale bar, 80 μm.
- Fig. 7.
Release of GSH from glia is both sufficient and necessary for conferring neuronal protection. A,B, Infection with ad-Nrf2 increases total intracellular GSH/GSSG as well as GSH released into the medium (MEM/5% FCS, no phenol red). C, Exogenous addition of reduced GSH concurrently with glutamate treatment protects neurons from oxidative glutamate toxicity. D, Glial GSH release is necessary for Nrf2-dependent neuronal protection. A membrane-delimited coculture (see Fig. 1 B) was used, allowing enriched glial cultures to be pretreated separately with the GSH synthesis inhibitor BSO and then to be washed and added to wells containing neurons. BSO pretreatment (200 μm) for 24 hr produces long-term reduction of intracellular GSH/GSSG and release of GSH from glia and abolishes glial-mediated neuronal protection. Data represent the mean ± SEM of at least three independent experiments performed in triplicate; #*p < 0.05.
- Fig. 8.
Neuronal protection can be achieved by activation of endogenous Nrf2 with the use of a small molecule inducer.A, Immature cortical cultures pretreated for 24 hr with 10 and 20 μm tBHQ are protected from 1–3 mmglutamate exposure. Partial protection is conferred by tBHQ treatment at 3 mm glutamate. B, Selective induction of endogenous Nrf2 in glia led to partial neuronal protection from oxidative glutamate toxicity. Glia pretreated with a range of tBHQ concentrations (0–20 μm) for 24 hr were transplanted into naive neuronal cultures at a plating density of 5% of the total cell number, using a coculture setup (see Fig.1 A). Data represent the mean ± SEM from three independent experiments performed in quadruplicate; *p < 0.05.
- Fig. 9.
Schematic diagram of GSH biosynthesis and release pathways that may be involved with Nrf2-dependent coupling of GSH between astrocytes and neurons. Astrocyte GSH synthesis and release are more robust with Nrf2 overexpression. Higher levels of xCT (system xc −) expression were detected, which promotes cystine uptake and provides a precursor for GSH synthesis. Microarray analyses indicate that all major enzymes involved in GSH biosynthesis are upregulated also. GSH release from astrocytes is increased, leading into several possible pathways of extracellular GSH metabolism, including an initial breakdown by γ-glutamylcysteine transpeptidase (γGT) and possible further breakdown by aminopeptidase (Apep) into the glutathione precursors Cys, Gly, and CysGly suitable for neuronal uptake (Dringen et al., 1999, 2001). Alternatively, extracellular GSH may be taken up by neurons directly or may contribute to the reduction of cystine into cysteine, which may be a source of sulfhydryl species for neuronal uptake (Sagara et al., 1993;Wang and Cynader, 2000). Neurons may also uptake cystine for glutathione synthesis (Murphy et al., 1990). Microarray analyses also indicate the upregulation of additional factors involved in detoxification, ROS scavenging, and NADPH production that may work together with GSH to protect neurons. Genes that are upregulated significantly by Nrf2 overexpression are underlined. Intracellular and extracellular concentrations of glutamate are average values from a combination of previous studies in the human brain and CSF (Siegel, 1981). CSF levels of cystine are typically very low at ∼0.2 μm (Lakke and Teelken, 1976; Araki et al., 1988; Wang and Cynader, 2000). The Invitrogen MEM used in this study is formulated to contain 100 μm cystine.
Tables
- Table 1.
Evaluation of Na+-independentl-[3H]-glutamate uptake by systemx
in ad-Nrf2-infected mixed immature cortical cultures: percentage increase and pharmacology of induced uptakead-GFP ad-Nrf2 ad-Nrf2DN 100% 372 ± 82%* 76.7 ± 7.1%* Compound ad-GFP (%) ad-Nrf2 (%) None 100 372 ± 82 l-Cystine 115.4 ± 13.2 86.5 ± 16.01-159 Quisqualic acid 77.8 ± 10.8 71.8 ± 10.51-159 Homocysteic acid 74.2 ± 22.0 80.0 ± 31.01-159 -
Data represent the mean ± SEM of five independent experiments performed in quadruplicate.
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↵* p < 0.05, compared with ad-GFP control.
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↵F1-159 p < 0.05, compared with ad-Nrf2 control with no compounds. Uptake assay performed on mixed neuron/glial immature cultures. The basal rate ofl-[3H]-glutamate uptake by ad-GFP-expressing cultures is 59.1 ± 5.7 fmol/mg protein per min.
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Cell type Ad-GFP Ad-Nrf2 Ad-Nrf2DN Infected neuron 21.1 ± 4.9 (60) 40.6 ± 10 (60)* 25.0 ± 5.2 (60) Uninfected neuron 19.0 ± 4.9 (60) 34.1 ± 10 (60)* 19.9 ± 4.5 (60) Infected glia 61.6 ± 11 (25) 119 ± 17 (59)* 35.1 ± 5.2 (40)*,2-159 -
Data represent the mean ± SEM, in arbitrary units of fluorescence intensity. Cell type was identified based on morphology (glial cells larger with web-like processes). The total number of cells evaluated for each group is listed in parentheses; with data collected from cells over three independent experiments performed in duplicate.
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↵* p < 0.001, compared with ad-GFP of same cell type.
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↵F2-159 p < 0.05, compared with ad-GFP of same cell type.
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- Table 3.
Common Nrf2 upregulated genes in glial-enriched cultures and mixed neuronal/glial cultures
GenBank accession number Gene Glial-enriched Neuron/glial mixed R Fold CV BS R Fold CV BS Detoxification M58495 NAD(P)H:quinone oxidoreductase 8 4.3 0.24 2621 18 5.4 0.56 141 K00136 Glutathione S-transferase A1 or 2 6 17.6 0.41 24 12 8.8 0.80 100 K01932 Glutathione S-transferase A3 8 12.3 0.77 4657 18 19.8 0.56 223 X62660 Glutathione S-transferase A4 8 21.7 0.56 443 18 4.1 0.42 769 X02904 Glutathione S-transferase P2 8 3.4 0.38 22952 18 2.4 0.25 7230 J02722 Heme oxygenase 1 8 2.8 0.18 24137 18 21.3 1.2 358 M11670 Catalase 8 4.1 0.34 1381 16 1.7 0.22 593 M11794 Metallothionein 1 or 2 4 3.7 0.56 15750 16 2.83 0.35 1274 AI010083 Peroxiredoxin 1 8 2.9 0.06 32993 18 2.5 0.25 6609 AF014009 Peroxiredoxin 6 (1-cys peroxiredoxin) 8 3.3 0.12 6153 14 1.8 0.20 1772 M21060 Cu/Zn superoxide dismutase 8 1.5 0.09 28627 16 1.9 0.27 14495 AF106563 p-glycoprotein/multi-drug resistance protein (MDR2) 6 1.9 0.23 1459 10 5.9 0.71 2671 X90642 Multi-drug resistance protein (MRPI) 8 2.3 0.33 3432 14 1.7 0.13 1317 Signal transduction U09583 Src-related tyrosine kinase 8 22.6 0.90 89 18 25.6 0.16 56 AI231354 Stress-activated protein kinase, α 8 3.1 0.10 1097 14 1.65 0.26 880 D78610 Protein tyrosine phosphatase receptor E 7 6.7 0.27 83 14 0.0 0.00 858 M15427 c-raf 8 3.6 0.25 3558 18 1.6 0.22 7427 U31668 E2F-5 transcription factor 8 2.8 0.06 2242 18 1.7 0.16 933 U04835 cAMP-responsive element modulator 8 2.0 0.20 2242 10 2.7 1.1 756 Antioxidant/reducing potential/metabolism AI233261 Glutamate-cysteine ligase, modifier subunit (γGCS) 8 13.0 0.13 945 18 5.1 0.21 208 U73174 Glutathione reductase 1 8 3.3 0.15 1667 14 2.0 0.32 1324 U63923 Thioredoxin reductase 1 8 2.6 0.16 10344 16 3.4 0.56 3426 M26594 Malic enzyme 1 8 8.3 0.23 1264 18 10.1 0.35 227 AI169802 Ferritin H subunit H 7 1.8 0.23 50557 10 1.3 0.13 18452 J02791 Acyl-coenzyme A dehydrogenase, medium chain 8 3.2 0.22 925 12 3.2 0.87 42 M60322 Aldose reductase 1 8 5.1 0.15 24812 18 2.4 0.18 9958 AA799452 Transaldolase 8 3.6 0.25 21356 18 1.8 0.23 19236 M96633 Mitochondrial intermediate peptidase 8 2.8 0.30 699 9 1.5 0.24 452 AA945054 Cytochrome b5 8 2.6 0.40 1902 14 1.6 0.27 2179 S53527 S-100 calcium binding protein β 8 78.7 1.15 1167 10 2.3 0.68 67 U26714 Solute carrier family 29 (iron-regulated transporter) 6 21.3 0.37 402 18 5.9 0.27 102 Inflammation AI176170 FK506 binding protein 6 1.5 0.20 19602 18 1.3 0.06 18354 AA818025 CD59 antigen 8 25.9 0.04 1128 18 1.4 0.08 10966 U03388 Cyclooxygenase 1 8 3.9 0.23 2083 18 5.6 0.50 312 Housekeeping V01217 β actin 186225 164621 X02231 Glyceraldehyde-3-phosphate-dehydrogenase 405 348 U07181 Lactate dehydrogenase B 13108 11253 -
Genes with fold change >1.3 are listed. R, Relative rank; CV, coefficient of variation; BS, basal signal intensity from GFP-infected cultures. Data shown are from n = 2 glial-enriched cultures and n = 3 mixed cultures.
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- Table 4.
Nrf2 upregulated genes specific to glial enriched cultures or mixed neuronal/glial cultures
GenBank accession number Gene R Fold CV BS Neuron increased genes Antioxidant/reducing potential AF090867 Guanosine monophosphate reductase 12 2.5 0.41 330 Signal transduction M54987 Corticotropin-releasing hormone 12 6.8 1.20 120 M19651 Fos-related antigen (Fra-1) 16 7.4 1.10 70 D15069 Adrenomedullin 12 12.7 0.74 106 Miscellaneous M84488 Vascular cell adhesion molecule-1 18 16.5 0.85 74 Astrocyte increased genes Detoxification/metabolism S83436 Glutathione S-transferase, mitochondrial 4 3.9 0.27 341 AF041105 Organic anion transporter protein 4 11.3 0.47 21 S56936 UDP-glucuronosyltransferase, bilirubin-specific 6 3.0 0.12 382 X56228 Rhodonase, thiosulfate sulphurtransferase 8 7.7 0.65 1686 J02852 Cytochrome P450 2A3 6 3.2 0.45 23 Antioxidant/reducing potential X03518 γ-Glutamyl transpeptidase 4 6.5 0.15 149 L38615 Glutathione synthetase 8 3.9 0.04 1328 Signal transduction/apoptosis AI228669 GABA transporter protein 8 25.2 0.40 26 L00981 Tumor necrosis factor 4 6.7 0.13 182 M91595 Insulin-like growth factor binding protein 2 8 3.5 0.28 4228 U63740 Protein kinase C-binding protein ζ 1 4 6.2 0.81 86 AI072770 Proteolipid protein 6 10.6 1.10 137 AI145444 Neurabin, actin-binding protein 4 3.3 0.13 23 AF061726 Calpain 3 8 3.3 0.24 176 Inflammation AF010464 Interleukin 7 4 7.8 0.26 32 U66322 Dithiolethione-inducible gene (DIG-1) 8 3.0 0.52 1515 M16410 Tachykinin 2 4 4.5 0.65 244 Miscellaneous AF098301 NFB42-neural F box 8 42.6 0.86 841 X99773 Neuroserpin, serine proteinase inhibitor 8 6.3 0.58 187 U09022 15 kDa perforational protein (PERF) 8 97.0 0.62 29 AF029690 8-Oxoguanine-DNA-glycosylase 8 4.7 0.13 1248 -
Genes with fold change >1.3 are listed. R, Relative rank; CV, coefficient of variation; BS, basal signal intensity from GFP-infected cultures. Data shown are from n = 2 glial-enriched cultures and n = 3 mixed cultures.
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