FIG. 5.

Lentiviral over-expression of PRX2 enhances Trx-ASK1 association and protects against oxygen and glucose deprivation (OGD)-induced neuronal injury in cortical neuronal cultures. Primary cortical neurons at 9 days in vitro were infected for 3 days with empty lentivirus or lentiviral (Ln) vector carrying GFP (LnGFP), or with hemagglutinin (HA)-tagged human PRX2 (PRX2) or mutant PRX2 in which Cys⁵¹ and Cys¹⁷² were replaced by alanine (PRX2_C/A). (A) Lentivirus-mediated overexpression of PRX2 in neurons was confirmed by Western blotting against the HA tag and PRX2, respectively, and by triple-label immunofluorescence [GFP (a,b) or HA (c, d), MAP2 (b,d,f), and DAPI (b,d,f)]. Scale bar, 50 μm. (B, C) Transfection of PRX2 enhances PRX and Trx activity after OGD. Infected neurons were subjected to OGD for 60 min and, at the indicated time points after OGD, cytosolic proteins were processed for PRX activity (B) and Trx activity (C) assays. Note that transfection of PRX2 but not of PRX2_C/A reinstated both PRX and Trx activities to nearly preischemia levels at 1, 3, and 6 h after OGD. Data are expressed as percentage changes over control (Con) cell cultures without OGD challenge. (D) Transfection of PRX2 but not PRX2_C/A enhances Trx-ASK1 association and inhibits ASK1 activation after OGD. Protein extracts were prepared from infected cells at 3 h after OGD and processed for ASK1 IP followed by immunoblotting against Trx or the ASK1 activity assay. Semi-quantitative data are expressed as the fractions of Trx that bound to ASK1 (right upper panel) or the fold changes of ASK1 activity over control cultures (right lower panel). Data are based on three to four independent experiments. (E, F) Transfection of PRX2 but not of PRX2_C/A protects against neuronal cell death at 24 h after 60 min of OGD. Representative triple-label fluorescence images for DNA damage are presented in (E), where neurons were triple-labeled for GFP (green, a, b) or HA (green, c, d) with TUNEL (red) and DAPI (blue). Scale bar, 50 μm. (F) Cell viability was quantified using the Alamar blue assay (left panel); cell death was assessed by measuring lactate dehydrogenase (LDH) release (upper right panel) and by counting TUNEL-positive cells (lower right panel). n=12 from 3 independent experiments. All quantitative data are presented as mean±SEM, *p<0.05, **p<0.01, ***p<0.001 versus Ln- or LnGFP-infected cell cultures under the indicated experimental conditions. Multiple comparisons were made using one-way ANOVA, followed by Bonferroni's/Dunn's post hoc analysis. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).