Tat-antioxidant 1 protects against stress-induced hippocampal HT-22 cells death and attenuate ischaemic insult in animal model

Abstract

Oxidative stress-induced reactive oxygen species (ROS) are responsible for various neuronal diseases. Antioxidant 1 (Atox1) regulates copper homoeostasis and promotes cellular antioxidant defence against toxins generated by ROS. The roles of Atox1 protein in ischaemia, however, remain unclear. In this study, we generated a protein transduction domain fused Tat-Atox1 and examined the roles of Tat-Atox1 in oxidative stress-induced hippocampal HT-22 cell death and an ischaemic injury animal model. Tat-Atox1 effectively transduced into HT-22 cells and it protected cells against the effects of hydrogen peroxide (H2O2)-induced toxicity including increasing of ROS levels and DNA fragmentation. At the same time, Tat-Atox1 regulated cellular survival signalling such as p53, Bad/Bcl-2, Akt and mitogen-activate protein kinases (MAPKs). In the animal ischaemia model, transduced Tat-Atox1 protected against neuronal cell death in the hippocampal CA1 region. In addition, Tat-Atox1 significantly decreased the activation of astrocytes and microglia as well as lipid peroxidation in the CA1 region after ischaemic insult. Taken together, these results indicate that transduced Tat-Atox1 protects against oxidative stress-induced HT-22 cell death and against neuronal damage in animal ischaemia model. Therefore, we suggest that Tat-Atox1 has potential as a therapeutic agent for the treatment of oxidative stress-induced ischaemic damage.

Keywords: Tat-Atox1; ischaemic injury; oxidative stress; protein therapy; protein transduction domain.

Figure 1

Purification and transduction of Tat-Atox1 protein into HT-22 cells. Overview of Tat-Atox1 protein (A). Expression and purification of Tat Atox1 protein (B) and control Atox1 protein (C) were detected by Western blot analysis using 15% SDS-PAGE and rabbit anti-polyhistidine antibody. Tat-Atox1 and control Atox1 proteins (0.5–3 μM) were added to the culture media for 1 hr (D), Tat-Atox1 and control Atox1 proteins (3 μM) were added to the culture media for 10–60 min. (E), Tat-Atox1 protein (3 μM) was transduced into cells for 1 hr and the cells were incubated for 36 hrs (F). Then the cells were treated with trypsin-EDTA, washed with PBS three times. Transduction of Tat-Atox1 protein was measured by Western blotting, and the intensity of the bands was measured by a densitometer. The localization of transduced Tat-Atox1 protein (G). After transduction of Tat-Atox1 protein (3 μM) for 1 hr, the localization of transduced Tat-Atox1 protein was examined by confocal fluorescence microscopy; scale bar = 20 μm.

Figure 2

Protective effects of transduced Tat-Atox1 protein against oxidative stress. Pre-treatment of HT-22 cells with Tat-Atox1 protein (0.5–3 μM) and control Atox1 protein for 1 hr and treatment with 1 mM hydrogen peroxide (H₂O₂) for 2 hrs. Then, cell viability was assessed by MTT assay (A). *P < 0.05 and **P < 0.01 compared with H₂O₂-treated cells. Effects of transduced Tat-Atox1 protein on H₂O₂-induced ROS production. Treatment with Tat-Atox1 protein (3 μM) and control Atox1 protein was followed by 10 min. treatment with H₂O₂ (1 mM). Intracellular ROS levels were measured by DCF-DA staining and fluorescence intensity was measured by ELISA plate reader (B); scale bar = 50 μm. **P < 0.01 compared with H₂O₂-treated cells. Effects of transduced Tat-Atox1 protein on H₂O₂-induced DNA fragmentation. One-hour pre-treatment of HT-22 cells with Tat-Atox1 protein (3 μM) and control Atox1 protein was followed with 4-hr treatment with H₂O₂ (1 mM). DNA fragmentation was measured by TUNEL staining and the fluorescent intensity was measured by ELISA plate reader (C); scale bar = 50 μm. **P < 0.01 compared with H₂O₂-treated cells.

Figure 3

Effects of Tat-Atox1 protein on H₂O₂-induced pro- and anti-apoptotic proteins. One-hour pre-treatment of HT-22 cells with Tat-Atox1 protein (0.5–3 μM) and control Atox1 protein was followed with treatment with H₂O₂ (1 mM) for 30 min. (Bcl-2/Bax) and 20 min. (p-p53/p53), respectively. The expression levels of Bcl-2/Bax (A) and p-p53/p53 (B) were determined by Western blot analysis and the band intensity was measured by densitometer. (*P < 0.01 compared with H₂O₂-treated cells).

Figure 4

Effects of Tat-Atox1 protein on oxidative stress-induced pAkt/Akt (ser-473), pBad/Bad (s112) and MPPKs. Pre-treatment of HT-22 cells with Tat-Atox1 protein (0.5–3 μM) and control Atox1 protein was followed with treatment with H₂O₂ (1 mM) for 10 min. (pAkt/Akt) and 10 min. (pBad/Bad), respectively. H₂O₂-induced pAkt/Akt (A) and pBad/Bad (B) expression levels were determined by Western blot analysis and the band intensity was measured by densitometer. The cells were stimulated with H₂O₂ (1 mM) for 30 min. with or without pre-treated with Tat-Atox1 protein (0.5–3 μM) for 1 hr. Then, the cells were prepared and analysed for phosphorylation of p38 (C), ERK (D), JNK (E) levels by Western blotting and the band intensities were measured by densitometer. (*P < 0.01 compared with H₂O₂-treated cells).

Figure 5

Effect of transduced of Tat-Atox1 protein on animal brain. Transduction of Tat-Atox1 protein into brain (A). Animals were treated with single i.p. injection of Tat-Atox1 protein and killed after 6 hrs. Transduction of Tat-Atox1 protein into the CA1 region was determined by immunohistochemistry with a rabbit anti-polyhistidine antibody and FITC-conjugated anti-rabbit IgG. Immunohistochemistry for NeuN in the hippocampal CA1 region (B). Control, ischaemia, Tat peptide, Atox1 protein and Tat-Atox1 protein-treated groups 4 days after ischaemia-reperfusion. SP, stratum pyramidale; SO, stratum oriens; SR, stratum radiatum; bar = 50 μm. The relative number of NeuN-immunoreactive neurons versus control group per section in all the groups (n = 5 per group; *P < 0.05, significantly different from the control group, ^#P < 0.05, significantly different from the ischaemia group). The bars indicate standard error of mean (SEM).

Figure 6

Inhibitory effects of transduced Tat-Atox1 protein in ischaemic animal models. Immunohistochemistry for GFAP (a, c, e, g and i) and Iba-1 (b, d, f, h and j) in the CA1 region of the control (a and b), ischaemia (c and d), Tat peptide (e and f), Atox1 protein (g and h) and Tat-Atox1 protein-treated (i and j) groups 4 days after ischaemia/reperfusion (A). SP, stratum pyramidale; SO, stratum oriens; SR, stratum radiatum; bar = 50 μm. The locomotor activity in gerbils before and 1 day after ischaemia-reperfusion in ischaemia, Tat peptide, Atox1 protein and Tat-Atox1 protein-treated groups. Spontaneous locomotor activity is evaluated in terms of entire distance (metres) travelled before and 1 day after ischaemia-reperfusion (B) (n = 5 per group; *P < 0.05, significantly different from the before group, ^#P < 0.05, significantly different from the ischaemia group). The bars indicate standard error (SE). Analysis of HNE levels in the hippocampus of control, ischaemia, Tat peptide, Atox1 protein and Tat-Atox1 protein-treated groups at 3 hrs after ischaemia-reperfusion (C) (n = 5 per group, *P < 0.05, significantly different from the control group, ^#P < 0.05, significantly different from the ischaemia group). The bars indicate SE.