Overexpression of copper/zinc superoxide dismutase in transgenic rats protects vulnerable neurons against ischemic damage by blocking the mitochondrial pathway of caspase activation

Abstract

Mitochondria are known to be involved in the early stage of apoptosis by releasing cytochrome c, caspase-9, and the second mitochondria-derived activator of caspases (Smac). We have reported that overexpression of copper/zinc superoxide dismutase (SOD1) reduced superoxide production and ameliorated neuronal injury in the hippocampal CA1 subregion after global ischemia. However, the role of oxygen free radicals produced after ischemia/reperfusion in the mitochondrial signaling pathway has not been clarified. Five minutes of global ischemia was induced in male SOD1-transgenic (Tg) and wild-type (Wt) littermate rats. Cytosolic expression of cytochrome c and Smac and activation of caspases were evaluated by immunohistochemistry, Western blot, and caspase activity assay. Apoptotic cell death was characterized by DNA nick end and single-stranded DNA labeling. In the Wt animals, early superoxide production, mitochondrial release of cytochrome c, Smac, and cleaved caspase-9 were observed after ischemia. Active caspase-3 was subsequently increased, and 85% of the hippocampal CA1 neurons showed apoptotic DNA damage 3 d after ischemia. Tg animals showed less superoxide production and cytochrome c and Smac release. Subsequent active caspase-3 expression was not evident, and only 45% of the neurons showed apoptotic DNA damage. A caspase-3 inhibitor (N-benzyloxycarbonyl-val-ala-asp-fluoromethyl ketone) reduced cell death only in Wt animals. These results suggest that overexpression of SOD1 reduced oxidative stress, thereby attenuating the mitochondrial release of cytochrome c and Smac, resulting in less caspase activation and apoptotic cell death. Oxygen free radicals may play a pivotal role in the mitochondrial signaling pathway of apoptotic cell death in hippocampal CA1 neurons after global ischemia.

Fig. 1.

SOD activity in non-ischemic tissue (A) and in the hippocampus (B) before and after ischemia. In Wt tissue, SOD activity in the striatum, spinal cord, and heart was significantly higher than in the hippocampus. SOD activity in the hippocampus was not altered after ischemia until 3 d in both the Wt and Tg animals.Wt, Wild-type littermates; Tg, SOD1 transgenic animals. n = 4 in each group.

Fig. 2.

Ethidium signals in the hippocampal CA1 pyramidal cell layer (A–D) and quantitative evaluation of the signals (E). In non-ischemic brains, ethidium signals (red) were observed as small particles in Wt (A) as well as in Tg (C) animals. One hour after ischemia, a marked increase in these punctate signals and also diffuse cytosolic signals were observed in the Wt animals (C), but the increase in signals in the Tg animals (D) was not as obvious as in the Wt rats. A quantitative analysis (n = 4) confirmed that the intensity of the ethidium signals was greater in the Wt animals 1 hr and 1 d after ischemia (*p < 0.001; **p < 0.01). Nuclei were counterstained with DAPI (blue).N, Non-ischemic. Scale bar, 20 μm.

Fig. 3.

Fluorescent double staining of ethidium signals (red) and NeuN (A–C), GFAP (D–F), or the mitochondrial marker MitoTracker (G–I). Results were confirmed by at least three independent studies. Ethidium signals 1 d after ischemia in Wt animals (A) were almost exclusively localized in the NeuN-positive pyramidal neurons (B). An overlapped photo from the same field (C) confirmed the colocalization. Most of the ethidium signals 1 d after ischemia in the Wt animals (D) were not in the GFAP-positive astrocytes (E). In the overlapped image (F), arrows indicate GFAP-positive astrocytes without the ethidium signals, and thearrowhead indicates one with the ethidium signals. MitoTracker visualized numerous small particles considered to be mitochondria (G), and many of them were colocalized with ethidium signals (H). An overlapped image (I) showed double-stained particles in yellow (arrows). Nuclei were also counterstained with DAPI (blue). Scale bars, 20 μm.

Fig. 4.

Representative photomicrographs of H&E, TUNEL, and ssDNA staining (A) and cell counting study of morphologically damaged, TUNEL-positive, and ssDNA-positive cells (B). Three days after ischemia, most of the CA1 neurons in the Wt animals showed shrunken, triangular-shaped, condensed nuclei on H&E-stained sections; however, many neurons preserved their normal features of the nuclei in the Tg animals. A majority of these damaged neurons became TUNEL- and ssDNA-positive at the same time (A). Cell-counting analyses (n = 6 each) confirmed that ∼85% of the hippocampal CA1 pyramidal neurons in the Wt rats and 45% in the Tg animals underwent delayed death, and most of these cells were positive for TUNEL and ssDNA. The neuronal damage matured by 3 d after ischemia. There was a significant difference between the Wt and Tg groups. *p < 0.01; **p < 0.001. Scale bar, 20 μm.

Fig. 5.

Representative photomicrographs of cytochromec and Smac immunohistochemistry in the hippocampal CA1 subregion (A) and Western blot analyses of cytochrome c, Smac, and cleaved caspase-9 in cytosolic (B) and mitochondrial (C) fractions. Cytochrome c staining appeared as an indistinct faint punctate staining in the non-ischemic hippocampus in both the Wt and Tg animals, and it became more intense and some cells showed a diffuse cytosolic staining pattern (arrows). Some of the cytosolic cytochrome c-positive cells showed a long, process-like structure resembling axons or dendrites. The number of the cells with a cytosolic cytochrome cpattern was greater in the Wt animals than in the Tg animals. Smac staining appeared as more diffuse cytosolic staining with a few small dots in non-ischemic brains, and it became more intense 1 d after ischemia. The staining intensity was stronger in Wt animals than in Tg animals. Ethidium signals (red) were overlapped in cytochrome c immunostaining, and nuclei were counterstained with DAPI (blue) in all photomicrographs. Western blot analysis of cytosolic fraction showed that cytochromec and Smac started to increase 6 hr after ischemia and remained elevated until 1 d after ischemia, and cleaved caspase-9 started to increase at 12 hr and also remained increased at 1 d (B). The increase of cytochrome cand Smac was greater in the Wt animals than in the Tg animals. Correspondingly, cytochrome c and Smac both decreased at the same time in the mitochondrial fraction (C). The decrease in cytochrome c in the Wt animals was greater than in the Tg animals. Consistent bands of β-actin and cytochrome oxidase are also shown. Results shown are representative of three independent studies. N, Non-ischemic;CyC, cytochrome c; COX, cytochrome oxidase. Scale bar, 20 μm.

Fig. 6.

Active caspase-3 immunohistochemistry (A), Western blot studies of caspase-3 and PARP (B), and caspase-3 activity assay (C). A majority of the CA1 pyramidal neurons became strongly active caspase-3 positive at 3 d only in the Wt animals (A). Western blot analysis also showed that procaspase-3, active caspase-3, and PARP were more abundant at 3 d in the Wt animals than in other samples (B). These data are representative of two independent studies. Caspase-3 activity was increased at 3 d compared with non-ischemic brains in Wt animals, but there was no increase in Tg animals (n = 4 each). *p < 0.001. N, Non-ischemic;Wt, wild-type; Tg, SOD1 transgenic. Scale bar, 20 μm.

Fig. 7.

Cell-counting study of damaged neurons (A) and Western blot analyses of cytosolic cytochrome c and caspase-3 (B) in the caspase-3 inhibitor study. The number of damaged CA1 neurons was significantly smaller in the Z-DEVD-FMK-treated Wt group than in the vehicle-treated Wt group (n = 6 each), whereas no difference was observed in the Tg group (A). Z-DEVD-FMK did not affect cytosolic cytochrome c at 1 d but reduced procaspase-3 and active caspase-3 only in the Wt animals at 3 d (B). The data shown are representative of two independent studies. *p < 0.05. N, Non-ischemic; Veh, vehicle;DEVD, Z-DEVD-FMK.