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FJ EXPRESS SUMMARY ARTICLE
The Full-length version of this article is also available, published online October 4, 2002 as doi:10.1096/fj.02-0251fje.
Published as doi: 10.1096/fj.02-0251fje.
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(The FASEB Journal. 2002;16:1997-1999.)
© 2002 FASEB

Overexpression of SOD1 protects vulnerable motor neurons after spinal cord injury by attenuating mitochondrial cytochrome c release 1

TAKU SUGAWARA, ANDERS LEWÉN, YVAN GASCHE, FENGSHAN YU and PAK H. CHAN2

Department of Neurosurgery, Department of Neurology and Neurological Sciences, and Program in Neurosciences, Stanford University School of Medicine, Stanford, California, USA

2Correspondence: Neurosurgical Laboratories, Stanford University, 1201 Welch Road, MSLS #P314, Stanford, CA 94305-5487, USA. E-mail: phchan{at}leland.stanford.edu

SPECIFIC AIMS

Ventral horn motor neurons (VMN) are selectively vulnerable to mild spinal cord injury (SCI) and die possibly through an apoptotic pathway. We used copper/zinc superoxide dismutase (SOD1) -overexpressing transgenic animals to investigate the involvement of reactive oxygen species (ROS) and cytochrome c-dependent apoptosis in their death after injury.

PRINCIPAL FINDINGS

1. Distribution of SOD1 in the spinal cord and superoxide production after mild SCI in wild-type (Wt) rats
In the ventral horn of the spinal cord, double immunostaining showed that astrocytes express much more SOD1 in their cytosol than VMN. The production of superoxide after SCI was investigated by in situ detection of oxidized hydroethidine (HEt). HEt is taken up by living cells and oxidized to a red fluorescent dye, ethidium (Et), specifically by superoxide but not by other ROS in the cells. After mild compression SCI by a vascular clip, an increase in the intensity of Et signals was detected in neurons (Fig. 1 a–c), whereas GFAP-positive astrocytes showed no obvious increase (Fig. 1d-f ). Double staining of Et and a mitochondrial marker showed that most of the punctate superoxide signals were colocalized with mitochondria, confirming mitochondria as a major source of superoxide production (Fig. 1g-i ).



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  		Figure 1. Fluorescent double labeling of ethidium and NeuN (a–c)/GFAP (d–f)/mitochondrial marker MitoTracker (g–i) in the Wt ventral horn 1 day after SCI. Clusters of intense ethidium signals (a) were mainly observed in NeuN-positive (b) cells. In the overlapped photo (c), a large NeuN-positive cell and an injured NeuN-positive cell with shrunken cytoplasm and a triangle-shaped nucleus are indicated by an arrow and an arrowhead, respectively. Ethidium signals in the cytoplasm (d) were not colocalized with GFAP-positive cells (e). Overlapped image (f) shows large GFAP-negative cells with ethidium signals (arrowheads) and GFAP-positive cells without signals (arrows). Most of the ethidium signals in the cytoplasm (g) were colocalized with the mitochondrial marker (MitoTracker) signals (h). An overlapped image (i) shows double-stained particles in orange/yellow. Insets are highly magnified photos of the rectangled areas. Nuclei were counterstained with DAPI (blue). Scale bars = 20 µm.

2. Superoxide production after SCI was ameliorated in SOD1-overexpressing transgenic (Tg) rats
In SOD1-overexpressing Tg rats, total SOD activity was 4.6-fold higher in the spinal cord than that in the Wt rats. SOD1 immunoreactivity was much more intense in the Tg than in the Wt animals in astrocytes and VMN. The signal intensity of Et was increased in the Tg spinal cord after SCI; however, a quantitative study showed that the relative Et signal intensity in Wt rats was significantly greater than in the SOD1 Tg rats 6 h after injury.

3. VMN are selectively vulnerable and undergo delayed DNA-fragmented death after mild SCI
At the lesion site, a majority of VMN disappeared between 1 and 3 days after SCI in Wt rats and morphologically fragmented nuclei appeared at 3 days in the ventral horn. In situ labeling of DNA fragmentation was performed using the terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL) reaction; TUNEL-positive large nuclei appeared at the same time in the ventral horn. The number of surviving VMN was significantly greater in SOD1 Tg rats vs. Wt rats at 3 days. DNA gel electrophoresis showed that fragmentation of DNA was evident 3 days after SCI in Wt and Tg rats. Astrocyte counting revealed that the number of GFAP-positive astrocytes was not changed after SCI in either the Wt or Tg rats.

4. Involvement of cytochrome c-dependent apoptosis in delayed death of VMN
An immunohistochemical study showed no cytosolic cytochrome c-positive cells in the uninjured ventral horn in either Wt or Tg rats; however, a majority of VMN in Wt rats and many VMN in the Tg animals became cytochrome c-positive at 6 h. Likewise, no active caspase-9-positive cells were observed in the uninjured animals, but many VMN became positive 6 h after SCI. Fluorescent double staining showed that most cytochrome c-positive VMN were positive for active caspase-9. Positive cell counting revealed that the number of cytosolic cytochrome c-positive cells increased 6 h after SCI and that the number was significantly greater in Wt than in the Tg animals at 6 h to 1 day. Western blot analysis of the cytosolic fraction showed that cytochrome c increased 6 h after SCI, and the increase was greater in the Wt rats than in the Tg animals at 1 day (Fig. 2 a). Correspondingly, cytochrome c decreased at the same time in the mitochondrial fraction and the decrease in cytochrome c in the Wt animals was greater than in the Tg animals (Fig. 2b ). Active caspase-9 increased at 6 h in the whole cell fraction and there was more active caspase-9 in the Wt animals than in the Tg animals at 1 day (Fig. 2c ). Statistical analysis confirmed the difference in optical density for cytosolic/mitochondrial cytochrome c and active caspase-9 between Wt and Tg animals 1 day after SCI (n=4 each; graphs in Fig. 2a-c ).



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  		Figure 2. Western blot analyses of cytochrome c and cleaved caspase-9. Western blot analysis of the cytosolic fraction showed that cytochrome c increased 1 day after SCI and that the increase was greater in Wt than in Tg animals (a, *P <0.05). Correspondingly, cytochrome c decreased in the mitochondrial fraction. The decrease in cytochrome c in Wt animals was greater than in Tg animals (b, *P <0.05). Cleaved caspase-9 increased in the whole cell fraction and there was a significant difference between the Wt and Tg animals (c, *P<0.05). Procaspase-9 also increased after SCI in both Wt and Tg animals. Consistent bands of ß-actin (a, c) and cytochrome oxidase (b) are shown. No cytochrome oxidase bands were in the cytosolic fraction (a). Results shown are representative of 3 independent studies. Wt, wild-type; Tg, transgenic; C, uninjured control; CyC, cytochrome c; COX, cytochrome oxidase IV; MW, molecular weight.

CONCLUSIONS AND SIGNIFICANCE

Our study shows that overexpression of SOD1 in Tg animals reduced superoxide production at an early stage after SCI and showed the subsequent death of VMN. The exclusive spatial distribution of superoxide signals and the selective vulnerability of VMN also suggest the involvement of oxidative stress in the VMN death pathway. Compared with astrocytes, VMN showed less constitutive expression of SOD1 and much more superoxide production after SCI, suggesting an uneven distribution of the SOD1 protein as a possible cause of the selective vulnerability of VMN (Fig. 3 ).



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  		Figure 3. Schematic diagram. VMN showed low constitutive SOD and an increase in superoxide production after SCI. Mitochondrial cytochrome c was released to their cytosol and caspases were activated. Finally, VMN underwent DNA fragmented cell death. In contrast, astrocytes showed high constitutive SOD and no change in superoxide production after SCI. No injury on astrocytes was observed subsequently. We propose that oxidative stress to the mitochondria is an initiator of cytochrome c-dependent apoptosis and that uneven distribution of SOD is responsible for the selective vulnerability of VMN after SCI.

We provide evidence that superoxide production was primarily in mitochondria in VMN. Since mitochondria are known as the site of superoxide production under normal or pathological conditions, excessive superoxide production may cause mitochondrial injury, which leads to the release of proteins such as cytochrome c and procaspase-9 from their intermembrane space. Our study also demonstrated that cytochrome c was released from mitochondria to the cytosol in VMN after SCI. Immunohistochemistry showed numerous faint punctate stainings of cytochrome c in normal VMN and a diffuse cytosolic staining pattern after SCI, conceivably showing the release of cytochrome c from the mitochondria to the cytosol. The translocation of cytochrome c was further confirmed by Western blot analyses.

We reported the apoptotic death of VMN after SCI; however, methods to detect TUNEL and DNA laddering depend on the detection of DNA strand breaks and their specificity for apoptosis are controversial. Nevertheless, the protective effects of the nonspecific caspase inhibitor on behavioral outcome and lesion size in previous studies strongly suggest the existence of a biochemical cascade in neuronal death after SCI.

In the present study, superoxide production was observed 6 h after SCI; at the same time, mitochondrial cytochrome c release and caspase-9 cleavage were increased, and subsequent death of VMN occurred after 1 day. Regarding the order in which signaling factors are activated in VMN, these data are compatible with the latest concept of the cytochrome c-dependent mitochondrial caspase cascade. Further, exclusive spatial expression of superoxide, cytochrome c, and caspase-9 in VMN supports this hypothesis. In the Wt rats, superoxide overproduction, cytochrome c release, activated caspase-9, and subsequent VMN death were all greater than in the Tg animals. These results indicate that oxidative stress to the mitochondria may be an initiator of the cytochrome c-dependent caspase cascade and that reduced oxidative stress may be responsible for the ameliorated injury of VMN in the Tg animals.

FOOTNOTES

1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0251fje; to cite this article, use FASEB J. (October 4, 2002) 10.1096/fj.02-0251fje




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