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SOD1 down-regulates NF-kappaB and c-Myc expression in mice after transient focal cerebral ischemia
C Y Huang1, M Fujimura, N Noshita
1Department of Neurosurgery, Stanford University School of Medicine, California, USA.
Abstract:
Reactive oxygen species (ROS) are implicated in reperfusion injury after focal cerebral ischemia (FCI). Reactive oxygen species regulate activity of transcription factors like NF-kappaB. The authors investigated the role of ROS in NF-kappaB activity after FCI using transgenic mice that overexpressed human copper/zinc-superoxide dismutase (SOD1) and that had reduced infarction volume after FCI. Superoxide dismutase transgenic and wild-type mice were subjected to 1 hour of middle cerebral artery occlusion (MCAO) and subsequent reperfusion. Immunohistochemistry showed SOD1 overexpression attenuated ischemia-induced NF-kappaB p65 immunoreactivity. Colocalization of NF-kappaB and the neuronal marker, microtubule-associated proteins (MAPs), showed that NF-kappaB was up-regulated in neurons after FCI. Electrophoretic mobility shift assays showed that SODI overexpression reduced ischemia-induced NF-kappaB DNA binding activity. Supershift assays showed that DNA-protein complexes contained p65 and p50 subunits. Immunoreactivity of c-myc, an NF-kappaB downstream gene, was increased in the ischemic cortex and colocalized with NF-kappaB. Western blotting showed that SOD1 overexpression reduced NF-kappaB and c-Myc protein levels in the ischemic brain. Colocalization of c-Myc and TUNEL staining was observed 24 hours after FCI. The current findings provide the first evidence that SOD1 overexpression attenuates activation of NF-kappaB after transient FCI in mice and that preventing this early activation may block expression of downstream deleterious genes like c-myc, thereby reducing ischemic damage.
Insights
Overexpressing superoxide dismutase 1 (SOD1) in mice reduces brain damage after focal cerebral ischemia. This protection is linked to decreased activity of the transcription factor NF-kappaB, a key player in inflammation and cell death.
Area of Science:
- Neuroscience
- Molecular Biology
- Biochemistry
Background:
- Reactive oxygen species (ROS) contribute to reperfusion injury following focal cerebral ischemia (FCI).
- ROS modulate the activity of crucial transcription factors, including NF-kappaB, which plays a role in inflammatory and cell death pathways.
Purpose of the Study:
- To investigate the role of ROS in NF-kappaB activation after FCI.
- To determine if overexpressing human copper/zinc-superoxide dismutase (SOD1) can mitigate NF-kappaB activity and subsequent ischemic damage.
Main Methods:
- Utilized transgenic mice overexpressing SOD1 and wild-type littermates subjected to middle cerebral artery occlusion (MCAO) for 1 hour followed by reperfusion.
- Employed immunohistochemistry to assess NF-kappaB p65 and c-Myc immunoreactivity, and neuronal markers.
- Performed electrophoretic mobility shift assays (EMSA) and supershift assays to evaluate NF-kappaB DNA binding activity.
- Used Western blotting to quantify protein levels of NF-kappaB and c-Myc.
- Conducted TUNEL staining to assess apoptosis.
Main Results:
- SOD1 overexpression significantly attenuated ischemia-induced NF-kappaB p65 immunoreactivity and DNA binding activity in neurons.
- NF-kappaB was found to be up-regulated in neurons after FCI, and its downstream gene, c-myc, showed increased expression.
- SOD1 overexpression reduced protein levels of NF-kappaB and c-Myc in the ischemic brain, with c-Myc colocalizing with apoptotic cells.
Conclusions:
- This study provides the first evidence that SOD1 overexpression attenuates NF-kappaB activation following transient focal cerebral ischemia in mice.
- Preventing early NF-kappaB activation by enhancing SOD1 activity may inhibit the expression of deleterious downstream genes like c-myc.
- This mechanism contributes to reducing ischemic brain damage and infarction volume.