Glutaredoxin Protects Cerebellar Granule Neurons from Dopamine-induced Apoptosis by Activating NF-κB via Ref-1

The neurotransmitter dopamine (DA) induces apoptosis via its oxidative metabolites. This study shows that glutaredoxin 2 (Grx2) from Escherichia coli and human glutaredoxin could protect cerebellar granule neurons from DA-induced apoptosis. E. coli Grx2, which catalyzes glutathione-disulfide oxidoreduction via its -Cys-Pro-Tyr-Cys- active site, penetrates into cerebellar granule neurons and exerts its activity via NF-κB activation. Analysis of single and double cysteine to serine substitutions in the active site of Grx2 showed that both cysteine residues were essential for activity. Although DA significantly reduced NF-κB binding activity, Grx2 could stimulate the binding of NF-κB to DNA by: (i) translocating NF-κB from the cytoplasm to the nucleus after promoting the phosphorylation and degradation of I-κBα, and (ii) activating the binding of pre existing nuclear NF-κB. The DNA binding activity of NF-κB itself was essential for neuronal survival. Overexpression of I-κB dominant negative gene (I-κB-ΔN) in granule neurons significantly reduced their viability, irrespective of the presence of Grx2. Ref-1 expression was down-regulated by DA but up-regulated by Grx2, while treatment of neurons with Ref-1 antisense oligonucleotide reduced the ability of Grx2 to activate NF-κB binding activity. These results show that Grx2 exerts its anti apoptotic activity through the activation of Ref-1, which then activates NF-κB.