Copper,zinc superoxide dismutase (SOD1) in mammals is activated principally via a copper chaperone (CCS) and to a lesser degree by a CCS-independent pathway of unknown nature. In this study, we have characterized the requirement for CCS in activating SOD1 from Drosophila. A CCS-null mutant (Ccs n29E ) of Drosophila was created and found to phenotypically resemble Drosophila SOD1-null mutants in terms of reduced adult life span, hypersensitivity to oxidative stress, and loss of cytosolic aconitase activity. However, the phenotypes of CCS-null flies were less severe, consistent with some CCS-independent activation of Drosophila SOD1 (dSOD1). Yet SOD1 activity was not detectable in Ccs n29E flies, due largely to a striking loss of SOD1 protein. In contrast, human SOD1 expressed in CCS-null flies is robustly active and rescues the deficits in adult life span and sensitivity to oxidative stress. The dependence of dSOD1 on CCS was also observed in a yeast expression system where the dSOD1 polypeptide exhibited unusual instability in CCS-null (ccs1⌬) yeast. The residual dSOD1 polypeptide in ccs1⌬ yeast was nevertheless active, consistent with CCS-independent activation. Stability of dSOD1 in ccs1⌬ cells was readily restored by expression of either yeast or Drosophila CCS, and this required copper insertion into the enzyme. The yeast expression system also revealed some species specificity for CCS. Yeast SOD1 exhibits preference for yeast CCS over Drosophila CCS, whereas dSOD1 is fully activated with either CCS molecule. Such variation in mechanisms of copper activation of SOD1 could reflect evolutionary responses to unique oxygen and/or copper environments faced by divergent species.

Nerve growth factor (NGF) can regulate nitric oxide synthase (NOS) expression and nitric oxide (NO) can modulate NGFmediated neurotrophic responses. In this study, the role of NO in NGF-stimulated amyloid precursor protein (APP) levels was studied. PC12 cells were treated with either the non-selective NOS inhibitor N x -nitro-L-arginine methylester (L-NAME) or the inducible NOS selective inhibitor s-methylisothiourea (S-MIU), and the effect on NGF-mediated increases in APP expression was determined. NGF significantly increased total APP protein levels following 96 h of treatment and this increase was prevented in cells pre-treated with S-MIU. Pre-treatment of cells with actinomycin D also blocked this NGF-mediated induction of APP, indicating de novo protein synthesis is necessary. Treatment with NGF increased APP promoter activity; however, this increase was only partially inhibited by pre-treatment with S-MIU and was increased in the presence of L-NAME. This suggests that NO may be modulating other aspects of APP expression in addition to transcription. Inhibition of NGF signaling pathways was also investigated using inhibitors of mitogen-activated protein (MAP) kinase (U0126), Akt (LY294002) and protein kinase C (PKC; U73122 and bisindolylmaleimide 1 (BIS-1)) activation. Inhibition of each of these pathways prevented NGF-mediated increases in APP protein expression; however, only BIS-1 attenuated NGFmediated increases in promoter activation. This study indicates that NO is involved in the NGF-mediated regulation of APP, in part at the level of APP transcription and could involve the modulation of NGF signal transduction pathways.