Inhibition of nerve growth factor signaling by peroxynitrite

Abstract

The reactive oxygen species peroxynitrite has been implicated in mediating oxidative damage within the brain, and in particular in those regions associated with the pathology of Alzheimer disease. Evidence for peroxynitrite damage includes the abundance of nitrated tyrosine residues within proteins of neural cells. Potential sites for peroxynitrite-induced cytotoxicity are the tyrosine residues of tyrosine kinase receptors that are crucial for the maintenance of cholinergic neurons. The peroxynitrite generator 3-morpholinosydnonmine (SIN-1) was used to examine the effects of peroxynitrite generation on nerve growth factor (NGF)/TrkA signaling in PC12 pheochromocytoma cells that express a cholinergic phenotype. NGF produced a concentration-dependent increase in PC12 cellular metabolism (EC(50) = 15.2 ng/ml) measured in a microphysiometer. This action of NGF was inhibited in a concentration-dependent manner up to 67% of control by a brief (20 min) exposure of the cells to SIN-1. This inhibition of the NGF cellular response by SIN-1 was not related to generalized cellular toxicity. In fact, the peroxynitrite scavenger uric acid significantly attenuated the inhibitory actions of SIN-1. Pretreatment with SIN-1 also resulted in a decrease in the NGF-induced phosphorylation of TrkA protein. Furthermore, SIN-1 treatment reduced the activity of mitogen activated protein kinase (MAPK), a downstream kinase activated by TrkA receptor stimulation. These data suggest that SIN-1 treatment inhibits NGF signaling by inactivating TrkA receptors through the formation of nitrotyrosine residues on the receptor. The inactivation of TrkA receptors may contribute to the initial insult that eventually leads to neuronal cell death.