The etiology of Parkinson's disease is still unknown, though current investigations support the notion of the pivotal involvement of oxidative stress in the process of neurodegeneration in the substantia nigra (SN). In the present study, we investigated the molecular mechanisms underlying cellular response to a challenge by dopamine, one of the local oxidative stressors in the SN. Based on studies showing that nuclear factor kappa B (NF-kB) is activated by oxidative stress, we studied the involvement of NF-kB in the toxicity of PC12 cells following dopamine exposure. We found that dopamine (0.1±0.5 mM) treatment increased the phosphorylation of the IkB protein, the inhibitory subunit of NF-kB in the cytoplasm. Immunoblot analysis demonstrated the presence of NF-kB-p65 protein in the nuclear fraction and its disappearance from the cytoplasmic fraction after 2 h of dopamine exposure. Dopamine-induced NF-kB activation was also evidenced by electromobility shift assay using radioactive labeled NF-kB consensus DNA sequence. Cell-permeable NF-kB inhibitor SN-50 rescued the cells from dopamineinduced apoptosis and showed the importance of NF-kB activation to the induction of apoptosis. Furthermore,¯ow cytometry assay demonstrated a higher level of translocated NF-kB-p65 in the apoptotic nuclei than in the unaffected nuclei. In conclusion, our ®ndings suggest that NF-kB activation is essential to dopamine-induced apoptosis in PC12 cells and it may be involved in nigral neurodegeneration in patients with Parkinson's disease.

Summary. Parkinson 's disease (PD) is a progressive neurological disorder caused by rather selective degeneration of the dopaminergic (DA) neurons in the substantia nigra . Though subject to intensive research, the etiology of this nigral neuronal loss is still enigmatic and treatment is basically symptomatic. The current major hypothesis suggests that nigraI neuronal death in PD is due to excessive oxidative stress generated by auto-and enzymatic oxidation of the endogenous neurotransmitter dopamine (DA ), the formation of neuromelanin and presence of high concentrations of iron.We have found that DA toxicity is mediated through its oxidative metabolites. Whereas thiol-containing antioxidants provided marked protection against DA toxicity, ascorbic acid accelerated DA-induced death. Using the differential display approach, we sought to isolate and characterize genes whose expression is altered in response to DA toxicity. We found an upregulation of the collapsin response mediator protein (CRM) and TCP-lb in sympathetic neurons, which undergo dopamine-induced apoptosis. The isolation of these genes led us to examine the expression and activity of CRM and TCP-lb related genes. Indeed, we found a significant induction of mRNAs of the secreted collapsin-l and the mitochondrial stress protein HSP60. Antibodies directed against collapsin -l provided marked and prolonged protection of several neuronal cell types from dopamine-induced apoptosis. In a parallel study, using antisense technology, we found that inhibition of TCP-lb expression significantly reduced DA-induced neuronal death. These findings suggest a functional role for collapsin-l and TCP-lb as positive mediators of DA-induced neuronal apoptosis.