Journal o/ Neurochemistry Lippincolt—Raven Publishers, Philadelphia © 1996 International Society for Neurochemistry Age-Dependent Sensitivity of Cultured Peripheral Sympathetic Neurons to I -Methyl-4-Phenylpyridinium: Role of Glutathione Sanjiv V. Bhave, *Jan N. Johannessen, Lawrence H. Lash, Taruna D. Wakade, and Arun R. Wakade Department of Pharmacology, WSU School 01 Medicine, Detroit, Michigan; and 5Di Vision of Toxicological Research, FDA/HFS-507, Laurel, Maryland, U.S.A. Abstract: We demonstrate that 1-methyl-4-phenylpyri- dinium (MPP~) is toxic to chick peripheral sympathetic neurons maintained in culture in the presence of nerve growth factor (NGF). When MPP~ was added to the cul- ture medium at the time the neurons were plated, cell loss after 3 days in culture was evident at concentrations as low as 3 nM, and near maximal at 1 p.M. Toxicity was blocked by brief preincubation with the norepinephrine (NE)-reuptake blocker desipramine (DM1; 10 p.M for 30 mi. MPP blocked the uptake of [3H]NE by sympa- thetic neurons in a dose-dependent manner with a po- tency roughly equal to DM1. At concentrations up to 10 pM, MPP~ had no neurotoxic effect on the survival of sensory neurons maintained in the presence of NGF. The sensitivity of sympathetic neurons to the toxic effects of MPP~ diminished gradually with increasing lengths of time in culture. When MPP~ was added to the culture medium 48 h after plating, concentrations up to 100 pM did not cause neuronal death. This increasing resistance of sympathetic neurons to MPP~-inducedcell death could not be explained by an increasing capacity for se- questration of MPP~ within synaptic vesicles. The loss of sensitivity with time in culture was, however, accompa- nied by a threefold increase in the levels of glutathione (GSH). Furthermore, addition of MPP~ (1 1.tM)to cultures previously maintained for 2 days in the presence of the GSH-synthesis inhibitor L-buthionine- [S,R] -sulfoximine (1 pM) caused the same degree of cell death as when added to freshly plated neurons. These results suggest that the observed toxicity of MPP in freshly plated chick sympathetic neurons may involve the formation of free radicals and that GSH plays a role in protecting sym- pathetic neurons in vivo from the toxicity of MPP Key Words: 1-Methyl-4-phenylpyridinium—Neuronal cultures—Sympathetic neurons—Glutathione—— Neuro- toxicity —Tetrabenazine — L - Buthionine - [S. R] - sulfoxi- mine. J. Neurochem. 67, 557—565 (1996). - Methyl - 4 - phenyl - 1. 2, 3, 6 - tetrahydropyridine (MPTP), a toxin selective for nigral dopamine cells, is used widely to elicit the symptoms of Parkinson’s disease in experimental animals (Jenner, 1989; Maret et al., 1990). MPTP is toxic to the nigral dopaminergic neurons of several species in vivo as well as in vitro (Burns et al.. 1983; Langston et al., 1983, 1984; Heik- kila et al., l984a; Mytilineou and Friedman, 1988; Johannessen et al., 1989). The neurotoxic effect of MPTP depends on its conversion to l-methyl-4-phe- nylpyridine (MPP~)by monoamine oxidase B (Chiba et al., 1984; Heikkila et al.. 1984h; Markey et al., 1984), localized mainly in the glial cells (Westlund et al., 1985; Barnes et al.. 1986; Ransom et al.. 1987). MPP destroys the mesencephalic dopaminergic neu- rons in vivo (Bradbury et al., 1986) and in vitro (Myti- lineou Ct al., 1985; Danias et al., 1989). The selective neurotoxicity of MPP~ for dopaminergic neurons is explained in part because it is a very good substrate for the catecholamine reuptake mechanisms (Javitch etal., 1985). The neurotoxic action of MPP 1 on cultured mesen- cephalic dopaminergic neurons has been studied exten- sively. The heterogeneous nature of’ these cultures makes it difficult to follow biochemical changes in- duced selectively within dopaminergic neurons by MPP (dopamine-containing neurons ccnstitute only —~~l% of the total cells; Sanchez-Ramos et al., 1988a). A relatively homogeneous population of catecholamin- ergic neurons (almost 95% of the cells) is present in the cultures of sympathetic neurons obtained from chick embryos. Although noradrenergic neurons, in- Resubmitted manuscript received Mardi 19, I 996: accepted March 26. 1996. Address correspondence and reprint reque~,1s 10 Dr. A. R. Wakade al Department of Pharmacology. WSIJ School of Medicine .540 E. Canlield, Detroit, Ml 48201, U.S.A. Abbrem-iat,on.s used: liSO, ,—hulhionine— I SR I —sulfoximine: DM1, desipramine: GSI-1. glulathione: GSSG. glutathione disultide: HPLC, high-pressure liquid chromatography: MPP - . I -methyl-4-phenyl- pyridine: MPTP, I -mclhyl-4-pheny]- I .2.3.6-letrahydropyridine: NE, norepinephrine; NGF. nerve growth factor: IBZ, tetrabcnai.ine. 557