Mutations in a-synuclein (A30P and A53T) are involved in some cases of familial Parkinson's disease (FPD), but it is not known how they result in nigral cell death. We examined the effect of a-synuclein overexpression on the response of cells to various insults. Wild-type a-synuclein and a-synuclein mutations associated with FPD were overexpressed in NT-2/D1 and SK-N-MC cells. Overexpression of wild-type a-synuclein delayed cell death induced by serum withdrawal or H 2 O 2 , but did not delay cell death induced by 1-methyl-4-phenylpyridinium ion (MPP 1 ). By contrast, wild-type a-synuclein transfectants were sensitive to viability loss induced by staurosporine, lactacystin or 4-hydroxy-2-transnonenal (HNE). Decreases in glutathione (GSH) levels were attenuated by wild-type a-synuclein after serum deprivation, but were aggravated following lactacystin or staurosporine treatment. Mutant a-synucleins increased levels of 8-hydroxyguanine, protein carbonyls, lipid peroxidation and 3-nitrotyrosine, and markedly accelerated cell death in response to all the insults examined. The decrease in GSH levels was enhanced in mutant a-synuclein transfectants. The loss of viability induced by toxic insults was by apoptosic mechanism. The presence of abnormal a-synucleins in substantia nigra in PD may increase neuronal vulnerability to a range of toxic agents.

The ubiquitin/proteasome pathway plays an essential role in protein turnover in vivo, and contributes to removal of oxidatively damaged proteins. We examined the effects of proteasome inhibition on viability, oxidative damage and antioxidant defences in NT-2 and SK-N-MC cell lines. The selective proteasome inhibitor, lactacystin (1 microM) caused little loss of viability, but led to significant increases in levels of oxidative protein damage (measured as protein carbonyls), ubiquitinated proteins, lipid peroxidation and 3-nitrotyrosine, a biomarker of the attack of reactive nitrogen species (such as peroxynitrite, ONOO(-)) upon proteins. Higher levels (25 microM) of lactacystin did not further increase the levels of carbonyls, lipid peroxidation, 3-nitrotyrosine, or ubiquitinated proteins, but produced increases in the levels of 8-hydroxyguanine (a biomarker of oxidative DNA damage) and falls in levels of GSH. Lactacystin (25 microM) caused loss of viability, apparently by apoptosis, and also increased production of nitric oxide (NO.) (measured as levels of NO2- plus NO3-) by the cells; this was inhibited by N-nitro-L-arginine methyl ester (L-NAME), which also decreased cell death induced by 25 microM lactacystin and decreased levels of 3-nitrotyrosine. The NO. production appeared to involve nNOS; iNOS or eNOS were not detectable in either cell type. Another proteasome inhibitor, epoxomicin, had similar effects.

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