Antioxidants N-acetylcysteine (NAC) and 2-mercaptoethanol (2-ME) affect the survival and differentiative potential of cholinergic precursors from the embryonic septal nuclei and basal forebrain: involvement of ras signaling.

We investigated the effects of antioxidants N-acetylcysteine (NAC) and 2-mercaptoethanol (2-ME) on the expression of choline acetyltransferase (ChAT) in cultured cholinergic precursors from the embryonic rat septal nuclei and basal forebrain. Carboxy-dichlorofluorescein fluorescence confirmed that 2-ME inhibited intracellular oxidation. Low micromolar concentrations of 2-ME produce as much as a 12-fold increase in ChAT; this is enhanced further by inclusion of nerve growth factor (NGF). NAC effects are biphasic: 0.15 mM produces profound increases in ChAT while 1.5 mM has no effect. Immature (E16) cultures respond with increases in ChAT while more highly differentiated cultures (E18) do not. Labeling of single precursors with a lacZ-expressing retrovirus reveals that the increase in ChAT is due primarily to an increased number and size of clones, not an increase in cholinergic neurons per clone, suggesting an effect on precursor survival. Inhibition of ras farnesylation inhibits both 2-ME and NAC induction of ChAT suggesting a ras-mediated pathway. Inclusion of the MEK inhibitor PD98059 does not affect low doses of NAC, but at doses of NAC that fail to increase ChAT activity, inhibition of the pathway actually raises ChAT. Immunocytochemical investigation of the cultures indicates that cells exposed to low doses of NAC develop healthy neuronal arbors in the apparent absence of glial support. At higher concentrations of NAC, neurons were found in association with astrocytes, making contact via elaborate varicose fibers. Treatment of the cultures with PD98059 to inhibit MEK returned cultures to a 'low-dose' phenotype. These data suggest that redox status of basal forebrain precursors affect both their survival and differentiative potential.