Effect of cycloheximide and mRNA synthesis inhibition on death of trophically deprived ciliary ganglion neurons in culture.

Abstract

1. The relationship between cycloheximide (CHX) and RNA synthesis inhibitors on trophic-deprived neuronal survival was studied with the use of primary cultures of stage (St) 34 chick ciliary ganglion (CG) neurons, to analyze the biological process of neuronal death caused by trophic factor withdrawal. Tissue culture conditions were refined by characterizing the additional medium components required to obtain 100% survival, for at least 1 wk, in the presence of an eye extract [choroid, ciliary body, iris, and pigment epithelium (CIPE)] as a trophic support for the neurons. Highly enriched neuronal cultures almost devoid of nonneuronal cells were used. 2. The time at which trophically deprived neurons cannot be rescued by the addition of trophic support, "commitment point," was established to be between 11 and 17 h after trophic deprivation. 3. CHX, an inhibitor of protein translation, reduced 3H-leucine incorporation by 90-95%, at a concentration of 10-100 micrograms/ml. The effect of the RNA transcription blockers actinomycin D (Act-D), alpha-amanitin, and 5.6 dichlorobenzimidazole riboside (DRB) on 3H-uridine incorporation into macromolecules was evaluated. Total RNA synthesis was inhibited by 10-25% by alpha-amanitin, whereas Act-D and DRB inhibited 80-97.5% of the 3H-uridine incorporation. 4. The effect of short- and long-term incubation with CHX on neuronal survival was analyzed. Continuous application of CHX promoted survival for 2-3 days, but thereafter neurons died regardless of whether CIPE was present or absent. Application of CHX for 6 h from the onset of the culture was enough to delay the commitment point up to 24 h after plating, and the addition of CIPE at this time maintained survival and promoted differentiation of CHX treated neurons. 5. The RNA transcription blockers Act-D, alpha-amanitin, and DRB were applied to both trophically deprived and trophically supported neurons, and the survival of each was evaluated. Neither drug was effective in supporting the survival of trophically deprived neurons in culture, and in most cases neurons even when cultured with CIPE died within 1-2 days in the presence of either drug. 6. Experiments using both CHX and mRNA synthesis blockers were performed to determine the effect of blocking mRNA transcription in trophically deprived neurons rescued by CHX. The addition of mRNA synthesis inhibitors precluded the effect of CHX on neuronal survival. 7. The effect of CHX (20 micrograms/ml) on RNA and protein synthesis was studied by measuring the incorporation of radiolabeled metabolic precursors (3H-leucine or 3H-uridine) into macromolecules. A 95% reduction in the protein synthesis was observed after 1 h of application of the drug, and by 24 h, 3H-leucine incorporation was reduced to 15-20% of the control values. Wash out of CHX after 6 h of incubation produced a recovery of protein synthesis up to 50% of control values 18 h later. CHX did not affect the synthesis of RNA for up to 12 h; however, it impaired the ability of the cell to take up metabolic precursors. 8. In conclusion, the present results support the hypothesis that the CHX effect on neuronal survival is due to its ability to induce the expression of survival or protective genes rather than to block the expression of killer proteins. This view is supported by 1) the 24-h delay of the commitment point following the short-term application of CHX, 2) the impaired ability of CHX to rescue trophic-deprived neurons by the addition of mRNA synthesis blockers, and 3) the fact that neuronal survival caused by trophic factors like CIPE, is blocked by blocking RNA transcription.