EFFECT OF CYCLOHEXIMIDE AND mRNA SYNTHESIS INHIBITION ON DEATH OF
TROPHICALLY-DEPRIVED CILIARY GANGLION NEURONS IN CULTURE.
Brus[acute]es, Juan L. and Guillermo R. Pilar.
University of Connecticut, Department of Physiology & Neurobiology, 75 N.
Eagleville Rd., U-42, Storrs, CT 06269.
APStracts 2:0216N, 1995.
SUMMARY AND CONCLUSIONS
1. The relationship between cycloheximide (CHX) and RNA synthesis inhibitors
on trophic-deprived neuronal survival was studied using primary cultures of
stage (St) 34 chick ciliary ganglion (CG) neurons, to analyse 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 week, in the presence of an
eye extract (CIPE) as a trophic support for the neurons. Highly enriched
neuronal cultures almost devoid of non-neuronal cells were used. 2. The time
at which trophically deprived neurons can not 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 3 H-leucine incorporation by 90-95%, at a concentration of 10-100
[mu]g/ml. The effect of the RNA transcription blockers actinomycin D (Act-D),
alpha-amanitin, and 5,6 dichlorobenzimidazole riboside (DRB) on 3 H-uridine
incorporation into macromolecules was evaluated. Total RNA synthesis was
inhibited by 10-25% by alpha-amanitin, whilst Act-D and DRB inhibited 80-97.5%
of the 3 H-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 hours from the
onset of the culture was enough to delay the committment 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 [mu] g/ml) on
RNA and protein synthesis was studied by measuring the incorporation of
radiolabeled metabolic precursors ( 3 H-leucine or 3 H-uridine) into
macromolecules. A 95% reduction in the protein synthesis was observed after 1
h of application of the drug, and by 24 h 3 H-leucine incorporation was
reduced to 15-20% of the control values. Wash-out of CHX after 6 h of
incubation produce 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 blocked the expression of killer
proteins. This view is supported by i) the 24 h delay of the commitment point
following the short term application of CHX, ii) the impaired ability of CHX
to rescue trophic-deprived neurons by the addition of mRNA synthesis blockers,
and iii) the fact that neuronal survival caused by trophic factors like CIPE,
is blocked by blocking RNA transcription.
Received 16 November 1994; accepted in final form 30 June 1995.
APS Manuscript Number J724-4.
Article publication pending J. Neurophysiol.
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 10 August 1995.