ANTISENSE OLIGODEOXYNUCLEOTIDES TO BAX MESSENGER-RNA PROMOTE SURVIVALOF RAT SYMPATHETIC NEURONS IN CULTURE

Previous in vitro studies have shown that the presence of high levels of Bar protein accelerated the rate of cell death following growth fac tor deprivation and that the ratio of cell death repressor Bcl-2 to ce ll death effector Bar may determine the susceptibility to apoptosis, B oth Bcl-2 and Bar protein expression has been detected in sympathetic neurons in vivo, and overexpression of bcl-2 in cultured sympathetic n eurons prevented apoptosis after deprivation of nerve growth factor (N GF). Tn the present study, we investigated the expression of bar and b cl-2 in primary cultures of sympathetic neurons from rat superior cerv ical ganglia, Furthermore, we tested the effects of a partially phosph orothioated bar antisense oligodeoxynucleotide (ODN) on the survival o f sympathetic neurons in cultures supplied with suboptimal concentrati ons of NGF (0.5 ng/ml), A constitutive expression of bar mRNA at high levels was detected by reverse transcription and polymerase chain reac tion which did not change significantly following NGF reduction or tre atment with bar antisense ODN, A decrease in Bcl-2 immunoreactivity wa s observed by immunocytochemistry in tyrosine hydroxylase-positive neu rons when cultured under suboptimal NGF concentrations, whereas Bcl-2 immunolabeled non-neuronal cells were not affected, Maximal number of neurons was obtained in control cultures containing 50 ng/ml of NGF, F ew neurons survived in cultures grown in 0.5 ng/ml of NGF for 2 days ( 12.0 +/- 1.5% of controls, mean +/- SEM), Addition of two control ODNs at 1 mu M had no effect on neuronal survival (10.1 +/- 1.2% and 11.0 +/- 1.3%, respectively), while the number of neurons was significantly increased in NGF-reduced cultures treated with a bar antisense ODNs ( 1 mu M) (31.5 +/- 1.9%), Administration of fluorescein-labeled ODNs de monstrated intracellular uptake into cultured neurons, Treatment with bar antisense ODNs caused a significant reduction of Bar protein level s in SCG neurons by 46 +/- 2.6% as assessed by immunocytochemistry and digital image analysis, Taken together, our data demonstrate a consti tutive expression of bar mRNA in sympathetic neurons suggesting that a ctivation of bar expression may not be required for neuronal cell deat h after NGF withdrawal, After changing to suboptimal NGF concentration s, the cell-specific reduction in Bcl-2 immunoreactivity preceded morp hological signs of degeneration indicating that growth factor starvati on may down-regulate neuronal bcl-2 expression, Treatment with bar ant isense ODNs indicated that suppression of Bar protein synthesis may pr omote neuronal survival in the threshold situation of insufficient tro phic support. (C) 1996 Wiley-Liss, Inc.