We have examined whether sympathetic neurones that have lost the potential to be rescued by protein and RNA synthesis inhibitors after a period of nerve growth factor (NGF) deprivation are irreversibly committed to die. We found that 15 h after withdrawal of NGF from 7-day cultures of neonatal rat superior cervical ganglion neurones, 50% of the neurones lost the potential to be rescued by cycloheximide but that NGF rescued most of the neurones. By 22 h after NGF withdrawal, only 10% of the neurones were rescued by inhibition of macromolecular synthesis with cycloheximide, puromycin, or actinomycin D, but as many as 60-80% of the neurones were rescued by NGF. This is after the time at which a DNA "ladder," consistent with cell death by apoptosis, was first detected (18 h). As long as 27 h of NGF withdrawal was required before 50% of the neurones lost the potential to be rescued by NGF. The survival-promoting agent 8-(4-chlorophenylthio)cyclic AMP (CPTcAMP) or depolarization with 50 mM KCl (HK) rescued neurones with kinetics similar to those of NGF, and rescue by all three agents did not require protein synthesis. Thus, NGF, CPTcAMP, and HK can rescue neurones deprived of NGF at much later times than either protein or RNA synthesis inhibitors by acting at the posttranslational level, a finding suggesting that initiation of the cell death programme in sympathetic neurones is not an irreversible step.

Madin-Darby canine kidney (MDCK) cells and Chinese hamster ovary (CHO) cells were transfected with wild-type and cytoplasmic deletion mutants of mouse syndecan-1 to study the requirements for transport and polarized expression of this proteoglycan. Expression in MDCK cells revealed that wild-type syndecan-1 is directed to the basolateral surface via a brefeldin A-insensitive route. A deletion of the last 12 amino acids of the syndecan-1 cytoplasmic tail (CT22) was sufficient to result in the appearance of mutant proteoglycans at both the basolateral and apical cell surfaces. Treatment with brefeldin A was able to prevent apical transport of the mutants. We thus propose that the C-terminal part of the cytoplasmic tail is required for steady-state basolateral distribution of syndecan-1. In CHO cells a deletion of the last 25 or 33 amino acids of the 34-residue cytoplasmic domain (CT9 and CT1, respectively) resulted in partial retention of the mutants in the endoplasmic reticulum (ER). A deletion mutant lacking the last 12 amino acids (CT22) was not retained. Interestingly, the unglycosylated core proteins of the CT9 and CT1 mutants showed a significantly lower apparent molecular weight when analyzed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis than wild-type syndecan-1. However, when CHO transfectants expressing the CT1 mutant were incubated with brefeldin A, causing fusion of the ER and Golgi, CT1 ran with an almost equally high apparent molecular weight as the wild-type molecule. This would suggest that syndecan-1 undergoes extensive posttranslational modifications or forms an SDS-resistant dimer/complex after transit from the ER.

Nerve growth factor (NGF) induces transient Fos-immunoreactivity (Fos-IR) independently of any other factor, both in newly isolated rat sympathetic neurons and in established cultures after NGF deprivation. The same proportion of neurons that express Fos-IR in response to NGF also survive. In addition to direct stimulation of Fos-IR expression, the presence or recent exposure to NGF is required to obtain Fos-IR expression by other stimuli. In newly isolated neurons no Fos-IR is detected in response to stimulation by serum alone and a response to depolarization or cyclic AMP is obtained only if neurons are stimulated within a short period after ganglion excision. In established cultures none of these stimuli, nor the trauma of cutting neurites or spiking cell bodies with a microinjection needle induce Fos-IR unless NGF is present or had been removed for <8 - 16 h. The lack of response is not due to a general decrease in the rate of protein or RNA synthesis. These findings show that in regenerating sympathetic neurons NGF induces c-Fos and suggest that NGF may activate a master trigger that is required for c-Fos expression to be induced by other stimuli.

Sympathetic neurons depend on nerve growth factor (NGF) for their survival and die by apoptosis when NGF is withdrawn, despite their post-mitotic state. Martin et al. (1990, J. Neurosci. 10, 184–193) showed that cytosine arabinoside, but no other arabinofuranosyl nucleoside, could induce cell death in the presence of NGF and they suggested that it may block a critical step in the NGF-signalling pathway. We show that cytosine arabinoside is not the only nucleoside capable of inducing apoptosis in sympathetic neurons in the presence of NGF. In newly isolated neurons from P0 rat pups cultured in the presence of NGF, all the arabinose nucleosides (adenine, cytosine, guanine and thymine) induce apoptosis at 10 microM when combined with 5-fluorodeoxyuridine treatment. Because 1-beta-arabinofuranosylcytosine is associated with double-strand breaks and chromosomal abberrations, we examined whether topoisomerase II inhibitors, which also cause double-strand breaks by stabilising the enzyme-DNA ‘cleavable complex’, were capable of promoting apoptosis in these neurons. Although P0 rat neurons are strictly postmitotic, topoisomerase II inhibitors teniposide and mitoxantrone induced them to die by apoptosis in the presence of NGF with the same apparent time-course as arabinose treatment or NGF withdrawal. By contrast, ICRF 193, a catalytic inhibitor of topoisomerase II, reduced the extent of apoptosis induced by mitoxantrone or teniposide by 80% if added simultaneously with the latter but by 2 hours it had no rescue effect, suggesting that topoisomerase II is highly active in these neurons. ICRF 193 also partially reduced the induction of fluorodeoxyuridine-dependent apoptosis by the arabinose nucleosides.(ABSTRACT TRUNCATED AT 250 WORDS)