INVOLVEMENT OF CASPASES IN SYMPATHETIC NEURON APOPTOSIS

In order to study the involvement of caspases in neuronal cell death, we have examined the effects of the viral caspase inhibitor p35 and pe ptide caspase inhibitors on sympathetic neurons isolated from the supe rior cervical ganglion (SCG), In these neurons, apoptosis can be induc ed by the withdrawal of nerve growth factor (NGF) and also by the addi tion of the kinase inhibitor staurosporine. p35 has been shown to be a broad spectrum inhibitor of the caspase family and promotes the survi val of SCG neurons withdrawn from NGF, We show that p35 is also protec tive when apoptosis is induced by staurosporine. In addition, p35 inhi bits a number of the morphological features associated with apoptosis, such as nuclear condensation, TUNEL labelling, and externalisation of phosphatidylserine, The tri-peptide caspase inhibitor benzyloxycarbon yl-Val-Ala-Asp (O-methyl)-fluoromethylketone (zVAD-fmk) was effective at inhibiting NGF withdrawal-induced and staurosporine-induced apoptos is of SCG neurons, Two other peptide inhibitors, acetyl-Tyr-Val-Ala-As p-aldehyde (Ac-YVAD-CHO) and acetyl-Asp-Glu-Ala-Asp-aldehyde (Ac-DEVD- CHO), also inhibited apoptosis induced by both means when microinjecte d into SCG neurons but peptides derived from the caspase cleavage site in p35 were not protective. We present data to suggest that apoptosis induced by separate death stimuli can result either in the activation of distinct caspases or in differences in the time of activation of t he family members.