RAIDD aggregation facilitates apoptotic death of PC12 cells and sympathetic neurons.

In human cell lines, the caspase 2 adaptor RAIDD interacts selectively with caspase 2 through its caspase recruitment domain (CARD) and leads to caspase 2-dependent death. Whether RAIDD induces such effects in neuronal cells is unknown. We have previously shown that caspase 2 is essential for apoptosis of trophic factor-deprived PC12 cells and rat sympathetic neurons. We report here that rat RAIDD, cloned from PC12 cells, interacts with rat caspase 2 CARD. RAIDD overexpression induced caspase 2 CARD- and caspase 9-dependent apoptosis of PC12 cells and sympathetic neurons. Apoptosis correlated with the formation of discrete perinuclear aggregates. Both death and aggregates required the expression of full-length RAIDD. Such aggregates may enable more effective activation of caspase 2 through close proximity. Following trophic deprivation, RAIDD overexpression increased death and aggregate formation. Therefore, RAIDD aggregation is important for its death-promoting effects and may play a role in trophic factor withdrawal-induced neuronal apoptosis.

Inhibitors of trypsin-like serine proteases prevent DNA damage-induced neuronal death by acting upstream of the mitochondrial checkpoint and of p53 induction.

We have previously shown that the pharmacological agents 4-(2-aminoethyl)=benzenesulfonylfluoride hydrochloride (AEBSF) and Na-p-tosyl-L-lysine chloromethylketone (TLCK), inhibitors of trypsin-like serine proteases, prevent the death of trophic factor-deprived PC12 cells and sympathetic neurons. Both AEBSF and TLCK inhibit caspase activation in this model, but it is unclear whether they do so indirectly or through a direct effect at the level of the caspases. In the current study, we have used these agents in another model of neuronal death that is induced by DNA damage. We find that both agents delay the death of DNA-damaged PC12 cells, neonatal rat sympathetic neurons and embryonic rat cortical neurons. As in the trophic deprivation model, they act upstream of the caspases. In addition, they prevent mitochondrial alterations, such as cytochrome c release or loss of transmembrane potential. In contrast, the general caspase inhibitor bok-asp-fmk does not prevent cytochrome c release and has only a partial and transient effect on loss of transmembrane potential. Interestingly, both AEBSF and TLCK prevent the induction and nuclear accumulation of p53 that is induced by DNA damage in cortical neurons. Therefore, these serine protease inhibitors act at a point upstream in the apoptotic pathway, prior to p53 induction and the mitochondrial checkpoint, to delay neuronal death in this model, and do not act at the level of the caspases. We conclude that therapeutic strategies based on serine protease inhibition may be useful in preventing neuronal cell death.