Role of protein phosphatases in inhibition of sympathetic neuron apoptosis by ceramide

We examined the anti-apoptotic effect of ceramide in rat sympathetic neurons deprived of NGF. Upon NGF withdrawal cultured sympathetic neurons undergo apoptosis (Deshmukh and Johnson, 1997). However, addition of ceramide prevents the appearance of classical trademarks of apoptosis such as nuclear fragmentation and caspase-3 activation (Nair et al. 2000; Song and Posse de Chaves, 2002). Here, we show that treatment with C₆-ceramide or threo-PPMP, an inhibitor of ceramide conversion to glucosylceramide also prevent loss of mitochondrial potential, a late hallmark in sympathetic neuron apoptosis.; We investigated the role of serine/threonine protein phosphatases as targets for ceramide action. We demonstrate that ceramide activates PP-1 and that PP1-activation is essential for C₆-ceramide anti-apoptotic activity. Additionally, we examined the phosphorylation status of pRb, a substrate of PP-1 implicated in neuronal apoptosis. We found that pRb is hyperphosphorylated upon NGF-deprivation and that ceramide can prevent such hyperphosphorylation by a mechanism dependent on PP-1 activation.