Mechanisms of apoptosis in embryonic cortical neurons (E6 and E7) in culture involve lipid signalling, protein phosphorylation and caspase activation

Cultured embryonic (E7) chick neurons, derived from cerebral hemispheres, u nderwent apoptosis in response to inhibitors of protein kinase C (staurospo rine) and phosphatidylinositol-3-kinase (wortmannin and LY294002), in a dos e- and time-dependent manner. This was monitored by loss of cell viability, increased DNA fragmentation, and activation of caspase-3-like activity, al l of which were partially reversed by elevating the level of cAMP in the ce lls with Bt(2)cAMP or (Sp)cAMPS. Further studies revealed that an early ste p in apoptosis was the formation of ceramide from sphingomyelin, resulting from the activation of a neutral pH sphingomyelinase activity. Thus inhibit ors of protein kinase C and phosphatidylinositol-3-kinase increased ceramid e levels in the same time-frame as caspase-3 activation and DNA fragmentati on. Neurons could also be killed by the addition of either water-soluble C2 -ceramide (30 mu M) or natural C22/24 ceramide (0.5 mu M). In contrast to t he apparent protective effect of ser/thr protein phosphorylation, a pro-apo ptotic role for tyrosine phosphate phosphorylation was suggested by the abi lity of protein tyrosine phosphate phosphatase inhibitor, Bis(maltolato)oxo vanadium (IV) (BMOV), to induce apoptosis in E7CH neurons. Thus BMOV (25 mu M) killed 50% of E7CH neurons and B lymphocytes but not glial cells, or T- lymphocytes, suggesting the existence of a common apoptotic pathway in neur ons and B-cells. We conclude that the major pathway for programmed cell death in embryonic c hick neurons has many elements in common with that described for other cell s but that there may be some unique aspects which can be used to protect em bryonic neurons from opioid and other drug-enhanced apoptosis. (C) 2000 ISD N. Published by Elsevier Science Ltd. All rights reserved.