PITUITARY ADENYLYL CYCLASE-ACTIVATING POLYPEPTIDE AND NERVE GROWTH-FACTOR USE THE PROTEASOME TO RESCUE NERVE GROWTH FACTOR-DEPRIVED SYMPATHETIC NEURONS CULTURED FROM CHICK-EMBRYOS

Removal of nerve growth factor (NGF) from sympathetic neurons initiate s a neuronal death program and apoptosis. We show that pituitary adeny lyl cyclase-activating polypeptide (PACAP) prevents apoptosis in NGF-d eprived sympathetic neurons. PACAP (100 nM) added to culture medium at the time of plating failed to support neuronal survival. However, in neurons grown for 2 days with NGF and then deprived of NGF, PACAP prev ented cell death for the next 24-48 h. Uptake of [H-3]norepinephrine ( [3H]NE) was used as an index of survival and decreased >50% in NGF-dep rived cultures within 24 h, PACAP(1-100 nM) restored [3H]NE uptake to 92 +/- 8% of that of NGF-supported controls. Depolarization-induced [H -3]NE release in neurons rescued by PACAP was the same as that in NGF- supported neurons. PACAP rescue was not mimicked by forskolin or 8-bro mo-cyclic AMP and was not blocked by the protein kinase A inhibitor Rp -adenosine 3',5'-cyclic monophosphothioate. Mobilization of phosphati dylinositol by muscarine failed to support NGF-deprived neurons. Thus, PACAP may use novel signaling to promote survival of sympathetic neur ons. The apoptosis-associated caspase CPP32 activity increased approxi mately fourfold during 6 h of NGF withdrawal (145 +/- 40 versus 38 +/- 17 nmol of substrate cleaved/min/mg of protein) and returned to even below the control level in NGF-deprived, PACAP-rescued cultures(14 +/- 7 nmol/min/mg of protein). Readdition of NGF or PACAP to NGF-deprived cultures reversed CPP32 activation, and this was blocked by lactacyst in, a potent and specific inhibitor of the 20S proteasome, suggesting that NGF and PACAP target CPP32 for destruction by the proteasome. As PACAP is a preganglionic neurotransmitter in autonomic ganglia, we pro pose a novel function for this transmitter as an apoptotic rescuer of sympathetic neurons when the supply of NGF is compromised.