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 initiates a neuronal death program and apoptosis. We show that pituitary adenylyl cyclase-activating polypeptide (PACAP) prevents apoptosis in NGF-deprived 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 prevented cell death for the next 24-48 h. Uptake of [3H]norepinephrine ([3H]NE) was used as an index of survival and decreased >50% in NGF-deprived cultures within 24 h. PACAP (1-100 nM) restored [3H]NE uptake to 92 +/- 8% of that of NGF-supported controls. Depolarization-induced [3H]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-bromo-cyclic AMP and was not blocked by the protein kinase A inhibitor Rp-adenosine 3',5'-cyclic monophosphothioate. Mobilization of phosphatidylinositol by muscarine failed to support NGF-deprived neurons. Thus, PACAP may use novel signaling to promote survival of sympathetic neurons. The apoptosis-associated caspase CPP32 activity increased approximately 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 lactacystin, 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 propose a novel function for this transmitter as an apoptotic rescuer of sympathetic neurons when the supply of NGF is compromised.
Przywara
D A
DA
Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Kulkarni
J S
JS
Wakade
T D
TD
Leontiev
D V
DV
Wakade
A R
AR
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D002642:Chick Embryo; D003546:Cysteine Endopeptidases / Q000502:physiology*; D005728:Ganglia, Sympathetic / Q000166:cytology / Q000196:embryology / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000502:physiology*; D009414:Nerve Growth Factors / Q000172:deficiency* / Q000502:physiology*; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D009479:Neuropeptides / Q000494:pharmacology / Q000502:physiology*; D018696:Neuroprotective Agents / Q000494:pharmacology; D018377:Neurotransmitter Agents / Q000494:pharmacology; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D046988:Proteasome Endopeptidase Complex; D015398:Signal Transduction / Q000187:drug effects
D009097:Multienzyme Complexes; D009414:Nerve Growth Factors; D009479:Neuropeptides; D018696:Neuroprotective Agents; D018377:Neurotransmitter Agents; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex