title: Involvement of caspase activation in azaspiracid-induced neurotoxicity in neocortical neurons.
abstract: Azaspiracids (AZAs) are a novel group of marine phycotoxins that have been associated with severe human intoxication. We found that AZA-1 exposure increased lactate dehydrogense (LDH) efflux in murine neocortical neurons. AZA-1 also produced nuclear condensation and stimulated caspase-3 activity with an half maximal effective concentration (EC(50)) value of 25.8 nM. These data indicate that AZA-1 triggers neuronal death in neocortical neurons by both necrotic and apoptotic mechanisms. An evaluation of the structure-activity relationships of AZA analogs on LDH efflux and caspase-3 activation demonstrated that the full structure of AZAs was required to produce necrotic or apoptotic cell death. The similar potencies of AZA-1 to stimulate LDH efflux and caspase-3 activation and the parallel structure-activity relationships of azaspiracid analogs in the two assays are consistent with a common molecular target for both responses. To explore the molecular mechanism for AZA-1-induced neurotoxicity, we assessed the influence of AZA-1 on Ca(2+) homeostasis. AZA-1 suppressed spontaneous Ca(2+) oscillations (EC(50) = 445 nM) in neocortical neurons. A distinct structure-activity profile was found for inhibition of Ca(2+) oscillations where both the full structure as well as analogs containing only the FGHI domain attached to a phenyl glycine methyl ester moiety were potent inhibitors. The molecular targets for inhibition of spontaneous Ca(2+) oscillations and neurotoxicity may therefore differ. The caspase protease inhibitor Z-VAD-FMK produced a complete elimination of AZA-1-induced LDH efflux and nuclear condensation in neocortical neurons. Although the molecular target for AZA-induced neurotoxicity remains to be established, these results demonstrate that the observed neurotoxicity is dependent on a caspase signaling pathway.
authors.affiliation: Department of Pharmacology, School of Medicine, Creighton University, Omaha, Nebraska 68178, USA.
authors.forename: Zhengyu
authors.initials: Z
authors.lastname: Cao
authors.forename: Keith T
authors.initials: KT
authors.lastname: LePage
authors.forename: Michael O
authors.initials: MO
authors.lastname: Frederick
authors.forename: Kyriacos C
authors.initials: KC
authors.lastname: Nicolaou
authors.forename: Thomas F
authors.initials: TF
authors.lastname: Murray
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D053148:Caspase 3 / Q000096:biosynthesis* / Q000187:drug effects*; D061945:Caspase Inhibitors; D002478:Cells, Cultured; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004622:Embryo, Mammalian / Q000166:cytology / Q000196:embryology; D006706:Homeostasis / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000187:drug effects / Q000378:metabolism; D008387:Marine Toxins / Q000737:chemistry / Q000633:toxicity*; D051379:Mice; D009336:Necrosis / Q000139:chemically induced; D019579:Neocortex / Q000166:cytology / Q000196:embryology; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D057096:Shellfish Poisoning; D015398:Signal Transduction / Q000187:drug effects; D013141:Spiro Compounds / Q000737:chemistry / Q000633:toxicity*; D013329:Structure-Activity Relationship; D000095702:Polyether Toxins
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D002118:Calcium; D053148:Caspase 3; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D007770:L-Lactate Dehydrogenase; D008387:Marine Toxins; D013141:Spiro Compounds; C406592:azaspiracid; D000095702:Polyether Toxins; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone