Early apoptotic and late necrotic components associated with altered Ca2+ homeostasis in a peptide-delivery model of polyglutamine-induced neuronal death.
The mechanisms by which polyglutamine expansion causes common features of neuronal death remain unclear. Here we describe an approach for delivering polyglutamine expansions directly into cultured sympathetic neurons. Glutamine (Q) residues (n = 10, 22, 30) were conjugated with a peptide possessing translocation properties across plasma membranes (PDP) and a nuclear localization signal (NLS). These peptides were rapidly incorporated into sympathetic neurons and showed neurotoxicity in a length- and dose-dependent manner. A robust induction of c-jun and cyclin D1 occurred following treatment with PDP-Q22-NLS. Enhanced c-Jun phosphorylation showed c-Jun N-terminal kinase (JNK) activation. Coincidentally, TrkA tyrosine phosphorylation was decreased in association with loss of phospho-Akt, the downstream target of PI-3 kinase. Despite such proapoptotic signals, neither release of cytochrome c from mitochondria nor caspase-3/7 activation was detected. TdT-mediated dUTP nick-end labeling-positive nuclear condensation, but no fragmentation, occurred. At 24 hr of treatment, cytoplasmic Ca2+ levels began to become elevated, and the cellular level of ATP was decreased. Cytoplasmic Ca2+ responses to KCl depolarization displayed a delayed recovery, providing evidence for lack of Ca2+ homeostasis. The neurons became committed to death at about 36 hr when mitochondrial Ca2+ uptake declined concurrently with loss of mitochondrial membrane potential. Collectively, these results show that, despite induction of early apoptotic signals, nonapoptotic neuronal cell death occurred via perturbed Ca2+ homeostasis and suggest that mitochondrial permeability transition may play important roles in this model of neuronal death.
Suzuki
Mari
M
Molecular Neurobiology Laboratory, Division of Biological Sciences, Graduate School of Science,Hokkaido University, Sapporo, Japan.
Koike
Tatsuro
T
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western / Q000379:methods; D002118:Calcium / Q000378:metabolism*; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases / Q000378:metabolism; D002452:Cell Count / Q000379:methods; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D019938:Cyclin D1 / Q000235:genetics / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D004097:Dihydrotachysterol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006706:Homeostasis / Q000187:drug effects; D007150:Immunohistochemistry / Q000379:methods; D020287:In Situ Nick-End Labeling / Q000379:methods; D007476:Ionophores / Q000494:pharmacology; D019344:Lactic Acid / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D008954:Models, Biological; D009336:Necrosis / Q000139:chemically induced / Q000378:metabolism; D020932:Nerve Growth Factor / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D018696:Neuroprotective Agents / Q000494:pharmacology; D010455:Peptides / Q000633:toxicity*; D010539:Permeability / Q000187:drug effects; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D019311:Staurosporine / Q000494:pharmacology; D017783:Superior Cervical Ganglion / Q000166:cytology; D013997:Time Factors
D000590:Amino Acid Chloromethyl Ketones; D004791:Enzyme Inhibitors; D007476:Ionophores; D018696:Neuroprotective Agents; D010455:Peptides; D011500:Protein Synthesis Inhibitors; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D019938:Cyclin D1; D005576:Colforsin; C097188:polyglutamine; D019344:Lactic Acid; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D020932:Nerve Growth Factor; D003513:Cycloheximide; C505410:Casp3 protein, rat; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases; D019311:Staurosporine; D004097:Dihydrotachysterol; D002118:Calcium