title: Distinct mechanisms of neuronal apoptosis are triggered by antagonism of Bcl-2/Bcl-x(L) versus induction of the BH3-only protein Bim.
abstract: Primary cerebellar granule neurons (CGNs) require depolarizing extracellular potassium for their survival. Removal of depolarizing potassium triggers CGN apoptosis that requires induction of Bim, a BH3-only Bcl-2 family member. Bim is classically thought to promote apoptosis by neutralizing pro-survival Bcl-2 proteins. To determine if this is the principal function of Bim in CGNs, we contrasted Bim-mediated apoptosis to neuronal death induced by HA14-1, a BH3-domain mimetic that antagonizes Bcl-2 and Bcl-x(L). HA14-1 elicited CGN apoptosis characterized by caspase 3 and 9 activation, cytochrome c release, conformational activation of Bax, and mitochondrial depolarization. HA14-1 provoked CGN apoptosis in the absence of Bim induction and negative regulators of Bim transcription did not prevent HA14-1-induced cell death. However, the antioxidant glutathione and its precursor, N-acetyl-l-cysteine, suppressed HA14-1-induced apoptosis. Similarly, apoptosis induced by either a structurally distinct Bcl-2/Bcl-x(L) inhibitor (compound 6) or Bcl-2 antisense oligonucleotides was diminished by glutathione. In contrast, antioxidants had no effect on CGN apoptosis provoked by either removal of depolarizing potassium or overexpression of a GFP-Bim fusion protein, two models of Bim-dependent death. These data show that antagonism of Bcl-2/Bcl-x(L) function elicits oxidative stress-dependent CGN apoptosis that is mechanistically distinct from Bim-mediated cell death. These results further indicate that, although Bcl-2/Bcl-x(L) antagonism is sufficient to induce neuronal apoptosis, Bim likely promotes neuronal death by interacting with additional proteins besides Bcl-2/Bcl-x(L).
authors.affiliation: Neuroscience Program, University of Colorado Health Sciences Center, Denver, Colorado, USA.
authors.forename: Angela K
authors.initials: AK
authors.lastname: Zimmermann
authors.forename: F Alexandra
authors.initials: FA
authors.lastname: Loucks
authors.forename: Shoshona S
authors.initials: SS
authors.lastname: Le
authors.forename: Brent D
authors.initials: BD
authors.lastname: Butts
authors.forename: Maria L
authors.initials: ML
authors.lastname: Florez-McClure
authors.forename: Ron J
authors.initials: RJ
authors.lastname: Bouchard
authors.forename: Kim A
authors.initials: KA
authors.lastname: Heidenreich
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Linseman
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins; D000072224:Bcl-2-Like Protein 11; D001578:Benzopyrans / Q000494:pharmacology; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D002452:Cell Count; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D009474:Neurons / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D009570:Nitriles / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D051020:bcl-X Protein
chemical_list: D051017:Apoptosis Regulatory Proteins; D000072224:Bcl-2-Like Protein 11; C494813:Bcl2l1 protein, rat; C000606778:Bcl2l11 protein, rat; D001578:Benzopyrans; D002352:Carrier Proteins; D008565:Membrane Proteins; D009570:Nitriles; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051020:bcl-X Protein; C456800:ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate