title: Effects of acute ethanol exposure on regulatory mechanisms of Bcl-2-associated apoptosis promoter, bad, in neonatal rat cerebellum: differential effects during vulnerable and resistant developmental periods.
abstract: BACKGROUND
  Prenatal alcohol exposure produces anatomical and behavioral abnormalities associated with fetal alcohol syndrome (FAS). Animal FAS models have demonstrated temporal windows of vulnerability in the developing cerebellum, with substantial ethanol (EtOH)-mediated apoptotic activation during these periods. In rodents, the cerebellum is most sensitive to EtOH on postnatal days 4 to 6 (P4 to P6). At slightly later ages (P7 and later), this region is less vulnerable to EtOH. The present study investigated EtOH effects on mechanisms related to activities of Bad, a proapoptotic member of the Bcl-2 gene family, to further characterize processes underlying these disparate EtOH sensitivities. In healthy cells, Bad is retained in the cytosol by association with 14-3-3, a primarily cytosolic protein. Bad promotes apoptosis by disassociating from 14-3-3 and sequestering Bcl-xL through heterodimerization. This dimerization prevents the neutralizing association of Bcl-xL with Bax, freeing Bax to perform in a prodeath manner. Caspase-dependent cleavage of Bad to a 15-kDa fragment increases its proapoptogenic capacity.
  
  
  METHODS
  Two hours following EtOH exposure of P4 and P7 animals via inhalation, we determined how exposure affects intracellular localization and proteolytic cleavage of Bad and expression of cerebellar 14-3-3, using subcellular fractionation and Western blot techniques. Ethanol effects on interactions between Bad and 14-3-3 or Bcl-xL at the more vulnerable and less vulnerable ages were determined using an enzyme-linked immunosorbent assay-based technique to detect native protein-protein interactions.
  
  
  RESULTS
  At P4, EtOH increased mitochondrial localization of Bad, expression of a 15-kDa fragment recognized by Bad antibody, and formation of Bad:Bcl-xL complexes. At that more vulnerable age, EtOH also decreased formation of Bad:14-3-3 complexes. At P7, EtOH increased Bad:14-3-3 complexes and reduced Bad:Bcl-xL complexes. Cytosolic 14-3-3 remained unchanged by EtOH at P4 and P7.
  
  
  CONCLUSIONS
  Ethanol-induced alterations of Bad-related mechanisms at P4 favor a prodeath response. EtOH does not influence these same mechanisms in a manner that promotes cell death at P7. Divergent Bad-related responses at these 2 developmental ages likely contribute to their differential EtOH vulnerability.
authors.affiliation: Department of Neuroscience, Center for Alcohol Research, McKnight Brain Institute, University of Florida, Gainesville, Florida 32611, USA. kendra@ufl.edu
authors.forename: Kendra I
authors.initials: KI
authors.lastname: Siler-Marsiglio
authors.forename: Irina
authors.initials: I
authors.lastname: Madorsky
authors.forename: Qun
authors.initials: Q
authors.lastname: Pan
authors.forename: Michael
authors.initials: M
authors.lastname: Paiva
authors.forename: Andy W
authors.initials: AW
authors.lastname: Neeley
authors.forename: Gerry
authors.initials: G
authors.lastname: Shaw
authors.forename: Marieta B
authors.initials: MB
authors.lastname: Heaton
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism; D000375:Aging; D000818:Animals; D000831:Animals, Newborn / Q000254:growth & development / Q000378:metabolism*; D015153:Blotting, Western; D002458:Cell Fractionation; D002531:Cerebellum / Q000737:chemistry / Q000187:drug effects* / Q000648:ultrastructure; D004797:Enzyme-Linked Immunosorbent Assay; D000431:Ethanol / Q000008:administration & dosage*; D005260:Female; D008297:Male; D008928:Mitochondria / Q000737:chemistry; D010446:Peptide Fragments / Q000032:analysis; D051381:Rats; D020318:Rats, Long-Evans; D051025:bcl-Associated Death Protein / Q000032:analysis* / Q000378:metabolism*; D051020:bcl-X Protein / Q000378:metabolism
chemical_list: D048948:14-3-3 Proteins; C494821:Bad protein, rat; C494813:Bcl2l1 protein, rat; D010446:Peptide Fragments; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein; D000431:Ethanol