ALTERED PROTEIN-BINDING TO THE OCTAMER MOTIF APPEARS TO BE AN EARLY EVENT IN PROGRAMMED NEURONAL CELL-DEATH

Electrophoretic mobility-shift assays were used to characterize binding of nuclear proteins to consensus sequences for Sp1, E2F, octamer, and cAMP responsive enhancer element (CRE) during neuronal death in vitro after removal of nerve growth factor (NGF). Molecular events occurring prior to cell death in terminally differentiated PC12 cells could be divided into three phases: (i) within 2 hr of removing NGF, binding to the octamer sequence decreased, (ii) after 5-7 hr an increase in binding to CRE occurred; and (iii) after 14 hr (the point at which 50% of the cells are committed to die) a decrease in binding to the Sp1 sequence occurred. Assays performed with extracts from sympathetic ganglia indicated that changes in binding to CRE and octamer motifs also occurred during the period of developmental cell death in vivo. Double-stranded oligonucleotides were delivered to neurons to act as dominant negative ''promoters'' unable to couple to transcriptional events but capable of binding and sequestering transcription factors. Double-stranded but not single-stranded octamer oligonudeotides increased cell death of primary cultures of sympathetic neurons. Most of the induced neuronal cell death could be blocked with NGF, which is consistent with oligonucleotides activating an endogenous death program rather than having a nonspecific toxic effect. Other double-stranded oligonucleotides as well as a mutant octamer oligonucleotide had tittle or no effect on cell death. These data are consistent with the hypothesis that cell death results from a cascade of cellular and molecular events and that an early event in programmed neuronal cell death is a decrease in binding of transcription factor(s) to octamer motif sequences.