Characterization of Early Molecular Events in Programmed Neuronal Cell Death
Abstract
To understand and elucidate biochemical and molecular mechanisms responsible for regulating neuronal cell death, an in vitro model system was established using differentiated pheochromocytoma cells (PC12). Introducing the proto-oncogene bcl-2 into these cells using a retroviral vector blocked cell death caused by growth factor deprivation. Electrophoretic mobility-shift assays were used to investigate early molecular changes occurring in programmed cell death induced by removing nerve growth factor (NGF). Within 2 hours of removing NGF, binding to the octamer sequence decreased, after 5-7 hours an increase in binding to cAMP responsive enhancer element (CRE) occurred, and after 14 hours (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 into neurons to sequester transcription factors and act as dominant negative "promoters". Double-stranded octamer oligonucleotides increased death of neurons, whereas other double-stranded oligonucleotides or a mutant octamer oligonucleotide had little or no effect on cell death. Most of the induced death could be blocked with NGF, which is consistent with oligonucleotides activating an endogenous death program rather than having a nonspecific toxic effect. A subtracted cDNA library was constructed, and a series of screens were performed to identify cDNAs increased in early stages of death. Several clones were selected and verified using Northern blots. Sequence analysis showed that some of the cDNAs were identical to known genes, while others appeared to be novel. One of the cDNAs, p180, increased in sympathetic ganglia during the period of neuronal death, while two others, p117 and p160, were up-regulated in PC12 cells induced to die with the neurotoxin A$\beta$25-35. Since these three cDNAs are up-regulated in dying cells in more than one cell death paradigm, they may represent components of a common cell death pathway.
Subject Area
Neurosciences|Cellular biology|Molecular biology
Recommended Citation
Wang, Songli, "Characterization of Early Molecular Events in Programmed Neuronal Cell Death" (1994). Dissertations available from ProQuest. AAI9521140.
https://repository.upenn.edu/dissertations/AAI9521140