Neurite outgrowth in peripherin-depleted PC12 cells.
Peripherin is the major neuronal intermediate filament (IF) protein in PC12 cells and both its synthesis and amount increase during nerve growth factor (NGF) promoted neuronal differentiation. To address the question of the biological function of peripherin in neurite initiation we have used an antisense oligonucleotide complementary to the 5' region of peripherin mRNA to specifically inhibit its transcription. The oligonucleotide blocks both the synthesis of peripherin and its increase in response to NGF. Peripherin was found to be a stable protein with a cellular half-life of approximately 7 d. 6 wk of incubation with the oligonucleotide decreases peripherin to 11% of the level in naive control cells and to 3% of that in NGF-treated control cells. Despite the depletion, NGF elicits apparently normal neurite outgrowth from the oligonucleotide-treated cells. As evaluated by EM, there are few IFs in these cells, either in the cell bodies or neurites. There is no compensatory increase in NF-M, NF-L, or vimentin levels as a result of the inhibition of peripherin synthesis. These findings suggest that peripherin is not required for neurite formation, but is necessary for the formation of a cellular IF network which could be involved in process stability. They also demonstrate the utility of antisense oligonucleotides for the study of proteins with long half-lives.
Troy
C M
CM
Department of Pathology, Alzheimer's Disease Research Center, College of Physicians and Surgeons, Columbia University, New York 10032.
Greene
L A
LA
Shelanski
M L
ML
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects; D007381:Intermediate Filament Proteins / Q000235:genetics / Q000502:physiology*; D007382:Intermediate Filaments / Q000378:metabolism; D008562:Membrane Glycoproteins; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000494:pharmacology*; D009419:Nerve Tissue Proteins; D016501:Neurites / Q000187:drug effects* / Q000502:physiology; D009479:Neuropeptides / Q000235:genetics / Q000502:physiology*; D016716:PC12 Cells; D064531:Peripherins; D016372:RNA, Antisense / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics
D007381:Intermediate Filament Proteins; D008562:Membrane Glycoproteins; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D009479:Neuropeptides; D064531:Peripherins; D016372:RNA, Antisense