The Use of Antisense Intervention to Decipher the Role of the Neuronal Growth-Associated Protein GAP-43

Rachael L. Neve, Kathryn J. Ivins, Kim Neve, Ben A. Bahr, Peter W. Vanderklish, Amy Arai, Gary Lynch

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Two forms of antisense technology were used to explore the role of the neuronal growth-associated protein GAP-43 (neuromodulin, B-50, Fl, P-57) in the brain. The first method exploited the ability of dividing cell lines to take up and stably integrate recombinant retrovirus vectors. We transfected PC12 cells with a recombinant expression vector containing a human GAP-43 cDNA in reverse (antisense) orientation. Retrieval and characterization of two stable transfectants, AS1 and AS2, expressing high levels of antisense GAP-43 RNA revealed that the level of GAP-43 protein in these cell lines was reduced relative to controls. in the presence of extraceilular calcium, a depolarizing concentration of K+ failed to stimulate dopamine release from AS1 and AS2 cells although evoked dopamine release from control cells was normal. Similarly, the calcium ionophore A23189 was ineffective in stimulating dopamine release from the cells expressing GAP-43 antisense cRNA even though it evoked dopamine release from control cells. These data suggest that GAP-43 plays an essential role in calcium-dependent neurotransmitter release in PC12 cells. The antisense GAP-43-transfected PC12 cells displayed processes of normal length when treated with NGF on polylysine or laminin substrates. in contrast, primary rat hippocampal cell cultures plated in the presence of GAP-43 antisense oligonucleotides, in a series of experiments exploiting a second antisense technology, were deficient In process outgrowth. This deficiency was dose-dependent. To venture further into the realm of GAP-43 function in the brain, we have designed a procedure to depress levels of GAP-43 protein in cultured hippocampal slices.

Original languageEnglish (US)
Pages (from-to)39-49
Number of pages11
JournalNeuroprotocols
Volume2
Issue number1
DOIs
StatePublished - Feb 1993

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GAP-43 Protein
Nerve Growth Factors
PC12 Cells
Dopamine
alaproclate
Calcium
Technology
Cell Line
Complementary RNA
Polylysine
Calcium Ionophores
Antisense Oligonucleotides
Brain
Laminin
Nerve Growth Factor
Retroviridae
Neurotransmitter Agents

Cite this

The Use of Antisense Intervention to Decipher the Role of the Neuronal Growth-Associated Protein GAP-43. / Neve, Rachael L.; Ivins, Kathryn J.; Neve, Kim; Bahr, Ben A.; Vanderklish, Peter W.; Arai, Amy; Lynch, Gary.

In: Neuroprotocols, Vol. 2, No. 1, 02.1993, p. 39-49.

Research output: Contribution to journalArticle

Neve, Rachael L. ; Ivins, Kathryn J. ; Neve, Kim ; Bahr, Ben A. ; Vanderklish, Peter W. ; Arai, Amy ; Lynch, Gary. / The Use of Antisense Intervention to Decipher the Role of the Neuronal Growth-Associated Protein GAP-43. In: Neuroprotocols. 1993 ; Vol. 2, No. 1. pp. 39-49.
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