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Transfection of C6 glioma cells with glia maturation factor upregulates brain-derived neurotrophic factor and nerve growth factor: trophic effects and protection against ethanol toxicity in cerebellar granule cells
Brain research
  • N. J. Pantazis
  • A. Zaheer
  • Donghai Dai, University of Iowa
  • S. Zaheer
  • S. H. Green
  • R. Lim
Document Type
Article
Peer Reviewed
1
Publication Date
5-1-2000
NLM Title Abbreviation
Brain Res
PubMed ID
10814733
Abstract

Glial cells play active roles in neuronal survival, as well as neuroprotection against toxic insult. Recent studies suggest that the brain protein glia maturation factor (GMF) is involved in intracellular signaling in glia. This study investigated whether or not GMF plays a role in the survival-promoting and neuroprotective functions of glia. C6 glioma cells were transfected in vitro with GMF utilizing an adenovirus vector. The transfected cells overexpressed GMF intracellularly, but did not secrete the protein. The conditioned medium (CM) was obtained from the GMF-transfected cells (CM-GMF) and tested on primary neuronal cultures, consisting of cerebellar granule cells (CGC). The CGC cultures were utilized because these cultures have a background level of cell death, and the survival-promoting, i.e. neurotrophic effect, of the CM could be tested. In addition, since CGC cultures are ethanol-sensitive (ethanol enhances neuronal death), the neuroprotective effect of the CM against ethanol-induced cell death was tested also. We demonstrated that the CM-GMF had an enhanced neurotrophic effect as well as an increased neuroprotective effect against ethanol-induced cell death compared to control CM obtained from untransfected C6 cells (CM-Mock) or CM obtained from cells transfected with an unrelated gene (CM-LacZ). Because neurotrophins have trophic and protective effects, we investigated whether GMF-transfection upregulated the expression of neurotrophins in C6 cells. RT-PCR verified that GMF-transfected C6 cells had increased mRNA levels for BDNF and NGF. Immunoblotting corroborated the RT-PCR results and indicated that CM-GMF contained greater concentrations of BDNF and NGF protein compared to CM-Mock and CM-LacZ. A soluble TrkB-IgG fusion protein, which selectively binds BDNF and prevents its binding to the neuronal TrkB receptor, eliminated the neurotrophic effect of CM-GMF; whereas anti-NGF antibody was ineffective in preventing this effect, suggesting that the neurotrophic effect was due to BDNF. On the other hand, both the TrkB-IgG fusion protein and anti-NGF reduced neuroprotection, suggesting that BDNF and NGF both contribute to the neuroprotective effect of CM-GMF. In conclusion, GMF upregulates the expression of BDNF and NGF in C6 cells, and these factors exert neurotrophic and neuroprotective functions on primary neurons.

Keywords
  • Adenoviridae/physiology,
  • Alcohol-Induced Disorders,
  • Nervous System/drug therapy/physiopathology,
  • Animals,
  • Antibody Specificity,
  • Brain-Derived Neurotrophic Factor/analysis/antagonists & inhibitors/metabolism/pharmacology,
  • Cell Death/drug effects/genetics,
  • Central Nervous System Depressants/toxicity,
  • Cerebellar Cortex/cytology/drug effects/metabolism,
  • Culture Media,
  • Conditioned/chemistry/pharmacology,
  • Ethanol/toxicity,
  • Gene Expression Regulation/physiology,
  • Genetic Vectors/physiology,
  • Glia Maturation Factor/genetics/metabolism,
  • Glioma,
  • Humans,
  • Nerve Growth Factor/antagonists & inhibitors/metabolism/pharmacology,
  • Neuroglia/cytology/metabolism,
  • Neurons/drug effects/metabolism,
  • Neuroprotective Agents/metabolism,
  • RNA,
  • Messenger/metabolism,
  • Rats,
  • Rats,
  • Sprague-Dawley,
  • Receptors,
  • Nerve Growth Factor/genetics,
  • Recombinant Proteins/pharmacology,
  • Transfection,
  • Tumor Cells,
  • Cultured
Published Article/Book Citation
Brain research, 865:1 (2000) pp.59-76.
Citation Information
N. J. Pantazis, A. Zaheer, Donghai Dai, S. Zaheer, et al.. "Transfection of C6 glioma cells with glia maturation factor upregulates brain-derived neurotrophic factor and nerve growth factor: trophic effects and protection against ethanol toxicity in cerebellar granule cells" Brain research Vol. 865 Iss. 1 (2000) p. 59 - 76 ISSN: 0006-8993
Available at: http://works.bepress.com/donghai_dai/15/