Co-expression of mRNA for neurotrophic factors in human neurons and glial cells in culture.
Enriched populations of neurons and astrocytes of 93-99% purity were obtained from mixed cultures of four human fetal brains. Total cellular RNA was extracted from cell pellets and reverse transcribed into cDNA. Five microliters of cDNA were subjected to PCR amplification using primers specific for sequences of NGF, BDNF, NT-3 and CNTF. PCR products were separated through 5% acrylamide gel and identified by DNA sequencing. Results showed that neurons expressed detectable levels of mRNA for NGF in all four cultures; BDNF and NT-3 mRNA was seen only in two cultures; CNTF mRNA was not detected in all four cultures. Astrocytes expressed mRNA for NGF, BDNF, and NT-3 but not for CNTF in all cultures examined. Astrocytic expression of mRNA for NGF, BDNF and NT-3 was found during the active cell proliferation as well as at a phase of mitotic quiescence. This study provides evidence that dissociated cell cultures of human neurons produce NGF, BDNF and NT-3 in early stages of their development and that astrocytes are constitutively committed to synthesize neurotrophic factors, NGF, BDNF and NT-3. The active synthesis of selected neurotrophic factors by neurons and astrocytes is relevant in supporting migration, survival and differentiation of developing neurons in vivo.
Moretto
G
G
Department of Medicine, University of British Columbia, Vancouver, Canada.
Xu
R Y
RY
Walker
D G
DG
Kim
S U
SU
D001253:Astrocytes / Q000166:cytology / Q000378:metabolism*; D001483:Base Sequence; D001921:Brain / Q000378:metabolism*; D019208:Brain-Derived Neurotrophic Factor; D002478:Cells, Cultured; D020934:Ciliary Neurotrophic Factor; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D017931:DNA Primers; D005333:Fetus; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D005904:Glial Fibrillary Acidic Protein / Q000032:analysis; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000096:biosynthesis; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000032:analysis / Q000096:biosynthesis; D008938:Mitosis; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000032:analysis / Q000096:biosynthesis*; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000096:biosynthesis; D009457:Neuroglia / Q000166:cytology / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis*
D019208:Brain-Derived Neurotrophic Factor; D020934:Ciliary Neurotrophic Factor; D017931:DNA Primers; D005904:Glial Fibrillary Acidic Protein; D008869:Microtubule-Associated Proteins; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; C071951:neurotrophin 5; D004247:DNA; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases