EXPRESSION AND REGULATION OF GLIAL-CELL-LINE-DERIVED NEUROTROPHIC FACTOR (GDNF) MESSENGER-RNA IN HUMAN ASTROCYTES IN-VITRO

The expression and modulation of mRNA for glial-cell-line-derived neur otrophic factor (GDNF) in human glial cells was investigated. Astrocyt e cell cultures were isolated from human fetal brains, characterized b y immunocytochemistry and maintained in vitro in conditions of high pu rity; sister cultures were exposed to protein kinase C (PKC) inhibitor s for 20 min. Total RNA was extracted from the cell pellets, reverse-t ranscribed into cDNA and amplified by the polymerase chain reaction (P CR) with primers specific for GDNF. A reverse-transcription/PCR proced ure was also performed on mRNA extracted from human fibroblast and lym phocyte cell lines. Human astrocytes grown in the absence of neurons e xpressed detectable amounts of mRNA for GDNF but no amplification prod ucts were observed in fibroblasts and lymphocytes, thus confirming tha t GDNF production was cell-type specific. After exposure to PKC inhibi tors, a dramatic down-regulation of GDNF mRNA was observed in astrocyt e cell cultures, Thus, human astrocytes are constitutively capable of producing GDNF, such trophic activity is restricted to neural cells, a nd PKC plays key roles in signal pathways that regulate the gene activ ation and production of GDNF.