Neurotrophic effects of immunophilin ligands have been shown in animal models of peripheral and central nervous system insult. To investigate the specific growth-promoting effects of these compounds, we examined the effects of various immunophilin ligands on primary dopamine (DA) neurons in culture and compared these with a well-known DA trophic factor, glial cell line-derived neurotrophic factor (GDNF). In neuronal cultures from Embryonic Day 14 ventral mesencephalon, enhanced elongation of DA neurites was observed with immunophilin ligands, which inhibited the phosphatase activity of calcineurin (FK506 and cyclosporin A) when compared to vehicle-treated cultures. This elongation was also observed with GDNF, known to exert its trophic effects through phosphorylation-dependent pathways. In contrast, immunophilin ligands that do not inhibit calcineurin (rapamycin and V-10,367) increased branching of DA neurites, suggesting that elongation is dependent upon maintained phosphorylation while branching is not. In addition, both V-10,367 and rapamycin antagonized the elongation effects of FK506 and induced branching. The antagonism of elongation (and reappearance of branching) illustrates the intrinsic abilities of developing DA neurons to either elongate or branch, but not both. We show that the immunophilin FKBP12 (12-kDa FK506-binding protein) is expressed in ventral mesencephalic neuronal cultures and colocalizes with DA neurons. This work elucidates the specific growth-promoting effects by which GDNF and immunophilin ligands modify developmental growth processes of DA neurons, via their interactions with intracellular targets.
Copyright 2000 Academic Press.