GDNF signaling in embryonic midbrain neurons in vitro

The glial cell line-derived neurotrophic factor (GDNF) exerts trophic actions on a number of cell types, including mesencephalic dopaminergic (mDA) neurons. Using rat mesencephalic primary cultures enriched in mDA neurons, we show that protracted GDNF stimulation increases their survival and neurite outgrowth. It modulates the expression of genes essential for DA function (tyrosine hydroxylase, TH and dopamine transporter, dat) and of DA high affinity uptake. To identify genes involved in GDNF signaling pathways, we have used DNA microarray on mDA cultures stimulated with GDNF for 3 h. Here we show that GDNF signaling sequentially activates the genes encoding for the transcription factors EGR1 and TIEG. In addition, it increases the expression of cav1, which encodes for the major component of caveolae. GDNF also modulates the expression of the genes encoding for the Calcineurin subunits ppp3R1 and ppp3CB, and inhibits calcium–calmodulin-dependent protein kinase II β isoform (CaMKIIβ) gene expression. These proteins are involved in neuronal differentiation and synaptic plasticity. Moreover, GDNF stimulation down regulates the expression of the glycogen synthase kinase 3β (gsk3β) gene, involved in neuronal apoptosis. Using inhibitors of specific intracellular signal transduction pathways we show that changes of egr1, tieg, cav1, CaMkIIβ and gsk3β genes expression are extracellular-signal regulated kinases 1/2 (ERK)-dependent, while the cAMP-dependent protein kinase (PKA) pathway influences the up-regulation of ppp3R1 and ppp3CB gene expression. These results demonstrate that GDNF stimulation results in the transcriptional modulation of genes involved in neuronal plasticity and survival and in mDA function, mediated in part by ERK and PKA signaling.