Inhibition of glial cell line-derived neurotrophic factor induced intracellular activity by K-252b on dopaminergic
1Department of Neuroscience, Amgen, Inc., Thousand Oaks, California 91320, U.S.A.
Abstract:
The c-ret protooncogene encodes Ret, the functional tyrosine kinase receptor for glial cell line-derived neurotrophic factor (GDNF). K-252b, a known protein tyrosine kinase inhibitor, has been shown earlier to inhibit the trophic activity of brain-derived neurotrophic factor on dopaminergic (DAergic) neurons and nerve growth factor on basal forebrain cholinergic neurons while potentiating neurotrophin-3 activity on central cholinergic and peripheral sensory neurons and PC12 cells. We tested whether K-252b would modulate GDNF-induced differentiation in DAergic neuron cultures. Exposure to 1 ng/ml GDNF increased dopamine (DA) uptake 80% above control, whereas treatment with 5 microM K-252b decreased the efficacy of GDNF by 60%. Concentrations of GDNF of <100 pg/ml were completely inhibited, whereas concentrations of >100 pg/ml were moderately active, between 10 and 20% above control. In addition, K-252b shifted the ED50 from 20 to 200 pg/ml. GDNF treatment increased soma size and neurite outgrowth in tyrosine hydroxylase-immunoreactive neurons. K-252b inhibited differentiation of these morphological parameters induced by GDNF. Furthermore, GDNF stimulated Ret autophosphorylation at maximal levels, whereas the inhibition of DA uptake and morphological differentiation by K-252b correlated with a significantly decreased level of Ret autophosphorylation. Therefore, K-252b is able to inhibit intracellular activities induced by GDNF on mesencephalic DAergic neurons.
Insights
The protein kinase inhibitor K-252b blocks glial cell line-derived neurotrophic factor (GDNF) from promoting dopamine (DA) neuron differentiation. K-252b reduces GDNF
Area of Science:
- Neuroscience
- Molecular Biology
- Cell Biology
Background:
- The c-ret protooncogene encodes the Ret tyrosine kinase receptor, crucial for glial cell line-derived neurotrophic factor (GDNF) signaling.
- K-252b is a known protein tyrosine kinase inhibitor with varied effects on neurotrophic factor activity.
Purpose of the Study:
- To investigate the effect of K-252b on GDNF-induced differentiation in dopaminergic (DAergic) neuron cultures.
- To determine if K-252b modulates Ret autophosphorylation in response to GDNF.
Main Methods:
- Primary cultures of mesencephalic DAergic neurons were treated with GDNF and K-252b.
- Dopamine uptake, morphological parameters (soma size, neurite outgrowth), and Ret autophosphorylation levels were measured.
Main Results:
- K-252b significantly inhibited GDNF-induced dopamine uptake and morphological differentiation.
- K-252b reduced GDNF-stimulated Ret autophosphorylation in a dose-dependent manner.
- The efficacy of GDNF was reduced by K-252b, shifting the ED50 value.
Conclusions:
- K-252b effectively inhibits GDNF-induced intracellular signaling pathways in DAergic neurons.
- The findings suggest K-252b interferes with Ret receptor activity, impacting neuronal differentiation.
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