Wnt-pathway perturbation
For further analysis of the role of the canonical Wnt-pathway for the development of ventral midbrain NPCs and the elucidation of new targets for therapeutical intervention perturbation experiments are performed using molecular genetical and biochemical tools.For this purpose, knockdown of the components of the canonical Wnt-pathway Fzd3, LRP5/6, Dvl1-3, GSK-3β and β-catenin is achieved by stable in vitro transfection of human NPCs with siRNA-eGFP-constructs using nucleofection technology. Over-expression of the components of the canonical Wnt-pathway LRP5/6, Dvl1-3, GSK-3β and S33A-β-catenin is accomplished by stable in vitro transfection of eGFP-constructs containing the respective genes using the same transfection technique.Candidate small molecule lead compounds (conventional GSK-3β inhibitors, SB216763-derivatives and resveratrol) are tested for their effect on GSK-3β activity (GS-phsphorylation) and cytosolic and nuclear β-catenin accumulation by quantitative Western blot (Odyssey system), on β-catenin-dependent transcriptional activity by TCF-luciferase reporter assay and on neuronal, especially dopaminergic differentiation, by quantitative immunofluorescence assessment of respective marker molecules (nestin for stem cells, GFAP for astrocytes, β-III-tubulin for neurons, MAP2ab for mature neurons and tyrosine hydroxylase for dopaminergic neurons). Experiments are performed by quantitative immunofluorescence in the transfected NPC line ReNcell VM. Furthermore,large compound libraries derived from combinatory chemistry are assessed in ReNcell VM cells, NeuroProgen cells and the human embryonic stem cells SA002.5 (Cellartis, Göteborg, Sweden) by applying microplate-based HTS using eGFP reporter gene constructs coupled to the T cell factor/lymphoid enhancer factor TCF1/LEF1. Subsequently, active compounds are tested in a high content setting for their influence on marker expression for NPCs, astroglial cells and dopaminergic neurons as for the above mentioned already established lead compounds. A special focus of investigations will be to elucidate the role of Wnt-8 for the differentiation of ventral midbrain cells. Wnt8 has been found to be transcribed and differentially expressed during the in vitro differentiation process in human ventral midbrain derived NPCs ReNcell VM cells by gene expression microarray analsis. To further elucidate the role of Wnt8 for neural differentiation, Wnt8 expression is investigated in ReNcell VM cells on transcriptional level (real-time RT-PCR, LightCycler technology) and on protein level by quantitative Western blot (Odyssey system). Now, the Wnt8 gene will be over-expressed and deleted in the same cells by transfection experiments. For this purpose pilot experiments are performed to establish the respective transfection methods in the conventional model system of non-neural HEK293 cells.