Wnt-pathway modelling
Analysis of the functional impact of the canonical Wnt/Fz/Dsh/GSK-3β/β-catenin-signal pathway for the differentiation of human neural progenitor cells.Key components of the canonical Wnt-pathway are assessed by quantitative Western blot (Odyssey system) in proliferating and differentiating transfected NPC lines for in silico modelling and simulation of the spatio-temporal intracellular signalling and evaluation of the predictions of modelling and simulation done in cooperation with the Depts. of Modelling and Simulation and of Systems Biology and Bioinformatics of the University of Rostock. Analysis of spatio-temporal changes of the Wnt receptor components LRPs, Fzds, Dkk, Ryk, Dvl, CK1γ and ε, β-Arr 1 and 2, Dpr 1 in differentiating ReNcell VM cells is performed by time course recording using quantitative Western blot (Odyssey system) of the membrane compartment and confocal immunofluorescence microscopy. Assessment of spatio-temporal changes of the Wnt degradation complex components β-catenin (cytosolic), CK1α, GSK-3β, APC, Axin1-2, PP2A, diversin and FRAT1-3 in differentiating ReNcells VM is performed by kinetic recording using quantitative Western blot of the cytosolic compartment and by confocal immunofluorescence microscopy.
Role of the non-canonical Wnt-dependent Ca++-signal pathway in the differentiation of human neural progenitor cells.
The role of intracellular Ca++-transients for the dopaminergic differentiation of immortalised and non-immortalised human NPCs will be elucidated by (1) assessment of the influence of intracellular Ca++-transients by Wnt molecules, (2) identification of target genes that are affected by Wnt-induced Ca++-transients, (3) modelling of Wnt-induced signal pathways that are frequency- and/or amplitude modulated by Ca++-transients and (4) analysis of the mechanisms of Wnt-induced Ca++-transients.
Experiments will be performed with human v-myc transfected immortalised ventral midbrain NPCs ReNcell VM cells (ReNeuron, Guildford, UK) and human non-transfected primary mesencephalic NPCs (NeuroProgen, Leipzig, Germany).