Functional relevance of the Wnt signaling pathway in adult neural stem cells

Introduction

Neural stem cells can be isolated from the adult rat brain. The mechanisms of their proliferation, self-renewal, and differentiation need clarification. We have used proteomic screening to identify new pathways critical to neurogenesis. In this context, a potential pathway is the canonical Wnt signaling pathway. It is known to be activated during embryonic development and neural crest formation. We hypothesize that the Wnt pathway becomes activated in adult neural stem cells when they differentiate into neurons and glia (see figure).

Figure 1

Material and Methods

Two groups of adult neural stem cells were compared: undifferentiated vs. in vitro differentiated cells. Protein extracts were separated by two-dimensional gel electrophoresis and identified by MALDI-TOF mass spectrometry (Proteosys, Mainz, Germany). RT-PCR was performed for specific sequences of the Wnt signaling pathway and its target genes. Immunostaining was used to determine the degree of the differentiation. Astrocytes were detected by anti-GFAP staining (1:500, BD Biosciences, Heidelberg, Germany), neurons were detected by anti-Map2b and anti-tubulin-IIIbeta staining (1:100, Chemicon, Temecula, CA, USA). Nuclei were counterstained with propidium iodide. The microscope images were analyzed by cell counting where the ratio of marker positive cells and total cell number was compared between the two groups.

Results

We were able to verify specific sequences of the Wnt signaling pathway and its target genes on the protein level by two-dimensional gel electrophoresis, Western blotting, and immunohistochemistry, and on the mRNA level by RT-PCR. Proteomic comparison of the undifferentiated vs. the in vitro differentiated neural stem cells identified the up-regulated expression of several members of the Wnt signaling pathway including the beta-catenin-binding protein Pontin 52, the Adenomatosis Polyposis Coli binding protein Eb1, and the Rho-binding protein C87222, and the down-regulation of some proteasome subunits. Western Blotting showed a decreased cytoplasmic concentration of beta-catenin in the differentiated neurospheres. RT-PCR confirmed the differential expression of Wnt signaling molecules and respective target genes, including the down-regulation of bmp4 and msx1, and the up-regulation of GFAP. Immunostaining showed an increased number of cells positive for the astrocytic marker protein GFAP, and the neuronal marker proteins MAP2b and tubulin-IIIbeta after in vitro differentiation.

Conclusions

These data are in accordance with the hypothesis that the Wnt signaling pathway is activated during in vitro differentiation of adult neural stem cells, resulting in an increased differentiation into neurons and astrocytes. The data indicate that the canonical Wnt signaling pathway is a main regulatory element for neural stem cell differentiation.