Wspólnotowy Serwis Informacyjny Badan i Rozwoju - CORDIS

Protocol - dopaminergic differentiation of neural stem cells

The Wnts are a family of glycoproteins that regulate cell proliferation, fate decisions and differentiation. In our study we examined the contribution of Wnts to the development of ventral midbrain (VM) dopaminergic (DA) neurons. Our results show that b-catenin is expressed in DA precursor cells and that b-catenin signaling takes place in these cells, as assessed in TOPGAL reporter mice. We also found that Wnt-1, -3a and -5a are differentially regulated and that partially purified Wnts distinctively regulate VM development. Wnt-3a promoted the proliferation of Nurr1+ precursor cells but did not increase the number of TH+ neurons. Instead, Wnt-1 and -5a increased the number of rat midbrain DA neurons in rat E14.5 precursor cultures by two distinct mechanisms. Wnt-1 predominantly increased the proliferation of Nurr1+ precursors, upregulated cyclin D1 and D3, and downregulated p27 and p57 mRNAs. In contrast, Wnt-5a primarily increased the proportion of Nurr1+ precursors that acquired a neuronal DA phenotype, which included the upregulation of Ptx3 and c-ret mRNA. Moreover, the soluble cysteine-rich domain of Frizzled 8 (a Wnt inhibitor) blocked endogenous Wnts and the effects of Wnt-1 and Wnt-5a on proliferation and the acquisition of a DA phenotype in precursor cultures. These findings indicate that Wnts are key regulators of proliferation and differentiation of DA precursors during VM neurogenesis and that different Wnts have specific and unique activity profiles.

We next explored the mechanism by which Wnt-5a promoted DA neuron differentiation and found that purified Wnt-5a protein induces the hyperphosphorylation of dishevelled but not the stabilization of b-catenin. Surprisingly however, we also found that stabilization of b-catenin after overexpression or treatment with GSK3beta inhibitors such as Indirubin 3 monoxime or kempaullone also results in the differentiation of Nurr1+ precursors into TH+ dopaminergic neurons. Thus, our results suggest that the differentiation of dopaminergic precursors into neurons can be enhanced by activation of both canonical and non-canonical Wnt pathways. Finally, we have recently found that Wnt-5a is secreted mainly by glial cells in the ventral midbrain and is part of the dopaminergic inductive activity derived from the glia. Moreover, we found that Wnt-5a blocking antibodies block the effects of ventral midbrain glia on the differentiation of neurospheres into dopaminergic neurons, indicating that Wnt-5a is in part required for the dopaminergic differentiation of stem cells. Thus, our results suggest that Wnt-5a could be applied to promote the differentiation of stem cells into dopaminergic neurons.

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Ernest ARENAS, (Professor)
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