Tiam1 Regulates the Wnt/Dvl/Rac1 Signaling Pathway and the Differentiation of Midbrain Dopaminergic Neurons

Abstract

Understanding the mechanisms that drive the differentiation of dopaminergic (DA) neurons is crucial for successful development of novel therapies for Parkinson's disease, in which DA neurons progressively degenerate. However, the mechanisms underlying the differentiation-promoting effects of Wnt5a on DA precursors are poorly understood. Here, we present the molecular and functional characterization of a signaling pathway downstream of Wnt5a, the Wnt/Dvl/Rac1 pathway. First, we characterize the interaction between Rac1 and Dvl and identify the N-terminal part of Dvl3 as necessary for Rac1 binding. Next, we show that Tiam1, a Rac1 guanosine exchange factor (GEF), is expressed in the ventral midbrain, interacts with Dvl, facilitates Dvl-Rac1 interaction, and is required for Dvl- or Wnt5a-induced activation of Rac1. Moreover, we show that Wnt5a promotes whereas casein kinase 1 (CK1), a negative regulator of the Wnt/Dvl/Rac1 pathway, abolishes the interactions between Dvl and Tiam1. Finally, using ventral midbrain neurosphere cultures, we demonstrate that the generation of DA neurons in culture is impaired after Tiam1 knockdown, indicating that Tiam1 is required for midbrain DA differentiation. In summary, our data identify Tiam1 as a novel regulator of DA neuron development and as a Dvl-associated and Rac1-specific GEF acting in the Wnt/Dvl/Rac1 pathway.

ACKNOWLEDGMENTS

We thank R. J. Lefkowitz (HHMI, Duke University, Durham, NC) for Dvl2-EGFP, S. Yanagawa (Kyoto University) for Dvl2-MYC, R. T. Moon (HHMI, University of Washington, Seattle, WA) for Dvl3 constructs, Alan Hall (Memorial Sloan-Kettering Cancer Center, New York, NY) for Rac1-MYC, M. Bienz (LMB, MRC, Cambridge, United Kingdom) for Dvl DIX mutants (M1 to M4), Pontus Aspenström (KI, Stockholm, Germany) for GST-PAK CRIB, K. Tolias (Baylor College of Medicine, Houston, TX) for Tiam1-FLAG constructs, and Ondřej Slabý for U78MG and T98G cells. We acknowledge Johnny Söderlund, Nad'a Bílá, Jakub Harnoš, and Alessandra Nanni for excellent assistance.

This work was supported by grants from the European Union (Neurostemcell), Swedish Foundation for Strategic Research (SRL Program and CEDB project), Swedish Research Council (VR2008:2811, VR2011:3116, and DBRM), Norwegian Research Council, and Karolinska Institute to E.A., as well as grants from the Ministry of Education, Youth, and Sports of the Czech Republic (MSM0021622430, CZ.1.07/2.3.00/20.0180), EMBO Installation Grant, and Czech Science Foundation (204/09/0498; 204/09/H058) to V.B. L.C. was supported by a KID Ph.D. student fellowship (6110/06-225) from Karolinska Institute. S.T. was supported by a VR fellowship for visiting scientists and short-term fellowship from the Onassis Foundation.

We declare no competing interests.