Identification of novel Wnt/PCP signaling regulators and their role in midbrain dopaminergic neuron development and Parkinson's disease

Sammanfattning: Wnt signaling controls a wide spectrum of complex cell responses during prenatal development, in the adulthood and during disease. In this doctoral study, we have identified and explored novel regulatory components of Wnt/Planar Cell Polarity (PCP) pathway and their function in various cellular processes during embryogenesis and central nervous system (CNS) development. We paid special attention to molecular mechanisms underlying the morphogenesis of the ventral midbrain (VM) and development of midbrain dopaminergic (mDA) neurons, a brain area that is strictly regulated by Wnt signaling. We also touched upon possible clinical applications of our findings in neurodegenerative disorders, such as Parkinson's disease (PD). We used a large number of traditional biochemical tools as well as more advanced methodologies such as proteomics and phospho-proteomics, RNA-scope in situ hybridization, confocal microscopy, electron microscopy and CRISPR/Cas9 technology (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9). We have also used different models such as cell lines and primary cultures, as well as genetically modified organisms, including Xenopus laevis (Frog), Danio rerio (zebrafish) and mouse embryos. To better understand the functional complexity of the Wnt/PCP signaling, we examined a number of transgenic mice models, which allowed us to uncover the function of Wnt/PCP protein complexes in the mammalian CNS. Finally, some of our observations were confirmed by using human prenatal brain tissue (study II). Please find below the main highlights of each study included in this thesis. In study I, we explored the molecular mechanism by which the crucial Wnt signaling integrator Dvl and the cell cycle protein kinase NEK2 regulate the progression of cells from the G2 to the M phase. We identified Dvl as a NEK2 substrate and described that they mediate disassembling of centrosomal linker proteins from the centrosome, a process essential for duplicating the centrioles and polarization of the mitotic spindle during mitosis. Such findings are of tremendous importance in cancer research and in the context of ciliopathies which show defects in the centrosomal structures. In study II, we investigated the expression of mammalian Wnts in developing choroid plexi. We discovered that biologically active Wnt5a is secreted to the cerebral spinal fluid (CSF) by the epithelial cells of the hindbrain, but not the telencephalic choroid plexus, in both mouse and in human embryos. We further describe that secreted Wnt5a forms a complex with high-density lipoprotein particles containing ApoE and ApoJ, but is not found in exosomes. Analysis of the Wnt5a deficient mice revealed a possible function of Wnt5a in the choroid plexus to inhibit progenitor proliferation in the neighbor ventricular zone. Our results suggest that Wnt5a gradients in the developing mammalian brain might be formed by diffusion of Wnt5a-lipoprotein complexes through the CSF. In study III, we tackled a molecular mechanism behind the Wnt5a signal transduction in the ventral midbrain. Analysis of Wnt5a-/-, Wnt5a overexpressing, Wnt5a-/-;Ror2-/- and Ror2-/-;Vangl2-/- mice identified a function of the Wnt5a-Ror2/Vangl2 signaling axis in the VM morphogenesis and in mDA neuron development. Our study shows that correct Wnt5a expression levels are crucial for VM morphogenesis, mDA neurogenesis and mDA neuron maturation. Moreover, we found a novel phenotype of bilateral asymmetry in Ror2-/-;Vangl2-/- animals which suggests that Vangl2 alone or in a complex with Ror2 controls the correct position, proliferation and differentiation of mDA progenitors into mDA neuroblasts and neurons. Our results additionally identify a novel role of Wnt/PCP signaling in controlling mDA neurogenesis, which may be of interest for the development of novel regenerative approaches to treat neurodegenerative diseases which affect mDA neurons, such as Parkinson's disease. In study IV, we performed a proteomic analysis of the core Wnt/PCP receptor Ror2, and discovered several novel binding partners which were verified in mDA cells and in the developing ventral midbrain. We selected SorCS2, a proneurotrophin receptor from the VPS10-domain containing sortilin receptor family, as a top candidate because of its specific expression in the mouse midbrain floorplate and its functional involvement in mDA neuron wiring. By using X. laevis and D. rerio, we found that the Ror2-SorCS2 receptor complex is required during embryogenesis to regulate convergent extension, somitogenesis and brain development. We also suggest that SorCS2 has the capacity to internalize Ror2 and its other co-receptors in a Wnt/PCP-dependent manner in vitro and in vivo, via an unknown pathway. These data reveal that the two pathways previously considered to be independent, Wnt/PCP and proneurotrophin receptor signaling, functionally interact. Moreover, our results identify SorCS2 as a novel regulator of Wnt/PCP signaling in vertebral embryogenesis. In study V, we investigated whether Leucine-rich repeat kinase 2 (Lrrk2), the protein product of the park8 gene, which is mutated in more than 40% of patients with inherited PD, can interact with the Wnt/PCP pathway by using a proteomic screening. We describe that Lrrk2 interacts with a number of Wnt/PCP components in dopaminergic cells, in the VM of E18.5 mice embryos, and in a human cell line. Particularly, we show the capacity of Lrrk2 to inhibit Wnt/β-catenin signaling in vitro and in vivo in X. laevis embryos. We observed that these regulatory changes depend on the presence of Prickle1 and Dvl. Our results thus provide novel insights into the molecular mechanisms by which Lrrk2 and Wnt signaling interact, and describe Lrrk2 and Prickle1 as novel dual regulators of Wnt/PCP and Wnt/β-catenin signaling. Moreover, we suggest that the pathogenesis of PD may involve an alteration in the balance between these two Wnt signaling pathways.