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

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

Martin Luther and Extra Ecclesiam Nulla Salus (“Outside of the Church There is No Salvation”): Did Luther Really Abandon Cyprian?

... careful study of the Luther’s writings reveals that, while he repudiated many Catholic ways of understanding and conducting church, and while he attempted to harmonize ecclesiastical structures and sacramental theology with the foundational principles of Protestantism, he was essentially unable to break away from medieval modes of thinking. Notwithstanding his rejection of the Catholic emphasis on the visible church, he struggled to free himself from reliance on institutional structures for salvation. Ultimately, Luther affirmed the necessity of the visible church for salvation. In His wisdom, Luther believed, God had decreed the church to be the means of grace, without which no one could be saved. As a result, while a person could be in the church and unsaved, the option of not being in the church was not open to those who were predestined for salvation by God. Abandoning the church was a sure sign that one had not been among the elect. While each of the Reformation’s solas represented some form of reaction against medieval Catholic soteriology, the fact that Luther’s soteriology developed within the context of Augustinian monergism resulted only in providing an alternative theological foundation for the Catholic doctrine of extra ecclesiam nulla salus. Such a vision of the church was congruent with the social and political milieu of early 16th century. While beginning well, Luther’s Reformation ultimately defaulted to its ecclesiastical and cultural surroundings

WNT5A is transported via lipoprotein particles in the cerebrospinal fluid to regulate hindbrain morphogenesis.

WNTs are lipid-modified proteins that control multiple functions in development and disease via short- and long-range signaling. However, it is unclear how these hydrophobic molecules spread over long distances in the mammalian brain. Here we show that WNT5A is produced by the choroid plexus (ChP) of the developing hindbrain, but not the telencephalon, in both mouse and human. Since the ChP produces and secretes the cerebrospinal fluid (CSF), we examine the presence of WNT5A in the CSF and find that it is associated with lipoprotein particles rather than exosomes. Moreover, since the CSF flows along the apical surface of hindbrain progenitors not expressing Wnt5a, we examined whether deletion of Wnt5a in the ChP controls their function and find that cerebellar morphogenesis is impaired. Our study thus identifies the CSF as a route and lipoprotein particles as a vehicle for long-range transport of biologically active WNT in the central nervous system.We thank Nadia Wänn for maintenance of mice colonies; the members of Bryja and Arenas lab for their help and suggestions; Martin Häring for help with in situ analysis; Johnny Söderlund and Alessandra Nanni for their technical and secretarial assistance; and the CLICK imaging facility at Karolinska Institutet for technical support. We thank MEYS CR for support to the following core facilities: Proteomics (CIISB research infrastructure project LM2015043), cellular imaging at CEITEC institution at Masaryk University (LM2015062 Czech-BioImaging) Czech Centre for Phenogenomics (LM2015040), Higher quality and capacity of transgenic model breeding (by MEYS and ERDF, OP RDI CZ.1.05/2.1.00/19.0395), Czech Centre for Phenogenomics: developing towards translation research (by MEYS and ESIF, OP RDE CZ.02.1.01/0.0/0.0/16_013/0001789). The collaboration between Masaryk University and Karolinska Institutet (KI-MU program), was co-financed by the European Social Fund and the state budget of the Czech Republic (CZ.1.07/2.3.00/20.0180). Funding to the VB lab was obtained from Neuron Fund for Support of Science (23/2016), and Czech Science Foundation (GA17-16680S). Work in the EA lab was supported by the Swedish Research Council (VR projects: DBRM, 2011-3116, 2011-3318 and 2016-01526), Swedish Foundation for Strategic Research (SRL program and SLA SB16-0065), European Commission (NeuroStemcellRepair), Karolinska Institutet (SFO Strat Regen, Senior grant 2018), Hjärnfonden (FO2015:0202 and FO2017-0059) and Cancerfonden (CAN 2016/572). Research in the JCV lab was supported by Karolinska Institutet Foundations. KK was supported by Masaryk University (MUNI/E/0965/2016). DP and ZZ were supported by the CEITEC 2020 (LQ1601) project from MEYS CR

Methodology of Landscape Conservation Zones' Impact Assessment

The methodology defines a procedure for the landscape conservation zones' impact assessment. The method is based on a precautionary principle and is designed so that would be possible to prevent heritage area from negative irreversible impact on the landscape cultural and historical values. The methodology is intended for the experts and competent staff of the National Heritage Institute responsible for landscape conservation zones planning and management

Methodology of Monitoring of the Landscape Conservation Zones

Cultural landscape presents with its diversity and uniqueness a significant part of the natural and cultural heritage of human society. They are the Landscape Conservation Zones (LCZ) are currently the only legal category, through which the state heritage preservation may enter the field of protection of unique historical cultural landscape. Basic problem of the current management of the Landscape Conservation Zones is an absence of systematically designed, structured and standardized system of area conservation, management and planning including aspects of identification and evidence of the landscape values and methods of landscape quality monitoring. The guidance presents main principles of the suitable monitoring indicators and way of the landscape heritage assessment in the Czech Republic. There is the special emphasis is put on indicators describing changes of the land use pattern, landscape visibility and effectiveness of the planning tools

Typology of the historical cultural landscape of the Czech Republic:methodology with a certificate from the Ministry of Culture

The new national typology of the historical cultural landscapes is created by 34 types of the historical cultural landscapes that exist in the Czech Republic. The typology is based on three categories of the cultural landscapes that were defined by the World Heritage Committee (UNESCO), i.e. designed, organically evolved and associative landscapes. The typology of the historical cultural landscapes could be used for identification, classification and evaluation of the historical cultural landscapes on the national level. The typology is applicable for the spatial identification of landscape with important cultural values that serve to the public interests in landscape protection. The typology could be also used for identification of the cultural landscapes in analysis for the spatial planning

Methodics of identification of designed landscape

The methodology of identification composed landscapes presents procedures, which are used to identify traces and signs of intentional creation of space according landscape architectural principles in today's cultural landscape . Landscape composition is still often involved in the organization of space, its identity and characteristics of the landscape, but ignorance of their composition and integrity leads to the disruption and damage caused by rough spatial development

Additional file 8: Figure S4. of A proteomic analysis of LRRK2 binding partners reveals interactions with multiple signaling components of the WNT/PCP pathway

Confirmation of the interaction between LRRK2 and CELSR1 using different conditions. (A) Western blotting analysis of co-IP of overexpressed V5-Lrrk2 and Celsr1-EGFP in HEK293T cells. Different vectors were used to confirm LRRK2-CELSR1 interaction. LRRK2 binds to CELSR1 even when pulled-down with different antibody. (B) Co-immunoprecipitation of overexpressed LRRK2 with CELSR1 by using different ratio of transfected DNA in HEK293T cells. Efficiency of the LRRK2-CELSR1 pulldown had improved when less CELSR1 and more LRRK2 was transfected. This not only confirms the interaction but also excludes the false positive interactions compared to the negative controls. (EPS 5517 kb