WNT/FZD Signaling: An Odyssey from Molecular Pharmacology to Brain (Patho)Physiology

The 19 members of the lipoglycoprotein family of WNTs interact with the highly conserved cysteine-rich domain (CRD) of ten members of the Frizzled (FZD1-10) family. The seven transmembrane-spanning surface receptors are listed as G protein-coupled receptors and are known to interact with heterotrimeric G proteins as well as the scaffolding protein disheveled. Upon ligand binding, activation of β-catenin-dependent and/or –independent pathways are induced. The WNT/Frizzled signaling pathway is highly conserved across species and plays an essential role in a plethora of physiological processes, such as embryonic stem cell differentiation, adult stem cell renewal, migration, proliferation and cell polarity. In humans, dysregulation of the pathway is related to developmental alterations, as well as numerous diseases, among which various types of cancers. The general aim of this thesis was to investigate the role of WNT/FZD signaling in physiological and pathophysiological processes of the central nervous system. Additionally, we attempted to dissect the molecular features of the WNT/FZD pathway regarding signaling specification, a contribution aimed at increasing the possibilities of future drug development. To date, WNT/FZD binding specificity, as well as the downstream signaling trigged by different ligand-receptor combinations, have not been systematically mapped. Furthermore, the involvement and role of heterotrimeric G proteins downstream of FZDs is unclear. In paper I, we demonstrated that WNT-5A is able to activate PTX-sensitive Gαi/o proteins in the microglialike cell line N13. Additionally, in paper II we concluded that WNT-3A, -4, and -5A have a putative functional selectivity for individual downstream signaling pathways depending on the FZD present. WNT-5A is known to be expressed in the central nervous system, where it plays a role in various neurological processes. However, it had not been fully delineated which cell type expresses and secretes WNT-5A in the brain. In paper IV, we identified a subpopulation of astrocytes that express high levels of WNT-5A in the brain of adult mice. These astrocytes are localized in the subventricular zone, the rostral migratory stream, and in the hippocampus. By studying a WNT-5A mouse model, we show that some astrocytes release WNT-5A and fulfill a crucial role in guiding the migration of neuronal precursor cells from the subventricular zone to the olfactory bulb. In paper V, we investigated the pathological implications of WNT-5A signaling to the prognostics of human glioblastomas, and described a strong association between WNT-5A expression in in the tumor microenvironment and increased infiltration of microglia. Besides the role in neuronal precursor migration, WNT-5A induced a proinflammatory response in mouse microglia cells, shown by the expression of several proinflammatory markers (paper III). Additionally, WNT-5A induced proliferation, invasion and calcium signaling in microglia. By employing the Gαi/o inhibitor PTX, as well as the MEK1/2 inhibitor, a part of these events could be blocked, suggesting G protein-dependent mechanisms downstream of WNT-5A. LIST OF SCIENTIFIC PAPERS I. WNT-5A stimulates the GDP/GTP exchange at pertussis toxin-sensitive heterotrimeric G proteins Kilander MBC, Dijksterhuis JP, Ganji RS, Bryja V, Schulte G Cellular Signalling (2011) 23: 550-554 II. Systematic mapping of WNT-Frizzled interactions reveals functional selectivity by distinct WNT-Frizzled pairs Dijksterhuis JP, Baljinnyam B, Stanger K, Sercan HO, Ji Y, Andres O, Rubin JS, Hannoush RN and Schulte G The Journal of Biological Chemistry (2015): In press III. Heterotrimeric G protein-dependent WNT-5A signaling to ERK1/2 mediates distinct aspects of microglia proinflammatory transformation Halleskog C, Dijksterhuis JP, Kilander MBC, Becerril-Ortega J, Villaescusa JC, Lindgren E, Arenas E, Schulte G Journal of Neuroinflammation (2012) 9:111. IV. Partial ablation of WNT-5A affects neuronal precursor migration along the rostral migratory stream Dijksterhuis JP, Kamuda K, Gyllborg D, Persson A, Uhlen M, Brismar H, Blom H, Arenas E, Mulder J and Schulte G Manuscript V. WNT-5A is upregulated in a subset of human glioblastoma and is associated with a distinct inflammatory phenotype caused by microglia invasion Dijksterhuis JP, Arthofer E, Marinescu V, Nelander S, Mathias Uhlen, Ponten F, Mulder J, Schulte G Manuscript ADDITIONAL PUBLICATIONS Related published papers and database entry involving the author that are not included in the thesis: I. Sequential activation and inactivation of Dishevelled in the Wnt/betacatenin pathway by casein kinases Bernatik O, Ganji RS, Dijksterhuis JP, Konik P, Cervenka I, Polonio T, Krejcin P, Schulte G, Bryja V The Journal of Biological Chemistry (2011) 25;286(12):10396-410. II. The Concise Guide to PHARMACOLOGY 2013/14: overview CGTP Collaborator British Journal of Pharmacology (2013) 170(8):1449-58. III. WNT/Frizzled signalling: receptor-ligand selectivity with focus on FZD-G protein signalling and its physiological relevance: IUPHAR Review 3 Dijksterhuis JP, Petersen J, Schulte G British Journal of Pharmacology (2014) 171(5):1195-209. IV. Frizzled Class GPCRs entry on the IUPHAR database Contributor www.iuphar-db.org * These authors contributed equally.