Dissecting Repulsive Guidance Molecule/Neogenin function and signaling during neural development

During neural development a series of precisely ordered cellular processes acts to establish a functional brain comprising millions of neurons and many more neuronal connections. Neogenin and its repulsive guidance molecule (RGM) ligands contribute to neuronal network formation by inducing axon repulsion. Furthermore, RGM-Neogenin signaling controls several other developmental processes in the nervous system including neurogenesis, neuronal differentiation, migration and apoptosis. From a clinical perspective, RGMa and Neogenin have been implicated in regeneration failure in the adult CNS and contribute to diseases such as multiple sclerosis, Alzheimer and Parkinson’s disease. Our understanding of Neogenin, RGM ligand and Unc5 coreceptor function in the developing brain is far from complete. In addition, the Neogenin signal transduction cascade that mediates neurite outgrowth inhibition and other Neogenin functions remains poorly characterized. In this thesis we expanded our knowledge on Neogenin function and signaling by performing a detailed expression study for Neogenin, RGM ligands and Unc5 coreceptors during mouse brain development. In addition, we conducted proteomics screens for novel Neogenin-interacting proteins in a cell line and in brain lysates of synapsin I-driven GFP-Neogenin transgenic mice. In the expression study, in situ hybridization and immunohistochemistry revealed prominent and specific, but also overlapping, expression of Neogenin, RGMa/b and Unc5A-D in the developing mouse brain. Characteristic cell layer-specific expression patterns were detected. Furthermore, strong expression of RGMa, RGMb and Neogenin protein was found on several major axon tracts. These data not only hint at a widespread role for RGM-Neogenin signaling during the development of multiple different neuronal systems, but also suggest that Neogenin may partner with different Unc5 family members to subserve different functions in different systems. The proteomics screens for Neogenin-interacting proteins identified 80 putative novel signaling proteins that can be linked to several cellular processes known to be regulated by Neogenin, including neurite formation, gene transcription and cell survival. The interaction of Neogenin and two novel Neogenin-interacting proteins: leucine-rich repeats and immunoglobulin-like domains protein 2 (Lrig2) and dedicator of cytokinesis 7 (Dock7), were studied in more detail using colocalization, in vivo co-immunoprecipitation and functional experiments. A functional neurite outgrowth assay revealed the requirement for Lrig2 and Dock7 in RGMa-induced neurite outgrowth inhibition. Lrigs are known inhibitors of growth factor tyrosine receptor signaling activity by inducing receptor degradation or by blocking receptor localization to lipid rafts. Lrig functions during neural development are largely unexplored. Lrig2 could possibly mediate RGMa-Neogenin signaling by inducing localization of the Neogenin receptor to lipid rafts or by terminating Neogenin receptor signaling by inducing receptor endocytosis. Dock7 is an activator of Cdc42 and Rac GTPases and functions in neurogenesis, cell polarity, axon formation, migration and myelination.Dock7 could mediate RGMa-Neogenin repulsive effects by regulating the activity of Rac or Cdc42 GTPase or by signaling through its binding partners TACC3, myosin VI and MICAL-1. The identification of novel Neogenin signaling proteins that function in Neogenin-mediated neurite outgrowth inhibition, as shown for Lrig2 and Dock in this thesis, generates novel opportunities for the development of therapies to promote axon regeneration