In the course of neuronal development, neurons undergo a well-defined maturation programme in order to establish their highly elaborate and characteristic morphology. Determined by extracellular cues and intracellular signalling pathways, an intrinsic sequence of developmental stages generates the formation of neuronal architecture. The PI3K signalling pathway is a key regulator of the neuronal growth program. Through phosphorylation of PI(4,5)P2 to PI(3,4,5)P3, PI3K activates downstream signalling processes mediating cytoskeletal reorganization and resulting in morphological changes. The phosphatase PTEN acts as the major suppressor of PI3K. PTEN predominantly functions at the membrane by dephosphorylating PI(3,4,5)P3 to PI(4,5)P2, thereby directly antagonises PI3K activity. PTEN is highly enriched in developing neurons and is known to dynamically localise to specific sub-domains of a cellular compartment. For example, PTEN is transiently recruited to the membrane of axonal growth cones mediating chemorepulsion induced morphological changes. However the function of PTEN within the axonal shaft has not been investigated yet. The present thesis characterised the transmembrane protein PRG2 as a novel interaction partner of PTEN. PRG2 is one out of five members of the PRG protein family, which all constitute of a core structure encompassing six transmembrane spanning domains connected by intra- and extracellular loops. Unique within this protein family is the accumulation of 20 glutamic acid residues in the long intracellular C-terminal domain of PRG2. In the course of this project, we were able to show that PRG2 is highly developmentally regulated, with highest protein expression coinciding with the phase of neuronal branching and outgrowth. Interestingly, depletion of PRG2 by shRNA mediated knock down in early stages of neuronal development, severely impaired the emergence of axonal branches. Immunocytochemical approaches demonstrated a striking periodic appearance of PRG2 puncta along the axonal plasma membrane, which was dependent on actin cytoskeletal dynamics. This combined results identify the transmembrane protein PRG2 as a novel mediator of axon branch formation in developing neurons. Further, biochemical data revealed self-association ability of PRG2 as well as the possibility to interact with other PRG family members. We also provided evidence that PRG2 multimers interact with PTEN in primary neurons and that in association with PRG2, PTEN lipid phosphatase activity is inhibited. We hypothesise that through its interaction with the lipid phosphatase PTEN, PRG2 spatiotemporally inhibits PTEN activity leading to local nanodomains of PI(3,4,5)P3 accumulations along the axonal plasma membrane and to the formation of axonal branches. PI(3,4,5)P3 domains are described to precede F-actin patch formation, which in turn drive the emergence of axonal filopodia. Through invading microtubules, filopodia eventually mature into collateral branches. In summary, this study presents a model, which describes PRG2 as novel regulator of PTEN activity and thereby expands the understanding of the signalling mechanisms of how neurons adopt their characteristic morphology.
