Endocytosis and Transport of Growth Factor Receptors in Peripheral Axon Regeneration: Novel Lessons from Neurons Expressing Lysine-Deficient FGF Receptor Type 1 in vitro.

In the course of peripheral nerve regeneration, axons encounter different extracellular growth factors secreted by non-neuronal cells at the injury site and retrogradely transported after binding to neuronal membrane receptor tyrosine kinases. The present study reviews the role of receptor transport in peripheral axon outgrowth and provides novel data on trafficking of fibroblast growth factor receptor type 1 (FGFR1). Differences in receptor transport are determined by different numbers of lysine residues acting as ubiquitination sites in the intracellular receptor domain. We previously demonstrated that overexpression of mutant FGFR1-25R (25 out of 29 intracellular lysines replaced with arginine) results in enhanced receptor recycling as compared to wild-type FGFR1 followed by strong stimulation of elongative axon growth in vitro. Here, the effects of lysine-deficient FGFR1 (FGFR1-29R lacking all 29 cytoplasmic lysine residues) or of only 15 lysine mutations (FGFR1-15R) on axon outgrowth and concomitant changes in signal pathway activation were investigated by immunocytochemistry and morphometry of cultured primary neurons. Overexpression of FGFR1-15R in adult sensory neurons resulted in enhanced receptor recycling, which was accompanied by increased axon elongation without stimulating axon branching. By contrast, FGFR1-29R was neither endocytosed nor axon outgrowth affected. Although overexpression of FGFR1-15R or FGFR1-25Ra strongly promoted elongation, we did not detect increased signal pathway activation (ERK, AKT, PLC, or STAT3) in neurons expressing mutant FGFR1 as compared with wild-type neurons raising the possibility that other signaling pathways or signaling independent mechanisms may be involved in the axon outgrowth effects of recycled FGF receptors. Anat Rec, 302:1268-1275, 2019. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Leupeptin enhances cell surface localization of fibroblast growth factor receptor 1 in adult sensory neurons by increased recycling.

Fibroblast growth factors (FGFs) act as trophic factors during development and regeneration of the nervous system. FGFs mediate their responses by activation of four types of FGF receptors (FGFR1-4). FGFR1 is expressed in adult sensory neurons of dorsal root ganglia (DRG), and overexpression of FGFR1 enhances FGF-2-induced elongative axon growth in vitro. Ligand-induced activation of FGFR1 is followed by endocytosis and rapid lysosomal degradation. We previously reported that the lysosomal inhibitor leupeptin prevents degradation of FGFR1 and promotes FGF-2-induced elongative axon growth of DRG neurons overexpressing FGFR1. Therefore, we analyzed the effects of leupeptin on intracellular sorting of FGFR1 in PC12 pheochromocytoma cells and DRG neurons. Leupeptin increased colocalization of FGFR1 with lysosomes. Furthermore, leupeptin enhanced the cell surface localization of FGFR1 by increased receptor recycling and this effect was abolished by the recycling inhibitor monensin. In addition, a lysine mutant of FGFR1, which is preferentially recycled back to the cell surface, promoted elongative axon growth of DRG neurons similar to leupeptin. In contrast, the lysosomal inhibitor bafilomycin had no effect on surface localization of FGFR1, inhibited axon growth of DRG neurons and abolished the effects of leupeptin on receptor recycling. Together, our results strongly imply that increased recycling of FGFR1 promotes axon elongation, but not axonal branching, of adult DRG neurons in vitro.