Inhibition of fibroblast growth factor receptor 1 endocytosis promotes axonal branching of adult sensory neurons.

Fibroblast growth factors (FGFs) promote axon growth during development and regeneration of the nervous system. Among the four types of FGF receptors (FGFRs), FGFR1 is expressed in adult sensory neurons of dorsal root ganglia (DRG), and overexpression of FGFR1 promotes FGF-2-induced elongative axon growth in vitro. Ligand-induced activation of FGFR1 is followed by endocytosis and lysosomal degradation, which leads to the termination of receptor signaling. We previously reported that the lysosomal inhibitor leupeptin enhances FGF-2-induced elongative axon growth of adult DRG neurons overexpressing FGFR1. To better understand the role of subcellular localization of FGFR1 in axon growth, we analyzed the effects of inhibition of endocytosis of FGFR1 on FGF-2-induced neurite outgrowth in PC12 pheochromocytoma cells and adult DRG neurons. The endocytosis inhibitors methyl-ß-cyclodextrin (MßCD) and chlorpromazine enhanced surface localization of FGFR1 in PC12 cells and DRG neurons. Furthermore, MßCD and chlorpromazine increased FGF-2-induced neurite outgrowth of PC12 cells and axonal branching of adult DRG neurons overexpressing FGFR1, whereas MßCD inhibited FGF-2-induced axonal elongation. Analysis of the signaling pathways involved in axon morphology revealed that FGF-2-induced phosphorylation of extracellular signal-regulated kinase (ERK) and Akt was increased by inhibition of FGFR1 endocytosis. Together, our results imply that inhibition of FGFR1 endocytosis by MßCD or chlorpromazine promotes FGF-2-induced axonal branching. The results of this study confirm that internalization of FGFR1 controls axon growth and morphology of adult sensory neurons via selective activation of intracellular signaling pathways.

Selective up-regulation of the vasodilator peptide apelin after dorsal root but not after spinal nerve injury.

Regeneration of sensory neurons is limited in response to lesion of their central axons when compared to lesion of their peripheral axons. To identify transcriptional changes underlying this differential regenerative response between dorsal root and spinal nerve axons, the L5 dorsal root ganglion (DRG) of adult rats was investigated three days after crushing the respective nerve branches by performing high density genome oligonucleotide microarrays. RT-PCR, in situ hybridization and immunohistochemistry confirmed the up-regulation of the vasodilator peptide apelin in non-neuronal cells of the DRG after dorsal root but not after spinal nerve lesion. Induction of apelin mRNA and peptide is accompanied by increased vascular permeability around neuronal cell bodies as demonstrated by Evans-blue albumin (EBA) leakage. Enhanced vasodilation and increased vascular permeability cause intraganglionic edema, which may play a key role in the reduced axonal regeneration rate after dorsal root injury.

Promotion of neurite outgrowth by fibroblast growth factor receptor 1 overexpression and lysosomal inhibition of receptor degradation in pheochromocytoma cells and adult sensory neurons.

Basic fibroblast growth factor (FGF-2) is up-regulated in response to a nerve lesion and promotes axonal regeneration by activation of the tyrosine kinase receptor fibroblast growth factor receptor 1 (FGFR1). To determine the effects of elevated FGFR1 levels on neurite outgrowth, overexpression was combined with lysosomal inhibition of receptor degradation. In pheochromocytoma (PC12) cells, FGFR1 overexpression resulted in flattened morphology, increased neurite outgrowth and activation of extracellular signal-regulated kinase (ERK) and AKT. Degradation of FGFR1 was inhibited by the lysosomal inhibitor leupeptin and by the proteasomal inhibitor lactacystin. In rat primary adult neurons, FGFR1 overexpression enhanced FGF-2-induced axon growth which was further increased by co-treatment with leupeptin. Lysosomal inhibition of receptor degradation concomitant with ligand stimulation of neurons overexpressing FGFR1 provides new insight in tyrosine kinase receptor-mediated promotion of axon regeneration and demonstrates that adult sensory neurons express sub-optimal levels of tyrosine kinase receptors for neurotrophic factors.