Brain Advance Access originally published online on September 25, 2009
Brain 2009 132(11):3102-3121; doi:10.1093/brain/awp240
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Off-target effects of epidermal growth factor receptor antagonists mediate retinal ganglion cell disinhibited axon growth
1 Molecular Neuroscience Group, School of Clinical and Experimental Medicine, College of Medical and Dental Sciences, University of Birmingham, Birmingham, B15 2TT, UK 2 Department of Neurology, Dudley Group of Hospitals NHS Foundation Trust, Russells Hall Hospital, Dudley, DY1 2HQ, UK
Correspondence to: Dr Zubair Ahmed, Molecular Neuroscience Group, School of Clinical and Experimental Medicine, College of Medical and Dental Sciences, University of Birmingham, Room WX2.17 Institute of Biomedical Research (West), Edgbaston, Birmingham B15 2TT, UK. These authors contributed equally to this work. E-mail: z.ahmed.1{at}bham.ac.uk
Inhibition of central nervous system axon growth is reportedly mediated in part by calcium-dependent phosphorylation of axonal epidermal growth factor receptor, with local administration of the epidermal growth factor receptor kinase inhibitors AG1478 and PD168393 to an optic nerve lesion site promoting adult retinal ganglion cell axon regeneration. Here, we show that epidermal growth factor receptor was neither constitutively expressed, nor activated in optic nerve axons in our non-regenerating and regenerating optic nerve injury models, a finding that is inconsistent with phosphorylated epidermal growth factor receptor-dependent intra-axonal signalling of central nervous system myelin-related axon growth inhibitory ligands. However, epidermal growth factor receptor was localized and activated within most glia in the retina and optic nerve post-injury, and thus an indirect glial-dependent mechanism for stimulated retinal ganglion cell axon growth by epidermal growth factor receptor inhibitors seemed plausible. Using primary retinal cultures with added central nervous system myelin extracts, we confirmed previous reports that AG1478/PD168393 blocks epidermal growth factor receptor activation and promotes disinhibited neurite outgrowth. Paradoxically, neurites did not grow in central nervous system myelin extract-containing cultures after short interfering ribonucleic acid-mediated knockdown of epidermal growth factor receptor. However, addition of AG1478 restored neurite outgrowth to short interfering ribonucleic acid-treated cultures, implying that epidermal growth factor receptor does not mediate AG1478-dependent effects. TrkA-/B-/C-Fc fusion proteins and the kinase blocker K252a abrogated the neuritogenic activity in these cultures, correlating with the presence of the neurotrophins brain derived neurotrophic factor, nerve growth factor and neurotrophin-3 in the supernatant and increased intracellular cyclic adenosine monophosphate activity. Neurotrophins released by AG1478 stimulated disinhibited retinal ganglion cell axon growth in central nervous system myelin-treated cultures by the induction of regulated intramembraneous proteolysis of p75NTR and Rho inactivation. Retinal astrocytes/Müller cells and retinal ganglion cells were the source of neurotrophins, with neurite outgrowth halved in the presence of glial inhibitors. We attribute AG1478-stimulated neuritogenesis to the induced release of neurotrophins together with raised cyclic adenosine monophosphate levels in treated cultures, leading to axon growth and disinhibition by neurotrophin-induced regulated intramembraneous proteolysis of p75NTR. These off-target effects of epidermal growth factor receptor kinase inhibition suggest a novel therapeutic approach for designing treatments to promote central nervous system axon regeneration.
Key Words: AG1478; disinhibition; RGC; EGFR; siRNA; neurite outgrowth; axon regeneration; glia
Abbreviations: BDNF, brain derived neurotrophic factor; cAMP, cyclic adenosine monophosphate; CME, central nervous system myelin extracts; CNS, central nervous system; CNTF, ciliary neurotrophic factor; EGFR, epidermal growth factor receptor; ELISA, enzyme linked immunosorbent assay; 5-FDU, 5-fluoro-2-deoxyuridine; NGF, nerve growth factor; NT-3, neurotrophin-3; ONC, optic nerve crush; PBS, phosphate buffered saline; PBS-T-BSA, phosphate buffered saline-Tween 20-bovine serum albumin; pEGFR, phosphorylated epidermal growth factor receptor; RGC, retinal ganglion cells; siRNA, short interfering ribonucleic acid; TACE, tumour necrosis factor alpha converting enzyme; TBS, Tris buffered saline
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Received June 10, 2009. Revised July 29, 2009. Accepted August 11, 2009.
*These authors contributed equally to this work.