Off-target effects of epidermal growth factor receptor antagonists mediate retinal ganglion cell disinhibited axon growth

Abstract

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 p75(NTR) 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 p75(NTR). 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.

Pigment Epithelium-Derived Factor Is Retinal Ganglion Cell Neuroprotective and Axogenic After Optic Nerve Crush Injury

“…The optic nerves (ON) of anesthetised adult female 200 to 250 g Sprague-Dawley rats were exposed surgically through a supraorbital approach and crushed bilaterally 2 mm from the lamina cribrosa, using calibrated watchmaker's forceps as described by us previously. [51][52][53][54][55][56][57] Experimental Design: In Vivo Experiments…”

“…For example, NGF administered to either the cell body or axon in separate Campenot chamber compartments of cultured sympathetic neurons supports survival and axon growth, but only the latter when applied to somata. 80 Similarly, application of NTF to RGC somata in vivo promotes both RGC survival and axon regeneration 51,54,55,57 and both intravitreal and intra-ON inflammation are RGC neuroprotective, but only the former promotes axon regeneration, 52 suggesting that a key RGC axon regeneration stimulatory factor is absent from the transected ON and exclusively secreted by retinal cells.…”

The effects of erythropoietin on RhoA/Rho-associated kinase expression in rat retinal explants cultured with glutamate

“…A number of studies have confirmed that Rho and its associated signaling molecules are involved in and mediate the biological processes of axon regeneration, extension and fiber projection (28)(29)(30)(31). Rho regulates the cell actin cytoskeleton by its downstream effective factor ROCK, which is extensively involved in the biological processes of cell migration, movement, apoptosis, gene transcription and nerve regeneration (32).…”

Restoration of optic neuropathy

“…These off-target effects of EGFR kinase inhibition suggest a novel therapeutic approach for the regeneration of CNS including RGC axons. 30 BDNF released from RGCs acts as a survival factor for postsynaptic neurons in retinal target fields. 31 BDNF protects RGCs from apoptosis mediated by transient ischemia through inhibition of c-Jun and caspase-2 activities.…”