A molecular mechanism of optic nerve regeneration in fish: the retinoid signaling pathway

Abstract

The fish optic nerve regeneration process takes more than 100 days after axotomy and comprises four stages: neurite sprouting (1-4 days), axonal elongation (5-30 days), synaptic refinement (35-80 days) and functional recovery (100-120 days). We screened genes specifically upregulated in each stage from axotomized fish retina. The mRNAs for heat shock protein 70 and insulin-like growth factor-1 rapidly increased in the retinal ganglion cells soon after axotomy and function as cell-survival factors. Purpurin mRNA rapidly and transiently increased in the photoreceptors and purpurin protein diffusely increased in all nuclear layers at 1-4 days after injury. The purpurin gene has an active retinol-binding site and a signal peptide. Purpurin with retinol functions as a sprouting factor for thin neurites. This neurite-sprouting effect was closely mimicked by retinoic acid and blocked by its inhibitor. We propose that purpurin works as a retinol transporter to supply retinoic acid to damaged RGCs which in turn activates target genes. We also searched for genes involved in the second stage of regeneration. The mRNA of retinoid-signaling molecules increased in retinal ganglion cells at 7-14 days after injury and tissue transglutaminase and neuronal nitric oxide synthase mRNAs, RA-target genes, increased in retinal ganglion cells at 10-30 days after injury. They function as factors for the outgrowth of thick, long neurites. Here we present a retinoid-signaling hypothesis to explain molecular events during the early stages of optic nerve regeneration in fish.

Keywords: 3-amino-9-ethylcarbazole; AEC; CNS; CRABPs; CYP26a1; FXIII; Fish retina; GAP43; GCL; HRP; HSF; HSP70; HSPs; IGF-1; INL; NADPH diaphorase; NADPHd; NO; NO signaling; NOS; OKR; OMR; ONL; Optic nerve regeneration; PI3K; PKG; Purpurin; RA; RAGs; RALDH; RARs; RBP; RGCs; Retinoid signaling; S-nitroso-N-acetyl penicillamine; SNAP; TG; TG(R); TUNEL; cFXIII; cGMP; cellular factor XIII; cellular retinoic acid-binding proteins; central nervous system; cyclic GMP; cytochrome P450/26a1; factor XIII; ganglion cell layer; growth-associated protein 43; heat shock factor; heat shock protein 70; heat shock proteins; horse radish peroxidase; inner nuclear layer; insulin-like growth factor-1; nNOS; neuronal NOS; nitric oxide; nitric oxide synthetase; optokinetic response; optomotor response; outer nuclear layer; p-Akt; p-Bad; phosphatidylinositol-3-kinase; phospho-Akt; phospho-Bad; protein kinase G; regeneration-associated genes; retinal ganglion cells; retinal tissue type TG; retinaldehyde dehydrogenase; retinoic acid; retinoic acid receptors; retinol-binding protein; terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; transglutaminase.