Functional characterization of fish neuroglobin: zebrafish neuroglobin is highly expressed in amacrine cells after optic nerve injury and can translocate into ZF4 cells

Abstract

Neuroglobin (Ngb) is a recently discovered vertebrate heme protein that is expressed in the brain and can reversibly bind oxygen. Mammalian Ngb is involved in neuroprotection under conditions of oxidative stress, such as ischemia and reperfusion. We previously found that zebrafish Ngb can penetrate the mammalian cell membrane. In the present study, we investigated the functional characteristics of fish Ngb by using the zebrafish cell line ZF4 and zebrafish retina. We found that zebrafish Ngb translocates into ZF4 cells, but cannot protect ZF4 cells against cell death induced by hydrogen peroxide. Furthermore, we demonstrated that a chimeric ZHHH Ngb protein, in which module M1 of human Ngb is replaced by that of zebrafish, is a cell-membrane-penetrating protein that can protect ZF4 cells against hydrogen peroxide exposure. Moreover, we investigated the localization of Ngb mRNA and protein in zebrafish retina and found that Ngb mRNA is expressed in amacrine cells in the inner nuclear layer and is significantly increased in amacrine cells 3days after optic nerve injury. Immunohistochemical studies clarified that Ngb protein levels were increased in both amacrine cells and presynaptic regions in the inner plexiform layer after nerve injury. Taken together, we hypothesize that fish Ngb, whose expression is upregulated in amacrine cells after optic nerve injury, might be released from amacrine cells, translocate into neighboring ganglion cells, and function in the early stage of optic nerve regeneration. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

Keywords: Amacrine; CNgb; Cell-membrane-penetrating activity; DIG; DMEM; Dulbecco's modified Eagle's medium; FBS; FITC; G protein; GAPDH; GCL; GDI; HNgb; INL; IPL; Mb; N-acetylcysteine; NAC; Neuroglobin; Ngb; ONL; PBS; ROS; Retina; Stx; ZNgb; Zebrafish; chimeric ZHHH neuroglobin; digoxigenin; fetal bovine serum; fluorescein isothiocyanate; ganglion cell layer; glyceraldehyde-3-phosphate dehydrogenase; guanine nucleotide dissociation inhibitor; guanine nucleotide-binding protein; human neuroglobin; inner nuclear layer; inner plexiform layer; myoglobin; neuroglobin; outer nuclear layer; phosphate-buffered saline; reactive oxygen species; syntaxin; zebrafish neuroglobin.