Monoclonal antibodies differentiate neurofilament and glial filament proteins in the goldfish visual pathway: probes for monitoring neurite outgrowth from retinal explants

Abstract

The expression of the neurofilament proteins of the goldfish visual pathway reflects the degeneration and regeneration of the optic nerve after nerve crush. To monitor these processes, monoclonal antibodies (mAb) were generated to the intermediate filament proteins of this pathway. The predominant goldfish visual pathway intermediate filament proteins have a molecular weight of 58K and can be separated into 4 isoelectric variants, 2 of which are neuronal (ON1 and ON2) and 2 of which are non-neuronal (ON3 and ON4). The specificities of the mAbs were characterized biochemically and histologically. Immunoblot analysis demonstrated that 2 of the antibodies reacted specifically with the neurofilament proteins (ON1/ON2) and another antibody reacted specifically with the glial filament proteins (ON3/ON4) and with a 48K optic nerve protein of non-neuronal origin. Chymotrypsin digestion of the ON proteins and immunoblotting of the resulting fragments showed that the anti-ON1/ON2 mAbs were directed toward the variable domains of the filament proteins. In contrast, the anti-ON3/ON4 mAb was directed toward the 40K chymotrypsin-resistant region of the glial filament proteins containing the conserved intermediate filament core. When sections of optic nerve tissue were incubated with anti-ON1/ON2 or anti-ON3/ON4 mAbs, the staining resulted in either axonal or glial patterns, respectively. In retina, after optic nerve crush, anti-ON1/ON2 labeled retinal ganglion cells and Müller fibers. In contrast, prior to optic nerve crush, only Müller fibers were labeled. One of the neuronal-directed mAbs was used to decorate growing neurites from retinal explants; anti-ON1/ON2 reactivity appeared in a time-dependent manner that paralleled the expression of ON1/ON2 in vivo. Thus, the antibodies can differentiate these 2 types of goldfish intermediate filament proteins and can be used to monitor optic nerve regeneration in the goldfish visual pathway both in vivo and in vitro.