Trauma to the brain of man often severs nerve fibers which then cannot regrow. Transection of the optic tract in the goldfish, however, initiates a remarkable process during which axons of retinal ganglion cells grow back into the optic tectum with the formation of new synapses and the recovery of sight. During this regeneration, glia of the optic tract show marked increases in new protein bio-synthesis and cellular proliferation. Many investigators have argued that glia play an important role in directing axonal growth. Previous experiments show soluble factors recovered from de-innervated tecta promote biosynthesis and cellular proliferation of optic tract glia. Importantly, these Glia Promoting Factors (GPFs) from fish also stimulate specific classes of mammalian glial. I detect 4 GPFs, designated 1-4, which are trypsin-sensitive peptides ranging in size from 3,000 to 14,000 daltons. GPF-1 and 3 selectively stimulate proliferation of rat brain oligodendroglia in culture while GPF-2 and 4 stimulate astroglia. Comparisons between factors recovered from de-innervated and intact tecta show that the activities of 6PF-1, GPF-3, and GPF-4 are increased during regeneration while GPF-2 remains unchanged. The proposed research is to isolate and characterize GPFs found within de-innervated goldfish optic tectum. Using cultures of rat brain glia, all 4 GPFs will be tested for their ability to recruit undifferentiated macroglia, to alter cell migration patterns, and to promote neurite outgrowth. I will attempt to isolate GPF-1 which acts specifically upon oligodendroglia. A crude soluble fraction recovered from tecta will be partially purified using P-10 columns and HPLC. Further purification will employ reverse-phase HPLC, iso-electric focusing, and ion exchange chromatography. Detection of glia promoting factors will be carried out by UV absorption, fluorescamine assays, and by silver stain of SDS-PAGE. Tissue culture of rat glial will offer an opportunity to test factor activity by examining oligodendroglial populations using immuno-histochemical markers, by quantitating incorporation of radiolabeled precursors, and by measuring the activity of a marker enzyme 2':'3-cyclic nucleotide 3'-phosphohydrolase. I hope that study of GPFs will help to uncover mechanisms which aid regeneration of the central nervous system.
