PURIFICATION AND CHARACTERIZATION OF "NEURITE OUTGROWTH-PROMOTING FACTORS" (EXTRACELLULAR MATRIX, LAMININ, AXON GROWTH AND GUIDANCES, CELL-SUBSTRATUM INTERACTION, NEURONAL CELL CULTURE)

The outgrowth of axons and their guidance to targets constitutes a crucial phase of neuronal development. The role that molecules in the neuronal environment play in influencing axonal growth and guidance may be studied in vitro, by examining the effects of particular substances on neurite outgrowth. The studies reported here concern the effects of substances known as "neurite outgrowth-promoting factors", which are found in media conditioned by many cell types. "Neurite outgrowth-promoting factors" act by adsorbing to appropriate culture substrata and enhancing the initiation and extension of neurites.

The "neurite outgrowth-promoting factor" in bovine corneal endothelial conditioned medium was purified, and characterized through physical and enzymatic treatments, metabolic labeling, denaturing gel electrophoresis, and immunochemical analysis. The "neurite outgrowth-promoting factors" in other conditioned media were also studied. Purified molecules derived from the extracellular matrix were screened for activity resembling that of "neurite outgrowth-promoting factors".

The results indicate that the bovine corneal endothelial "neurite outgrowth-promoting factor" consists of three associated molecules: the basement membrane glycoprotein laminin, a proteoglycan of the heparan sulfate class, and entactin, another extracellular matrix glycoprotein. Of these molecules, laminin appears primarily responsible for mediating neurite outgrowth-promoting activity, although the proteoglycan plays an important role in attaching the "neurite outgrowth-promoting factor" to the culture substratum. Studies of the "neurite outgrowth-promoting factors" in media conditioned by other cells indicated they were similar in composition. Interestingly, immunochemical differences between the neurite outgrowth-promoting activity of a purified preparation of laminin and the neurite outgrowth-promoting activity in conditioned media suggested that different forms of laminin exist, or that molecules bound to laminin alter its immunoreactivity.

These findings suggest that laminin may play a role in axonal growth and guidance in vivo.