Abstract
Neuronal survival factors in the central nervous system were investigated by using a primary culture of embryonic rat neocortical neurons. Bovine hippocampus was homogenized, and the supernatant from high-speed centrifugation was used as the starting material. At the step of DE-52 ion-exchange chromatography, neuronal survival activity was recovered in two fractions, fraction 14 (F14) and fraction 23 (F23). Antisera to the crude F14 and F23 fractions were raised in rabbits. These two antisera completely inhibited the neurotrophic activity of both fractions. Western blotting analysis revealed that anti-F14 antiserum recognized mainly a 30-kDa protein in F14 and anti-F23 antiserum recognized mainly a 44-kDa protein in F23. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis of F23, the 44-kDa protein was cut out from the gel and partial amino acid sequences of the protein fragments were determined. A GenBank data bank indicated that the amino acid sequence of the fragment was identical to that of neuron-specific enolase (NSE). In our assay system, commercially available NSE itself possessed neuronal survival activity for the cultured neocortical neurons. The effects of NSE and F23 were inhibited completely by anti-NSE polyclonal antibody. Furthermore, highly purified NSE supported the survival of cultured neurons in a dose-dependent manner, and the neurotrophic effect was inhibited by monoclonal antibody to the NSE. These results strongly suggest that NSE is one of the neuronal survival factors in the central nervous system.
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