Promotion of neuronal survival in vitro by thermal proteins and poly(dicarboxylic)amino acids

Evaluating molecules for their ability to promote survival and growth of neurons, we tested thermal proteins on cultures of dissociated fetal rat forebrain neurons. (Thermal proteins are polyamino acids formed when mixtures of amino acids with minimal proportions of glutamic or aspartic acid are heated.) Thermal proteins, added to low-density cultures in serum-free medium, stimulated neurite outgrowth and induced the formation of neuronal networks which survived for 6-10 days. Neurons in control cultures failed to grow and degenerated completely within 2-4 days. Effective concentrations (EC50) of thermal proteins ranged from 3 to 100 micrograms/ml. They were equally effective when present in the medium during the culture time or after precoating of the culture dishes. A single preparation which contained only aspartic and glutamic acid was effective, and similar survival promoting actions were then found for polyglutamic acid and mixed polyamino acids containing glutamic or aspartic acid. Thermal proteins and polyglutamic acid acted in a specific manner since, under the same experimental conditions, many control peptides, proteins and growth hormones failed to promote survival of neurons. Furthermore, their effects were antagonized by heparin, but not heparan sulfate nor chondroitin sulfate. These findings suggest that sequences of successive dicarboxylic amino acid residues are able to promote survival and neurite elongation of cultured neurons and that such sequences are responsible for the survival promoting action of thermal proteins. They invite the speculation that sequences of successive dicarboxylic amino acids, while occur in many proteins and show a high degree of evolutionary conservation, may have functional role in molecular recognition processes during neuronal development.