Susan K. Burgess

A sensitive solution-hybridization assay was used to investigate the expression of genes encoding insulin-like growth factors I and II (IGF-I and -II) in the rat central nervous system (CNS). mRNAs for both IGFs are synthesized throughout the CNS of adult rats but exhibit distinct regional differences for each growth factor. IGF-I mRNA is 8-10 times more abundant in the cervical-thoracic spinal cord and in the olfactory bulb than in whole brain and is enriched 3-fold in the midbrain and cerebellum. IGF-II mRNA is minimally enriched in the medulla-pons and cerebellum but is 3-5 times less abundant in the midbrain and corpus striatum than in total brain. During CNS development the content of IGF-I and IGF-II mRNAs is highest at embryonic day 14 and declines by a factor of 3-4 at birth, to values found in adult brain. Embryonic neurons and glia synthesize IGF-I mRNA during short-term cell culture; only glia produce IGF-II mRNA. These observations show that IGF-I and IGF-II are differentially expressed in the developing and adult CNS and suggest that each growth factor may play a unique role in the mammalian nervous system.

Primary neuronal cultures from fetal rat brain were utilized to investigate the possible role of insulin-like growth factor I (IGF-I) in neuronal growth and differentiation. 125I-IGF-I binding to intact cultured neurons was specific and saturable with an apparent Kd of 7.0 +/- 1.2 nM and a Bmax of 1.8 +/- 0.3 pmol/mg protein. Binding of 125I-IGF-I to neurons was inhibited by IGF-I, followed by IGF-II and insulin. 7 S nerve growth factor, but not beta-nerve growth factor, also inhibited 125I-IGF-I binding. A similar binding site was detected on brain membranes. Affinity cross-linking of 125I-IGF-I to intact cultured neurons revealed, under reducing conditions, a major binding moiety with an Mr of 115,000 and a minor component at Mr 260,000. The former represents a neuronal type of the IGF-I receptor alpha subunit, whereas the latter probably represents an alpha dimer. The Mr = 115,000 binding component for 125I-IGF-I was also present in membranes prepared from postnatal whole brain. In contrast, the binding moiety in cultured glial cells was of Mr = 135,000, which was identical to the IGF-I receptor alpha subunit of placenta. Thus mature brain, despite its cellular heterogeneity, expresses a structural subtype of IGF-I receptor which appears to be unique to differentiated neurons. Moreover, glial and neuronal cultures secreted a polypeptide which specifically bound IGF-I; the apparent Mr of this binding protein was determined by affinity cross-linking to be approximately 35,000. The presence of neuronal IGF-I receptors and binding proteins suggested that IGF-I may exert neurotrophic effects on developing neurons. This possibility was supported by the observation that IGF-I markedly stimulated neuronal RNA synthesis.

Embryonic rat neurons cultured in defined medium, essentially in the absence of glia, were highly enriched in phorbol ester receptors. The neurons displayed a single class of phorbol 12,13-dibutyrate binding sites with a maximum binding capacity, after 10 d in culture, of 18.6 pmol/mg protein and an apparent dissociation constant of 7.1 nM. Phorbol ester binding sites were associated with protein kinase C, which represented a major protein kinase activity in primary neuronal cultures. Ca2+-phosphatidylserine-sensitive phosphorylation of endogenous substrates was more marked than that observed in the presence of cyclic AMP or Ca2+ and calmodulin. Phorbol ester receptors and protein kinase C levels were critically dependent on the culture age. Thus, about a 20-fold increase in binding sites occurred during the first week in culture and was accompanied by a corresponding increase in Ca2+-phosphatidylserine-sensitive protein phosphorylation in soluble neuronal extracts. These changes largely paralleled a similar rise in phorbol ester binding during fetal development in vivo. The apparent induction of phorbol ester receptors was specific relative to other cellular proteins and could be inhibited by cycloheximide or Actinomycin D. Phosphorylation of endogenous substrates in intact cultured neurons paralleled the age-dependent increase in protein kinase C. Furthermore, 32P incorporation into several major phosphoproteins was markedly augmented by treating the neuronal cultures with phorbol esters. Such phosphorylation events may provide a clue to the significance of protein kinase C in developing neurons.